Antibody-pyrrolobenzodiazepine derivative conjugate

Abstract

To provide a novel antibody-drug conjugate having strong antitumor activity.SOLUTION: The present invention provides a novel antibody-pyrrolodiazepine derivative and a novel antibody-pyrrolodiazepine derivative conjugate using the same, and a novel CLDN6 and / or CLDN9 antibody.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] Antibody-drug conjugates (ADCs) are antibodies that bind to antigens expressed on the surface of cancer cells and internalize the antigens into the cells, and are combined with drugs that have cytotoxic activity. ADCs can efficiently deliver drugs to cancer cells, and are expected to accumulate the drugs in the cancer cells and kill them. For example, ADCs such as Adcetris™ (brentuximab vedotin), which combines monomethyl auristatin E with an anti-CD30 monoclonal antibody, have been approved for the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma. In addition, Kadcyla™ (trastuzumab emtansine), which combines emtansine with an anti-HER2 monoclonal antibody, is used to treat HER2-positive advanced or recurrent breast cancer. One of the useful drugs to be bound to ADCs is pyrrolobenzodiazepine (PBD). PBDs exhibit cytotoxicity by binding to the PuGPu sequence in the DNA minor groove. Anthramycin, a naturally occurring PBD, was first discovered in 1965, and since then, various naturally occurring and analogous PBDs have been discovered (Non-Patent Documents 1 to 4). The general structural formula of PBD is as follows: [ka] PBDs differ in the number, type, and position of the substituents in the A and C rings, and are known to have different degrees of unsaturation in the B and C rings. It is known that the cytotoxicity of PBD is dramatically improved by forming a dimer structure (Non-Patent Documents 5 and 6), and various ADCs of dimeric PBDs have been reported (Patent Documents 1 to 13). However, no PBDs having a spiro ring at the C2 position or their ADCs are known. Human CLDN6 (Claudin-6, hereinafter referred to as hCLDN6) is a member of the Claudin (CLDN) family of proteins, and is a four-transmembrane protein consisting of 220 amino acid residues. It has been suggested that hCLDN6 is overexpressed in some cancers and is an attractive target for cancer therapy (Non-Patent Documents 7 to 9). In addition, CLDN family proteins are taken up into cells by endocytosis, and some family proteins with short turnover times have been reported (Non-Patent Document 10), making it suitable as a target for antibody-drug conjugates (ADCs). Based on information suggesting such an association with cancer, monoclonal antibodies that specifically recognize hCLDN6 have been discovered (Patent Documents 14 and 15), and ADCs have been reported in which tubulin polymerization inhibitors monomethylauristatin E (MMAE) or maytansinoid (DM1) are linked to a CLDN6-specific monoclonal antibody (Non-Patent Document 11). On the other hand, an ADC has been disclosed in which a pyrrolobenzodiazepine (PBD), which has a strong cytocidal effect, is linked to an antibody that recognizes CLDN6 and CLDN9 (Patent Document 16), as an antibody that recognizes multiple CLDN families may be able to broaden the range of therapeutic applications (Patent Document 17). However, their activity is still not sufficient, and there exists an unmet medical need to target hCLDN6 therapeutically. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2013 / 173496 [Patent Document 2] International Publication No. 2014 / 130879 [Patent Document 3] International Publication No. 2017 / 004330 [Patent Document 4] International Publication No. 2017 / 004025 [Patent Document 5] International Publication No. 2017 / 020972 [Patent Document 6] International Publication No. 2016 / 036804 [Patent Document 7] International Publication No. 2015 / 095124 [Patent Document 8] International Publication No. 2015 / 052322 [Patent Document 9] International Publication No. 2015 / 052534 [Patent Document 10] International Publication No. 2016 / 115191 [Patent Document 11] International Publication No. 2015 / 052321 [Patent Document 12] International Publication No. 2015 / 031693 [Patent Document 13] International Publication No. 2011 / 130613 [Patent Document 14] International Publication No. 2009 / 087978 [Patent Document 15] International Publication No. 2011 / 057788 [Patent Document 16] International Publication No. 2015 / 069794 [Patent Document 17] International Publication No. 2017 / 096163 [Non-patent literature]

[0004] [Non-Patent Document 1] Julia Mantaj,et al.,Angewandte Chemie Internationl Edition 2016,55,2-29 [Non-Patent Document 2] Dyeison Antonow.et al., Chemical Reviews 2010,111, 2815-2864 [Non-Patent Document 3] In Antibiotics III. Springer Verlag, New York, pp.3-11 [Non-Patent Document 4] Accounts of Chemical Research 1986, 19, 230 [Non-Patent Document 5] Journal of the American Chemical Society 1992,114, 4939 [Non-Patent Document 6] Journal of Organic Chemistry 1996, 61, 8141 [Non-Patent Document 7] BMC Cancer, 2006, 6, 186. [Non-Patent Document 8] Histopathology, 2012, 61, 1043-1056. [Non-Patent Document 9] Int J Cancer, 2014, 135, 2206-2214. [Non-Patent Document 10] J Membrane Biol, 2004, 199, 29-38. [Non-Patent Document 11] 14th Annu Meet Cancer Immunother(CIMT)(May 10-12, Mainz) 2016, Abst 185 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugates and novel pyrrolobenzodiazepine (PBD) derivatives. The present invention provides a novel anti-CLDN6 antibody. The present invention also provides a pharmaceutical composition containing the antibody-PBD derivative conjugate, PBD derivative, or anti-CLDN6 antibody having anti-tumor activity. Furthermore, the present invention provides a method for treating cancer using the antibody-PBD derivative conjugate, the PBD derivative, or the anti-CLDN6 antibody. [Means for solving the problem]

[0006] As a result of extensive investigations, the present inventors have found that a novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate has strong antitumor activity, and have completed the present invention. That is, the present invention relates to the following:

[0007] [1] The following formula: [ka] (In the formula, m 1 represents an integer from 1 to 10, preferably an integer from 2 to 8), Ab refers to an antibody or a functional fragment of the antibody, and the glycan of the antibody may be remodeled. L represents a linker connecting Ab and D, Ab may be linked to L directly through its amino acid residue or through a glycan or remodeled glycan of Ab; D represents a drug represented by the following formula: [ka] In the formula, the asterisk indicates a bond to L. n 1 denotes an integer from 2 to 8, A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or 3- to 5-membered saturated heterocycle which may be substituted with 1 to 4 halogen atoms; R 1 and R 2each independently represents a C1-C6 alkoxy group, a C1-C6 alkyl group, a hydrogen atom, a hydroxyl group, a thiol group, a C1-C6 alkylthio group, a halogen atom, or -NR'R'', Here, R' and R'' each independently represent a hydrogen atom or a C1-C6 alkyl group. R 3 , R 4 and R 5 is selected from the following (i) to (iii): (i)R 3 and R 4 together form a double bond with the carbon atom to which each group is attached, R 5 represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 1, or a C1-C6 alkyl group which may have one or more substituents selected from Group 2, (ii)R 3 indicates a hydrogen atom, R 4 and R 5 But R 4 and R 5 together with the carbon atom to which it is bonded, form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring or CH 2 = Form, (iii)R 3 , R 4 and R 5 But R 3 The carbon atom to which R is bonded 4 and R 5 is combined with the carbon atom to which it is bonded to form a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from Group 3. R 6 and R 7 Both represent hydrogen atoms or R 6 and R 7 together indicate an imine bond (C=N), R 8 is a hydroxyl group or a C1-C3 alkoxy group, X and Y are each independently an oxygen atom, a nitrogen atom, or a sulfur atom; Group 1 includes: a) a C1-C6 alkoxy group optionally substituted by 1 to 3 halogen atoms; b) a C1-C6 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3 to C5 cycloalkoxy group, e) a C1 to C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i) -NHCOR', or j) represents a hydroxyl group; where R' and R'' are as defined above, and R''' each independently represents a hydrogen atom or a C1-C6 alkyl group; Group 2 represents a halogen atom, a hydroxyl group, or a C1-C6 alkoxy group, and Group 3 represents a halogen atom, or a C1-C6 alkyl group or a C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms. The antibody-drug conjugate shown in FIG. [2] A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 1 and R 2 each independently represents a C1-C3 alkoxy group; R 3 and R 4 together form a double bond with the carbon atom to which each group is attached, R 5 represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 4, or a C1-C3 alkyl group which may have one or more substituents selected from Group 5, X and Y are oxygen atoms, Group 4 includes a) a C1-C3 alkoxy group optionally substituted by 1 to 3 halogen atoms, b) a C1-C3 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR'', -C(=NR')-NR''R'''', and -NHC(=NR')-NR''R'''', c) a C3 to C5 cycloalkoxy group, d)-C(=NR')-NR''R'''', e) -NHC(=NR')-NR''R'''', or f) represents a hydroxyl group; where R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, and Group 5 represents a halogen atom, a hydroxyl group, or a C1-C3 alkoxy group. The antibody-drug conjugate according to [1]. [3] A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 1 and R 2 each independently represents a C1-C3 alkoxy group; R 3 represents a hydrogen atom, R 4 and R 5 But R 4 and R 5 together with the carbon atom to which it is attached form a 3-5 membered saturated hydrocarbon ring or =CH 2 and The antibody-drug conjugate according to [1], wherein X and Y are oxygen atoms. [4] A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 1 and R 2 each independently represents a C1-C3 alkoxy group; R 3 , R 4 and R 5 But R 3The carbon atom to which R is bonded 4 and R 5 is bonded to the carbon atom to form a benzene ring which may have one or more substituents selected from Group 6, X and Y are oxygen atoms, and Group 6 represents a halogen atom, or a C1-C3 alkyl group or a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms. The antibody-drug conjugate according to [1]. [5] D is expressed by one of the following two formulas: [ka] In the formula, an asterisk indicates binding to L. An antibody-drug conjugate according to [1] or [2]. [6] D is represented by one of the following two formulas: [ka] In the formula, an asterisk indicates binding to L. An antibody-drug conjugate according to [1] or [3]. [7] L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, In the formula, the asterisk indicates a bond to D. B represents a phenyl group or a heteroaryl group; Lp represents a linker consisting of an amino acid sequence cleavable in a target cell; La is the following group: -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2-C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -NH-C(=O)-(CH 2 CH 2 O)n 3 -CH 2 CH 2 -C(=O)- and -(CH 2 )n 4 -OC(=O)-, indicates one selected from n 2 is an integer between 1 and 3, n 3 is an integer between 1 and 5, n 4 represents an integer from 0 to 2, The antibody-drug conjugate according to any one of [1] to [6], wherein Lb represents a spacer that bonds La to the glycan or remodeled glycan of Ab. [8] The antibody-drug conjugate according to [7], wherein B is any one selected from a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, and a 2,5-thienyl group. [9] The antibody-drug conjugate according to [8], wherein B is a 1,4-phenyl group.

[10] The antibody-drug conjugate according to any one of [7] to [9], wherein Lp is an amino acid residue consisting of 2 to 7 amino acids.

[11] The antibody-drug conjugate according to any one of [7] to

[10] , wherein Lp is an amino acid residue consisting of an amino acid selected from glycine, valine, alanine, phenylalanine, glutamic acid, isoleucine, proline, citrulline, leucine, serine, lysine and aspartic acid.

[12] Lp is in the following groups: The antibody-drug conjugate according to any one of [7] to

[11] , which is selected from -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, and -GGPL-.

[13] La is selected from the group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)- and -OC(=O)- The antibody-drug conjugate according to any one of [7] to

[12] , selected from the following:

[14] Lb is expressed by the following formula: [ka] [ka] or [ka] The antibody-drug conjugate according to any one of [7] to

[13] , wherein in the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the glycan or remodeled glycan of Ab.

[15] L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)-* is shown in In the formula, B is a 1,4-phenyl group; Lp is a member of the following groups: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-, indicates one selected from La is the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)- and -OC(=O)-, and Lb is expressed by the following formula: [ka] [ka] or [ka] Here, in the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the glycan or remodeled glycan of Ab, an antibody-drug conjugate according to any one of [7] to

[14] .

[16] L is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)-、and -Z 3 -CH 2 -OC(=O)-GGVA-NH-B-CH 2 selected from -OC(=O)-, where Z 1 has the following structural formula: [ka] indicates, Z 2 has the following structural formula: [ka] indicates, Z 3 has the following structural formula: [ka] indicates, Here, Z 1 , Z 2 and Z 3 In the structural formula of the formula, the asterisk indicates binding to La, the wavy line indicates binding to the glycan of Ab or the remodeled glycan, and The antibody-drug conjugate according to any one of [7] to

[15] , wherein B represents a 1,4-phenyl group.

[17] L is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2-C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, where B is a 1,4-phenyl group and Z 1 has the following structural formula: [ka] indicates, Here, Z 1 In the structural formula, the asterisk represents Z 1 The antibody-drug conjugate described in

[16] , wherein the wavy line indicates binding to the C(=O) adjacent to the A, and the wavy line indicates binding to the glycan of the Ab or a remodeled glycan.

[18] L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, In the formula, the asterisk indicates a bond to D. B represents a 1,4-phenyl group; Lp represents -GGVA- or -VA; La is -(CH 2 )n 9 -C(=O)- or -(CH 2 CH 2 )n10 -C(=O)-NH-(CH 2 CH 2 O)n 11 -CH 2 CH 2 -C(=O)-, n 9 is an integer between 2 and 7, n 10 is an integer between 1 and 3, n 11 represents an integer between 6 and 10, The antibody-drug conjugate according to any one of [1] to [6], wherein Lb is -(Succinimid-3-yl-N)-.

[19] L is one of the following groups: -(Succinimid-3-yl-N)-(CH 2 ) 5 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -(Succinimid-3-yl-N)-(CH 2 ) 5 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, and -(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- indicates that the number is one selected from the The antibody-drug conjugate according to

[18] , wherein B is a 1,4-phenyl group.

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

[19] , wherein the antibody is an IgG.

[21] The antibody-drug conjugate according to

[20] , wherein the antibody is IgG1, IgG2 or IgG4.

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

[21] , wherein the antibody binds to a tumor cell and is taken up and internalized within the tumor cell.

[23] The antibody-drug conjugate according to

[22] , wherein the antibody further has an antitumor effect.

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

[17] and

[20] to

[23] , wherein the antibody is bound to L via a glycan that binds to Asn297 of the antibody (N297 glycan).

[25] The antibody-drug conjugate according to

[24] , wherein the N297 glycan is a remodeled glycan.

[26] The antibody-drug conjugate according to

[24] or

[25] , wherein the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has a structure represented by the following formula: [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, where the amino group at the right end is amide bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 side of the branched chain of β-Man in the N297 glycan, The asterisk indicates the connection to the linker L, and n 5 indicates an integer from 2 to 10; [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, where the amino group at the right end is amide-bonded to the 2-position carboxylic acid of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 glycan, The asterisk indicates the connection to the linker L, and n 5 represents an integer of 2 to 10; and [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, where the amino group at the right end is amide-bonded to the 2-position carboxylic acid of the sialic acid at the non-reducing end of both the 1-3 and 1-6 chains of the branched β-Man chain of the N297 sugar chain, The asterisk indicates the connection to the linker L, and n 5 represents an integer from 2 to 10.

[27] 5 The antibody-drug conjugate according to

[26] , wherein

[28] The following formula: [ka] (In the formula, m 2 indicates 1 or 2, L is a linker connecting the N297 glycan of Ab and D, and is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- is any one of the linkers selected from where B is a 1,4-phenyl group and Z 1 has the following structural formula: [ka] indicates, Here, Z shown above 1 In the structural formula, the asterisk represents Z 1 The wavy line indicates that the glycan is bound to the N297 glycan of Ab. Ab represents an IgG antibody or a functional fragment of said antibody; The N297 sugar chain of Ab is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula: [ka] [ka] [ka] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 )n 5 - indicates *, where n 5 indicates an integer of 2 to 5, the amino group at the left end is amide-bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side or / and 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, and an asterisk indicates Z in the linker L. 1 represents that the ring is bonded to the nitrogen atom at the 1st or 3rd position on the triazole ring of D is any one selected from the following group: [ka] In the formula, the asterisk indicates a bond to L. The antibody-drug conjugate according to any one of

[25] to

[28] ,

[30] The following groups: [ka] [ka] [ka] [ka] Selected from In each of the structural formulas shown above, m 2 indicates 1 or 2, Ab indicates an IgG antibody or a functional fragment thereof; The N297 sugar chain of Ab is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each having the structure shown in the following formula: [ka] [ka] [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 -* indicates that Here, the amino group at the left end indicates that it is amide-bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man of the N297 glycan, and the asterisk indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring in each structural formula, antibody-drug conjugate.

[30] An antibody or a functional fragment of the antibody that binds to CLDN6 and / or CLDN9.

[31] The antibody or functional fragment of the antibody described in

[30] , wherein CLDN6 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 1, and CLDN9 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 3.

[32] The antibody or functional fragment of the antibody according to

[30] or

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

[33] The antibody or a functional fragment of the antibody according to

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

[34] The antibody or a functional fragment of the antibody according to any one of

[30] to

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

[35] The antibody or functional fragment of the antibody according to any one of

[30] to

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

[36] The antibody or a functional fragment of the antibody according to

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

[37] The antibody or a functional fragment of the antibody according to any one of

[30] to

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

[38] The antibody or a functional fragment of the antibody according to any one of

[30] to

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

[39] The antibody or a functional fragment of the antibody according to any one of

[30] to

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

[40] The antibody or functional fragment of the antibody according to

[38] or

[39] , comprising a heavy chain and a light chain selected from the group consisting of the following (a) to (e): (a) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52 and a light chain (H1L1) consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 36, (b) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56, and a light chain (H2L2) consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 40; (c) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52 and a light chain (H1L3) consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44; (d) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56 and a light chain (H2L4) consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 48; and (e) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 60, and a light chain (H3L3) consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44.

[41] An antibody or a functional fragment of the antibody described in

[30] or

[31] , which binds to a site on an antigen recognized by the antibody described in any one of

[32] to

[36] and

[40] .

[42] The antibody or functional fragment of the antibody described in

[30] or

[31] , which competes with the antibody described in any one of

[32] to

[36] and

[40] in binding to CLDN6 and / or CLDN9.

[43] A polynucleotide encoding the antibody described in any one of

[30] to

[42] .

[44] The polynucleotide according to

[43] , comprising a polynucleotide selected from the group consisting of the following (a) to (j): (a) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 38; (b) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42; (c) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 54, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 46; (d) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 50; (e) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 62, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 46; (f) a polynucleotide encoding a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52, and a polynucleotide encoding a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 36; (g) a polynucleotide encoding a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56, and a polynucleotide encoding a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 40; (h) a polynucleotide encoding a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 52, and a polynucleotide encoding a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44; (i) a polynucleotide encoding a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 56, and a polynucleotide encoding a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 48, and (j) a polynucleotide encoding a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 60, and a polynucleotide encoding a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 44.

[45] An expression vector comprising the polynucleotide described in any one of

[43] and

[44] .

[46] A host cell transformed with an expression vector according to

[45] .

[47] The host cell according to

[46] , wherein the host cell is a eukaryotic cell.

[48] ​​The host cell according to

[47] , wherein the host cell is an animal cell.

[49] A method for producing an antibody or a functional fragment of the antibody described in

[30] to

[42] , comprising the steps of culturing a host cell described in any one of

[46] to

[48] , and collecting the antibody of interest from the culture obtained in the step.

[50] An antibody or a functional fragment of the antibody, characterized in that it is obtained by the production method described in

[49] .

[51] The antibody or functional fragment of the antibody according to any one of

[30] to

[42] and

[50] , comprising 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.

[52] The antibody or functional fragment of the antibody according to

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

[53] The antibody or functional fragment of the antibody according to

[52] , wherein one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains.

[54] The antibody or functional fragment of the antibody according to any one of

[50] to

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

[55] The following steps: i) culturing the host cell according to any one of

[46] to

[48] and collecting the antibody of interest from the resulting culture; ii) treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; and iii)-1 A step of reacting a glycan donor molecule obtained by introducing an azide group-containing PEG linker into the carbonyl group of the carboxylate at the 2-position of the sialic acid of MSG(9) or SG(10) and oxazolinating the reducing end with a (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase, or iii)-2 A method for producing a glycan-remodeling antibody, comprising the steps of: introducing a PEG linker having an azide group into the carbonyl group of the carboxylic acid at the 2-position of the sialic acid of (MSG-)Asn or (SG-)Asn, whose α-amino group may be protected, and the carbonyl group of the carboxylic acid of the Asn; treating the resulting product with a hydrolase; and then oxazolinating the reducing end to obtain a glycan donor molecule, and reacting the resulting molecule with a (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase.

[56] The method according to

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

[57] The following steps: i) producing a glycan remodeling antibody by the method according to

[55] or

[56] ; and ii) a method for producing an antibody-drug conjugate according to any one of [1] to

[29] , comprising a step of reacting a drug linker (production intermediate) having DBCO with an azide group in the glycan of the glycan remodeling antibody produced in step i).

[58] A glycan remodeling antibody, characterized in that it is obtained by the production method described in

[55] or

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

[59]

[57] .

[60] Next group: [ka] [ka] [ka] or [ka] Selected from In each of the structural formulas shown above, m 2 indicates 1 or 2, Ab indicates that the antibody or a functional fragment of the antibody according to any one of

[30] to

[42] ,

[50] to

[54] , and

[58] , or an anti-HER2 antibody, The N297 sugar chain of Ab is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, each of which has the structure shown in the following formula: [ka] [ka] [ka] In the formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 -* indicates that the amino group at the left end is amide-bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side of the branched chain of β-Man in the N297 glycan, and the asterisk indicates that the bond is to the 1- or 3-position nitrogen atom on the triazole ring in each structural formula, antibody-drug conjugate.

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

[29] ,

[59] to

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

[62] The following formula [ka] (In the formula, l represents an integer of 2 to 8, E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle which may be substituted with 1 to 4 halogen atoms; R 9 and R 10 each independently represents a C1-C6 alkoxy group, a C1-C6 alkyl group, a hydrogen atom, a hydroxyl group, a thiol group, a C1-C6 alkylthio group, a halogen atom, or -NR'R'', Here, R' and R'' each independently represent a hydrogen atom or a C1-C6 alkyl group. R 11 , R 12 and R 13 is selected from the following (i) to (iii): (i)R 11 and R 12together form a double bond with the carbon atom to which each group is attached, R 13 represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 7, or a C1-C6 alkyl group which may have one or more substituents selected from Group 8, (ii)R 11 indicates a hydrogen atom, R 12 and R 13 are taken together to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, or CH 2 = or (iii)R 11 and R 12 are taken together to form a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from Group 9; R 13 indicates a single bond, R 14 and R 15 Both represent hydrogen atoms or R 14 and R 15 together indicate an imine bond (C=N), R 16 and R 17 indicates either (a) or (b) below, (a)R 16 and R 17 together form an imine bond (N=C), (b)R 16 is J-La'-Lp'-NH-B'-CH 2 -O(C=O)-* where the asterisk represents R 16 indicates that the nitrogen atom is adjacent to the B' represents a phenyl group or a heteroaryl group; Lp' represents a linker consisting of an amino acid sequence cleavable in a target cell, La' is the following group: -C(=O)-(CH 2 CH 2 )n 6-C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -C(=O)-NH-(CH 2 CH 2 )n 7 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -C(=O)-NH-(CH 2 CH 2 O)n 7 -CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -NH-C(=O)-(CH 2 CH 2 O)n 7 -CH 2 CH 2 -C(=O)-, -(CH 2 )n 8 -OC(=O)-, -(CH 2 )n 12 -C(=O)- and -(CH 2 CH 2 )n 13 -C(=O)-NH-(CH 2 CH 2 O)n 14 -CH 2 CH 2 -C(=O)- indicates one selected from Wherein, n 6 is an integer between 1 and 3, n 7 is an integer between 1 and 5, n 8 is an integer between 0 and 2, n 12 is an integer between 2 and 7, n 13 is an integer between 1 and 3, n 14 represents an integer between 6 and 10, J represents one of the following: [ka] In the structural formula of J shown above, the asterisk indicates that it is bonded to La', R 17 represents a hydroxyl group or a C1-C3 alkoxy group, V and W are each independently an oxygen atom, a nitrogen atom, or a sulfur atom; Group 7 includes: a) a C1-C6 alkoxy group optionally substituted by 1 to 3 halogen atoms, b) a C1-C6 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3 to C5 cycloalkoxy group, e) a C1 to C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i) -NHCOR', or j) represents a hydroxyl group; where R' and R'' are as defined above, and R''' each independently represents a hydrogen atom or a C1-C6 alkyl group; Group 8 represents a halogen atom, a hydroxyl group, or a C1-C6 alkoxy group, and Group 9 represents a halogen atom, or a C1-C6 alkyl group or a C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms. A compound represented by the formula:

[63] E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 9 and R 10 each independently represents a C1-C3 alkoxy group; R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, R13 represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 10, or a C1-C3 alkyl group which may have one or more substituents selected from Group 11, V and W are oxygen atoms; Group 10 includes: a) a C1-C3 alkoxy group optionally substituted by 1 to 3 halogen atoms; b) a C1-C3 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR'', -C(=NR')-NR''R'''', and -NHC(=NR')-NR''R'''', c) a C3 to C5 cycloalkoxy group, d)-C(=NR')-NR''R'''', e) -NHC(=NR')-NR''R'''', or f) represents a hydroxyl group; where R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, and Group 11 is the compound according to

[62] , a salt thereof, or a hydrate of the compound or the salt, wherein R represents a halogen atom, a hydroxyl group, or a C1-C3 alkoxy group.

[64] E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 9 and R 10 each independently represents a C1-C3 alkoxy group; R 11 represents a hydrogen atom, R 12 and R 13 But R 12 and R 13 together with the carbon atom to which it is attached form a 3- to 5-membered saturated hydrocarbon ring or CH 2 = and The compound, salt, or hydrate of the compound or salt according to

[62] , wherein V and W are oxygen atoms.

[65] E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring optionally substituted with 1 to 2 halogen atoms; R 9 and R 10 each independently represents a C1-C3 alkoxy group; R 11 , R 12 and R 13 But R 11 The carbon atom to which R is bonded 12 and R 13 is bonded to the carbon atom to form a benzene ring which may have one or more substituents selected from Group 12, V and W are oxygen atoms, and Group 12 represents a halogen atom, or a C1-C3 alkyl group or a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms, the compound according to

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

[66] The compound, its salt, or a hydrate of the compound or the salt according to any one of

[62] to

[65] , wherein B' is any one selected from a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, and a 2,5-thienyl group.

[67] The compound, salt thereof, or hydrate thereof according to

[66] , wherein B' is a 1,4-phenyl group.

[68] Lp' is selected from the group consisting of: The compound, salt thereof, or hydrate thereof according to any one of

[62] to

[67] , wherein the amino acid residue is selected from -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, and -GGPL-.

[69] La' is a member of the group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)-, -OC(=O)-, -(CH 2 ) 5 -C(=O)- and -CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)- The compound, salt thereof, or hydrate thereof according to any one of

[62] to

[68] , selected from:

[70] R 16 But J-La'-Lp'-NH-B'-CH 2 -O(C=O)-* is shown in In the formula, B' is a 1,4-phenyl group; Lp' represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-, La' represents one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)-, -OC(=O)-, -(CH 2 ) 5 -C(=O)- and -CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)- ,and, J represents one of the following: [ka] The compound, salt, or hydrate thereof according to any one of

[62] to

[69] , wherein in the structural formula of J, an asterisk indicates that J is bonded to La'.

[71] R 16 But the following group: J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B'-CH 2 -OC(=O)-, J 1-C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O)2 -CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 2 -OC(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 3 -CH 2 -OC(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, and J 4 -CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)- is selected from Here, J 1 , J 2 , J 3 and J 4 has the following structural formula: [ka] indicates, Here, J1 , J 2 , J 3 and J 4 In the structural formula of The compound, salt, or hydrate of any one of

[62] to

[70] , wherein B' is a 1,4-phenyl group.

[72] R 16 But the following group: J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, and J 4 -(CH 2 ) 5 -C(=O)-VA-NH-B'-CH 2 -OC(=O)- is selected from where B' is a 1,4-phenyl group; J 1 , J 4 has the following structural formula: [ka] indicates, Here, J 1 and J 4 In the structural formula, an asterisk indicates a bond to an adjacent group, or a compound, a salt thereof, or a hydrate thereof according to any one of

[62] to

[71] .

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

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

[75] D is a group having the formula: [ka] The antibody-drug conjugate according to any one of [1] to

[29] and

[59] to

[61] ,

[76] The following formula: [ka] The compound, its salt, or a hydrate of the compound or the salt according to any one of

[62] to

[72] and

[74] ,

[77] D is a group having the formula: [ka] The antibody-drug conjugate according to any one of [1] to

[29] and

[59] to

[61] ,

[78] The following formula: [ka] A compound, a salt thereof, or a hydrate thereof according to any one of

[62] to

[72] and

[74] , which is represented by the following formula:

[79] A pharmaceutical composition comprising any one selected from the group consisting of an antibody-drug conjugate described in any one of [1] to

[29] ,

[59] to

[61] ,

[75] and

[77] , a salt thereof, or a hydrate thereof, an antibody or a functional fragment of the antibody described in any one of

[30] to

[42] ,

[50] to

[54] and

[58] , and a compound described in any one of

[62] to

[74] ,

[76] and

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

[80] The pharmaceutical composition according to any one of

[79] , which is an antitumor drug.

[81] The pharmaceutical composition described in

[80] , wherein the tumor is a tumor expressing CLDN6 and / or CLDN9.

[82] The pharmaceutical composition according to

[80] or

[81] , wherein the tumor is ovarian cancer (superficial epithelial tumor, stromal tumor, germ cell tumor), lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, uterine body cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, testicular cancer, placental choriocarcinoma, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer or esophageal cancer.

[83] A method for treating a tumor, comprising administering to an individual any one selected from the antibody-drug conjugate described in any one of [1] to

[29] ,

[59] to

[61] ,

[75] and

[77] , a salt thereof, or a hydrate thereof, the antibody or a functional fragment of the antibody described in any one of

[30] to

[42] ,

[50] to

[54] and

[58] , and the compound described in any one of

[62] to

[74] ,

[76] and

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

[84] The method for treating a tumor described in

[83] , wherein the tumor is a tumor expressing CLDN6 and / or CLDN9.

[85] The method of treatment according to

[83] or

[84] , wherein the tumor is ovarian cancer (superficial epithelial tumor, stromal tumor, germ cell tumor), lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, uterine body cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, testicular cancer, placental choriocarcinoma, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer or esophageal cancer.

[86] A method for treating a tumor, comprising administering to an individual simultaneously, separately or consecutively a pharmaceutical composition comprising at least one selected from the antibody-drug conjugate described in any one of [1] to

[29] ,

[59] to

[61] ,

[75] and

[77] , a salt thereof, or a hydrate thereof, the antibody or a functional fragment of the antibody described in any one of

[30] to

[42] ,

[50] to

[54] and

[58] , and the compound described in any one of

[62] to

[74] ,

[76] and

[78] , a salt thereof, or a hydrate thereof, and at least one antitumor drug.

[87] A compound that exhibits a proton NMR spectrum having peak positions substantially similar to those listed in Table 1 or Table 2. Effect of the Invention

[0008] The novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate provided by the present invention has excellent antitumor activity and safety, and is therefore useful as an antitumor agent. In addition, the PBD derivative of the present invention has antitumor activity and is useful as a drug for the conjugate. Furthermore, the antibody of the present invention recognizes or binds to tumor cells, and is therefore useful as an antibody for the conjugate. [Brief description of the drawings]

[0009] [Figure 1] Schematic representation of the drug-conjugate of the present invention (molecule (I)). (a) represents drug D, (b) represents linker L, (c) represents N3-L(PEG)-, and (d) represents N297 sugar chain (wherein the white oval represents NeuAc(Sia), the white hexagon represents Man, the filled hexagon represents GlcNAc, the white diamond represents Gal, and the white inverted triangle represents Fuc). (b) and (c) are linked by reaction of the azide group (black teardrop) in (c) with the alkyne structure in the spacer (white semicircle) in (b) to form a triazole ring. The Y-shape represents the antibody Ab. In addition, in this schematic diagram, the N297 sugar chain is conveniently displayed as N297-(Fuc)MSG, and an embodiment is shown in which only one branched chain of each N297 sugar chain has sialic acid bound to a PEG linker having an azide group (N3-L(PEG)-) and the other branched chain has no sialic acid at the non-reducing end, but by adopting N297-(Fuc)SG, an embodiment in which both branched chains have sialic acid bound to a PEG linker having an azide group at the non-reducing end may also be used. Such a display method is applied throughout the present specification unless otherwise specified. [Diagram 2]2A and 2B are schematic diagrams showing the structures of (Fucα1,6)GlcNAc-antibody (molecule (II) in A of FIG. 2) and MSG-type glycan remodeling antibody (molecule (III) in B of FIG. 2), which are intermediates in the production of the drug-conjugate of the present invention. In both figures, the Y-shape represents the antibody Ab, as in FIG. 1. In A of FIG. 2, (e) represents the N297 glycan consisting only of GlcNAc linked by α-glycosidic bond at position 1 and position 6 of Fuc. In B of FIG. 2, (d) represents the same N297 glycan as in FIG. 1, and (f) represents the structure of the PEG linker portion having an azide group, which is used for binding to the linker L at the end. The binding mode of the PEG linker having an azide group is the same as that described in FIG. 1. [Diagram 3] This is a schematic diagram of the process of producing an MSG-type glycan remodeling antibody from an antibody produced in an animal cell. Molecules (II) and (III) in the figure represent (Fucα1,6)GlcNAc-antibody and MSG-type glycan remodeling antibody, respectively, as in FIG. 2. Molecule (IV) is an antibody produced in an animal cell, and is a mixture of molecules with heterogeneous N297 glycans. FIG. 3A shows the process of producing a homogeneous (Fucα1,6)GlcNAc-antibody (II) by treating the heterogeneous N297 glycan of (IV) with a hydrolase such as EndoS. FIG. 3B shows the process of producing an MSG-type glycan remodeling antibody (III) by glycosyltransferring the glycan of an MSG-type glycan donor molecule to the GlcNAc of the N297 glycan of antibody (II) using a glycosyltransferase such as EndoS D233Q / Q303L mutant. The MSG-type glycan donor molecule used here is one in which the sialic acid at the non-reducing end of MSG is modified with a PEG linker having an azide group, and the sialic acid at the non-reducing end of the MSG-type N297 glycan remodeling antibody to be produced will also be similarly modified, as explained in Figure 2 B. In Figure 3 B, MSG is shown as the donor molecule for convenience, but by using SG (10) as the glycan donor, the remodeling antibody (III) is synthesized, in which a linker molecule having an azide group is bound to both non-reducing ends of the N297 glycan. [Figure 4]The effects of anti-HER2 antibody-drug conjugates ADC26, ADC19, and ADC54 on subcutaneously implanted human gastric cancer cell line NCI-N87 cells are shown. [Diagram 5] The effects of anti-HER2 antibody-drug conjugates ADC49, Trastuzumab, and anti-LPS antibody-drug conjugates ADC53 on subcutaneously implanted human gastric cancer cell line NCI-N87 cells are shown. [Figure 6] 1 shows the effects of anti-HER2 antibody-drug conjugate ADC49, anti-LPS antibody-drug conjugate ADC53, or Trastuzumab-Tesirine (Reference Example 1) on subcutaneously implanted KPL-4 cells, a human breast cancer line. [Figure 7] 1 shows the effect of anti-HER2 antibody-drug conjugate ADC49 or Trastuzumab-Tesirine (Reference Example 1) on subcutaneously implanted JIMT-1 cells, a human breast cancer line. [Figure 8] 1 shows the effect of anti-CLDN6 antibody-drug conjugate ADC40, or anti-CLDN6 antibody (H1L1)-Tesirine (Reference Example 1) on subcutaneously implanted OV-90 cells, a human ovarian cancer line. [Figure 9] 1 shows the effect of anti-CLDN6 antibody-drug conjugate ADC40, or anti-CLDN6 antibody (H1L1)-Tesirine (Reference Example 1) on subcutaneously implanted human ovarian cancer line, NIH:OVCAR-3 cells. [Figure 10] 1 shows the effect of antibody-drug conjugates, anti-TROP2 antibody-drug conjugate ADC50, or anti-LPS antibody-drug conjugate ADC53, on subcutaneously implanted FaDu cells, a human head and neck cancer cell line. [Figure 11] The full-length amino acid sequence of human CLDN6 (SEQ ID NO: 1) and the full-length cDNA nucleotide sequence (SEQ ID NO: 2) are shown. [Figure 12] The full-length amino acid sequence of human CLDN9 (sequence number 3) and the full-length cDNA nucleotide sequence (sequence number 4) are shown. [Figure 13] The amino acid sequences of CDRL1 to 3 of the B1 antibody light chain (SEQ ID NOs: 5 to 7) are shown. [Figure 14]The amino acid sequence of CDRL3 of humanized B1 antibody light chain L4 (sequence number 8) is shown. [Figure 15] The amino acid sequences of CDRH1 to 3 of the B1 antibody heavy chain (SEQ ID NOs: 9 to 11) are shown. [Figure 16] The amino acid sequences of CDRL1 to 3 of the C7 antibody light chain (SEQ ID NOs: 12 to 14) are shown. [Figure 17] The amino acid sequences of CDRH1 to 3 of the C7 antibody heavy chain (SEQ ID NOs: 15 to 17) are shown. [Figure 18] The nucleotide sequence of cDNA encoding the variable region of the B1 antibody light chain (SEQ ID NO: 18) and the amino acid sequence of the variable region of the B1 antibody light chain (SEQ ID NO: 19) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 19] The nucleotide sequence of cDNA encoding the variable region of the heavy chain of B1 antibody (SEQ ID NO: 20) and the amino acid sequence of the variable region of the heavy chain of B1 antibody (SEQ ID NO: 21) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 20] The nucleotide sequence of cDNA encoding the variable region of the C7 antibody light chain (SEQ ID NO: 22) and the amino acid sequence of the variable region of the C7 antibody light chain (SEQ ID NO: 23) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 21] The nucleotide sequence of cDNA encoding the variable region of the heavy chain of the C7 antibody (SEQ ID NO: 24) and the amino acid sequence of the variable region of the heavy chain of the C7 antibody (SEQ ID NO: 25) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 22] The amino acid sequence of the chB1 light chain (SEQ ID NO: 28) and a DNA fragment containing a DNA sequence encoding the amino acid sequence of the chB1 light chain (SEQ ID NO: 29) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 23] The amino acid sequence of the variable region of the chB1 light chain (SEQ ID NO: 30) and the nucleotide sequence encoding the chB1 light chain variable region (SEQ ID NO: 31) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 24]The amino acid sequence of the chB1 heavy chain (SEQ ID NO: 32) and the nucleotide sequence encoding the chB1 heavy chain (SEQ ID NO: 33) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Diagram 25] The amino acid sequence of the variable region of the chB1 heavy chain (SEQ ID NO: 34) and the nucleotide sequence encoding the variable region of the chB1 heavy chain (SEQ ID NO: 35) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 26] The amino acid sequence of the humanized antibody light chain hL1 (SEQ ID NO: 36) and the nucleotide sequence encoding the humanized antibody light chain hL1 (SEQ ID NO: 37) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Figure 27] The amino acid sequence of the variable region of the humanized antibody light chain hL1 (SEQ ID NO: 38) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL1 (SEQ ID NO: 39) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Figure 28] The amino acid sequence of the humanized antibody light chain hL2 (SEQ ID NO: 40) and the nucleotide sequence encoding the humanized antibody light chain hL2 (SEQ ID NO: 41) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 29] The amino acid sequence of the variable region of the humanized antibody light chain hL2 (sequence number 42) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL2 (sequence number 43) are shown. [Diagram 30] The amino acid sequence of the humanized antibody light chain hL3 (SEQ ID NO: 44) and the nucleotide sequence encoding the humanized antibody light chain hL3 (SEQ ID NO: 45) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 31] The amino acid sequence (SEQ ID NO: 46) of the variable region of the humanized antibody light chain hL3 and the nucleotide sequence (SEQ ID NO: 47) encoding the variable region of the humanized antibody light chain hL3 are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 32] The amino acid sequence of the humanized antibody light chain hL4 (SEQ ID NO: 48) and the nucleotide sequence encoding the humanized antibody light chain hL4 (SEQ ID NO: 49) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 33]The amino acid sequence of the variable region of the humanized antibody light chain hL4 (SEQ ID NO: 50) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL4 (SEQ ID NO: 51) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 34] The amino acid sequence of the humanized antibody heavy chain hH1 (SEQ ID NO: 52) and the nucleotide sequence encoding the humanized antibody heavy chain hH1 (SEQ ID NO: 53) are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 35] The amino acid sequence of the variable region of the humanized antibody heavy chain hH1 (SEQ ID NO: 54) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH1 (SEQ ID NO: 55) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Diagram 36] The amino acid sequence of the humanized antibody heavy chain hH2 (SEQ ID NO: 56) and the nucleotide sequence encoding the humanized antibody heavy chain hH2 (SEQ ID NO: 57) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 37] The amino acid sequence of the variable region of the humanized antibody heavy chain hH2 (sequence number 58) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH2 (sequence number 59) are shown. [Figure 38] The amino acid sequence of the humanized antibody heavy chain hH3 (SEQ ID NO: 60) and the nucleotide sequence encoding the humanized antibody heavy chain hH3 (SEQ ID NO: 61) are shown. The underlines in the amino acid sequence indicate the CDR sequences. [Figure 39] The amino acid sequence (SEQ ID NO: 62) of the variable region of the humanized antibody heavy chain hH3 and the nucleotide sequence (SEQ ID NO: 63) encoding the variable region of the humanized antibody heavy chain hH3 are shown. The underlines in the amino acid sequence indicate CDR sequences. [Diagram 40] 1 shows the binding ability of B1 antibody and C7 antibody to human CLDN6 and family molecules CLDN3, CLDN4, and CLDN9 by flow cytometry. [Diagram 41] 1 shows the antibody internalization activity of B1 antibody and C7 antibody by Mab-ZAP. [Diagram 42]1 shows the binding ability of humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 to CLDN6 and family molecules by flow cytometry. [Diagram 43] The amino acid sequence of the Trastuzumab light chain (SEQ ID NO:64) and heavy chain (SEQ ID NO:65) are shown. [Diagram 44] The amino acid sequence of the light chain (SEQ ID NO: 73) and the heavy chain (SEQ ID NO: 75) of a Trastuzumab variant are shown. [Diagram 45] A diagram showing a comparison of the amino acid sequences of chB1_H, which is the heavy chain of human chimeric anti-CLDN6 antibody chB1, and the humanized antibody heavy chains hH1, hH2, and hH3. "·" indicates an amino acid residue that is identical to that in chB1_H, and the positions where amino acid residues are described indicate substituted amino acid residues. [Figure 46] A diagram showing a comparison of the amino acid sequences of chB1_L, which is the light chain of human chimeric anti-CLDN6 antibody chB1, and hL1, hL2, hL3, and hL4, which are humanized antibody light chains. "·" indicates an amino acid residue that is identical to that in chB1_L, and the positions where amino acid residues are described indicate substituted amino acid residues. [Figure 47] 1 shows the effects of anti-HER2 antibody-drug conjugates ADC49 and ADC55 on subcutaneously implanted KPL-4 cells, a human breast cancer line. [Figure 48] 1 shows the effect of anti-HER2 antibody-drug conjugate ADC55 on subcutaneously implanted JIMT-1 cells, a human breast cancer line. [Figure 49] 1 shows the effects of anti-HER2 antibody-drug conjugates ADC49 and ADC55, and anti-LPS antibody-drug conjugate ADC53 on subcutaneously implanted CFPAC-1 cells, a human pancreatic cancer line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is bound via a linker structure to an antibody capable of recognizing or binding to tumor cells.

[0011] In the present invention, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0012] In the present invention, the term "C1 to C6 alkyl group" refers to a straight or branched alkyl group having 1 to 6 carbon atoms. Examples of the "C1 to C6 alkyl group" include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, an s-butyl group, a t-butyl group, an n-pentyl group, and an n-hexyl group.

[0013] In the present invention, the term "C1 to C6 alkoxy group" refers to an alkoxy group having a straight or branched alkyl group having 1 to 6 carbon atoms. Examples of the "C1 to C6 alkoxy group" include a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, an i-butoxy group, an s-butoxy group, an n-pentyloxy group, and an n-hexyloxy group.

[0014] In the present invention, the term "C1 to C6 alkylthio group" refers to an alkylthio group having a straight or branched alkyl group having 1 to 6 carbon atoms. Examples of the "C1 to C6 alkylthio group" include a methylthio group, an ethylthio group, an n-propylthio group, an i-propylthio group, an n-butylthio group, an i-butylthio group, an s-butylthio group, a t-butylthio group, an n-pentylthio group, and an n-hexylthio group.

[0015] In the present invention, the term "3- to 5-membered saturated hydrocarbon ring" refers to a saturated cyclic hydrocarbon group having a carbon number of 3 to 5. Examples of the "3- to 5-membered saturated hydrocarbon ring" include a cyclopropyl group, a cyclobutyl group, and a cyclopentyl group.

[0016] In the present invention, the term "C3 to C5 cycloalkoxy group" refers to a cycloalkoxy group having a saturated cyclic hydrocarbon group having a carbon number of 3 to 5. Examples of the "C3 to C5 cycloalkoxy group" include a cyclopropoxy group, a cyclobutoxy group, and a cyclopentyloxy group.

[0017] In the present invention, examples of the "3- to 5-membered saturated heterocycle" include 1,3-propylene oxide, azacyclobutane, trimethylene sulfide, tetrahydrofuran, and pyrrolidine.

[0018] In the present invention, the "aryl group" includes a phenyl group, a benzyl group, an indenyl group, a naphthyl group, a fluorenyl group, an anthranyl group, a phenanthrenyl group and the like.

[0019] In the present invention, examples of the "heteroaryl group" include a thienyl group, a pyrrolyl group, a pyrazolyl group, a triazolyl group, an oxazolyl group, an oxadiazolyl group, a thiazolyl group, a pyridyl group, a pyrimidyl group, a pyridazyl group, a pyrazinyl group, a quinolyl group, a quinoxalyl group, a benzothiophenyl group, a benzimidazolyl group, a benzotriazolyl group, and a benzofuranyl group.

[0020] In the present invention, the "6-membered heterocycle" includes a pyridine ring, a pyrimidine ring, a pyridazine ring, and the like.

[0021] In the present invention, the term "spiro-bonded" means that, as illustrated in the Examples, a pyrrolidine ring to which A and A are bonded or a pyrrolidine ring to which E and E are bonded form a spiro ring.

[0022] [Antibody-drug conjugates] The antibody-drug conjugate of the present invention is represented by the following formula: [ka] m 1 indicates the number of drugs bound per antibody molecule in the antibody-drug conjugate, Ab indicates an antibody or a functional fragment of the antibody, L indicates a linker connecting Ab and D, and D indicates a drug. <Drugs> The following describes drug D bound to the antibody-drug conjugate of the present invention. Drug D of the present invention is preferably an antitumor compound. In this antitumor compound, the linker is partially or entirely cleaved in tumor cells, releasing the antitumor compound portion and exerting the antitumor effect. When the linker is cleaved at the binding portion with the drug, the antitumor compound is released in its original structure and exerts its original antitumor effect. The antitumor compound in the antibody-drug conjugate of the present invention is represented by the general formula (V):

[0023] [ka] It is a pyrrolobenzodiazepine derivative (PBD derivative) represented by the following formula: The asterisk indicates the bond with the linker L.

[0024] n 1 represents an integer of 2 to 8, preferably an integer of 2 to 6, and more preferably an integer of 3 to 5. Above n 1 is an integer of from 2 to 8, preferably from 2 to 6, and more preferably from 3 to 5. The alkyl chain represented by this integer may contain a double bond.

[0025] A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, preferably a 3- to 5-membered saturated hydrocarbon ring (cyclopropane, cyclobutane, cyclopentane), more preferably cyclopropane, cyclobutane, and most preferably cyclopropane. The spiro-bonded 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, and preferably with 1 or 2 fluorine atoms (for example, 2,2-difluorocyclopropane, etc.).

[0026] R 1 , R 2each independently represents a C1 to C6 alkoxy group, a C1 to C6 alkyl group, a hydrogen atom, a hydroxyl group, a thiol group, a C1 to C6 alkylthio group, a halogen atom or -NR'R'' and is preferably a C1 to C6 alkoxy group, a C1 to C6 alkyl group or a hydroxyl group, more preferably a C1 to C3 alkoxy group, and most preferably a methoxy group.

[0027] R 3 , R 4 and R 5 indicates the following (i) to (iii). (i)R 3 and R 4 when taken together, form a double bond with the carbon atom to which each group is attached, as shown below: [ka] R 5 represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 1, or a C1-C6 alkyl group which may have one or more substituents selected from Group 2, and is preferably an aryl group which may have one or more substituents selected from the above Group 1.

[0028] R 5 The "aryl group" in the "aryl group or heteroaryl group which may have one or more substituents selected from Group 1" is preferably a phenyl group or a naphthyl group, and more preferably a phenyl group.

[0029] R 5 The "heteroaryl group" in the "aryl group or heteroaryl group which may have one or more substituents selected from Group 1" is preferably a thienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, a quinoxalyl group, or a benzothiophenyl group, more preferably a 2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridyl group, or a 4-pyridyl group, and even more preferably a 3-pyridyl group or a 3-thienyl group.

[0030] Above R 5 Examples of the substituent on the aryl group or heteroaryl group include the following a) to j). a) a C1-C6 alkoxy group optionally substituted with 1 to 3 halogen atoms, b) a C1-C6 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3 to C5 cycloalkoxy group, e) a C1 to C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i)-NHCOR' j) Hydroxyl group Here, R', R'', and R''' in b) and f) to i) each independently represent a hydrogen atom or a C1 to C6 alkyl group, and preferably each independently represent a hydrogen atom or a C1 to C3 alkyl group.

[0031] a) to j) are preferably as follows: a) a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms, More preferred are a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, and a trifluoromethoxy group, further preferred are a methoxy group, an ethoxy group, and a trifluoromethoxy group, and most preferred is a methoxy group. b) A C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -C(=NR')-NR''R''', or -NHC(=NR')-NR''R'''', where R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxyl group, or any group selected from -OCOR', -C(=NR')-NR''R''', or -NHC(=NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a methyl group, further preferably a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group, or -CH 2 OCOMe, -CH 2 -NHC(=NH)-NH 2 , -CH 2 -NHC(=NMe)-NH 2 It is. c) A halogen atom, preferably a fluorine atom or a chlorine atom. d) A C3 to C5 cycloalkoxy group, more preferably a cyclopropoxy group. e) A C1 to C3 alkylthio group, more preferably a methylthio group or an ethylthio group. f) -NR'R'', where R' and R'' are each independently a hydrogen atom or a C1 to C3 alkyl group, and more preferably -NH 2 , -NHMe, -NMe 2 , -NHEt, -NEt 2 It is. g) -C(=NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a C1 to C3 alkyl group, more preferably -C(=NH)-NH 2 , -C(=NMe)-NH 2 It is. h) -NHC(=NR')-NR''R''', where R', R'' and R''' are each independently a hydrogen atom or a C1 to C3 alkyl group, more preferably -NHC(=NH)-NH 2、-NHC(=NMe)-NH 2 It is. i) -NHCOR', where R' is a hydrogen atom or a C1 to C3 alkyl group, more preferably -NHCOMe 、 -NHCOEt, j) A hydroxyl group.

[0032] R 5 The position of the substituent in the aryl group (preferably a phenyl group) or heteroaryl group (preferably a pyridyl group) may be any position, and when there are multiple substituents, they may be the same or different.

[0033] R 5 When is an aryl group, the substituent is preferably a), b), d), g), h), or j), more preferably a), b), d), or j).

[0034] R 5 When is a phenyl group, the substituent may be at any position and may have multiple substituents, but preferably the substituent is at the 3-position or / and 4-position and has one or two substituents, and more preferably the substituent is at the 4-position and has one substituent. R 5 When is a naphthyl group, the position of the substituent may be any position and there may be multiple substituents, but preferably the position of the substituent is the 6-position and there is one substituent.

[0035] R 5When is a phenyl group, it is more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a 4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, or a 4-carbamimidamimethyl-phenyl group, and even more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, a 4-carbamimidamimethyl-phenyl group, or a 4-trifluoromethylphenyl group. R 5 When is a naphthyl group, it is more preferably a naphthyl group or a 6-methoxy-2-naphthyl group. Most preferably, it is a 4-methoxyphenyl group.

[0036] R 5 When is a heteroaryl group, the substituent is preferably a), b), d), g), h), or j), more preferably a) or b).

[0037] R 5 When is a heteroaryl group, the positions of the substituents may be any; in the case of a 3-pyridyl group, they are preferably the 6-positions or / and 5-positions; in the case of a 2-pyridyl group, they are preferably the 5-positions or / and 4-positions, or the 5-positions or / and 6-positions; and in the case of a 4-pyridyl group, they are preferably the 2-positions or / and 6-positions.

[0038] R 5 When is a heteroaryl group, the number of substituents may be multiple, but is preferably one or two, and more preferably one.

[0039] R 5 When is a pyridyl group, it is preferably a 6-methoxy-3-pyridyl group or a 6-methyl-3-pyridyl group. R 5 When is a 3-thienyl group or a 6-quinoxalyl group, it is preferably unsubstituted.

[0040] R 5 The "C1 to C6 alkyl group" in the "C1 to C6 alkyl group which may have one or more substituents selected from Group 2" is preferably a C1 to C3 alkyl group, and more preferably a methyl group or an ethyl group.

[0041] R 5 The substituent of the "C1 to C6 alkyl group which may have one or more substituents selected from Group 2" is a halogen atom, a hydroxyl group, a C1 to C6 alkoxy group (preferably a C1 to C3 alkoxy group), preferably a hydroxyl group, a methoxy group, or an ethoxy group, more preferably a hydroxyl group.

[0042] (ii)R 3 When represents a hydrogen atom, R 4 and R 5 is R 4 and R 5 together with the carbon atom to which it is attached, forms a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring as shown below, or CH 2 = form. [ka] or [ka] The 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms. R 4 and R 5 are preferably taken together to form a 3- to 5-membered saturated hydrocarbon ring or CH 2 =, more preferably cyclopropane, cyclobutane or CH 2 = (exomethylene group), and more preferably cyclopropane. R 4 and R 5and R are taken together to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, it is preferable that A is the same 3- to 5-membered saturated hydrocarbon ring or 3- to 5-membered saturated heterocyclic ring as A. More preferably, A is a 3- to 5-membered saturated hydrocarbon ring, and R 4 and R 5 together form a 3- to 5-membered saturated hydrocarbon ring, more preferably A is a cyclopropane ring and R 4 and R 5 together form a cyclopropane ring.

[0043] (iii)R 3 , R 4 and R 5 But R 3 The carbon atom to which R is bonded 4 and R 5 is joined to the carbon atom to form a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from Group 3. The following formula is R 3 and R 4 may be taken together to form a benzene ring which may have one or more substituents. [ka] The positions of the substituents on the benzene ring or heterocycle may be any, and when there are a plurality of substituents, they may be the same or different.

[0044] The substituent of the benzene ring or the heterocycle is a halogen atom, a C1-C6 alkyl group or a C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms, preferably a halogen atom, a C1-C3 alkyl group or a C1-C3 alkoxy group which may be substituted with 1 to 3 halogen atoms, and more preferably a halogen atom, a methyl group or a methoxy group.

[0045] The "benzene ring or 6-membered heterocycle which may have one or more substituents" is preferably an unsubstituted benzene ring.

[0046] R 3 , R 4 and R 5 is most preferably the above (i).

[0047] R 6 , R 7 Both represent hydrogen atoms or R 6 and R 7 together indicate an imine bond (C=N).

[0048] R 8 R is a hydroxyl group or a C1 to C3 alkoxy group, preferably a hydroxyl group or a methoxy group, and more preferably a hydroxyl group. 8 is a bisulfite adduct (OSO 3 M, where M is a metal cation. R 8 is bonded to an asymmetric carbon atom, and therefore has the configuration shown by the following partial structure (Va) or (Vb). The wavy line indicates that it is bonded to Y in general formula (V), and the asterisk indicates that it is bonded to L. [ka]

[0049] X and Y each independently represent an oxygen atom, a nitrogen atom, or a sulfur atom, and are preferably an oxygen atom.

[0050] The drug D of the present invention is preferably a compound selected from the following group: [ka] , [ka] [ka]

[0051] <Linker structure> The linker structure that binds the antitumor drug to the antibody in the antibody-drug conjugate of the present invention will be described. The linker L has the formula: -Lb-La-Lp-NH-B-CH 2 -O(C=O)-* As shown in the figure. The asterisk indicates binding to the nitrogen atom at N10' position of drug D, and Lb indicates a spacer that links La to the glycan or remodeled glycan of Ab, or a spacer that links La to the side chain of an amino acid residue (e.g., cysteine, lysine, etc.) of antibody Ab.

[0052] B represents a phenyl group or a heteroaryl group, and is preferably a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a 1,4-phenyl group.

[0053] Lp represents a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells, for example, by the action of an enzyme such as esterase or peptidase. Lp is a peptide residue composed of 2 to 7 (preferably 2 to 4) amino acids, i.e., composed of a residue of an oligopeptide in which 2 to 7 amino acids are peptide-bonded. Lp binds to the carbonyl group of La in Lb-La- at the N-terminus and to the linker -NH-B-CH at the C-terminus. 2 An amide bond is formed with the amino group (-NH-) of the -O(C=O)- portion. The bond between the C-terminus of Lp and -NH- is cleaved by an enzyme such as the esterase.

[0054] The amino acids constituting Lp are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids, and may be, in addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid, or may be non-natural amino acids such as N-methylated amino acids.

[0055] The amino acid sequence of Lp is not particularly limited, but the constituent amino acids include glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline (Cit) are preferred. These amino acids may overlap, and the amino acid sequence may include any amino acid selected from the group consisting of 1 to 3. Furthermore, the drug release pattern can be controlled by the type of amino acid.

[0056] 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-, -GGGFG-, -DDGGFG-, -KDGGFG-, -GGGFGGF- The following can be mentioned. Here, the above "(D-)V" means D-valine, "(D-)P" means D-proline, and "(D-)D" means D-aspartic acid.

[0057] The linker Lp is preferably: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL-

[0058] The linker Lp is more preferably: -GGVA-, -GGVCit-, -VA-

[0059] Lb represents i) a spacer that bonds La to the glycan or remodeled glycan of the antibody Ab, or ii) a spacer that bonds La to the side chain of an amino acid residue (e.g., cysteine, lysine, etc.) of the antibody Ab. When Lb is i), La represents any one selected from the following group. -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 2 -NH-C(=O)-(CH 2 CH 2 O)n 3 -CH 2 CH 2 -C(=O)-, -(CH 2 )n 4 -OC(=O)- Wherein, n 2 is an integer of 1 to 3 (preferably 1 or 2), n 3 is an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably 2 or 4), n 4 represents an integer of 0 to 2 (preferably 0 or 1).

[0060] When Lb is i), La preferably represents any one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)- -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)- and -OC(=O)- La is more preferably -C(=O)-CH 2 CH 2 -C(=O)- or -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-.

[0061] The spacer of Lb is not particularly limited, but examples thereof include spacers represented by the following formula: [ka] [ka] [ka] In each of the structural formulas of Lb shown above, the asterisk represents the -(C=O) or -(CH 2 )n 4 The wavy lines indicate binding to the glycan of Ab or the remodeled glycan. In each of the structural formulas of Lb (Lb-1, Lb-2, or Lb-3) shown above, the triazole ring moiety formed by the click reaction of the azide group with DBCO has a geometric isomeric structure, and one Lb has either one of these two types of structures or a mixture thereof. 1 There are m "-LD"s, 1 Each Lb (Lb-1, Lb-2 or Lb-3) in L in each "-LD" has either one of these two types of structures or a mixture of both.

[0062] When Lb is i), L is preferably -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, B is a 1,4-phenyl group; Lp represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- La represents one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)-, -OC(=O)- Lb represents any one of the structural formulas of Lb shown above.

[0063] When Lb is i), L is more preferably any one selected from the following group: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)-、-Z 3 -CH 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)- Here, Z 1 represents the structural formula shown below for the above Lb:

Chemical formula

[0064] L is most preferably any of the following: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2-C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- where B is a 1,4-phenyl group and Z 1 is the structural formula of Lb shown below: [ka] It is.

[0065] When Lb is ii) and the amino acid residue is a cysteine ​​residue, the spacer of Lb is not particularly limited and examples thereof include -(Succinimid-3-yl-N)-. "-(Succinimid-3-yl-N)-" is the following formula

[0066] [ka] In the above structural formula, the asterisk indicates that it is bound to La. The wavy line indicates that it is bound to the thiol group of the cysteine ​​residue of the antibody via a thiol bond, and site-specific cysteine ​​conjugation is also acceptable (RSC Adv., 2017, 7, 24828-24832 etc.).

[0067] When Lb is ii), L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)-*, B is a 1,4-phenyl group; Lp represents any one of -GGVA-, -GG-(D-)VA-, -VA-, and -GGFG-; La is -(CH 2 )n9 -C(=O)-, or -(CH 2 CH 2 )n 10 -C(=O)-NH-(CH 2 CH 2 O)n 11 -CH 2 CH 2 -C(=O)- n 9 is an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably 2 or 5), n 10 is an integer of 1 to 3 (preferably 1), n 11 represents an integer of 6 to 10 (preferably, 8), Lb represents -(Succinimid-3-yl-N)-.

[0068] When Lb is ii), L is preferably any one of the following: -(Succinimid-3-yl-N)-(CH 2 ) 5 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, -(Succinimid-3-yl-N)-(CH 2 ) 5 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)-, or -(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- Here, B is a 1,4-phenyl group.

[0069] It is believed that when the antibody-drug conjugate of the present invention is largely transported into tumor cells and then the linker portion (e.g., Lp) is cleaved and activated by an enzyme or the like, the drug D portion is released (hereinafter referred to as the free drug (described below)), thereby exerting antitumor activity. Therefore, it is preferred that the antibody-drug conjugates of the present invention are stable outside tumor cells. <Free drugs and manufacturing intermediates> The intermediate and free drug of the antibody-drug conjugate of the present invention can be represented by the following formula:

[0070] [ka] This is explained below.

[0071] The free drug of the present invention is generated by cleavage of the linker L moiety in the antibody-drug conjugate after transfer into tumor cells, for example, drugs 1 to 16 in Examples 45 to 54 and 150 to 152. The antibody-drug conjugate of the present invention is produced using this production intermediate.

[0072] The free drug in the antibody-drug conjugate of the present invention is 16 , R 17 is R 16 and R 17 together to form an imine bond (N=C). The production intermediate of the antibody-drug conjugate of the present invention is (b) R 16 is J-La'-Lp'-NH-B'-CH 2 -O(C=O)-* This is the case when Therefore, in the formula, l and the n 1 , E and the above A, R 9 and the above R 1 , R 10 and the above R 2 , R 11 and the above R 3 , R 12 and the above R 4 , R 13 and the above R 5 , R 14 and the above R 6 , R 15 and the above R 7, V and X, W and Y, Group 7 and Group 1, Group 8 and Group 2, Group 9 and Group 3, Group 10 and Group 4, Group 11 and Group 5, and Group 12 and Group 6 have the same meaning.

[0073] l represents an integer of from 2 to 8, preferably an integer of from 2 to 6, and more preferably an integer of from 3 to 5. The alkyl chain in which l is an integer of 2 to 8, preferably an integer of 2 to 6, more preferably an integer of 3 to 5, may contain a double bond.

[0074] E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, preferably a 3- to 5-membered saturated hydrocarbon ring (cyclopropane, cyclobutane, cyclopentane), more preferably cyclopropane, cyclobutane, and most preferably cyclopropane. The spiro-bonded 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, and preferably with 1 or 2 fluorine atoms (for example, 2,2-difluorocyclopropane, etc.).

[0075] R 9 , R 10 each independently represents a C1 to C6 alkoxy group, a C1 to C6 alkyl group, a hydrogen atom, a hydroxyl group, a thiol group, a C1 to C6 alkylthio group, a halogen atom or -NR'R'' and is preferably a C1 to C6 alkoxy group, a C1 to C6 alkyl group or a hydroxyl group, more preferably a C1 to C3 alkoxy group, and most preferably a methoxy group.

[0076] R 11 , R 12 and R 13 indicates the following (i) to (iii). (i)R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, R 13represents an aryl group or heteroaryl group which may have one or more substituents selected from Group 7, or a C1-C6 alkyl group which may have one or more substituents selected from Group 8, and is preferably an aryl group which may have one or more substituents selected from the above Group 7.

[0077] R 13 The "aryl group" in the "aryl group or heteroaryl group which may have one or more substituents selected from Group 7" is preferably a phenyl group or a naphthyl group, more preferably a phenyl group.

[0078] R 13 The "heteroaryl group" in the "aryl group or heteroaryl group which may have one or more substituents selected from Group 7" is preferably a thienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, a quinoxalyl group, or a benzothiophenyl group, more preferably a 2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridyl group, or a 4-pyridyl group, and even more preferably a 3-pyridyl group or a 3-thienyl group.

[0079] Above R 13 Examples of the substituent on the aryl group or heteroaryl group include the following a) to j). a) a C1-C6 alkoxy group optionally substituted with 1 to 3 halogen atoms, b) a C1-C6 alkyl group optionally substituted with any one selected from 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3 to C5 cycloalkoxy group, e) a C1 to C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i) -NHCOR', j) Hydroxyl group Here, R', R'', and R''' in b) and f) to i) each independently represent a hydrogen atom or a C1 to C6 alkyl group, and preferably each independently represent a hydrogen atom or a C1 to C3 alkyl group.

[0080] a) to j) are preferably as follows: a) a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms, More preferably, it is a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, or a trifluoromethoxy group. More preferred are a methoxy group, an ethoxy group, and a trifluoromethoxy group, and most preferred is a methoxy group. b) a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''', where R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group; More preferably, it is a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxyl group, -OCOR', -C(=NR')-NR''R''', or -NHC(=NR')-NR''R'''', where R', R'', and R''' are each independently a hydrogen atom or a methyl group; More preferably, the group is a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group, or a -CH 2 OCOMe, -CH 2 -NHC(=NH)-NH 2、 -CH 2 -NHC(=NMe)-NH 2 It is. c) A halogen atom, preferably a fluorine atom or a chlorine atom. d) A C3 to C5 cycloalkoxy group, more preferably a cyclopropoxy group. e) A C1 to C3 alkylthio group, more preferably a methylthio group or an ethylthio group. f) -NR'R'', where R' and R'' are each independently a hydrogen atom or a C1-C3 alkyl group; More preferably, -NH 2 , -NHMe, -NMe 2 , -NHEt, -NEt 2 It is. g) -C(=NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group; More preferably, -C(=NH)-NH 2 , -C(=NMe)-NH 2 It is. h) -NHC(=NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group; More preferably, -NHC(=NH)-NH 2、 -NHC(=NMe)-NH 2 It is. i) -NHCOR', R' is a hydrogen atom or a C1-C3 alkyl group; More preferably, -NHCOMe 、 -NHCOEt, j) A hydroxyl group.

[0081] R 13 The position of the substituent in the aryl group (preferably a phenyl group) or heteroaryl group (preferably a pyridyl group) may be any position, and when there are multiple substituents, they may be the same or different.

[0082] R 13 When is an aryl group, the substituent is preferably a), b), d), g), h), or j), more preferably a), b), d), or j).

[0083] R 13When is a phenyl group, the substituent may be at any position and may have multiple substituents, but preferably the substituent is at the 3-position or / and 4-position and has one or two substituents, and more preferably the substituent is at the 4-position and has one substituent. R 5 When is a naphthyl group, the position of the substituent may be any position and there may be multiple substituents, but preferably the position of the substituent is the 6-position and there is one substituent.

[0084] R 13 is a phenyl group, more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a 4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, More preferred are a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, and a 4-trifluoromethylphenyl group. R 13 When is a naphthyl group, it is more preferably a naphthyl group or a 6-methoxy-2-naphthyl group. Most preferably, it is a 4-methoxyphenyl group.

[0085] R 13 When is a heteroaryl group, the substituent is preferably a), b), d), g), h), or j), more preferably a) or b). R 13 When is a heteroaryl group, the positions of the substituents may be any; in the case of a 3-pyridyl group, they are preferably the 6-positions or / and 5-positions; in the case of a 2-pyridyl group, they are preferably the 5-positions or / and 4-positions, or the 5-positions or / and 6-positions; and in the case of a 4-pyridyl group, they are preferably the 2-positions or / and 6-positions. R 13When is a heteroaryl group, the number of substituents may be multiple, but is preferably one or two, and more preferably one. R 13 When is a pyridyl group, it is preferably a 6-methoxy-3-pyridyl group or a 6-methyl-3-pyridyl group. R 13 When is a 3-thienyl group or a 6-quinoxalyl group, it is preferably unsubstituted.

[0086] R 13 The "C1 to C6 alkyl group" in the "C1 to C6 alkyl group which may have one or more substituents selected from Group 8" is preferably a C1 to C3 alkyl group, and more preferably a methyl group or an ethyl group. R 13 The substituent of the "C1 to C6 alkyl group which may have one or more substituents selected from Group 8" is a halogen atom, a hydroxyl group, a C1 to C6 alkoxy group (preferably a C1 to C3 alkoxy group), preferably a hydroxyl group, a methoxy group, or an ethoxy group, more preferably a hydroxyl group.

[0087] (ii)R 11 When represents a hydrogen atom, R 12 and R 13 is R 12 and R 13 forms a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring together with the carbon atom to which it is bonded, or CH 2 = form. The 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms. R 12 and R 13 are preferably taken together to form a 3- to 5-membered saturated hydrocarbon ring or CH 2 =, more preferably cyclopropane, cyclobutane or CH 2 = (exomethylene group), and more preferably cyclopropane. R 12 and R13 When R and R are taken together to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, it is preferable that the same 3- to 5-membered saturated hydrocarbon ring or 3- to 5-membered saturated heterocyclic ring as E is. More preferably, E is a 3- to 5-membered saturated hydrocarbon ring, and R 12 and R 13 are taken together to form a 3- to 5-membered saturated hydrocarbon ring, more preferably E is a cyclopropane ring and R 12 and R 13 together form a cyclopropane ring.

[0088] (iii)R 11 , R 12 and R 13 But R 11 The carbon atom to which R is bonded 12 and R 13 is joined to the carbon atom to form a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from Group 9. The positions of the substituents on the benzene ring or heterocycle may be any, and when there are a plurality of substituents, they may be the same or different. The substituent of the benzene ring or the heterocycle is a halogen atom, a C1-C6 alkyl group or a C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms, preferably a halogen atom, a C1-C3 alkyl group or a C1-C3 alkoxy group which may be substituted with 1 to 3 halogen atoms, and more preferably a halogen atom, a methyl group or a methoxy group. The "benzene ring or 6-membered heterocycle which may have one or more substituents" is preferably an unsubstituted benzene ring.

[0089] R 11 , R 12 and R 13 is most preferably the above (i).

[0090] R 14 , R 15 Both represent hydrogen atoms or R 14 and R 15together indicate an imine bond (C=N).

[0091] V and W each independently represent an oxygen atom, a nitrogen atom or a sulfur atom, and preferably an oxygen atom.

[0092] R 16 , R 17 teeth, (a)R 16 , R 17 is R 16 and R 17 together form an imine bond (N=C), or (b)R 16 is J-La'-Lp'-NH-B'-CH 2 -O(C=O)-*, R 17 is represented by a hydroxyl group or a C1 to C3 alkoxy group.

[0093] Above (b)R 16 But J-La'-Lp'-NH-B'-CH 2 In the case of -O(C=O)-*, the asterisk indicates attachment to the N10' position of the pyrrolobenzodiazepine ring shown in the formula above.

[0094] B' represents a phenyl group or a heteroaryl group, and is preferably a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a 1,4-phenyl group.

[0095] Lp' represents a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells, for example, by the action of an enzyme such as esterase or peptidase.

[0096] Specific examples of the linker Lp' include Examples of such genes 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-. Here, the above "(D-)V" means D-valine, "(D-)P" means D-proline, and "(D-)D" means D-aspartic acid.

[0097] The linker Lp' is preferably: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- More preferred are -GGVA-, -GGVCit- and -VA-.

[0098] La′ represents any one selected from the following group: -C(=O)-(CH 2 CH 2 )n 6 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -C(=O)-NH-(CH 2 CH 2 )n 7 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -C(=O)-NH-(CH 2 CH 2 O)n 7 -CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 )n 6 -NH-C(=O)-(CH 2 CH 2 O)n 7 -CH 2 CH2 -C(=O)-, -(CH 2 )n 8 -OC(=O)-, -(CH 2 )n 12 -C(=O)- and -(CH 2 CH 2 )n 13 -C(=O)-NH-(CH 2 CH 2 O)n 14 -CH 2 CH 2 -C(=O)- Wherein, n 6 is an integer of 1 to 3 (preferably 1 or 2), n 7 is an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably 2 or 4), n 8 is an integer of 0 to 2 (preferably 0 or 1), n 12 is an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably 2 or 5), n 13 is an integer of 1 to 3 (preferably 1), n 14 represents an integer of 6 to 10 (preferably 8).

[0099] La' preferably represents any one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2-NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-, -CH 2 -OC(=O)-, -OC(=O)-, -(CH 2 ) 2 -C(=O)-, -(CH 2 ) 5 -C(=O)-, and -CH 2 CH 2 -C(=O)-NH -(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)-

[0100] La' is more preferably -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)- or -(CH 2 ) 5 -C(=O)-.

[0101] There are no particular limitations on J, so long as it is a cyclic structure containing an alkyne structure that reacts with an azide group to form a 1,2,3-triazole ring. Examples of J include compounds represented by the following formula: [ka]

[0102] In each of the structural formulas of J shown above, the asterisk represents the -(C=O) or -(CH 2 )n 8 This indicates that it is bound to

[0103] Alternatively, J may be a compound or a halogen atom that bonds to the side chain of an amino acid residue (eg, cysteine, lysine, etc.) of the antibody Ab, and examples include a maleimidyl group represented by the following formula: [ka] In the maleimidyl group shown above, the asterisk represents the -(CH 2 )n 12 , or -(CH 2 CH 2 )n 13 This indicates that it is bound to

[0104] R 16 is preferably J-La'-Lp'-NH-B'-CH 2 -O(C=O)-*, B' is a 1,4-phenyl group; Lp' represents any one selected from the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- La' represents one selected from the following group: -C(=O)-CH 2 CH 2 -C(=O)-, -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-, -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-, -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2-C(=O)-, -OC(=O)-, -CH 2 -OC(=O)-, -(CH 2 ) 5 -C(=O)- and -CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)- J represents one of the following structural formulas: [ka] Here, in the structural formula of J, the asterisk indicates that it is bonded to La'.

[0105] R 16 is more preferably any one selected from the following group: J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B'-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-、 J 2 -OC(=O)-GGVA-NH-B’-CH2 -OC(=O)-, J 3 -CH 2 -OC(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-GGVA-NH-B'-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-VA-NH-B'-CH 2 -OC(=O)-, and J 4 -CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 8 -CH 2 CH 2 -C(=O)-VA-NH-B'-CH 2 -OC(=O)- Here, J 1 , J 2 , J 3 , J 4 represents the structural formula shown below, [ka] Here, J 1 , J 2 , J 3 and J 4 In the structural formula, the asterisk represents J 1 , J 2 , J 3 Or J 4 indicates that the bond is bonded to the adjacent group, B' is a 1,4-phenyl group.

[0106] R 16 is most preferably one of the following: J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B'-CH 2-OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-, J 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B’-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-, J 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B’-CH 2 -OC(=O)-, J 4 -(CH 2 ) 5 -C(=O)-VA-NH-B’-CH 2 -OC(=O)- Here, B’ is a 1,4-phenyl group, J 1 , J 4 is the structural formula of J shown below: [ka] Here, J 1 and J 4 In the structural formula, the asterisk represents J 1 Or J 4 indicates that the group is bonded to the adjacent group.

[0107] R 17 is a hydroxyl group or a C1 to C3 alkoxy group, preferably a hydroxyl group or a methoxy group. R 17 is a bisulfite adduct (OSO 3 M, where M is a metal cation. R 17 is bonded to an asymmetric carbon atom and therefore has the configuration shown in partial structure (VIa) or (VIb) below. The wavy line indicates that it is bonded to W in the intermediate and free drug represented by general formula (VI). [ka]

[0108] The free drug is preferably a compound selected from one of the following groups: [ka]

[0109] This free drug may be released in tumor cells with a portion of the linker L still bound, but even in this state, it is an excellent drug that exhibits excellent antitumor effects. After being transferred to tumor cells, this free drug is further oxidized to form R 16 , R 17 However, even under such conditions, it still exerts an excellent antitumor effect.

[0110] The intermediate is preferably a compound selected from the following group: [ka] [ka]

[0111] The intermediate is preferably a compound selected from the following group: [ka] [ka] [ka] [ka]

[0112] <Antibody> In the present invention, the terms "cancer" and "tumor" are used interchangeably. In the present invention, the term "gene" refers to a nucleotide or nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof, and for example, a polynucleotide, oligonucleotide, DNA, mRNA, cDNA, RNA, etc., which is a nucleotide sequence containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof, is included in the meaning of "gene". Examples of the "CLDN6 gene" of the present invention include DNA, mRNA, cDNA, cRNA, etc., which contain a nucleotide sequence encoding the amino acid sequence of the CLDN6 protein. In the present invention, "nucleotide", "polynucleotide", or "nucleotide sequence" is synonymous with "nucleic acid", and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence". In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably. In the present invention, "CLDN6" is used synonymously with CLDN6 protein.

[0113] In the present invention, the term "cell" includes cells in an animal individual and cultured cells. In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0114] In the present invention, the term "functional antibody fragment", also called "antigen-binding fragment of antibody", refers to a partial fragment of an antibody that has binding activity to an antigen, and includes Fab, F(ab')2, Fv, scFv, diabody, linear antibody, and multispecific antibody formed from antibody fragments. In addition, Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions, is also included in the antigen-binding fragment of an antibody. However, it is not limited to these molecules as long as it has the ability to bind to an antigen. In addition, these antigen-binding fragments include not only those obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using an antibody gene modified by genetic engineering. The functional fragments of the present invention include functional fragments that retain an aspartic acid (Asn297) and its surrounding amino acids that are modified by N-linked glycans that are well conserved in the Fc region of an IgG heavy chain, and have the ability to bind to an antigen.

[0115] In the present invention, the term "epitope" refers to a partial peptide or partial three-dimensional structure of an antigen (e.g., a partial peptide or partial three-dimensional structure of CLDN6) to which a specific antibody (e.g., an anti-CLDN6 antibody) binds. The epitope, which is the partial peptide (e.g., a partial peptide of CLDN6), can be determined by a method well known to those skilled in the art, such as an immunoassay method.

[0116] In the present invention, "CDR" means a complementarity determining region (CDR). It is known that the heavy and light chains of an antibody molecule each have three CDRs. CDRs are also called hypervariable regions, and are located in the variable regions of the heavy and light chains of an antibody, and are sites with particularly high variability in the primary structure, and are separated into three sites on the primary structure of the heavy and light chain polypeptide chains. In this specification, the CDRs of the heavy chain are represented as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are represented as CDRL1, CDRL2, and CDRL3 from the amino terminal side of the light chain amino acid sequence. These sites are close to each other in the three-dimensional structure, and determine the specificity for the antigen to which they bind.

[0117] In the present invention, "hybridizing under stringent conditions" means hybridizing at 68°C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (Clontech), or hybridizing using a DNA-fixed filter at 68°C in the presence of 0.7-1.0 M NaCl, followed by washing at 68°C using a 0.1-2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate), which can be identified by hybridization under equivalent conditions. In the present invention, "one to several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0118] In the present invention, antibodies that recognize or bind to CLDN6 or CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibodies" and "anti-CLDN6 / CLDN9 antibodies", respectively. Such antibodies include chimeric antibodies, humanized antibodies, human antibodies, etc. Antibodies that recognize or bind to CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibodies".

[0119] The antibody used in the antibody-drug conjugate of the present invention means an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody of the present invention may be of any class, IgG, IgE, IgM, IgD, IgA, or IgY, but is preferably IgG. The subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, but is preferably IgG1, IgG2, or IgG4. When IgG1 or IgG4 is used, the effector function can be adjusted by substituting a portion of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, and WO94 / 29351). The antibody may be derived from any species, but preferably, human, rat, mouse, and rabbit are examples. When derived from a species other than human, it is preferable to chimerize or humanize it using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable. 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.

[0120] The antibody of the present invention is preferably an antibody capable of targeting tumor cells. In other words, since a drug having antitumor activity is bound to the antibody via a linker, the antibody preferably has one or more of the following properties: the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be taken up and internalized in tumor cells, and the ability to damage tumor cells. The binding of the antibody to the tumor cells can be confirmed by flow cytometry. The uptake of the antibody into the tumor cells can be confirmed by (1) an assay in which the antibody taken up into the cells is visualized by a fluorescent microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which the amount of fluorescence taken up into the cells is measured using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is used to release the toxin when taken up into the cells, thereby suppressing cell proliferation (Bio Techniques 28:162-165, January 2000). A recombinant complex protein of the catalytic domain of diphtheria toxin and protein G can also be used as the immunotoxin. In the present invention, "high internalization ability" means that the viability (expressed as a relative rate, with the cell viability in the absence of antibody being taken as 100%) of target antigen-expressing cells (e.g., CLDN6-expressing cells) to which the antibody and a saporin-labeled anti-mouse or rat IgG antibody have been added is preferably 70% or less, more preferably 60% or less.

[0121] Since the antibody-drug conjugate of the present invention is bound to a compound that exerts an antitumor effect, it is preferable, but not essential, that the antibody itself has an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxicity of an antitumor compound in tumor cells, it is important and preferable that the antibody has the property of being internalized and transferred into tumor cells. From the viewpoint of exerting an antitumor effect, it is important and preferable that the antibody has the property of being internalized and transferred into tumor cells in order to specifically and selectively damage tumor cells with a drug. The antitumor activity of an antibody refers to the cytotoxic activity and anticellular effect on tumor cells. The antitumor activity can be confirmed using a known in vitro or in vivo evaluation system. Examples of such antibodies include antibodies against tumor-associated antigens, and examples of such antibodies include, but are not limited to, anti-CLDN6 antibodies, anti-CLDN6 / CLDN9 antibodies, anti-HER2 antibodies, anti-DLL3 (Delta like protein 3) antibodies, anti-A33 antibodies, anti-CanAg antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD98 antibodies, anti-TROP2 antibodies, anti-CEA antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-FGFR2 antibodies (WO201315206, etc.), anti-G250 antibodies, anti-MUC1 antibodies (WO2011012309, etc.), anti-GPNMB antibodies, anti-integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-Mesothelin antibodies, anti-EGFR antibodies, and anti-DR5 antibodies. The antibody of the present invention is preferably an anti-CLDN6 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 antibody, an anti-CD98 antibody, or an anti-TROP2 antibody, and more preferably an anti-CLDN6 antibody or an anti-HER2 antibody (eg, Trastuzumab, a Trastuzumab mutant).

[0122] The antibody of the present invention can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibody produced in the body using a method commonly used in this field. The origin of the antigen is not limited to human, and an animal can also be immunized with an antigen derived from an animal other than human, such as a mouse or a rat. In this case, an antibody applicable to a human disease can be selected by testing the cross-reactivity of the obtained antibody that binds to a heterologous antigen with a human antigen. Alternatively, according to known methods (e.g., Nature (1975) 256, p. 495-497; Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)), antibody-producing cells that produce antibodies against an antigen can be fused with myeloma cells to establish hybridomas, thereby obtaining monoclonal antibodies (described below). The antigen can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce the antigen.

[0123] The chimeric and humanized antibodies of the present invention can be obtained according to known methods (for example, Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984); Nature (1986) 321, p. 522-525; WO90 / 07861).

[0124] Anti-HER2 antibodies (such as U.S. Pat. No. 5,821,337), anti-TROP2 antibodies (such as WO2003 / 074566), and anti-CD98 antibodies (such as WO2015 / 146132) can be obtained by known means.

[0125] The anti-CLDN6 antibody used in the present invention is described below. Note that the embodiment described below is one representative embodiment of the present invention, and the scope of the present invention is not to be construed narrowly by this embodiment.

[0126] 1. CLDN6 and CLDN9 CLDN6 is a four-transmembrane protein consisting of 220 amino acids and belonging to the Claudin family, with both N- and C-termini inside the cell. The amino acid sequence and DNA sequence of human CLDN6 have been published on public databases and can be referenced by accession numbers such as NP_067018 (SEQ ID NO: 1 (FIG. 11)) and NM_021195 (SEQ ID NO: 2 (FIG. 11) (both NCBI)). Regarding the amino acid sequence of human CLDN6 protein (hereinafter referred to as "CLDN6 amino acid sequence"), the extracellular region is composed of an extracellular domain (EC1) consisting of amino acid numbers 29 to 81 of SEQ ID NO: 1 in the sequence listing, and an extracellular domain (EC2) consisting of amino acid numbers 138 to 160. CLDN9 is a 4-transmembrane protein consisting of 217 amino acids, belonging to the Claudin family, and has intracellular N- and C-termini. CLDN9 has high homology to CLDN6. The amino acid sequence and DNA sequence of human CLDN9 are published on public databases and can be referenced by accession numbers such as NP_066192 (sequence number 3 (Figure 12)) and NM_020982 (sequence number 4 (Figure 12)) (both NCBI).

[0127] 2. Anti-CLDN6 antibody An example of an anti-CLDN6 antibody of the present invention is an anti-CLDN6 antibody that recognizes a higher-order structure consisting of two extracellular domains, the amino acid sequence from 29 to 81 and the amino acid sequence from 138 to 160 from the N-terminus of CLDN6 shown in sequence number 1 in the sequence listing, and has internalization activity. The anti-CLDN6 antibody of the present invention is an antibody capable of targeting tumor cells, i.e., it has the properties of recognizing tumor cells, binding to tumor cells, and being taken up and internalized in tumor cells, etc. Therefore, the anti-CLDN6 antibody of the present invention can be bound to a compound having antitumor activity via a linker to form an antibody-drug conjugate. The anti-CLDN6 antibody of the present invention may have anti-tumor activity.

[0128] The anti-CLDN6 antibody can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibody produced in the body using a method commonly used in this field. Since CLDN6 is a four-transmembrane protein, the protein that retains its three-dimensional structure can be used as an antigen, and one such method is the cellular immunization method. Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce an antibody against an antigen with myeloma cells according to the above-mentioned known method, and a monoclonal antibody can be obtained.

[0129] A method for obtaining an antibody against CLDN6 will be specifically described below. 1) Preparation of antigen CLDN6 can be directly purified from human tumor tissues or tumor cells, or prepared from the cell membrane fraction of the tumor cells. CLDN6 can also be synthesized in vitro (e.g., using the Rapid Translation System (RTS) from Roche Diagnostics) or produced in host cells by genetic engineering. Specifically, in genetic engineering, the antigen can be obtained by inserting CLDN6 cDNA into an expression vector, synthesizing it in a solution containing enzymes, substrates and energy materials required for transcription and translation, or by expressing CLDN6 by transforming other prokaryotic or eukaryotic host cells. It is also possible to use the above-mentioned genetically engineered CLDN6-expressing cells or cell lines expressing CLDN6 as the CLDN6 protein. It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein linking the extracellular domain of the membrane protein CLDN6 with the constant domain of an antibody in an appropriate host vector system. The above-mentioned transformant itself can also be used as an antigen. Furthermore, cell lines expressing CLDN6 can also be used as antigens. Examples of such cell lines include human pancreatic cancer cell line NOR-P1 cells, human ovarian cancer lines NIH:OVCAR-3, OV-90, or OAW28, human ovarian teratocarcinoma cell line PA-1, human liver cancer line HuH-7, human gestational choriocarcinoma line JEG-3, and human pluripotent embryonal carcinoma line NTERA-2 clone D1, but are not limited to these cell lines as long as they express CLDN6. The CLDN9 protein used in the present invention can also be prepared and used in the same manner. 2) Production of anti-CLDN6 monoclonal antibody The anti-CLDN6 antibody used in the present invention is not particularly limited, and for example, an antibody specified by the amino acid sequence shown in the sequence listing of the present application can be preferably used. The anti-CLDN6 antibody used in the present invention preferably has the following characteristics: (1) An antibody characterized by having the following properties (a) and (b): (a) Recognizes or binds to the CLDN family. The antibody of the present invention recognizes the CLDN family. In other words, the antibody of the present invention binds to the CLDN family. The antibody of the present invention preferably binds to CLDN6, and more preferably specifically binds to CLDN6. Furthermore, the antibody of the present invention may recognize or bind to CLDN9. In the present invention, "specific recognition", i.e., "specific binding" means binding that is not nonspecific adsorption. The criterion for determining whether or not binding is specific can be, for example, the dissociation constant (hereinafter referred to as "KD"). The KD value of a suitable antibody of the present invention for CLDN6 and / or CLDN9 is 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less or 1×10 -6 M or less, more preferably 5×10 -7 M or less, 2×10 -7 M or less or 1×10 -7 It is below M. In the present invention, the binding between an antigen and an antibody can be measured or determined by ELISA, RIA, Surface Plasmon Resonance (hereinafter referred to as "SPR") analysis, etc. The binding between an antigen expressed on a cell surface and an antibody can be measured by flow cytometry, etc. (b) Having an activity of being internalized into CLDN6 and / or CLDN9 expressing cells by binding to CLDN6 and / or CLDN9. (2) The antibody according to (1) above, wherein CLDN6 and / or CLDN9 is human CLDN6 and / or human CLDN9.

[0130] The method for obtaining the antibody against CLDN6 of the present invention usually involves the steps described below, but is not limited thereto. (Method using hybridomas) (a) purifying a biopolymer to be used as an antigen, or preparing an antigen-expressing cell, and administering the biopolymer or the antigen-expressing cell to an animal; (b) collecting tissues (e.g., lymph nodes) containing antibody-producing cells from the above-mentioned animals in which an immune response has been induced; (c) preparation of myeloma cells (hereinafter referred to as "myeloma") (e.g., mouse myeloma SP2 / 0-ag14 cells); (d) cell fusion between antibody-producing cells and myelomas; (e) selection of hybridomas producing the desired antibody; (f) division into single cell clones (cloning); (g) In some cases, culturing hybridomas or rearing animals implanted with hybridomas for the large-scale production of monoclonal antibodies; (h) Examination of the physiological activity (internalization activity) and binding specificity of the monoclonal antibody thus produced, or testing of its properties as a labeling reagent. The antibody titer measurement method used here includes, for example, flow cytometry or Cell-ELISA, but is not limited to these methods.

[0131] Examples of the monoclonal anti-CLDN6 antibodies obtained in this manner include mouse anti-CLDN6 antibodies B1 and C7. In the present invention, the "B1" may be referred to as the "B1 antibody" and the "C7" may be referred to as the "C7 antibody." The nucleotide sequence of the heavy chain variable region of the B1 antibody is shown in SEQ ID NO: 20 (Figure 19) in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 21 (Figure 19). The nucleotide sequence of the light chain variable region of the B1 antibody is shown in SEQ ID NO: 18 (Figure 18) in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 19 (Figure 18). The amino acid sequence of CDRH1 of the B1 antibody is set forth in SEQ ID NO:9 (Figure 15), the amino acid sequence of CDRH2 is set forth in SEQ ID NO:10 (Figure 15), the amino acid sequence of CDRH3 is set forth in SEQ ID NO:11 (Figure 15), the amino acid sequence of CDRL1 is set forth in SEQ ID NO:5 (Figure 13), the amino acid sequence of CDRL2 is set forth in SEQ ID NO:6 (Figure 13), and the amino acid sequence of CDRL3 is set forth in SEQ ID NO:7 (Figure 13). The nucleotide sequence of the heavy chain variable region of the C7 antibody is shown in SEQ ID NO: 24 (Figure 21) in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 25 (Figure 21). The nucleotide sequence of the light chain variable region of the C7 antibody is shown in SEQ ID NO: 22 (Figure 20) in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 23 (Figure 20). The amino acid sequence of CDRH1 of the C7 antibody is set forth in SEQ ID NO: 15 (Figure 17), the amino acid sequence of CDRH2 is set forth in SEQ ID NO: 16 (Figure 17), the amino acid sequence of CDRH3 is set forth in SEQ ID NO: 17 (Figure 17), the amino acid sequence of CDRL1 is set forth in SEQ ID NO: 12 (Figure 16), the amino acid sequence of CDRL2 is set forth in SEQ ID NO: 13 (Figure 16), and the amino acid sequence of CDRL3 is set forth in SEQ ID NO: 14 (Figure 16).

[0132] Furthermore, even if the steps of "production of anti-CLDN6 antibody" (a) to (h) are carried out again to obtain a separate and independent monoclonal antibody, or if a monoclonal antibody is obtained by another method, it is possible to obtain an antibody having an internalization activity equivalent to that of the B1 or C7 antibody. An example of such an antibody is an antibody that binds to the same epitope as the B1 or C7 antibody. If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure to which the B1 or C7 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the B1 or C7 antibody. Furthermore, by confirming that the monoclonal antibody competes with the binding of the B1 or C7 antibody to CLDN6 (i.e., the monoclonal antibody inhibits the binding of the B1 or C7 antibody to CLDN6), it can be determined that the monoclonal antibody binds to the same epitope as the anti-CLDN6 antibody, even if the specific sequence or structure of the epitope has not been determined. If it is confirmed that the epitope is the same, it is strongly expected that the monoclonal antibody has antigen-binding ability, biological activity and / or internalization activity equivalent to those of the B1 antibody or C7 antibody.

[0133] The antibodies of the present invention include not only the above-mentioned monoclonal antibodies against CLDN6, but also genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity against humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods. (1) Chimeric antibody Chimeric antibodies include antibodies whose variable and constant regions are heterologous, for example, chimeric antibodies in which the variable region of a mouse or rat-derived antibody is joined to a constant region of human origin (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). A chimeric antibody derived from a mouse anti-human CLDN6 antibody B1 antibody, exemplified as a chimeric antibody of the present invention, is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 21 (Figure 19) and a light chain comprising a light chain variable region shown in SEQ ID NO: 19 (Figure 18), and may have a constant region of any human origin. A specific example of a chimeric antibody derived from mouse anti-human CLDN6 antibody B1 antibody is the chimeric antibody chB1 antibody (hereinafter also referred to as "chB1") derived from mouse anti-human CLDN6 antibody B1 antibody. The amino acid sequence of the chB1 antibody includes an antibody having a heavy chain having an amino acid sequence consisting of amino acid residues at positions 20 to 471 of SEQ ID NO: 32 (FIG. 24) in the sequence listing, and a light chain having an amino acid sequence consisting of amino acid residues at positions 21 to 234 of SEQ ID NO: 28 (FIG. 22) in the sequence listing. In the heavy chain sequence shown in SEQ ID NO:32 (FIG. 24) in the sequence listing, the amino acid sequence consisting of amino acid residues at positions 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues at positions 20 to 141 is a heavy chain variable region, and the amino acid sequence consisting of amino acid residues at positions 142 to 471 is a heavy chain constant region. In the light chain sequence shown in SEQ ID NO:28 (FIG. 22) in the sequence listing, the amino acid sequence consisting of amino acid residues at positions 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues at positions 21 to 127 is a light chain variable region, and the amino acid sequence consisting of amino acid residues at positions 128 to 234 is a light chain constant region. The amino acid sequences of the heavy and light chain variable regions of the chB1 antibody are set forth in SEQ ID NO: 34 (Figure 25) and SEQ ID NO: 30 (Figure 23) of the sequence listing. The heavy chain amino acid sequence of the chB1 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 33 of the Sequence Listing (FIG. 24). The nucleotide sequence consisting of 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 consisting of 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 consisting of 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. The base sequence of the heavy chain variable region of the chB1 antibody is shown in SEQ ID NO: 35 of the sequence listing (Figure 25). The light chain amino acid sequence of the chB1 antibody is encoded by the nucleotide sequence shown in SEQ ID NO:29 (FIG. 22) in the sequence listing. The nucleotide sequence consisting of nucleotides 26 to 85 of the nucleotide sequence shown in SEQ ID NO:29 in the sequence listing encodes the signal sequence of the chB1 antibody light chain, the nucleotide sequence consisting of nucleotides 86 to 406 of the nucleotide sequence shown in SEQ ID NO:29 in the sequence listing encodes the light chain variable region of the chB1 antibody, and the nucleotide sequence consisting of nucleotides 407 to 727 of the nucleotide sequence shown in SEQ ID NO:29 in the sequence listing encodes the light chain constant region of the chB1 antibody. The nucleotide sequence of the light chain variable region of the chB1 antibody is shown in SEQ ID NO: 31 of the sequence listing (Figure 23). (2) Humanized antibodies Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) has been incorporated into an antibody of human origin (see Nature (1986) 321, pp. 522-525), antibodies in which not only the CDR sequence but also some framework amino acid residues have been grafted into a human antibody by CDR grafting (WO90 / 07861), and antibodies in which the amino acid sequence of some of the CDRs has been modified while maintaining the antigen-binding ability. However, the humanized antibody derived from the B1 antibody or the C1 antibody is not limited to a specific humanized antibody as long as it retains all six CDR sequences of the B1 antibody or the C1 antibody and has CLDN6 binding activity, and furthermore, a humanized antibody mutant in which the amino acid sequence of one to several (preferably one to two, more preferably one) CDRs has been modified is also not limited to a specific humanized antibody as long as it recognizes the CLDN6 protein or has the CLDN6 protein binding activity of the antibody. Examples of the anti-CLDN6 humanized antibody or a functional fragment thereof of the present invention include: CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:9 in the sequence listing (FIG. 15) or the amino acid sequence in which one or several (preferably one or two) amino acids have been substituted; CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing (FIG. 15) or the amino acid sequence in which one or several (preferably one or two) amino acids have been substituted; and A heavy chain having a variable region including CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:11 (FIG. 15) in the sequence listing or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:5 in the sequence listing ( FIG. 13 ) or the amino acid sequence in which one or several (preferably one or two) amino acids have been substituted; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:6 in the sequence listing ( FIG. 13 ) or the amino acid sequence in which one or several (preferably one or two) amino acids have been substituted, and a light chain having a variable region including CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing ( FIG. 13 ) or an amino acid sequence in which one or several (preferably one or two) amino acids in said amino acid sequence have been substituted, Examples of such antibodies include those that recognize the CLDN6 protein of the present invention or that retain the CLDN6 protein-binding activity of the antibody, or functional fragments of the antibody. An example of an amino acid substitution in the CDR in the above-mentioned anti-CLDN6 humanized antibody or a functional fragment thereof preferably includes one to several (preferably 1 to 2) amino acid substitutions in the above-mentioned CDRL3, and an example of the CDRL3 shown in SEQ ID NO:8 in the sequence listing (Figure 14) in which amino acids 4 and 5 of SEQ ID NO:7 in the sequence listing have been substituted.

[0134] Examples of the heavy chain variable region of the humanized antibody having the above-mentioned CDRH include the amino acid sequence shown in SEQ ID NO: 54 (Figure 35) of the Sequence Listing, the amino acid sequence shown in SEQ ID NO: 58 (Figure 37) of the Sequence Listing, and the amino acid sequence shown in SEQ ID NO: 62 (Figure 39) of the Sequence Listing, and examples of the light chain variable region of the humanized antibody having the above-mentioned CDRL include the amino acid sequence shown in SEQ ID NO: 38 (Figure 27) of the Sequence Listing, the amino acid sequence shown in SEQ ID NO: 42 (Figure 29) of the Sequence Listing, the amino acid sequence shown in SEQ ID NO: 46 (Figure 31) of the Sequence Listing, and the amino acid sequence shown in SEQ ID NO: 50 (Figure 33) of the Sequence Listing.

[0135] As the humanized antibody comprising the above-mentioned combination of a heavy chain variable region and a light chain variable region, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:54 (Figure 35) in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:38 (Figure 27) in the sequence listing; A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:58 (Figure 37) in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:42 (Figure 29) in the sequence listing; A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:54 (Figure 35) in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:46 (Figure 31) in the sequence listing; A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:58 (Figure 37) in the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:50 (Figure 33) in the sequence listing; A suitable example is a humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62 (Figure 39) of the Sequence Listing, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 46 (Figure 31) of the Sequence Listing.

[0136] The full-length sequence of a humanized antibody comprising the above-mentioned combination of heavy chain variable region and light chain variable region is: A humanized antibody (H1L1) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 52 (Figure 34) in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 36 (Figure 26) in the sequence listing; A humanized antibody (H2L2) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 56 (Figure 36) in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 40 (Figure 28) in the sequence listing; A humanized antibody (H1L3) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 52 (Figure 34) in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 44 (Figure 30) in the sequence listing; A humanized antibody (H2L4) comprising a heavy chain consisting of the amino acid sequence shown in amino acid numbers 20 to 471 of SEQ ID NO: 56 (Figure 36) in the sequence listing, and a light chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 of SEQ ID NO: 48 (Figure 32) in the sequence listing, or An example is a humanized antibody (H3L3) comprising a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 471 of SEQ ID NO: 60 (Figure 38) in the sequence listing, and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 234 of SEQ ID NO: 44 (Figure 30) in the sequence listing. In the heavy chain amino acid sequence shown in SEQ ID NO: 52 (Figure 34), 56 (Figure 36), or 60 (Figure 38) in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is a heavy chain variable region, and the amino acid sequence consisting of amino acid residues 142 to 471 is a heavy chain constant region. In addition, among the light chain amino acid sequences shown in SEQ ID NO: 36 (Figure 26), 40 (Figure 28), 44 (Figure 30), or 48 (Figure 32) in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is a light chain variable region, and the amino acid sequence consisting of amino acid residues 128 to 234 is a light chain constant region.

[0137] The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H1L1 is SEQ ID NO: 53 (FIG. 34), the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 37 (FIG. 26), The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L2 is SEQ ID NO: 57 (Figure 36), the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 41 (Figure 28), The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H1L3 is SEQ ID NO: 53 (Figure 34), the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 45 (Figure 30), The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L4 is SEQ ID NO: 57 (Figure 36), the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 49 (Figure 32), The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H3L3 is a polynucleotide shown in SEQ ID NO: 61 (Figure 38), and the base sequence encoding the light chain amino acid sequence is a polynucleotide shown in SEQ ID NO: 45 (Figure 30). The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L1 is SEQ ID NO: 55 (FIG. 35), the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 39 (FIG. 27), The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L2 is SEQ ID NO: 59 (FIG. 37), the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 43 (FIG. 29), The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H1L3 is SEQ ID NO: 55 (Figure 35), the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 47 (Figure 31), The base sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H2L4 is SEQ ID NO: 59 (Figure 37), the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 51 (Figure 33), The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H3L3 is a polynucleotide shown in SEQ ID NO: 63 (Figure 39), and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is a polynucleotide shown in SEQ ID NO: 47 (Figure 31). In addition, the nucleotide sequence consisting of nucleotides 1 to 57 of each of the nucleotide sequences shown in SEQ ID NOs: 53 (Figure 34), 57 (Figure 36), and 61 (Figure 38) in the sequence listing encodes the signal sequence of the humanized antibody heavy chain, the nucleotide sequence consisting of nucleotides 58 to 423 encodes the amino acid sequence of the humanized antibody heavy chain variable region, and the nucleotide sequence consisting of nucleotides 424 to 1,413 encodes the antibody heavy chain constant region. Furthermore, the nucleotide sequence consisting of nucleotides 1 to 60 of each of the nucleotide sequences shown in SEQ ID NOs: 37 (Figure 26), 41 (Figure 28), 45 (Figure 30), and 49 (Figure 32) in the sequence listing encodes the signal sequence of the humanized antibody light chain, the nucleotide sequence consisting of nucleotides 61 to 381 encodes the amino acid sequence of the humanized antibody light chain variable region, and the nucleotide sequence consisting of nucleotides 382 to 702 encodes the antibody light chain constant region.

[0138] The antibodies of the present invention also include antibodies comprising the combination of the heavy chain variable region and light chain variable region described above, or antibodies having an identity or homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 99% or more with the amino acid sequence of an antibody comprising the combination of the heavy chain and light chain described above, as long as they have binding activity to CLDN6. Furthermore, antibodies comprising a combination of the above-mentioned heavy chain variable region and light chain variable region, or antibodies having a CDR consisting of the same amino acid sequence as the CDR of an antibody comprising the above-mentioned combination of a heavy chain and a light chain, and having an identity or homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 99% or more, with respect to the amino acid sequence excluding the amino acid sequence of the CDR of said antibody, are also included in the antibodies of the present invention, as long as they have binding activity to CLDN6. Furthermore, it is possible to select antibodies having biological activity equivalent to each of the above antibodies by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the amino acid sequence of the heavy chain or light chain. Furthermore, conservative amino acid substitutions are preferred for the amino acid substitutions herein (WO2013154206). Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their side chains. It is preferable that such amino acid substitutions are made within a range that does not impair the properties of the substance having the original amino acid sequence. The homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be accessed on the Internet at www.ncbi.nlm.nih.gov / blast. (3) Human antibodies The antibody of the present invention further includes a human antibody that binds to CLDN6 and / or CLDN9. An anti-CLDN6 and / or CLDN9 human antibody means a human antibody having only the gene sequence of an antibody derived from a human chromosome. An anti-CLDN6 human antibody can be obtained by a method using a human antibody-producing mouse having a human chromosome fragment containing the heavy and light chain genes of a human antibody (see 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, etc.). Specifically, such human antibody-producing mice can be produced as genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci have been destroyed and instead the human immunoglobulin heavy and light chain gene loci have been introduced via a yeast artificial chromosome (YAC) vector or the like, by producing knockout animals and transgenic animals and crossing these animals with each other. Alternatively, eukaryotic cells can be transformed by recombinant gene technology with cDNA encoding each of the heavy and light chains of such a human antibody, preferably with a vector containing the cDNA, and the transformed cells that produce the recombinant human monoclonal antibody can be cultured to obtain the antibody from the culture supernatant. Here, examples of hosts that can be used include eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, and myeloma cells. In addition, methods for obtaining phage display-derived human antibodies selected from a human antibody library are also known (see, for example, Investigative Ophthalmology & Visual Science. (2002) 43(7), p. 2301-2308; Briefings in Functional Genomics and Proteomics (2002), 1(2), p. 189-203; Ophthalmology (2002) 109(3), p. 427-431). For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105-1116) can be used in which the variable region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv) and a phage that binds to the antigen is selected. By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of an antigen-binding scFv has been elucidated, an expression vector carrying that sequence can be prepared and introduced into an appropriate host for expression to obtain a human antibody (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23(9), p. 1105-1116).

[0139] The chimeric antibodies, humanized antibodies, human antibodies, and the like obtained by the above-mentioned methods can be evaluated for their antigen-binding ability by known methods, and suitable antibodies can be selected. Another index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good index of the relative structural stability of a protein. By measuring Tm values ​​using DSC and comparing the values, differences in thermal stability can be compared. It is known that the storage stability of antibodies shows a certain degree of correlation with their thermal stability. (Pharmaceutical Development and Technology (2007) 12, p. 265-273), suitable antibodies can be selected using thermal stability as an index. Other indices for selecting antibodies include high yield in appropriate host cells and low aggregation tendency in aqueous solution. For example, an antibody with the highest yield does not necessarily exhibit the highest thermal stability, so it is necessary to make a comprehensive judgment based on the above-mentioned indices and select an antibody that is most suitable for administration to humans.

[0140] The antibody of the present invention also includes "antibodies that bind to the same site as the anti-CLDN6 antibody provided by the present invention." In other words, antibodies that bind to the site on the CLDN6 protein recognized by B1 or C7 of the present invention are also included in the present invention.

[0141] The antibody of the present invention also includes modified antibodies. The modified antibody means an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those in which a chemical moiety is bonded to the amino acid backbone, and those in which an N- or O-linked carbohydrate chain is chemically modified. Biological modifications include those that have been post-translationally modified (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, and oxidation of methionine), and those in which a methionine residue has been added to the N-terminus by expression using a prokaryotic host cell. In addition, those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled, are also included in the meaning of such modified antibodies. Such modified antibodies of the present invention are useful for improving the stability and blood retention of the antibody, reducing antigenicity, and detecting or isolating the antibody or antigen, and the like. Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by regulating the sugar chain modification bound to the antibody of the present invention (glycosylation, defucosylation, etc.). Techniques for regulating the sugar chain modification of antibodies are known, including, but not limited to, WO1999 / 54342, WO2000 / 61739, and WO2002 / 31140. The antibody of the present invention also includes an antibody in which the sugar chain modification has been regulated. Such modifications may be made at any or desired position in the antibody or functional fragment thereof, and the same or two or more different modifications may be made at one or more positions. In the present invention, the term "modified antibody fragment" also includes within its meaning "a fragment of a modified antibody".

[0142] When an antibody gene is isolated and then introduced into a suitable host to produce an antibody, a suitable combination of a host and an expression vector can be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence, etc., of an antibody described herein and a gene encoding the light chain sequence, etc. When transforming a host cell, the heavy chain sequence gene, etc. and the light chain sequence gene, etc. can be inserted into the same expression vector, or they can be inserted into separate expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, examples of animal cells include mammalian cells, such as monkey cells, COS cells (Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220), and FreeStyle 293F cells (Invitrogen). When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis. The antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody. In this culture, the yield may vary depending on the antibody sequence, and it is possible to select antibodies that are easy to produce as a pharmaceutical using the yield as an indicator from among antibodies with equivalent binding activity. Thus, the antibody of the present invention also includes an antibody obtained by a production method for the antibody, which is characterized by comprising the steps of culturing the transformed host cells and recovering the antibody of interest or a functional fragment of the antibody from the culture obtained in this step.

[0143] The above-mentioned antibody gene is preferably a polynucleotide comprising any one of the polynucleotides (a) to (e) below: (a) a combination of a polynucleotide encoding a heavy chain amino acid sequence and a polynucleotide encoding a light chain amino acid sequence of any one of the B1 or C7 antibody, the chB1 antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3; (b) a combination of a polynucleotide encoding a heavy chain amino acid sequence including CDRH1 to CDRH3 and a polynucleotide encoding a light chain amino acid sequence including CDRL1 to CDRL3 of any one of the B1 or C7 antibody, the chB1 antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3; (c) a combination of a polynucleotide encoding a heavy chain amino acid sequence including the amino acid sequence of the heavy chain variable region of any one of the B1 or C7 antibody, the chB1 antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3, and a polynucleotide encoding a light chain amino acid sequence including the amino acid sequence of the light chain variable region. (d) a polynucleotide that hybridizes under stringent conditions with a nucleotide consisting of a polynucleotide complementary to any one of the polynucleotides (a) to (c) and encodes an amino acid sequence of an antibody that binds to CDLN6; and (e) A polynucleotide encoding the amino acid sequence of a polypeptide in which 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid has been substituted, deleted, added, or inserted in the polynucleotide according to any one of (a) to (c), and encoding the amino acid sequence of an antibody that binds to CLDN6. The present invention includes nucleotides encoding the antibodies of the present invention or functional fragments thereof, or modified forms thereof, recombinant vectors into which the genes have been inserted, and cells into which the genes or the vectors have been introduced. The present invention also includes a method for producing an antibody, a functional fragment thereof, or a modified antibody, comprising the steps of culturing the cells and recovering the antibody, a functional fragment thereof, or a modified antibody from the culture.

[0144] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibody according to the present invention also includes antibodies and functional fragments of the antibodies that have been modified in this way, and also includes deletion products in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and deletion products that have been amidated (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated), etc. However, as long as the antigen-binding ability and effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above deletions, or a combination of any two types. The quantitative ratio of each deletion may be influenced by the type and culture conditions of the cultured mammalian cells producing the antibody according to the present invention, but the main component of the antibody according to the present invention may be one in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains.

[0145] The isotype of the anti-CLDN6 antibody of the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), etc., and preferably IgG1, IgG2 or IgG4. When IgG1 is used as the isotype of the antibody of the present invention, it is possible to adjust the effector function by substituting a portion of the amino acid residues in the constant region. Examples of IgG1 mutants with reduced or weakened effector function include IgG1 LALA (IgG1-L234A, L235A) and IgG1 LAGA (IgG1-L235A, G237A), and IgG1 LALA is preferred. The L234A and L235A represent the substitution of leucine at positions 234 and 235 with alanine as specified by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85), and G237A represents the substitution of glycine at position 237 with alanine as specified by the EU index.

[0146] The biological activities of an antibody generally include antigen-binding activity, activity of internalizing into cells expressing the antigen by binding to the antigen, activity of neutralizing antigen activity, activity of enhancing antigen activity, antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP), but the function of the antibody according to the present invention is binding activity to CLDN6, preferably activity of internalizing into CLDN6-expressing cells by binding to CLDN6. Furthermore, the antibody of the present invention may have ADCC activity, CDC activity, and / or ADCP activity in addition to the cell-internalizing activity.

[0147] The obtained antibody can be purified to homogeneity. The antibody can be separated and purified by the separation and purification method usually used for proteins. For example, the antibody can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (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. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatographies can be carried out using liquid chromatography such as HPLC or FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. It is also possible to purify antibodies by using a carrier on which an antigen is immobilized, utilizing their binding to the antigen.

[0148] The anti-HER2 antibody of the present invention is not particularly limited, but preferably has the following properties, for example. (1) An anti-HER2 antibody characterized by the following properties: (a) Specifically binds to HER2. (b) It has the activity of being internalized into HER2-expressing cells by binding to HER2. (2) The antibody described in (1) above, which binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) The antibody according to any one of (1) to (3) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and contains a mutation that results in reduced ADCC and / or CDC activity. (7) The antibody according to (6) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and leucine at positions 234 and 235 as determined by the EU index is substituted with alanine. (8) The antibody according to any one of (1) to (4) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 65 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 64. (9) The antibody according to any one of (1) to (3), (6) or (7) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 469 of SEQ ID NO: 75 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 73. (10) The antibody according to any one of (1) to (9) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (11) The antibody according to any one of (1) to (3), (8) or (10), comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 65 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 64. (12) The antibody according to any one of (1) to (3), (6), (7), (9) or (10) above, comprising a heavy chain consisting of an amino acid sequence set forth in amino acid numbers 20 to 468 of SEQ ID NO: 75 and a light chain consisting of an amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 73. (13) An antibody obtained by a method for producing the antibody, the method comprising the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (12) above, and collecting the antibody of interest from the culture obtained in the above step.

[0149] <Glycan remodeling> In recent years, a method has been reported in which heterogeneous antibody glycoproteins are remodeled by enzymatic reactions or the like to uniformly introduce functional glycans (ACS Chemical Biology 2012, 7, 110, ACS Medicinal Chemistry Letters 2016, 7, 1005). Attempts have also been made to use this glycan remodeling technology to site-specifically introduce drugs and synthesize homogeneous ADCs (Bioconjugate Chemistry 2015, 26, 2233, Angew.Chem.Int.Ed. 2016,55,2361-2367, US2016361436).

[0150] In the sugar chain remodeling of the present invention, first, a hydrolase is used to remove non-uniform sugar chains attached to a protein (such as an antibody) leaving only the terminal GlcNAc, and a uniform protein portion with GlcNAc attached is prepared (hereinafter referred to as "acceptor"). Next, a separately prepared sugar chain of any kind is prepared (hereinafter referred to as "donor"), and this acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a uniform glycoprotein with any sugar chain structure.

[0151] In the present invention, the term "glycan" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and glycans may be represented by abbreviations, such as "GlcNAc-" or "MSG-". When these abbreviations are used in structural formulae, the oxygen atom or nitrogen atom that belongs to the glycosidic bond with another structural unit at the reducing end is not included in the abbreviation representing the glycan, unless otherwise defined.

[0152] In the present invention, unless otherwise specified, monosaccharides that are the basic units of sugar chains are described by assuming that the carbon atom that is bonded to the oxygen atom constituting the ring and that is directly bonded to a hydroxyl group (or an oxygen atom belonging to a glycosidic bond) in the ring structure is in the 1st position (only in the case of sialic acid, the 2nd position). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0153] In the present invention, when a glycan is described as a symbol (e.g., GLY, SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O belonging to the N- or O-glycosidic bond.

[0154] In the present invention, unless otherwise specified, a partial structure in which an amino acid is linked to a glycan at its side chain will be represented by displaying the side chain portion in parentheses, for example, as "(SG-)Asn."

[0155] The antibody-drug conjugate of the present invention has the following formula: [ka] and the antibody Ab or a functional fragment thereof may be bound to L directly from a side chain of an amino acid residue (e.g., cysteine, lysine, etc.) thereof, or may be bound to L through a glycan or a remodeled glycan of the Ab, preferably through a glycan or a remodeled glycan of the Ab, and more preferably through a remodeled glycan of the Ab.

[0156] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain. N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0157] The Ab of the present invention is an IgG, preferably an IgG1, IgG2 or IgG4.

[0158] IgG has a well-conserved N-linked glycan at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, which is known to contribute to the activity and dynamics of antibody molecules (Biotechnol. Prog., 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem., 2013, 85, 715-736).

[0159] The amino acid sequence in the constant region of IgG is well conserved, and each amino acid is identified by Eu number (Eu INDEX) in Edelman et al. (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85). For example, Asn297, to which N-linked glycan is added in the Fc region, corresponds to position 297 in the Eu numbering, and even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by displaying it by Eu numbering.

[0160] In the antibody-drug conjugate of the present invention, more preferably, the antibody or functional fragment thereof is bound to L through a glycan attached to the side chain of Asn297 (hereinafter referred to as "N297 glycan"), and even more preferably, the antibody or functional fragment thereof is bound to L through the N297 glycan, and the N297 glycan is a remodeled glycan. The following formula shows the case where the antibody-drug conjugate of the present invention is bound to L from the N297 sugar chain of an antibody or a functional fragment thereof. [ka] An antibody having such a remodeled glycan is called a glycan-remodeled antibody.

[0161] SGP is an abbreviation for sialylated glycopeptide, and is a representative N-linked complex glycan. SGP can be isolated and purified from egg yolk of chicken eggs, for example, according to the method described in WO2011 / 0278681. In addition, purified products of SGP are commercially available (Tokyo Kasei Co., Ltd., Fushimi Pharmaceutical Co., Ltd.) and can be purchased. Disialooctasaccharide (Tokyo Kasei Co., Ltd.), which consists only of a glycan lacking one GlcNAc at the reducing end in the glycan portion of SG (hereinafter referred to as "SG(10)"), is commercially available.

[0162] In the present invention, a glycan structure in which sialic acid is deleted from the non-reducing end of only one of the branched chains of β-Man of SG(10) is referred to as MSG(9), and one having sialic acid only in the 1-3 glycan of the branched chain is referred to as MSG1, and one having sialic acid only in the 1-6 glycan of the branched chain is referred to as MSG2.

[0163] The remodeled glycan of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, or N297-(Fuc)SG, preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

[0164] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula: [ka] [ka] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2-NH-, and the amino group on the right end is amide bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 side of the branched chain of β-Man in the N297 glycan. The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, in particular, where n 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0165] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula: [ka] [ka] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, and the amino group on the right end is amide bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-6 side of the branched chain of β-Man in the N297 glycan. The asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, in particular, where n 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0166] N297-(Fuc)SG is represented by the following structural formula or sequence formula: [ka] [ka] In the above formula, the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH-, which indicates that the amino group at the right end is amide-bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain, and the asterisk indicates that it is bonded to the 1- or 3-position nitrogen atom on the 1,2,3-triazole ring of Lb in the linker L, particularly the linker L; where n 5 is an integer of 2 to 10, and preferably an integer of 2 to 5.

[0167] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which two linkers L and two drugs D are bound (the above m 2 =1) (see Figure 1). For example, Example 74: ADC8 is a case where the N297 sugar chain is N297-(Fuc)MSG1, and Example 67: ADC1 is a case where the N297 sugar chain is a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2.

[0168] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, the antibody is a dimer, and therefore the antibody-drug conjugate is a molecule to which four linkers L and four drugs D are bound (see above m 2 =2). For example, Example 72: ADC6 is a case where the N297 sugar chain is N297-(Fuc)SG.

[0169] The N297 sugar chain is preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or N297-(Fuc)SG, and more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2. 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, a homogeneous ADC can be obtained.

[0170] The present invention provides a method for producing a remodeling antibody or a functional fragment of the antibody, comprising the following steps i) to iii): i) culturing the host cell (e.g., animal cell (CHO cell, etc.)) according to any one of the above

[46] to

[48] , and collecting the antibody of interest from the obtained culture; ii) treating the antibody obtained in step i) with a hydrolase to produce an antibody in which the N297 sugar chain is (Fucα1,6)GlcNAc ((Fucα1,6)GlcNAc-antibody) ( FIG. 3A ); Preferably, the method further comprises the step of purifying the (Fucα1,6)GlcNAc-antibody from the reaction solution by a step including purification using a hydroxyapatite column; and Either step iii)-1 or iii)-2 (Figure 3B). iii)-1 A PEG linker (N) having an azide group is attached to the carbonyl group of the carboxyl group at the 2-position of the sialic acid of MSG(9) or SG(10). 3 a step of reacting a glycan donor molecule having an oxazolinyl group introduced thereinto (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase, thereby synthesizing a glycan remodeling antibody having an azide group introduced into the sialic acid thereof; iii)-2 PEG linker (N) having an azide group at the carbonyl group of the carboxylic acid at the 2-position of the sialic acid of (MSG-)Asn or (SG-)Asn, whose α-amino group may be protected or modified, and an azide group at the carbonyl group of the carboxylic acid of the Asn 3 This is a process in which a glycan remodeling antibody in which an azide group has been introduced into the sialic acid is synthesized by reacting a glycan donor molecule in which the reducing end has been oxazolinated with a (Fucα1,6)GlcNAc-antibody in the presence of a glycosyltransferase, after which a hydrolase is applied to the glycan donor molecule, and an azide group is introduced into the sialic acid. Furthermore, the present invention also includes the sugar chain remodeling antibody or a functional fragment thereof obtained by such a production method, or modified versions thereof.

[0171] The intermediate in the production of the antibody-drug conjugate has an alkyne structure that reacts with an azide group such as DBCO (dibenzocyclooctyne). Therefore, the antibody-drug conjugate of the present invention can be produced by reacting the intermediate in the production with an MSG1 type, MSG2 type or SG type glycan remodeling antibody or a functional fragment of the antibody, in which a PEG linker having an azide group has been introduced into the sialic acid of the glycan obtained in the steps i) to iii).

[0172] In the N297 sugar chain of the present invention, the fucose-added GlcNAc (Fucα1,6)GlcNAc) at the reducing end is derived from an antibody produced in animal cells, and the sugar chain on the non-reducing end side is preferably one remodeled to the same sugar chain structure as the above-mentioned MSG (MSG1, MSG2) or SG. In either case, L(PEG) is bound to the carboxylic acid bound to the 2-position of the sialic acid at the non-reducing end. Such glycan remodeling antibodies having MSG (MSG1, MSG2) or SG-type N297 glycans can be produced, for example, according to the method described in WO2013 / 120066, as shown in Figure 3. When an antibody is produced as a recombinant protein using an animal cell as a host according to a known method (step i above), the N297 glycan has a fucose-added N-linked glycan structure as a basic structure, but is obtained as a mixture of antibodies or fragments thereof having glycans of various structures with various modifications to the non-reducing terminal structure or the constituent sugars (IV in Figure 3A). When the antibody produced in animal cells in this manner is treated with a hydrolase such as EndoS, the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure is hydrolyzed to obtain an antibody molecule having a single glycan structure containing only (Fucα1,6)GlcNAc as the N297 glycan (referred to as "(Fucα1,6)GlcNAc-antibody" (see A in Figure 2) (Figure 3A) (step ii) above).

[0173] The enzyme used in the hydrolysis reaction of the N297 sugar chain may be EndoS or a mutant enzyme that retains its hydrolysis activity.

[0174] The (Fucα1,6)GlcNAc-antibody obtained by the above hydrolysis reaction can be reacted as a glycan acceptor molecule with an MSG (MSG1, MSG2) or SG-type glycan donor molecule using a glycosyltransferase such as EndoS D233Q or EndoS D233Q / Q303L mutant (WO2017010559, etc.) to obtain an antibody having an MSG (MSG1, MSG2) or SG-type N297 glycan having the above structure (see B in Figure 2) (Figure 3B) (steps iii)-1 and iii)-2 above).

[0175] The number of drugs bound per antibody molecule in an antibody-drug conjugate, m 2 When is 1, a glycan donor molecule having MSG, MSG1 or MSG2 as the glycan is used. Such glycans can be used by separating (MSG-)Asn1 or (MSG2-)Asn from commercially available monosialo-Asn free (1S2G / 1G2S-10NC-Asn, Glycotechnology Institute, hereinafter referred to as "(MSG-)Asn") as a raw material in accordance with the method described in Example 56, or can be used as a mixture without separation.

[0176] The number of drugs bound per antibody molecule in an antibody-drug conjugate, m 2 In the case of 2, a glycan donor molecule having SG(10) as a glycan is used in this glycosyltransfer reaction. Such an SG(10) glycan may be obtained, for example, from SGP by hydrolysis or the like, or a commercially available SG(10) glycan such as disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) may be used.

[0177] The MSG (MSG1, MSG2) or SG-type glycan contained in the donor molecule is linked to a PEG linker (N 3 -L(PEG)) containing an azide group at the 2-position of sialic acid.3 For the introduction of N-L(PEG), MSG (MSG(9)), MSG1 or MSG2, or disialooctasaccharide (SG(10)) and an azide group-containing PEG linker (N 3 -L(PEG) 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 (n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.) can be obtained by utilizing a reaction (condensation reaction, etc.) known in the field of organic synthesis science. That is, the 2-position of sialic acid is substituted with a carboxylic acid and N 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 The amino group at the right end of the amine group forms an amide bond through a condensation reaction.

[0178] In addition, the MSG (MSG1, MSG2) or SG type sugar chain is prepared by, for example, condensing a carboxylic acid at the 2-position of a raw material sialic acid such as (MSG1-)Asn, (MSG2-)Asn, or (SG-)Asn (Glycoscience Institute) whose α-amino group may be protected or modified, and a PEG linker (N) having an azide group to the carboxylic acid at the Asn position. 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 ) and reacting with a hydrolase such as EndoM or EndoRp to obtain the above (iii)-2). Examples of the protecting group for the α-amino group include, but are not limited to, an acetyl (Ac) group, a t-butyloxycarbonyl (Boc) group, a benzoyl (Bz) group, a benzyl (Bzl) group, a carbobenzoxy (Cbz) group, and a 9-fluorenylmethoxycarbonyl (Fmoc) group. The protecting group for the α-amino group is preferably an Fmoc group. Examples of the modifying group for the α-amino group include a hydroxyacetyl group, or a modifying group having a PEG structure or the like that improves water solubility. In (MSG1-)Asn, (MSG-2)Asn, or (SG-)Asn, the α-amino group is preferably protected with the above-mentioned protecting group. When the α-amino group is protected with a protecting group (e.g., Fmoc group), the protecting group can be removed, if necessary, after introducing a PEG linker having an azide group and before acting with a hydrolase.

[0179] It is preferable to use GlcNAc at the reducing end of the MSG (MSG1, MSG2) or SG-type glycan contained in the molecule that has been activated, for example, by oxazolination via 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride treatment.

[0180] As the enzyme (glycosyltransferase) used in the glycosyltransferase reaction, various enzymes can be used as long as they have the activity of transferring a complex-type glycan to the N297 glycan, but a preferred one is EndoS D233Q, a modified form in which the 233rd Asp of EndoS is replaced with Gln to suppress hydrolysis. The glycosyltransferase reaction using EndoS D233Q is described in WO2013 / 120066 and the like. In addition, a modified enzyme such as EndoS D233Q / Q303L, which is further mutated in EndoS D233Q (WO2017010559), may be used.

[0181] The purification procedure of the antibody after the glycoremodeling (glycolysis and transglycosylation) of the antibody is aimed at separating it from the low molecular weight compounds and enzymes used in the reaction, and for such purification, gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. are usually used, but additional purification using a hydroxyapatite column may also be performed. That is, the present invention provides a method for producing a drug-conjugate, which further includes a purification step using a hydroxyapatite column in the purification step of the intermediate from the reaction solution after glycolysis of the antibody. According to reports on glycan remodeling (J. Am. Chem. Soc. 2012, 134, 12308-12318., Angew. Chem. Int. Ed. 2016, 55, 2361-2367), the reaction mixture in which the antibody was treated with hydrolases was purified using a protein A column (affinity chromatography column). However, this purification method did not completely remove hydrolases (EndoS, etc.), and it was found that the residual enzymes affected the next glycosyltransfer reaction. As a result of examining the purification method, the reaction efficiency of the next glycosyltransfer reaction was improved without the influence of the residual enzymes by purifying the reaction mixture in which the antibody was treated with hydrolases using a protein A column and then a hydroxyapatite column (CHT column, Bio-Rad Laboratories, Inc.).

[0182] The antibody-drug conjugate of the present invention is most preferably an antibody-drug conjugate selected from the following group: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] In each of the structural formulas shown above, m 2 is 1 or 2 (preferably, m 2 indicates that 1), the antibody Ab is an IgG antibody (preferably, IgG1, IgG2, IgG4, more preferably, IgG1) or a functional fragment thereof; The N297 sugar chain is any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, and N297-(Fuc)SG (preferably, N297-(Fuc)MSG1), L(PEG) is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 -* indicates that the amino group at the left end is amide-bonded to the 2-position carboxylic acid of the non-reducing terminal sialic acid on the 1-3 chain side and / or the 1-6 chain side (preferably the 1-3 chain side) of the branched chain of β-Man in the N297 sugar chain, and the asterisk indicates that it is bonded to the 1- or 3-position nitrogen atom on the triazole ring of Lb in the linker L. For convenience, the most preferred antibody-drug conjugate is one in which one molecule of the conjugate contains two or four (m) units of "-(N297 sugar chain)-LD" ("(N297 sugar chain)-(N1Lb)LD") in which the N297 sugar chain is bound to the nitrogen atom at position 1 on the triazole ring of Lb in L. 2 = 1 or 2), or has two or four "-(N297 sugar chain)-LD" ("(N297 sugar chain)- (N3Lb)LD") bound to the nitrogen atom at position 3 (m 2 = 1 or 2), but one molecule of the conjugate contains "(N297 sugar chain)-(N1Lb)LD" (m 2 If =1, 1 piece, m 2 In the case of =2, 1, 2, 3) and "(N297 sugar chain)- (N3Lb)LD" (m 2 If =1, 1 piece, m 2 In other words, one molecule of the conjugate contains either "(N297 glycan)-(N1Lb)LD" or "(N297 glycan)-(N3Lb)LD", or both.

[0183] Furthermore, the Ab is preferably an anti-CLDN6 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 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-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD98 antibody, an anti-TROP2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-FGFR2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-In Examples of the antibody include a tegrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-Mesothelin antibody, an anti-EGFR antibody, and an anti-DR5 antibody. More preferred examples of the antibody include an anti-CLDN6 antibody, an anti-CLDN6 / CLDN9 antibody, an anti-HER2 antibody, an anti-CD98 antibody, and an anti-TROP2 antibody. Even more preferred examples of the antibody include the anti-CLDN6 antibody (e.g., Examples 106, 107, 108, and 109) and an anti-HER2 antibody (e.g., Trastuzumab and Trastuzumab variants).

[0184] The antibody-drug conjugate of the present invention and the anti-CLDN6 antibody or anti-HER2 antibody-drug conjugate of the present invention exhibit strong tumor activity (in vivo anti-tumor activity, in vitro anti-cellular activity), favorable pharmacokinetics and physical properties, and are highly safe, and therefore are useful as pharmaceuticals.

[0185] The antibody-drug conjugate of the present invention, its free drug or its manufacturing intermediate may have stereoisomers or optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention. The PBD derivative (V) or (VI) of the present invention has an optical isomer because it has an asymmetric carbon at the 11' position. In the present specification, these isomers and mixtures of these isomers are all represented by a single formula, that is, general formula (V) or (VI). Therefore, (V) or (VI) includes all optical isomers and mixtures of optical isomers in any ratio. The absolute configuration at the 11' position of (V) or (VI) can be determined by X-ray crystal structure analysis of a crystalline product or intermediate or a derivative thereof or NMR such as the Mosher method. In this case, the absolute configuration may be determined using a crystalline product or intermediate derivatized with a reagent having an asymmetric center with a known configuration. Stereoisomers can be obtained by isolating the synthesized compound according to the present invention by using a conventional optical resolution or separation method, if desired.

[0186] In the antibody-drug conjugate of the present invention, the number of drugs bound to one antibody molecule is an important factor that affects its efficacy and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions such as the amount of raw materials and reagents used to react so that the number of drugs bound is a certain number, but unlike chemical reactions of low molecular weight compounds, it is usually obtained as a mixture in which different numbers of drugs are bound. The number of drugs bound to one antibody molecule can be specified as an average value, that is, the average drug binding number (DAR: Drug to Antibody Ratio). The number of pyrrolobenzodiazepine derivatives bound to an antibody molecule can be controlled, and the average drug binding number (DAR) per antibody can be in the range of 1 to 10 pyrrolobenzodiazepine derivatives, but is preferably 1 to 8, and more preferably 1 to 5. In the antibody-drug conjugate of the present invention, when the antibody is bound to L via a remodeled sugar chain of the antibody, the number of drugs bound per antibody molecule in the antibody-drug conjugate m 2 is an integer of 1 or 2. When the glycan is an N297 glycan and the glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2). When the N297 sugar chain is N297-(Fuc)SG, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, and more preferably in the range of 3.5 to 4.2). Furthermore, a person skilled in the art would be able to design a reaction for binding a required number of drugs to an antibody from the description of the examples of the present application, and would be able to obtain an antibody in which the number of pyrrolobenzodiazepine derivatives bound is controlled.

[0187] Furthermore, the antibody-drug conjugate, free drug, or production intermediate of the present invention may absorb moisture and become adsorbed water or become a hydrate when left in the air or when recrystallized, and such compounds and salts containing water are also encompassed by the present invention.

[0188] When the antibody-drug conjugate, free drug, or production intermediate of the present invention has a basic group such as an amino group, it can be converted into a pharma- ceutically acceptable salt, if desired. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide salts; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate salts; lower alkanesulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate salts; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate salts; organic acid salts such as formate, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate, and maleate salts; and amino acid salts such as ornithine, glutamate, and aspartate salts.

[0189] When the antibody-drug conjugate, free drug, or production intermediate of the present invention has an acidic group such as a carboxy group, it is generally possible to form a base addition salt. Examples of pharma-ceutically acceptable salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt, and other organic amine salts.

[0190] The antibody-drug conjugate, free drug, or production intermediate of the present invention may exist as a hydrate by absorbing moisture in the air, etc. The solvate of the present invention is not particularly limited as long as it is medicamentously acceptable, and specifically, hydrates, ethanol solvates, 2-propanol solvates, etc. are preferred. In addition, when a nitrogen atom is present in the antibody-drug conjugate, free drug, or production intermediate of the present invention, it may be in the form of an N-oxide, and these solvates and N-oxides are also included in the scope of the present invention.

[0191] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate, free drug, or production intermediate of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 C) and the like. In addition, the compound of the present invention can be used, for example, with tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 The antibody-drug conjugate of the present invention may be radiolabeled with a radioisotope such as C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo imaging diagnostic agents. All isotopic variants of the antibody-drug conjugate of the present invention, whether radioactive or not, are within the scope of the present invention.

[0192] [Manufacturing method] Next, a representative method for producing the antibody-drug conjugate and free drug of the present invention or their production intermediates will be described. In the following, the compound numbers shown in each reaction scheme will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)", "compound (1)", etc. Compounds with other numbers will also be described in the same manner.

[0193] 1. Manufacturing method 1 Compound (1) of the present invention can be prepared according to Methods A to Q described below. [ka] Methods A to M are methods for producing intermediates in the production of the antibody-drug conjugate of the present invention. Methods N to Q are methods for producing the free drug of the present invention.

[0194] In each step of the following Methods A to Q, the desired reactions can be carried out using known organic chemistry techniques. The solvent used in the reactions in each step of the following Methods A to Q is not particularly limited as long as it does not inhibit or adversely affect the reaction and dissolves the starting materials to some extent. In the reactions in each step of the following Methods A to Q, the reaction temperature varies depending on the solvent, starting materials, reagents, etc., and the reaction time varies depending on the solvent, starting materials, reagents, reaction temperature, etc. In the reactions of each step of the following Methods A to Q, after the reaction is completed, each target compound is collected from the reaction mixture in a conventional manner. For example, the reaction mixture is appropriately neutralized, and if any insoluble matter is present, it is removed by filtration, and then water and an immiscible organic solvent such as ethyl acetate are added, and the organic layer containing the target compound is separated and washed with water or the like, and then dried over anhydrous magnesium sulfate, anhydrous sodium sulfate, or the like, filtered, and the solvent is distilled off to obtain the target compound. If necessary, the obtained target compound can be isolated by a conventional method, such as recrystallization, reprecipitation, chromatography (e.g., silica gel, alumina, magnesium-silica gel-based Florisil, SO 3The target compound can be separated and purified by appropriately combining methods commonly used for separating and purifying organic compounds, such as adsorption column chromatography using a carrier such as H-silica (manufactured by Fuji Silica); partition column chromatography using a carrier such as Sephadex LH-20 (manufactured by Pharmacia), Amberlite XAD-11 (manufactured by Rohm and Haas), and Diaion HP-20 (manufactured by Mitsubishi Chemical); ion exchange chromatography; normal phase and reverse phase column chromatography using silica gel or alkylated silica gel (preferably high performance liquid chromatography) appropriately combined and eluted with an appropriate eluent). For target compounds that are insoluble in a solvent, the obtained solid crude product can be washed with a solvent and purified. In addition, the target compound of each step can be used as it is in the next reaction without purification.

[0195] In the reactions of each step of the following Methods A to Q, J, La', Lp', B', E, V, W, and R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , l, n 7 , n 6 , m 1 has the same meaning as above. (Lp')' represents any of the dipeptide residues represented by -VA-, -FG-, -PI-, -VCit-, -VK-, -(D-)PI-, -PL-, -(D-)VA-, and -GF-. (R 13 )' is R 13 If there is a hydroxyl group or an amino group on the substituent of R, a protecting group may be used. If there is no protecting group, R 13 (R 17 )' is a state in which the hydroxyl group is protected by a protecting group such as tert-butyldimethylsilyloxy group, or R 17 Indicates one of the following.

[0196] PRO 1 , PRO 4 , PRO 6 , PRO8 , PRO 9 represents a protecting group for an amino group. 1 , PRO 4 , PRO 8 , PRO 9 is an allyloxycarbonyl group, a 2,2,2-trichloroethyloxycarbonyl group, a trimethylsilylethoxymethoxy group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, or the like. 6 is preferably a 2-(trimethylsilyl)ethoxymethyl group, a methoxymethyl group, or the like. Also, PRO 2 , PRO 3 , PRO 5 , PRO 7 , PRO 10 , PRO 11 , PRO 12 represents a protecting group for a hydroxy group, a phenol group, or a carboxyl group used in the field of organic synthetic chemistry, and is preferably an acetyl group, a benzyl group, a tert-butyldimethylsilyl (TBDMS) group, a triisopropylsilyl group, or a tert-butyl group.

[0197] X 2 represents a leaving group used in the field of organic synthetic chemistry, and is preferably a chlorine atom, a bromine atom, an iodine atom, a methanesulfonyl group, or a p-toluenesulfonyl group. R a , R c represents a substituent bonded to a carboxyl group, and is preferably a methyl group, an ethyl group, a benzyl group, a tert-butyl group, or the like. R b represents a leaving group that forms an enol sulfonate, and is preferably a trifluoromethanesulfonyl group, etc. In addition, amino groups and hydroxy groups not specifically protected in Methods A to Q may be protected using a protecting group as necessary. In addition, they may be deprotected as necessary, or after protection, deprotection may be performed and another protecting group may be added.

[0198] A method This production method is a method for producing compound (12a), which is a synthetic intermediate necessary for producing compound (1). [ka]

[0199] A-1 Step (1a) → (2a): Reduction reaction The reaction is carried out by treating compound (1a) with a reducing agent (lithium aluminum hydride, diborane, lithium borohydride, sodium borohydride, borane-tetrahydrofuran complex, sodium bis(2-methoxyethoxy)aluminum hydride, etc.) in a solvent (diethyl ether, tetrahydrofuran (THF), dichloromethane, ethanol, etc., or a mixture thereof) at a temperature ranging from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 50°C. The reducing agent is used in an amount of 1 to an excess molar amount, preferably 1 to 5 molar amounts, relative to compound (1a). If necessary, a Lewis acid (lithium chloride, calcium chloride, tin chloride, trifluoroborane ether complex, etc.) is added. The reaction time is 1 minute to 60 hours, preferably 5 minutes to 24 hours.

[0200] A-2 Step (2a) → (3a): Introduction of a protecting group (tert-butyldimethylsilyl group, etc.) PRO 2 When is a TBDMS group, compound (2a) is reacted with a silylation agent (tert-butyldimethylsilyl chloride, tert-butyldimethylsilyl trifluoromethanesulfonate, etc.) in a solvent (dichloromethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide (DMF), etc., or a mixture thereof) at -20°C to 120°C, preferably 0°C to 100°C. If necessary, a base (imidazole, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, sodium hydride, etc.) is added. The silylation agent is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, and the base is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (2a). The reaction time is 1 minute to 72 hours, preferably 5 minutes to 24 hours.

[0201] A-3 step (3a)→(4a): Deprotection reaction PRO 1 When is a benzyloxycarbonyl group, the reaction is carried out by catalytic hydrogenation of compound (3a) in a solvent (ethanol, propanol, methanol, ethyl acetate, THF, 1,4-dioxane, etc., or a mixture thereof) in the presence of a transition metal catalyst (palladium on carbon, etc.) at a temperature ranging from 0° C. to the boiling point of the solvent used in the reaction, preferably at 0° C. to 50° C. The reaction is usually carried out under a hydrogen atmosphere, but cyclohexene, 1,4-cyclohexadiene, etc. may be used as a hydrogen donor, if necessary. The reaction time is 10 minutes to 100 hours, preferably 30 minutes to 72 hours.

[0202] A-4 Step (4a) → (5a): Condensation reaction The reaction is carried out by reacting compound (4a) with a carboxylic acid (compound (A)) in a solvent (benzene, toluene, diethyl ether, dichloromethane, THF, DMF, water, etc., or a mixture thereof) at a temperature ranging from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C, in the presence of a condensing agent such as N,N-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoroborate, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, etc. Condensation agent is 0.3 to 5 moles, preferably 0.4 to 2 moles of carboxylic acid (compound (A)) per mole of compound (4a), and 1 to excess moles, preferably 1 to 5 moles of condensation agent. If necessary, a base (triethylamine, diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine, etc.) and an additive (1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, etc.) are added. The base is used in a catalytic amount to excess moles, preferably 0.2 to 3 moles, per mole of compound (4a). The additive is used in a catalytic amount to excess amount, preferably 0.01 to 3 moles, per mole of compound (4a). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. In addition, when the condensation reaction is carried out by converting the carboxylic acid (compound (A)) into an acid halide, compound (4a) is reacted with the acid halide of the carboxylic acid (compound (A)) in a solvent (benzene, toluene, diethyl ether, dichloromethane, tetrahydrofuran, dichloromethane, etc. or a mixture thereof) at a temperature ranging from -78°C to the boiling point of the solvent used in the reaction, preferably from -50°C to 100°C, in the presence of a base (triethylamine, diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine, etc.). For 1 mole of compound (4a), 0.3 to 5 moles, preferably 0.4 to 2 moles of the acid halide are used, and a catalytic amount to an excess mole, preferably 0.2 to 5 moles of the base are used. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. To prepare an acid halide of a carboxylic acid (compound (A)), the carboxylic acid (compound (A)) is treated with oxalyl chloride, thionyl chloride, or the like in a solvent (benzene, toluene, dichloromethane, dichloroethane, or the like, or a mixture thereof) at a temperature ranging from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 100°C. If necessary, a catalytic amount of N,N-dimethylformamide, or the like is added. Oxalyl chloride or thionyl chloride is used in an amount of 1 to an excess molar amount, preferably 1 to 10 molar, relative to the carboxylic acid (compound (A)). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0203] A-5 Step (5a) → (6a): Reduction reaction The reaction is carried out by catalytic hydrogenation of compound (5a) in a solvent (ethanol, propanol, methanol, ethyl acetate, THF, 1,4-dioxane, DMF, etc. or a mixture thereof) in the presence of a transition metal catalyst (palladium on carbon, nickel, etc.) at a temperature ranging from 0° C. to the boiling point of the solvent used in the reaction, preferably at 0° C. to 50° C. The reaction is usually carried out under a hydrogen atmosphere, but cyclohexene, 1,4-cyclohexadiene, hydrazine, etc. may be used as a hydrogen donor, if necessary. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. The reduction of the nitro group can also be carried out under the following conditions. The reaction is carried out by reacting compound (5a) with a reducing agent (iron, zinc, tin chloride, etc.) in a solvent (ethanol, methanol, diethyl ether, ethyl acetate, water or a mixture thereof) at a temperature ranging from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 90°C. If necessary, an acid (acetic acid, formic acid, ammonium chloride, etc.) is added. The reducing agent is used in an amount of 1 to an excess molar amount, preferably 1 to 100 moles, per mole of compound (5a), and the acid is added in an amount of 1 to an excess molar amount. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0204] A-6 Step (6a) → (7a): Carbamate reaction The reaction is carried out by reacting the compound (6a) with triphosgene (isocyanation agent) in a solvent (THF, dichloromethane, DMF, etc., or a mixture thereof) at a temperature ranging from -30°C to the boiling point of the solvent used in the reaction, preferably at 0°C to 50°C, generating an isocyanate intermediate in the system, and then treating it with an alcohol represented by general formula (B). If necessary, a base (triethylamine, diisopropylethylamine, sodium carbonate, sodium hydroxide, etc.) is added. 0.3 to an excess of moles of triphosgene (isocyanation agent), preferably 0.35 to 3 moles, are used per mole of the compound (6a), and 0.5 to 5 moles of base are added. The reaction time until the isocyanate intermediate is formed is 10 minutes to 24 hours, preferably 30 minutes to 1 hour. The reaction time between the isocyanate intermediate and the alcohol (B) is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The alcohol (B) used in this step can be produced according to Process L described below.

[0205] A-7 Step (7a) → (8a): Deprotection reaction PRO 2When is a TBDMS group, the reaction is carried out by reacting compound (7a) with either an acid (acetic acid, etc.) or a desilylation agent (hydrofluoric acid-pyridine, hydrofluoric acid-triethylamine, hydrofluoride salt, hydrofluoric acid, tetra-n-butylammonium fluoride, etc.) or a mixture thereof in a solvent (dichloromethane, chloroform, acetonitrile, methanol, ethanol, THF, water, etc., or a mixture thereof) at -20°C to 100°C, preferably 0°C to 50°C. The acid is used in an amount of 1 to an excess mole, and the acid and desilylation agent are used in an amount of 1 to an excess mole, preferably 1 to 10 moles, per mole of compound (7a). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0206] A-8 Step (8a) → (9a): Oxidation reaction The reaction is carried out by reacting compound (8a) with an oxidizing agent (chlorosulfonium salt, Dess-Martin reagent, tetrabutylammonium ruthenate, pyridinium chlorochromate, nitroxy radical oxidation catalyst, etc.) in a solvent (acetone, dichloromethane, pyridine, etc., or a mixture thereof) at a temperature ranging from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 30°C. If necessary, a base (triethylamine, diisopropylethylamine, sodium bicarbonate, sodium carbonate, sodium hydroxide, etc.) and a reoxidizing agent (N-methylmorpholine N-oxide, iodobenzene diacetate, sodium hypochlorite, etc.) or an additive (tetrabutylammonium bromide, potassium bromide, etc.) are added. The oxidizing agent is used in an amount of 0.005 to an excess molar amount, preferably 0.005 to 10 moles, per mole of compound (8a). In addition, 1 to 10 moles of base or reoxidizing agent and 0.02 to 1 mole of additive are added per mole of compound (8a). The reaction time is from 10 minutes to 72 hours, preferably from 30 minutes to 24 hours.

[0207] A-9 step (9a) → (10a): Protecting group introduction (R 17 When )' is a tert-butyldimethylsilyloxy group, it is prepared according to Step A-2.

[0208] A-10 Step (10a) → (11a): Deprotection reaction PRO 3 When is a triisopropylsilyl group, compound (10a) is treated with lithium acetate in a solvent (DMF, water, etc., or a mixture thereof) at a temperature ranging from 0° C. to the boiling point of the solvent used in the reaction, preferably from 0° C. to 50° C. Lithium acetate is used in an amount of 1 to an excess mole, preferably 1 to 5 moles, per mole of compound (10a). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0209] A-11 Step (11a) → (12a): Alkylation reaction The compound (11a) is reacted with an alkylating agent (C) (1,5-dibromopentane, 1,3-dibromopropane, etc.) in a solvent (THF, DMF, N,N-dimethylacetamide or a mixture thereof) at a temperature between -20°C and the boiling point of the solvent used in the reaction, preferably between 0°C and the boiling point. If necessary, a base (potassium carbonate, cesium carbonate, etc.) is added. For 1 mole of compound (11a), 1 mole to an excess of the alkylating agent, preferably 1 to 10 moles, and 0.4 to an excess of the base, preferably 0.5 to 5 moles, are used. The reaction time is 1 minute to 60 hours, preferably 5 minutes to 24 hours.

[0210] B method In this production method, R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, and R 14 , R 15 is hydrogen, which is a necessary intermediate for producing the compound (10b). [ka]

[0211] B-1 step (1b) → (2b): Deprotection group reaction PRO 7When is a triisopropylsilyl group, it can be prepared according to Method A, Step A-10. PRO 7 When is a benzyl group, it can be prepared according to Method A, Step A-3.

[0212] B-2 Step (2b) → (3b): Alkylation reaction Produced according to Method A, Step A-11.

[0213] B-3 step (3b) → (4b): Deprotection group reaction PRO 5 When is a TBDMS group, it can be prepared according to Method A, Step A-7. Pro 5 When is an acetyl group, the reaction is carried out by reacting compound (3b) with an appropriate base (potassium carbonate, sodium methoxide, sodium hydroxide, etc.) in a solvent (methanol, ethanol, THF, water, etc. or a mixture thereof) at a temperature ranging from -20°C to the boiling point of the solvent used in the reaction, preferably 0°C to 50°C. The amount of base used ranges from a catalytic amount to an excess molar amount, preferably 0.1 to 10 moles. The reaction time ranges from 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0214] B-4 Step (4b) → (5b): Oxidation reaction Produced according to Method A, Step A-8.

[0215] B-5 Step (5b) → (6b): Enol sulfonylation reaction R b When is a trifluoromethanesulfonyl group, the reaction is carried out by reacting compound (5b) with trifluoromethanesulfonic anhydride or the like in a solvent (dichloromethane, etc.) at a temperature ranging from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 30°C. If necessary, a base (2,6-lutidine, etc.) is added. Trifluoromethanesulfonic anhydride is used in an amount of 1 mole to an excess mole, preferably 1 mole to 5 moles, per mole of compound (5b). The base is used in an amount of 1 mole to 10 moles. The reaction time is 10 minutes to 24 hours, preferably 30 minutes to 6 hours.

[0216] B-6 Step (6b) → (7b): Transition metal catalyzed cross-coupling reaction (e.g., Suzuki-Miyaura reaction, etc.) The reaction is carried out using an organic boron compound (4-methoxyphenylboronic acid, etc.) in the presence of a transition metal catalyst (tetrakis(triphenylphosphine)palladium, dichlorobis(benzonitrile)palladium(II), etc.) in a solvent (ethanol, toluene, 1,4-dioxane, DMF, tetrahydrofuran, water, etc., or a mixture thereof) at 0°C to the boiling point of the solvent used in the reaction, preferably at 0°C to 120°C. If necessary, a base (sodium carbonate, potassium carbonate, cesium carbonate, sodium hydrogen carbonate, sodium hydroxide, etc.) and an additive (silver oxide, triphenylarsine, etc.) are added. The palladium catalyst is used in an amount of 0.01 to 1 mole, preferably 0.01 to 0.5 moles, per mole of compound (6b). In addition, the organic boron compound is used in an amount of 1 mole to an excess mole, preferably 1 to 10 moles, per mole of compound (6b), and the base is used in an amount of 1 to 5 moles and the additive is used in an amount of 0.1 to 5 moles. The reaction time is from 10 minutes to 72 hours, preferably from 30 minutes to 24 hours.

[0217] B-7 Step (7b) → (8b): Reduction reaction For example, PRO 6 When is 2-(trimethylsilyl)ethoxymethyl group, the reduction is carried out by treating compound (7b) with a reducing agent (lithium borohydride, sodium borohydride, etc.) in a solvent (diethyl ether, THF, dichloromethane, ethanol, etc. or a mixed solvent thereof) at from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 50°C. The reducing agent is used in an amount of 1 to an excess molar amount, preferably 1 to 30 molar amounts, relative to compound (7b). The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Compound (8b) can be produced by adding silica gel to a solution of the crude product obtained by the reduction reaction (dichloromethane, ethanol, water, or a mixed solvent thereof) and stirring the solution. Silica gel is used in an excess amount relative to compound (7b). The treatment time is 12 hours to 150 hours, preferably 12 hours to 100 hours.

[0218] B-8 Step (8b) → (9b): Reduction of imino group The reaction is carried out by treating compound (8b) with a reducing agent (sodium borohydride, cyanoborohydride, sodium triacetoxyborohydride, 2-picoline borane, pyridine borane, etc.) in a solvent (THF, dichloromethane, N,N-dimethylformamide, etc., or a mixture thereof) at a temperature between -78°C and the boiling point of the solvent used in the reaction, preferably between -78°C and 50°C. The reducing agent is used in an amount of 1 to an excess molar amount, preferably 1 to 5 molar amounts, relative to compound (8b). The reaction time is 1 minute to 60 hours, preferably 5 minutes to 24 hours.

[0219] B-9 Step (9b) → (10b): Introduction of Protective Group PRO 8 When is an allyloxycarbonyl group, (9b) is reacted with allyl chloroformate, diallyl dicarbonate, or the like in a solvent (benzene, toluene, pyridine, diethyl ether, dichloromethane, THF, 1,4-dioxane, water, or the like or a mixture thereof) at a temperature ranging from -30°C to the boiling point of the solvent used in the reaction, preferably 0°C to 50°C. If necessary, a base (triethylamine, diisopropylethylamine, pyridine, sodium carbonate, potassium carbonate, sodium hydroxide, or the like) is added. Allyl chloroformate is used in an amount of 1 mole to an excess mole, preferably 1 mole to 10 moles, and a base is used in an amount of 1 mole to an excess amount, preferably 1 to 10 moles, relative to 1 mole of compound (9b). The reaction time is 10 minutes to 72 hours, preferably 10 minutes to 48 hours. PRO 8 When is a 2'2'2'-trichloroethoxycarbonyl group, The reaction is carried out by reacting compound (9b) with 2,2,2-trichloroethyl chloroformate or the like in a solvent (benzene, toluene, pyridine, diethyl ether, dichloromethane, THF, 1,4-dioxane, water, or the like or a mixture thereof) at a temperature ranging from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. If necessary, a base (triethylamine, diisopropylethylamine, pyridine, sodium carbonate, sodium hydroxide, or the like) is added. 1 mole to an excess of 2,2,2-trichloroethyl chloroformate, preferably 1 mole to 10 moles, is used relative to 1 mole of compound (9b), and 1 mole to an excess of base, preferably 1 to 10 moles, is used. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 48 hours.

[0220] C method In this production method, R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, and R 14 , R 15 This is a method for producing compound (14c), an intermediate required for producing a compound in which is hydrogen. Compound (10b) can also be produced by this method. [ka]

[0221] C-1 Step (1c) → (2c): Introduction of Protective Group PRO 5When is an acetyl group, the reaction is carried out by reacting compound (1c) with an acetylating agent (acetic anhydride, acetyl chloride, etc.) in a solvent (dichloromethane, DMF, pyridine, THF, 1,4-dioxane, etc. or a mixture thereof) at a temperature ranging from -20°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 100°C. If necessary, a base (triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, etc.) is added. The acetylating agent is used in an amount ranging from 1 mole to an excess mole, preferably from 1 mole to 20 moles, and the base is used in a catalytic amount to an excess mole, preferably from 0.1 to 20 moles, per mole of compound (1c). The reaction time is from 10 minutes to 72 hours, preferably from 30 minutes to 24 hours. PRO 5 When is a TBDMS group, it is produced according to step A-2 of Method A.

[0222] C-2 process ~ C-5 process and C-7 process ~ C-14 process Process C-2 is manufactured in accordance with process A-5 of Method A, process C-3 is process B-9 of Method B, process C-4 is process A-7 of Method A, process C-5 is process A-8 of Method A, process C-7 is process B-8 of Method B, process C-8 is process B-9 of Method B, process C-9 is process B-3 of Method B, process C-10 is process A-8 of Method A, process C-11 is process B-5 of Method B, process C-12 is process B-6 of Method B, process C-13 is process A-10 of Method A, and process C-14 is process A-11 of Method A.

[0223] C-6 step (6c)→(7c): Deprotection group reaction PRO 9 When is 2'2'2'-trichloroethoxycarbonyl group, the reaction is carried out by reacting compound (6c) with a metal reagent (zinc, zinc-lead alloy, cadmium, cadmium lead, etc.) in a solvent (THF, acetic acid, aqueous ammonium acetate solution, water, etc. or a mixture thereof) at -20°C to the boiling point of the solvent, preferably 0°C to 40°C. The metal reagent is used in an amount of 1 to an excess mole, preferably 1 to 10 moles, per mole of compound (6c). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. PRO 9When is an allyloxycarbonyl group, the reaction is carried out with compound (6c) in a solvent (dichloromethane, DMF, THF, etc. or a mixture thereof) at a temperature ranging from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 30°C, using a palladium catalyst (e.g., tetrakis(triphenylphosphine)palladium) and an allyl group scavenger (e.g., pyrrolidine, morpholine, barbituric acid, etc.). The palladium catalyst is used in an amount of 0.005 mol to 1 mol, preferably 0.005 mol to 0.5 mol, per mol of compound (6c). The allyl group scavenger is used in an amount of 1 mol to an excess molar amount, preferably 1 mol to 10 mol. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0224] D method Compound (13c) can also be prepared by this method. [ka]

[0225] D-1 process ~ D-6 process, D-9 process, D-10 process Process D-1 is manufactured in accordance with process B-6 of Method B, process D-2 is process A-5 of Method A, process D-3 is process B-9 of Method B, process D-4 is process A-7 of Method A, process D-5 is process A-8 of Method A, process D-6 is process C-6 of Method C, process D9 is process B-8 of Method B, and process D-10 is process B-9 of Method B.

[0226] D-7 process and D-8 process Compound (7d) can also be prepared according to Step D-7, which is similar to Step B-6 in Method B, or Step D-8, which is similar to Step B-7 in Method B.

[0227] E method Method E is a method of producing compound (4e) by combining compounds (11a) and (12a) produced by Method A with compounds (10b) and (14c) produced by Method B or Method C. [ka]

[0228] E-1 process This step is a step of producing compound (1e) by coupling reaction of compound (11a) produced by Method A and compound (10b) produced by Method B. Compound (11a) is produced by coupling reaction with compound (10b) in a solvent (THF, DMF, N,N-dimethylacetamide or a mixture thereof) in the presence of a base (potassium carbonate, cesium carbonate, etc.) at a temperature between -20°C and the boiling point of the solvent used in the reaction, preferably between 0°C and 50°C. Compound (10b) is used in an amount of 1 mole to an excess mole, preferably 0.7 to 1.5 moles, per 0.5 mole of compound (11a). 1 to 5 moles of the base are used. The reaction time is 1 minute to 60 hours, preferably 5 minutes to 24 hours.

[0229] E-2 process Compound (1e) can also be produced by subjecting compound (12a) produced by Method A and compound (14c) produced by Method C to a coupling reaction in the same manner as in Step E-1.

[0230] E-3 process Step E-3 is produced according to Step A-7 of Method A.

[0231] E-4 process In this step, compound (2e) is reacted with PRO 4 and PRO 8 When the protecting group is the same as that of Step C-6 of Method C, compound (4e) is produced by deprotection reaction.

[0232] PRO of compound (2e) 4 and PRO 8 When is a different protecting group, compound (4e) can also be produced by carrying out the deprotection reaction stepwise via steps E-5 and E-6.

[0233] Steps E-5 and E-6 are produced according to Step C-6 of Method C.

[0234] F method Compound (4e) can also be produced from intermediate compound (10f) of this synthesis method. This synthesis method shows a method for producing compound (10f) and compound (4e). [ka]

[0235] F-1 process ~ F-10 process, F-15 process Process F-1 is manufactured in accordance with Process A-2, Process F-2 is Process B-3, Process F-3 is Process A-8 of Process A, Process F-4 is Process B-5 of Process B, Process F-5 is Process B-6 of Process B, Process F-6 is Process A-2 of Process A, Process F-7 is Process A-10 of Process A, Process F-8 is Process A-11 of Process A, Process F-9 is Process E-1 of Process E, Process F-10 is Process E-1 of Process E, and Process F-15 is Process B-8 of Process B.

[0236] F-11 process PRO 10 and (R 17 When both of the hydroxyl-protecting groups in (a) and (b) are TBDMS groups, the preparation can be carried out in accordance with Method A, Step A-7.

[0237] F-12 process In this step, the protecting group PRO is 4 and PRO 9 When the protecting groups are the same, compound (10f) is prepared by deprotection reaction in the same manner as in step C-6 of Method C.

[0238] F-13 and F-14 Processes PRO of compound (9f) 4 and PRO 9 When is a different protecting group, compound (10f) can be produced by carrying out the deprotection reaction stepwise through steps F-13 and F-14. Steps F-13 and F-14 are produced according to step C-6 of Method C.

[0239] G method In this production method, R 11 indicates a hydrogen atom, and R 12 and R 13together form a spiro ring, and R 14 , R 15 This is a method for producing compound (11g), which is an intermediate for producing a compound in which R represents a hydrogen atom. [ka]

[0240] G-1~G-2 process, G-5~G-11 process, Process G-1 is produced in accordance with process A-4 of Method A, process G-2 is produced in accordance with process A-5 of Method A, process G-5 is produced in accordance with process A-11 of Method A, process G-6 is produced in accordance with process B-7 of Method B, process G-7 is produced in accordance with process B-8 of Method B, process G-8 is produced in accordance with process B-9 of Method B, process G-9 is produced in accordance with process E-1 of Method E, process G-10 is produced in accordance with process A-7 of Method A, and process G-11 is produced in accordance with process C-6 of Method C.

[0241] Step G-3: Introduction of a protecting group The reaction is carried out by reacting compound (2g) with a chloromethoxy ether reagent (2-(chloromethoxy)ethyltrimethylsilane, chloromethyl methyl ether, benzyl chloromethyl ether, etc.) in a solvent (THF, DMF, dioxane, etc., or a mixture thereof) at a temperature ranging from -78°C to the boiling point of the solvent, preferably from 0°C to 50°C. If necessary, a base (sodium hydride, n-butyllithium, lithium hexamethyldisilazane) is added. The reagent is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (2g). The base is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (2g). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0242] G-4 process PRO 7 When is a benzyl group, it can be prepared according to Method A, Step A-3. PRO 7 When is a triisopropylsilyl group, it can be prepared according to Method A, Step A-10.

[0243] H method In this production method, R 11indicates a hydrogen atom, and R 12 and R 13 together form a spiro ring, and R 14 , R 15 In this method, R 12 and R 13 The spiro ring formed by is synonymous with E, so it is also represented by E. [ka]

[0244] H-1 process~H-10 process Step H-1 is manufactured in accordance with step A-4 of Method A, step H-2 is step A-1 of Method A, step H-3 is step C-1 of Method C, step H-4 is step A-4 of Method A, step H-5 is step A-5 of Method A, step H-6 is step B-9 of Method B, step H-7 is step A-6 of Method A, step H-8 is step B-3 of Method B, step H-9 is step A-8 of Method A, and step H-10 is step C-6 of Method C.

[0245] I Method In this production method, R 11 and R 12 together form a benzene ring, and R 13 is a single bond, and R 14 , R 15 The present invention relates to a method for producing compound (11i), which is an intermediate for producing a compound in which the above react with an imine. [ka]

[0246] I-1 process ~ I-11 process Step I-1 is manufactured in accordance with step A-4 of Method A, step I-2 is step A-5 of Method A, step I-3 is step B-9 of Method B, step I-4 is step A-7 of Method A, step I-5 is step A-8 of Method A, step I-6 is step A-2 of Method A, step I-7 is step A-10 of Method A, step I-8 is step A-11 of Method A, step I-9 is step E-1 of Method E, step I-10 is step A-7 of Method A, and step I-11 is step C-6 of Method C.

[0247] J method In this production method, R 12 and R 13 Together, CH 2 = Form R 11 is hydrogen, R 14 , R 15 The present invention relates to a method for producing compound (12j), which is an intermediate for a compound which reacts with the compound (12j) to form an imine. [ka]

[0248] J-1 process This step is a step of producing compound (2j) by subjecting compound (1j) to a Wittig reaction.

[0249] J-2 step: Protecting group introduction PRO 7 When is a triisopropylsilyl group, the reaction is carried out by reacting compound (2j) with a silylation agent (triisopropylsilyl chloride, triisopropylsilyl triflate, etc.) in a solvent (dichloromethane, acetonitrile, THF, DMF, etc. or a mixture thereof) at -20°C to 120°C, preferably 0°C to 100°C. If necessary, a base (imidazole, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, sodium hydride, etc.) is added. The silylating agent is used in an amount of 1 to an excess molar amount, preferably 1 to 3 moles, and the base is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (2a). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0250] J-3 process~J-11 process Process J-3 is manufactured in accordance with Process A-5 of Method A, Process J-4 is manufactured in accordance with Process B-9 of Method B, Process J-5 is manufactured in accordance with Process A-7 of Method A, Process J-6 is manufactured in accordance with Process A-8 of Method A, Process J-7 is manufactured in accordance with Process A-2 of Method A, Process J-8 is manufactured in accordance with Process A-10 of Method A, Process J-9 is manufactured in accordance with Process E-1 of Method E, Process J-10 is manufactured in accordance with Process A-7 of Method A, and Process J-11 is manufactured in accordance with Process C-6 of Method C.

[0251] K method In the K method, R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, and R 13 is a hydroxymethyl group, and R 14 , R 15 This is a method for producing compound (7k), which is an intermediate required for producing a compound in which the above react with the compound (7k) to form an imine. [ka]

[0252] K-1 process This step is a step of producing compound (1k) by subjecting compound (6b) to a carbonylation reaction.

[0253] K-2 process This step is a step of producing compound (2k) by subjecting compound (1k) to an aldehyde-selective reduction reaction.

[0254] K-3 process ~ K-7 process Process K-3 is manufactured in accordance with Process A, Process A-2, Process K-4 is manufactured in accordance with Process B, Process B-7, Process K-5 is manufactured in accordance with Process E, Process E-1, Process K-6 is manufactured in accordance with Process A, Process A-7, and Process K-7 is manufactured in accordance with Process C, Process C-6.

[0255] L method Method L is a representative method for producing compound (B). [ka]

[0256] The peptide residue represented by the general formula (Lp')' can be produced by a condensation reaction of amino acids. PRO 4 is N-terminally protected with a peptide residue (Lp')', and PRO 12 protects the C-terminus.

[0257] L-1 process Step L-1 is produced according to Step B-9 of Method B.

[0258] Step L-2: Deprotection reaction PRO 12 When is a tert-butyl group, the reaction is carried out by reacting compound (2l) with an acid (trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, acetic acid, etc.) in a solvent (dichloromethane, etc.) at a temperature ranging from 0° C. to the boiling point of the solvent used in the reaction, preferably from 0° C. to 40° C. The acid is used in a catalytic amount to an excess molar amount relative to 1 mole of compound (2l). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0259] L-3 process~L-4 process Step L-3 is produced in accordance with step B-9 of Method B, and step L-4 is produced in accordance with step A-4 of Method A.

[0260] L-5 process Compound (B) can also be produced by Step L-5 following Step B-9 of Method B.

[0261] M method Method M is a method for producing compound (2). Compound (2) shown in this production method is a compound (1) having R 16 J-La'-Lp'-NH-B'-CH 2 Equivalent to -O(C=O)-*. [ka]

[0262] Compound (1m) shown in the above preparation method represents compounds (4e), (4f), (11g), (9h), (11i), (12j), (7k), and (8k) prepared by methods E to K.

[0263] The (PBD)' shown in the above manufacturing method is [ka] indicates, PBD is [ka] Shows. (PBD)' is R in PBD 13 The above substituents (hydroxyl group, etc.) may be protected with a protecting group. When there is no protecting group, it is synonymous with PBD (R 13 =(R 13 )'). During the above manufacturing process, (Lp')" t In the peptide residue represented by -(Lp')', the functional group (such as an amino group) on the side chain of the amino acid residue represented by Lp' may be protected with a protecting group, and when the protecting group is unsubstituted, it is synonymous with Lp'. Here, (Lp')' represents a sequence of two amino acids as shown below, and if there are functional groups (amino group, hydroxyl group) in the side chains, they may be protected. -VA-, (D-)VA-, -FG-, -PI-, -VCit-, -VK-, -PL-, -(D-)PI-, or -GF- (Lp')'' represents a sequence of 2 to 4 amino acids as shown below, and if functional groups (amino group, hydroxyl group) are present in the side chains, they may be protected. -GG-, -EGG-, -DG-, -(D-)DG-, -EG-, -GGF-, -SG-, -KG-, -DGG-, -GGF-, -DDGG-, -KDGG-, or -GGFG-

[0264] (La′)′ represents any one selected from the following group: -C(=O)-(CH 2 CH 2 )n 6-C(=O), -C(=O)-(CH 2 CH 2 )n 6 -NH-C(=O)-(CH 2 CH 2 O)n 7 -CH 2 CH 2 -C(=O)-, -(CH 2 )n 8 -OC(=O)-, -(CH2)n 12 -C(=O)-, and -(CH 2 CH 2 )n 13 -C(=O)-NH-(CH 2 CH 2 O)n 14 -CH 2 CH 2 -C(=O)-

[0265] (La′)″ represents any one selected from the following group: -NH-(CH 2 CH 2 )n 7 -C(=O)- or -NH-(CH 2 CH 2 O)n 7 -CH 2 -C(=O)-, Each of s and t independently represents 0 or 1. For example, in the following step M-1, s and t are 0, in step M-3, s is 1 and t is 0, and in step M-5, s is 0 and t is 1.

[0266] (La')'-(La')'' s is synonymous with La'. (Lp')'' t When -(Lp')' has a protecting group, it becomes Lp' by deprotection, but when there is no protecting group, it has the same meaning as Lp'. In the above production methods, Lx represents a hydrogen atom or a leaving group (hydroxysuccinimide, etc.). In the above-mentioned production methods, 1m, 9m, 10m, 11m, and PBD or (PBD)' in compound (2) indicate that the asterisk (N10' position) is bonded to the right-most C(=O)- of -OC(=O)-.

[0267] M-1 process This step is a method for producing compound (11m) by carrying out a condensation reaction between compound (1m) produced by Methods E to K and compound (2m). When Lx=H and compound (2m) is a carboxylic acid, compound (2m) can be produced according to Method A, Step A-4. When Lx is a leaving group (hydroxysuccinimide, p-nitrophenoxy group, etc.), the reaction is carried out by reacting compound (1m) with compound (2m) in a solvent (benzene, toluene, diethyl ether, dichloromethane, THF, DMF, methanol, water, etc., or a mixture thereof) at temperatures ranging from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. Compound (2m) is used in an amount of 0.9 to an excess molar amount, preferably 0.9 to 2 moles, per mole of compound (1m). If necessary, a base (triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine, diazabicycloundecene, etc.) is added. Base is used in an amount of 1 mole to an excess amount, preferably 1 to 5 moles, per mole of compound (1m). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 36 hours.

[0268] M-2 process ~ M-5 process and M-8 process Process M-2 is produced in accordance with process M-1, process M-3 is produced in accordance with process A-4, process M-4 is produced in accordance with process M-1, process M-5 is produced in accordance with process A-4, and process M-8 is produced in accordance with process A-4.

[0269] M-6 process This step is a step of carrying out a condensation reaction of the compound (6m) to produce an active ester intermediate (7m). The reaction is carried out by reacting compound (6m) with hydroxysuccinimide or the like in the presence of a condensing agent such as N,N-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a solvent (benzene, toluene, diethyl ether, dichloromethane, THF, DMF, or the like or a mixture thereof) at a temperature ranging from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. The condensing agent is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (6m). Hydroxysuccinimide is used in an amount of 1 to an excess molar amount, preferably 1 to 5 moles, per mole of compound (6m). The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0270] M-7 process This step is a step of producing compound (11m) by carrying out a condensation reaction between compound (1m) and compound (7m) in the same manner as in step M-1.

[0271] M-9 process In this step, compound (9m) is subjected to a deprotection reaction to produce compound (10m). 4 When is a 9-fluorenylmethyloxycarbonyl group, the reaction is carried out by reacting compound (9m) with a base (1,8-diazabicyclo[5.4.0]-7-undecene, piperidine, etc.) in a solvent (THF, dichloromethane, DMF, etc. or a mixture thereof) at a temperature of from -20°C to the boiling point of the solvent, preferably from 0°C to 40°C. The base is used in an amount of 1 to an excess mole, preferably 1 to 10 moles, per mole of compound (9m). The reaction time is 1 minute to 72 hours, preferably 5 minutes to 24 hours.

[0272] M-10 process This step is a step of producing compound (11m) by carrying out a condensation reaction of compound (10m) with compound (2m) or (4m) in the same manner as in Step A-4 of Method A.

[0273] M-11 process In this process, compound (11m) is converted to (Lp')" tWhen -(Lp')' or PBD' has a protecting group, deprotection is carried out to produce compound (2), which is produced according to step B-3 of Method B and step C-6 of Method C. Furthermore, when (Lp')' or PBD' does not have a protecting group, step M-11 is omitted, and in that case, compound (11m) has the same meaning as (2).

[0274] N method The N method is to determine the free drug R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, and R 14 , R 15 is hydrogen and R 16 and R 17 This shows a method for synthesizing compounds in which the above are combined to form an imine bond. [ka]

[0275] N-1 process ~ N-8 process N-1 process is manufactured according to B-9 process of Method B, N-2 process is A-7 process of Method A, N-3 process is A-8 process of Method A, N-4 process is A-2 process of Method A, N-5 process is A-10 process of Method A, N-6 process is A-11 process of Method A, N-7 process is E-1 process of Method E, and N-8 process is E-1 process of Method E.

[0276] (R 13 )'=R 13 In this case, the production is carried out according to the following steps N-9 and N-10.

[0277] N-9 process Step N-9 is produced according to Step A-7 of Method A.

[0278] N-10 process PRO 4 and PRO 8 When the protecting groups are the same, the compound can be prepared according to step E-4 of Method E. 4 and PRO 8When is a different protecting group, the compound can be prepared according to Steps E-5 and E-6 of Method E.

[0279] (R 13 When N-11 has a protecting group, the compound can be prepared according to the following steps N-11 and N-12.

[0280] N-11 process It is produced according to Process B-3 of Method B.

[0281] N-12 process PRO 4 and PRO 8 When the protecting groups are the same, the compound can be prepared according to steps E-3 and E-4 of Method E. 4 and PRO 8 When is a different protecting group, the compound can be prepared according to steps E-3, E-5 and E-6 of Method E.

[0282] O method The O method is to determine the free drug R 11 and R 12 together form a double bond with the carbon atom to which each group is attached, and R 14 and R 15 and R 16 and R 17 are combined to produce compound (6o), which has an imine bond (C=N). [ka]

[0283] O-1 process ~ O-6 process Process O-1 is manufactured in accordance with Process E, Process E-1; Process O-2 is manufactured in accordance with Process B, Process B-3; Process O-3 is manufactured in accordance with Process A, Process B-5; Process O-5 is manufactured in accordance with Process B, Process B-6; and Process O-6 is manufactured in accordance with Process B, Process B-7.

[0284] P method This manufacturing method is for the free drug R 11 indicates a hydrogen atom, and R 12 and R 13 together form a spiro ring, and R14 and R 15 and R 16 and R 17 In this method, the starting material R represented by compound (4h) is 12 and R 13 The spiro ring formed by is synonymous with E, so it is also represented by E. [ka]

[0285] P-1 process~P-4 process Process P-1 is manufactured in accordance with process B-9 of Method B, process P-2 is manufactured in accordance with process B-3 of Method B, process P-3 is manufactured in accordance with process A-8 of Method A, and process P-4 is manufactured in accordance with process C-6 of Method C.

[0286] Q Method This manufacturing method is for the free drug R 11 , R 14 and R 15 indicates a hydrogen atom, and R 12 and R 13 taken together form a spiro ring, and R 16 and R 17 are combined to produce a compound that contains an imine bond (C=N). [ka]

[0287] Q-1 process~Q-6 process Step Q-1 is produced in accordance with step A-1 of Method A, step Q-2 is produced in accordance with step A-8 of Method A, step Q-3 is produced in accordance with step A-5 of Method A, step Q-4 is produced in accordance with step E-1 of Method E, step Q-5 is produced in accordance with step A-7 of Method A, and step Q-6 is produced in accordance with step C-6 of Method C.

[0288] In the above, the optionally protected amino and hydroxyl protecting groups refer to protecting groups which can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, or photolysis, and refer to protecting groups generally used in organic synthesis (see, for example, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc. (1999)). In the above, a "protecting group" for a hydroxy group which may be protected (e.g., an alkylcarbonyl group, a silyl group, or an aralkyl group), a "protecting group" for a carboxy group which may be protected (e.g., 1 -C 6 The "protecting group" of an optionally protected amino group (e.g., an alkyl group or an aralkyl group) is not particularly limited as long as it is a protecting group for a hydroxy group, a carboxy group, or an amino group used in the field of organic synthetic chemistry. Steps requiring protection and deprotection are carried out in accordance with known methods (for example, the methods described in "Protective Groups in Organic Synthesis" (Theodora W. Greene, Peter GMWuts, 1999, Wiley-Interscience Publication)).

[0289] R method: Preparation of antibodies Glycochain remodeling antibodies can be produced, for example, by a method similar to that described in WO2013 / 120066, as shown in FIG. 3. [ka]

[0290] R-1 process Hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure This step involves cleaving the N-linked glycan (N297-linked glycan) bound to aspartic acid at position 297 of the amino acid sequence of the antibody using a known enzymatic reaction to prepare a glycan-cleaved antibody. The antibody of interest (20 mg / ml) is placed in a buffer solution (such as 50 mM phosphate buffer solution) at 0°C to 40°C to carry out hydrolysis of the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure at the reducing end using a hydrolase such as EndoS enzyme. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. 0.1 to 10 mg, preferably 0.1 to 3 mg of wild-type EndoS enzyme is used per 100 mg of antibody. After completion of the reaction, affinity chromatography purification and / or hydroxyapatite column purification described below can be carried out to produce a (Fucα1,6)GlcNAc antibody in which the sugar chain between GlcNAcβ1 and 4GlcNAc has been hydrolyzed.

[0291] R-2 process: Glycosylation reaction This process involves binding MSG (MSG1, MSG2) or SG-type glycan oxazoline (hereinafter referred to as "azido glycan oxazoline") having an azide group-containing PEG linker to the above-mentioned (Fucα1,6)GlcNAc antibody using an enzymatic reaction to produce a glycan remodeling antibody. The glycosylation reaction is carried out by reacting the above-mentioned glycosylated antibody with an azido-glycosylated oxazoline in the presence of a catalytic amount of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (such as a phosphate buffer solution) at 0°C to 40°C. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. EndoS enzyme (D233Q / Q303L) is used in an amount of 1 to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody, and the azido-glycosylated oxazoline is used in an amount of 2 to an excess equivalent, preferably 2 to 20 equivalents. After completion of the reaction, a purified glycan remodeling antibody can be obtained by carrying out affinity chromatography purification and hydroxyapatite column purification. Azide sugar chain oxazoline compounds can be prepared according to the methods described in Examples 55 to 57. A PEG linker (N) containing an azide group is added to MSG (MSG1, MSG2) or disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) using a reaction (condensation reaction, etc.) known in the field of organic synthesis science. 3 -L(PEG)3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 That is, a carboxyl group and N-acetylglucosyl group can be introduced into the 2-position of sialic acid. 3 -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 -NH 2 The amino group at the right end of the amine group forms an amide bond through a condensation reaction. When a condensation reaction is used, examples of the condensation agent include N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), carbonyldiimidazole (CDI), 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (BOP), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and examples of the solvent used in the reaction include, but are not limited to, dichloromethane, DMF, THF, ethyl acetate, and the like, or a mixture thereof. The reaction temperature is usually in the range of -20°C to 100°C or the boiling point of the solvent, and preferably in the range of -5°C to 50°C. If necessary, an organic base such as triethylamine, diisopropylethylamine, N-methylmorpholine, or 4-dimethylaminopyridine, or an inorganic base such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, or sodium hydrogen carbonate, can be added. Furthermore, 1-hydroxybenzotriazole, N-hydroxysuccinimide, or the like can be added as a reaction promoter. MSG, MSG1 or MSG2 can be obtained by hydrolyzing the above-mentioned (MSG-)Asn or isolated and purified (MSG1-)Asn or (MSG2-)Asn (Example 56) with a hydrolase such as EndoM. Oxazolination can be carried out from the reducing terminal GlcNAc of MSG (MSG1, MSG2) or SG-type glycan according to the known literature (J. Org Chem., 2009, 74 (5), 2210-2212. Helv. Chim. Acta, 2012, 95, 1928-1936.).

[0292] In the preparation of the above-mentioned sugar chain remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the following common procedures A to C. Common procedure A: Concentration of antibody aqueous solution The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (30,000 to 50,000 MWCO, Millipore Co.) container, and the antibody and the antibody-drug conjugate solution described below were concentrated by centrifugation (at 2000 G to 4000 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.). Common Procedure B: Antibody concentration measurement The antibody concentration was measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used. Common Procedure C: Buffer exchange for antibodies The aqueous antibody solution was concentrated by adding a buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) using common procedure A. After repeating this procedure several times, the antibody concentration was measured using common procedure B, and the antibody concentration was adjusted to 10 mg / mL using a buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.).

[0293] S Method: Conjugation This production method is a method for producing an antibody-drug conjugate by binding the above-mentioned glycan remodeling antibody and production intermediate (2) by SPAAC (strain-promoted alkyne azide cycloaddition: J. AM. CHEM. SOC. 2004, 126, 15046-15047) reaction. In the formula, Ab represents the glycan remodeling antibody. [ka] The SPAAC reaction proceeds by mixing a buffer solution of the antibody Ab (sodium acetate solution, sodium phosphate solution, sodium borate solution, etc., or a mixture thereof) with a solution of compound (2) dissolved in an appropriate solvent (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP), propylene glycol (PG), etc., or a mixture thereof). The amount of compound (2) is 2 moles to an excess molar amount, preferably 1 mole to 30 moles, per mole of antibody, and the ratio of the organic solvent to the antibody buffer is preferably 1 to 200% v / v. The reaction temperature is 0° C. to 37° C., preferably 10° C. to 25° C., and the reaction time is 1 to 150 hours, preferably 6 hours to 100 hours. The pH during the reaction is preferably 5 to 9.

[0294] The antibody-drug conjugate can be subjected to buffer exchange, purification, antibody concentration, and measurement of the average number of drugs bound per antibody molecule by common procedures A to C described above and common procedures D to F described below, and the antibody-drug conjugate compound (ADC) can be identified.

[0295] Common Procedure D: Purification of Antibody-Drug Conjugates The NAP-25 column was equilibrated with one of the commercially available acetate buffers (10 mM, pH 5.5; referred to as ABS in this specification) containing sorbitol (5%). The antibody-drug conjugate reaction solution (about 1.5 to 2.5 mL) was loaded onto this NAP-25 column, and the antibody fraction was separated by eluting with the amount of buffer specified by the manufacturer. The separated fraction was again loaded onto the NAP-25 column, and the gel filtration purification procedure of eluting with buffer was repeated 2 to 3 times in total to obtain an antibody-drug conjugate from which unbound drug linkers, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-drug conjugate solution was adjusted by common procedures A and C. Common Procedure E: Measurement of antibody concentration in antibody-drug conjugates The bound drug concentration in an antibody-drug conjugate can be calculated using the Beer-Lambert law as shown below. The formula (I) using Beert's Lambert's law is shown below.

number

number

number

number

[0296] Common Procedure F: Measurement of the average number of drugs bound per antibody molecule in antibody-drug conjugates The average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high performance liquid chromatography (HPLC) analysis using the following method. [F-1. Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to cleave the disulfide bond between the L chain and the H chain of the antibody-drug conjugate, and the resulting sample is used for HPLC analysis. [F-2.HPLC analysis] The HPLC analysis is carried out under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet spectrophotometer (measurement wavelength: 280 nm, 329 nm) Column: BEH Phenyl (2.1 x 50 mm, 1.7 μm, Waters Acquity) Column temperature: 75℃ Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA, 15% isopropyl alcohol in acetonitrile Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17 min), 80%-14% (17 min-17.1 min), 14%-14% (17.1 min-23 min) Sample injection volume: 5μL

[0297] [F-3. Data Analysis] [F-3-1] Drug-free antibody light chain (L 0 ) and H chain (H 0 ) to the L chain with the drug bound to it (L chain with one drug bound to it: L 1 ) and H chain (H chain with one drug bound: H 1 , H chain with two drugs bound: H 2 , H chain with three drugs bound: H 3) increases in hydrophobicity in proportion to the number of bound drugs, and the retention time increases. 0 , L 1 , H 0 , H 1 , H 2 , H 3 They are eluted in the order of L 1 ,H 0 The order of the above may vary, but the H 0 There is no characteristic 329 nm wavelength absorption for drugs. Therefore, by checking the 329 nm wavelength absorption, 1 , H 0 It is possible to distinguish between 0 and H 0 The detected peaks were identified by comparing the retention times with L 0 , L 1 , H 0 , H 1 , H 2 , H 3 can be assigned to either [F-3-2] Since the drug linker has UV absorption, the peak area value is corrected according to the molar absorption coefficients of the L chain, H chain, and drug linker according to the number of drug linkers bonded, according to the following formula.

number

number

[0298] <Medicine> The antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells and can therefore be used as a pharmaceutical, particularly as a therapeutic and / or prophylactic agent for cancer.

[0299] Examples of cancer types to which the antibody-drug conjugate of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (superficial epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, etc., uterine body cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, and metastatic forms thereof, but are not limited to these, so long as the cancer cells to be treated express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0300] The antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0301] Substances used in pharmaceutical compositions containing the antibody-drug conjugates of the present invention can be appropriately selected and applied in terms of dosage amount and administration concentration from formulation additives and others commonly used in this field.

[0302] The antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing one or more pharma- ceutical compatible components. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The formulation corresponds to the mode of administration.

[0303] A variety of delivery systems are known and can be used to administer the antibody-drug conjugate of the present invention.Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.Administration can be, for example, by infusion or bolus injection.In certain preferred embodiments, administration of the ligand-drug conjugate is by infusion.Parenteral administration is the preferred administration route.

[0304] In a representative embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the medicament may also include a solubilizing agent and a local anesthetic to ease pain at the site of injection (e.g., lignocaine). Generally, the ingredients are supplied either separately or mixed together in a unit dosage form (e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent). If the medicament is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. If the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.

[0305] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present application, or may be a pharmaceutical composition containing the antibody-drug conjugate and at least one other cancer therapeutic agent. The antibody-drug conjugate of the present invention may be administered together with another cancer therapeutic agent, thereby enhancing the anti-cancer effect. The other anti-cancer agent used for such a purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or consecutively, or may be administered at different administration intervals. Examples of such cancer therapeutic agents include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, or agents described in WO2003 / 038043, as well as LH-RH analogues (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and the like, but are not limited thereto as long as they have antitumor activity.

[0306] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, the preparation may contain appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0307] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its medicinal effect even at a small dose, as the affinity of the antibody-drug conjugate to the antigen, i.e., the dissociation constant (Kd value) to the antigen, of the antibody-drug conjugate is higher (the Kd value is lower). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0308] The antibody of the present invention or a functional fragment of the antibody can also be used as a medicine. In this case, the description of the "antibody-drug conjugate" in the above <medicine> can be appropriately read as the description of the "antibody or a functional fragment of the antibody".

[0309] Furthermore, the free drug (novel PBD derivative compound), its salt, or their hydrates of the present invention can also be used as a medicine. In this case, the description of the "antibody-drug conjugate" in the above <medicine> can be appropriately read as a description of the "free drug (novel PBD derivative compound), its salt, or their hydrates." EXAMPLES

[0310] The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. Furthermore, the examples should not be construed as being limiting in any sense. Furthermore, reagents, solvents and starting materials not specifically described herein are readily available from commercial sources.

[0311] Reference Example 1: Trastuzumab-Tesirine Step 1: Conjugation of antibody and drug linker A 5 mM tetraacetate ethylenediamine-phosphate buffered saline (pH 6.5) solution (9.91 mg / mL, 0.70 mL) of Trastuzumab (Reference Example 3) was added with a dipotassium phosphate aqueous solution (1.0 M, 0.0112 mL) and a tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0086 mL) at 20° C., and reacted for 60 minutes at 20° C. and 30 minutes at room temperature. A dimethylacetamide solution (0.0415 mL) of tesirine (0.36 mg) synthesized with reference to a literature (Med. Chem. Lett. 2016, 7, 983-987) was added to the reaction solution, and the reaction was allowed to proceed for 1 hour at room temperature. An N-acetylcysteine ​​aqueous solution (100 mM, 0.0024 mL) was added to the reaction solution, and the reaction was stopped by reacting for 30 minutes. Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.40mg / mL, antibody yield: 4.90mg (71%), average number of drugs bound per antibody molecule (n): 2.0

[0312] Reference Example 2: Anti-CLDN6 (H1L1)-Tesirine Step 1: Conjugation of antibody and drug linker A 5 mM tetraacetate ethylenediamine-phosphate buffered saline (pH 6.5) solution (9.87 mg / mL, 0.45 mL) of anti-CLDN6 (H1L1) antibody was added to a dipotassium phosphate aqueous solution (1.0 M, 0.0072 mL) and tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0041 mL) at 20 ° C., and the reaction was allowed to proceed for 90 minutes at 20 ° C. Tesirine (0.15 mg) synthesized with reference to the literature (Med. Chem. Lett. 2016, 7, 983-987) was added to the reaction solution in N, N-dimethylacetamide solution (0.0277 mL), and the reaction was allowed to proceed for 1 hour at 20 ° C. An N-acetylcysteine ​​aqueous solution (100 mM, 0.001 mL) was added to the reaction solution, and the reaction was stopped by reacting for 30 minutes. Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound. Characterization: The following characteristic values ​​were obtained using common procedures E and F. Antibody concentration: 1.56 mg / mL, antibody yield: 3.90 mg (88%), average number of drugs bound per antibody molecule (n): 2.1

[0313] Reference example 3: Anti-HER2 antibody Trastuzumab The anti-HER2 antibody was prepared with reference to US 5821337. The amino acid sequences of the light chain and heavy chain of Trastuzumab are shown in SEQ ID NO:64 and SEQ ID NO:65.

[0314] Reference example 4: Anti-LPS antibody h#1G5-H1L1 The anti-LPS antibody was produced with reference to WO2015 / 046505. The amino acid sequences of the light chain and heavy chain of h#1G5-H1L1 are shown in SEQ ID NO:66 and SEQ ID NO:67.

[0315] Reference example 5: Anti-TROP2 antibody hRS7 The anti-TROP2 antibody was produced with reference to WO2003 / 074566 and WO2015 / 098099 (Reference Example 1). The amino acid sequences of the light chain and heavy chain of hRS7 are shown in SEQ ID NO:68 and SEQ ID NO:69.

[0316] Reference example 6: Anti-CD98 antibody hM23-H1L1 The anti-CD98 antibody was produced with reference to WO2015 / 146132. The amino acid sequences of the light chain and heavy chain of hM23-H1L1 are shown in SEQ ID NO:70 and SEQ ID NO:71.

[0317] [Synthesis of intermediates] Example 1: Intermediate 1 [ka] Step 1: Benzyl (6S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate Lithium borohydride (4.30 g, 178 mmol) was added in small portions to a solution of 5-benzyl 6-methyl(6S)-5-azaspiro[2.4]heptane-5,6-dicarboxylate (104 mmol, WO2012087596) in THF (500 mL) at 0° C. After stirring for 30 minutes at 0° C., the mixture was stirred at room temperature for 2 hours. Water (180 mL) and 2N hydrochloric acid (186 mL) were added at 0° C., and the mixture was evaporated under reduced pressure. The resulting residue was extracted four times with ethyl acetate, and the organic layer was washed with saturated saline and then dried over anhydrous sodium sulfate. The residue (27.9 g, 90%) obtained by evaporation under reduced pressure was used as it was in the next reaction.

[0318] Step 2: Benzyl (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane-5-carboxylate To a solution of the compound (27.9 g, 107 mmol) obtained in the above step 1 and imidazole (14.5 g, 214 mmol) in dichloromethane (300 mL), tert-butyldimethylsilyl chloride (24.2 g, 160 mmol) was added at room temperature, and the mixture was stirred at room temperature for 18 hours. The reaction solution was washed with a saturated aqueous solution of citric acid, a saturated aqueous solution of sodium bicarbonate, and a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 50:50 (v / v)] to obtain the target product (32.5 g, 81%). 1 H-NMR (CDCl 3 )δ:7.39-7.34(5H,m),5.23-5.11(2H,m),4.10-3.48(4H,m),3.16-3.14(1H,m),2.15-2 .04(1H,m),1.81-1.77(1H,m),0.91-0.88(9H,m),0.65-0.55(4H,m),0.08-0.01(6H,m). MS(APCI)m / z:376(M+H) +

[0319] Step 3: (6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane To a solution of the compound (32.5 g, 86.5 mmol) obtained in the above step 2 in ethanol (400 mL), 7.5% palladium carbon catalyst (54% water, 5.00 g) was added at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. The reaction solution was filtered through Celite, and the filtrate was evaporated under reduced pressure to obtain the target product (21.3 g, quantitative). 1 H-NMR (CDCl 3 )δ:3.79-3.77(1H,m),3.71-3.69(1H,m),3.65-3.60(1H,m),3.01-2.98(2H,m) ,1.81-1.71(2H,m),0.90(9H,s),0.65-0.57(4H,m),0.08(3H,s),0.07(3H,s). MS (APCI, ESI) m / z: 242 (M+H) +

[0320] 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 To a solution of 5-methoxy-2-nitro-4-{tri(propan-2-yl)silyl]oxy}benzoic acid (52.2 g, 141 mmol, US20150283262) and 1-hydroxybenzotriazole monohydrate (23.8 g, 155 mmol) in dichloromethane (500 mL), N,N'-dicyclohexylcarbodiimide (35.0 g, 170 mmol) was added under ice cooling. The reaction mixture was stirred at room temperature. After the carboxylic acid disappeared, a dichloromethane (100 mL) solution of the compound (34.1 g, 141 mmol) obtained in the above step 3 and triethylamine (29.4 mL, 212 mmol) was slowly added dropwise at -60 ° C. The reaction solution was stirred overnight at room temperature, and then a saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the reaction mixture was extracted with chloroform. The organic layer was washed with water and saturated saline, and dried over anhydrous magnesium sulfate. Ethyl acetate and diethyl ether were added to the residue obtained by distillation under reduced pressure, the solid components were removed by filtration, the filtrate was distilled under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 25:75 (v / v)] to obtain the target product (55.0 g, 66%). 1 H-NMR (CDCl 3 )δ:7.72-7.66(1H,m),6.80-6.73(1H,m),4.53-4.49(1H,m),4.04-3.95(1H,m),3.91-3.88(3H,m),3 .59-3.54(1H,m),3.36-3.25(0.5H,m),3.01-2.96(1.5H,m),2.24-2.20(0.3H,m),2.09-2.05(0.7H, m),2.00-1.97(0.7H,m),1.69-1.67(0.3H,m),1.32-1.24(3H,m),1.12-1.05(18H,m),0.93-0.91(6H ,m),0.79-0.77(3H,m),0.71-0.62(2H,m),0.57-0.40(2H,m),0.12-0.10(4H,m),0.11-0.15(2H,m). MS (APCI, ESI) m / z: 593 (M+H) +

[0321] 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 To a solution of the compound (55.0 g, 92.8 mmol) obtained in step 4 above in ethanol (300 mL), 7.5% palladium carbon (10.0 g) was added under a nitrogen atmosphere. The nitrogen balloon was immediately replaced with a hydrogen balloon, and the reaction mixture was vigorously stirred at room temperature under a hydrogen atmosphere. After the raw materials had disappeared, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure, and the obtained target product (52.2 g, 100%) was used as it was in the next reaction. 1 H-NMR (CDCl 3 )δ:6.71(1H,s),6.25(1H,s),4.55-4.28(2H,m),3.97(1H,m),3.75-3.62(3H,m),3.70(3H,s),3.09-3.07(1H,m),2.24-2 .19(1H,m),1.81-1.68(1H,m),1.27-1.22(3H,m),1.09-1.05(18H,m),0.90(9H,s),0.65-0.46(4H,m),0.07-0.03(6H,m). MS (APCI, ESI) m / z: 563 (M+H) +

[0322] Step 6: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide To a solution of the compound (18.6 g, 33.0 mmol) obtained in the above step 5 and triethylamine (6.26 mL, 45.2 mmol) in THF (300 mL), triphosgene (4.22 g, 14.2 mmol) was slowly added on an ethanol-ice bath. After the addition, a mixed solution of N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide (11.4 g, 30.2 mmol, WO2011130598) and triethylamine (6.26 mL, 45.2 mmol) in THF (100 mL) and N,N-dimethylformamide (30 mL) was slowly added dropwise to the ice-cooled reaction mixture. After the dropwise addition, the ice bath was removed, and the reaction mixture was stirred at 40° C. under a nitrogen atmosphere. After the raw materials disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 40:60 (v / v)] to obtain the target product (23.5 g, 74%). 1 H-NMR (CDCl 3 )δ:8.99(1H,m),8.58(1H,s),7.80(1H,s),7.55-7.53(2H,m),7.34-7.32(2H,m),6.77-6.75(2H,m),5.94-5.87(1 H,m),5.40-5.38(1H,m),5.33-5.29(1H,m),5.23-5.21(1H,m),5.13(1H,m),5.10(2H,m),4.69-4.64(1H,m),4.62 -4.52(2H,m),4.06-4.03(1H,m),3.98(1H,m),3.76-3.65(6H,m),3.04(1H,m),2.28-2.26(1H,m),2.18-2.13(1H, m),1.46(3H,m),1.32-1.25(3H,m),1.11-1.09(18H,m),0.99-0.84(15H,m),0.65-0.40(4H,m),0.08-0.00(6H,m). MS (APCI, ESI) m / z: 966 (M+H) +

[0323] Step 7: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide Acetic acid (200 mL) was added to a solution of the compound (23.5 g, 24.3 mmol) obtained in the above step 6 in THF (50 mL), methanol (50 mL), and water (44 mL) at room temperature. The reaction mixture was stirred at room temperature. After the raw materials disappeared, the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 0:100 (v / v)] to obtain the target product (18.0 g, 87%). 1 H-NMR (CDCl 3 )δ:8.64-8.62(1H,m),8.50(1H,m),7.69(1H,m),7.55-7.53(2H,m),7.34-7.32(2H,m),6.79-6.75(3H,m),5 .91-5.89(1H,m),5.39(1H,m),5.32-5.29(1H,m),5.23-5.21(1H,m),4.68-4.54(4H,m),4.31(1H,m),4.06-4 .04(1H,m),3.81-3.79(3H,m),3.76(3H,s),3.63-3.61(1H,m),3.13-3.11(1H,m),2.16-2.13(1H,m),1.87-1 .81(2H,m),1.46-1.43(3H,m),1.30-1.24(3H,m),1.12-1.08(18H,m),0.98-0.91(6H,m),0.63-0.45(4H,m). MS (APCI, ESI) m / z: 852 (M+H) +

[0324] Step 8: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a'S)-11'-hydroxy-7'-methoxy-5'-oxo-8'-{[tri(propan-2-yl)silyl]oxy}-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide Oxalyl chloride (2.17 mL, 25.3 mmol) was slowly added dropwise to a solution of dimethyl sulfoxide (3.75 mL, 52.8 mmol) in dichloromethane (300 mL) at -78°C under a nitrogen atmosphere. After the dropwise addition, the reaction mixture was stirred at -78°C. A solution of the compound (18.0 g, 21.1 mmol) obtained in the above step 7 in dichloromethane (50.0 mL) was slowly added dropwise to the reaction mixture. Triethylamine (14.6 mL, 105 mmol) was added to the reaction solution at -78°C. After the addition, the cooling bath was removed and the temperature was slowly raised to room temperature. After the raw materials disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with chloroform (200 mL). The organic layer was washed with water and saturated saline, and dried over anhydrous magnesium sulfate. After filtration, the residue obtained by evaporation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 0:60 (v / v)] to obtain the target product (16.5 g, 92%). 1 H-NMR (CDCl 3)δ:8.51-8.36(1H,m),7.54-7.38(2H,m),7.22-7.07(3H,m),6.73-6.64(1H,m),5.94-5.87(2H,m),5 .33-5.22(3H,m),5.09(1H,m),4.97(1H,m),4.64-4.58(4H,m),4.02-4.00(1H,m),3.86-3.83(3H,m) ,3.75-3.70(1H,m),3.61-3.54(2H,m),3.38-3.29(1H,m),2.40(1H,m),2.16-2.14(1H,m),1.74-1.7 1(1H,m),1.44(3H,m),1.18-1.16(3H,m),1.05-1.00(18H,m),0.97-0.92(6H,m),0.72-0.60(4H,m). MS (APCI, ESI) m / z: 850 (M+H) +

[0325] Step 9: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-5'-oxo-8'-{[tri(propan-2-yl)silyl]oxy}-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide To a dichloromethane (200 mL) solution of the compound (12.0 g, 14.1 mmol) obtained in the above step 8 and 2,6-lutidine (6.58 mL, 56.5 mmol), tert-butyldimethylsilyl trifluoromethylsulfonate (9.73 mL, 42.3 mmol) was slowly added dropwise at 0°C under a nitrogen atmosphere. After stirring for 10 minutes under ice cooling, the ice bath was removed and the mixture was stirred at room temperature. After the raw materials 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 saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 25:75 (v / v)] to obtain the target product (8.12 g, 60%). 1 H-NMR (CDCl 3 )δ:8.67-8.45(1H,m),7.50-7.44(2H,m),7.19(1H,s),7.13(2H,m),6.95(2H,m),6.62-6.57(2H,m),6.01(1H, m),5.95-5.86(1H,m),5.33-5.13(3H,m),4.82(1H,m),4.65-4.54(3H,m),4.03-4.01(1H,m),3.84-3.82(3H,m) ,3.73-3.66(1H,m),3.50-3.48(1H,m),3.27(1H,m),2.37-2.33(1H,m),2.19-2.13(1H,m),1.54-1.43(3H,m), 1.22-1.13(3H,m),1.10-1.00(18H,m),0.97-0.91(6H,m),0.81(9H,s),0.76-0.59(4H,m),0.19--0.09(6H,m). MS (APCI, ESI) m / z: 964 (M+H) +

[0326] Step 10: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a'S)-11'-{[tert-butyl(dimethyl)silyl]oxy}-8'-hydroxy-7'-methoxy-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide Lithium acetate (0.611 g, 9.26 mmol) was added to a solution of the compound (8.12 g, 8.42 mmol) obtained in the above step 9 in N,N-dimethylformamide (90 mL) and water (2 mL), and the mixture was stirred at room temperature. After the raw materials disappeared, water was added to the reaction mixture, and it was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) to 0:100 (v / v)] to obtain the target product (5.48 g, 81%). 1 H-NMR (CDCl 3 )δ:8.76-8.60(1H,m),8.02-7.56(1H,m),7.45-7.44(2H,m),7.21(1H,s),7.10-7.09(2H,m),6.81-6.74(1H,m ),6.65(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(3H,m),0.97-0.92(6H,m),0.81(9H,s),0.76-0.61(4H,m),0.20--0.06(6H,m). MS (APCI, ESI) m / z: 808 (M+H) +

[0327] Step 11: N-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a'S)-8'-(3-bromopropoxy)-11'-{[tert-butyl(dimethyl)silyl]oxy}-7'-methoxy-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide The compound (2.40 g, 2.97 mmol) obtained in the above step 10 was reacted in the same manner as in step 1 of Example 4 to obtain the target compound (2.73 g, 99%). 1 H-NMR (DMSO-D 6 )δ:10.01-9.86(1H,m),8.24-8.04(2H,m),7.64-7.54(2H,m),7.32-7.14(4H,m),6 .59-6.48(1H,m),5.94-5.88(2H,m),5.32-4.76(5H,m),4.44-4.38(3H,m),3.87-3 .81(5H,m),3.64-3.55(2H,m),3.41(1H,m),3.14(1H,m),2.45-2.09(4H,m),1.97- 1.94(1H,m),1.44-1.30(4H,m),0.89-0.53(9H,m),0.79(9H,s),0.13-0.06(6H,m). MS (APCI, ESI) m / z: 930 81 Br, (M+H) + ],928[ 79 Br, (M+H) + ].

[0328] Example 2: Intermediate 2 [ka] Step 1: N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycine Glycylglycine (0.328g, 2.49mmol), N,N-diisopropylethylamine (0.433mL, 2.49mmol) in N,N-dimethylformamide (20mL) was added with 1-{[4-(11,12-didehydrodibenzo[b,f]azocine-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (1.00g, 2.49mmol, Click Chemistry Tools), and water (10mL) at room temperature, and the mixture was stirred at room temperature overnight. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [chloroform:CMW=100:0(v / v) to 0:100(v / v)] to obtain the target product (0.930g, 89%). CMW is the organic phase partitioned with chloroform:methanol:water = 7:3:1 (v / v / v). 1 H-NMR (DMSO-D 6 )δ:12.58(1H,s),8.14-8.12(1H,m),8.08-8.07(1H,m),7.69-7.68(1H,m),7.62-7.61(1H,m),7.53-7.45(3H,m),7.40-7.29(3H,m), 5.05-5.01(1H,m),3.73-3.72(2H,m),3.66-3.60(3H,m),2.66-2.60(1H,m),2.33-2.24(1H,m),2.08-2.04(1H,m),1.81-1.77(1H,m). MS (APCI, ESI) m / z: 420 [(M+H) + ].

[0329] Step 2: 2,5-dioxopyrrolidin-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycinate 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.420g, 2.19mmol) was added to a dichloromethane (6mL) solution of the compound (0.612g, 1.46mmol) obtained in the above step 1) and N-hydroxysuccinimide (0.168g, 1.459mmol), and the mixture was stirred at room temperature for 21 hours. The residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [chloroform:CMW=100:0(v / v) to 0:100(v / v)] to obtain the target product (0.375g, 50%). CMW is the organic phase partitioned with chloroform:methanol:water = 7:3:1 (v / v / v).

[0330] Example 3: Drug Linker 1 [ka] 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 (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 (25.5g, 41.6mmol, WO2016149546) in THF (150mL) and ethanol (150mL) were added under a nitrogen atmosphere, and the reaction solution was stirred at room temperature for three days under a hydrogen atmosphere. Chloroform was added to the reaction solution, and the solution was filtered through Celite, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 50:50 (v / v)] to obtain the target product (19.4 g, 89%). 1 H-NMR (CDCl 3)δ:7.36(1H,s),7.25(1H,s),6.01(1H,s),5.45-5.43(1H,m),4.69-4...

Claims

[Claim 1] The invention described herein.

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