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

[0002] Antibody-drug conjugates (ADCs) are constructed by attaching a cytotoxic drug to an antibody that can bind to an antigen expressed on the surface of cancer cells, thereby internalizing the antigen into the cell. ADCs are expected to efficiently deliver drugs to cancer cells, leading to drug accumulation within the cancer cells and ultimately killing them. For example, Adcetris (brentuximab vedotin), which is an anti-CD30 monoclonal antibody conjugated with monomethyl auristatin E, is approved as a treatment for Hodgkin lymphoma and anaplastic large cell lymphoma. In addition, Kadcyla (trastuzumab emtansine), which is an anti-HER2 monoclonal antibody conjugated with emtansine, is used to treat HER2-positive advanced and recurrent breast cancer. One useful drug for binding to ADCs is pyrrolobenzodiazepine (PBD). PBDs exhibit cytotoxicity by binding to PuGPu sequences in DNA grooves. Anthramycin, a naturally occurring PBD, was first discovered in 1965, and since then, various naturally occurring PBDs and their analogues have been discovered (Non-Patent Documents 1-4). The general structural formula for PBD is shown below. [ka] As shown, PBDs are known to differ in the number, type, and location of substituents in the A and C rings, and also in the degree of unsaturation of the B and C rings. It is known that the cytotoxicity of PBD is dramatically improved by creating a dimeric structure (Non-Patent Documents 5, 6), and various ADC variants of dimeric PBD have been reported (Patent Documents 1-13). However, no PBD or its ADC variant having a spiro ring at the C2 position is known. Human CLDN6 (Claudin-6, hereinafter referred to as hCLDN6) is a type of Claudin (CLDN) family protein, a four-transmembrane membrane protein consisting of 220 amino acid residues. Previous studies have suggested that hCLDN6 is an attractive cancer treatment target, as it is overexpressed in some cancers (Non-Patent Documents 7-9). Furthermore, since CLDN family proteins are taken up into cells via endocytosis, and some family proteins have short turnover times (Non-Patent Document 10), they are considered suitable targets for antibody-drug conjugates (ADCs). Based on information suggesting a link to cancer, monoclonal antibodies that specifically recognize hCLDN6 have been discovered (Patent Documents 14, 15), and ADCs (artificial diuretics) in which CLDN6-specific monoclonal antibodies are conjugated with tubulin polymerization inhibitors such as monomethyl auristatin E (MMAE) or maytansinoid (DM1) have been reported (Non-Patent Document 11). On the other hand, antibodies that recognize multiple CLDN family members may broaden the range of therapeutic applications, and therefore, an ADC (antibody-drug conjugate) has been disclosed in which an antibody that recognizes CLDN6 and CLDN9 (Patent Document 16) is conjugated with pyrrolobenzodiazepine (PBD), which has a potent cytotoxic effect (Patent Document 17). However, the strength of their activity is still insufficient, and there is an unmet medical need for therapeutic targeting of hCLDN6. [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 [Overview of the project] [Problems that the invention aims to solve]

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

[0006] As a result of diligent research, the inventors discovered that a novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate possesses strong antitumor activity, thus completing the present invention. In other words, the present invention relates to the following:

[0007] [1] The following formula: [ka] (In the formula, m 1 (where is an integer from 1 to 10, preferably an integer from 2 to 8), Ab represents an antibody or a functional fragment of the antibody, and the glycans of the antibody may be remodeled. L represents the linker connecting Ab and D. Ab may be directly bound to L from its amino acid residue, or it may be bound to L from the glycan of Ab or a remodeled glycan. D represents the drug shown by the following formula: [ka] In the formula, the asterisk indicates that it is bonded to L. n 1 This represents integers from 2 to 8, A represents a 3-5 member saturated hydrocarbon ring or 3-5 member saturated heterocycle with a spiro bond that may be substituted with 1-4 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'', where R' and R'' each independently represent a hydrogen atom or a C1-C6 alkyl group, R 3 、R 4 and R 5 are selected from the following (i)-(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 represents a hydrogen atom, R 4 and R 5 form, together with the carbon atom to which R 4 and R 5 are attached, a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring or CH2=, (iii)R 3 、R 4 and R 5 form, together with the carbon atom to which R 3 is attached and the carbon atoms to which R 4 and R 5 are attached, a benzene ring or 6-membered heterocyclic ring 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 represent an imine bond (C=N), R 8 is a hydroxyl group or a C1-C3 alkoxy group, X and Y each independently represent an oxygen atom, a nitrogen atom or a sulfur atom, Group 1 consists of a) C1-C6 alkoxy groups which may be substituted with 1-3 halogen atoms. b) A C1-C6 alkyl group which may be substituted with one of the following: 1-3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''. c) Halogen atom, d) C3-C5 cycloalkoxy groups, e) C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R''', h)-NHC(=NR')-NR''R''', i) -NHCOR', or j) Showing a hydroxyl group, Here, R' and R'' are as defined above, and R'''' each independently represents a hydrogen atom or a C1-C6 alkyl group. Group 2 represents halogen atoms, hydroxyl groups, or C1-C6 alkoxy groups, and, Group 3 represents a halogen atom, or a C1-C6 alkyl group or C1-C6 alkoxy group which may be substituted with 1-3 halogen atoms. Antibody-drug conjugates represented by [this symbol]. [2] A represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 1 and R 2 However, each independently exhibits a C1-C3 alkoxy group, R 3 and R 4 Together, they form a double bond with the carbon atom to which each group is bonded. R 5 However, it is indicated that it is 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 consists of a) C1-C3 alkoxy groups which may be substituted with 1-3 halogen atoms. b) C1-C3 alkyl groups which may be substituted with one or more halogen atoms, hydroxyl groups, -OCOR'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''. c) C3-C5 cycloalkoxy group, d)-C(=NR')-NR''R''', e)-NHC(=NR')-NR''R''', or f) Showing a hydroxyl group, Here, R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, and, Group 5 is the antibody-drug conjugate described in [1], which represents a halogen atom, a hydroxyl group, or a C1-C3 alkoxy group. [3] A represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 1 and R 2 However, each independently exhibits a C1-C3 alkoxy group, R 3 However, it shows a hydrogen atom. R 4 and R 5 However, R 4 and R 5 Together with the bonded carbon atom, it forms a 3-5 membered saturated hydrocarbon ring or =CH2, and, The antibody-drug conjugate described in [1], wherein X and Y are oxygen atoms. [4] A represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 1 and R 2 However, each independently exhibits a C1-C3 alkoxy group, R 3 , R 4 R 5 However, R 3 The carbon atoms to which it is bonded, and R 4 and R 5Together with the bonded carbon atom, it forms 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 C1-C3 alkoxy group which may be substituted with 1-3 halogen atoms, as described in [1]. [5] D is given by one of the following two equations: [ka] In the formula, the asterisk indicates that it is bound to L, as described in [1] or [2], an antibody-drug conjugate. [6] D is given by one of the following two equations: [ka] In the formula, the asterisk indicates that it is bound to L, as described in [1] or [3], an antibody-drug conjugate. [7] L is represented as -Lb-La-Lp-NH-B-CH2-O(C=O)-*, In the formula, the asterisk indicates that it is bound to D. B represents a phenyl group or a heteroaryl group. Lp represents a linker consisting of an amino acid sequence that can be cleaved in target cells. La belongs to the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -NH-C(=O)-(CH2CH2O)n 3 -CH2CH2-C(=O)- and -(CH2)n 4-OC(=O)-, Show one of the following to be selected: n 2 n is an integer between 1 and 3. 3 n is an integer from 1 to 5. 4 This represents an integer between 0 and 2. Lb represents a spacer that connects the glycans or remodeled glycans of La and Ab in the antibody-drug conjugate described in any one of [1] to [6]. [8] The antibody-drug conjugate according to [7], wherein B is selected from one of the following: 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. [9] The antibody-drug conjugate described in [8], wherein B is a 1,4-phenyl group.

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

[11] An 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 group: An antibody-drug conjugate selected from -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, and -GGPL-, as described in any one of [7] to

[11] .

[13] La belongs to the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, and -OC(=O)- An antibody-drug conjugate selected from any one of the following [7] to

[12] .

[14] Lb is shown by the following equation, [ka] [ka] , or, [ka] In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or remodeled sugar chain of Ab, as described in any one of [7] to

[13] .

[15] L is -Lb-La-Lp-NH-B-CH2-O(C=O)-* As shown, In the formula, B is a 1,4-phenyl group. Lp belongs to the following group: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-, Show one of the following to be selected: La belongs to the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, and -OC(=O)-, It indicates one of the following to be selected, and also, Lb is given by the following equation: [ka] [ka] , or, [ka] Herein, in the structural formula of Lb shown above, the asterisk indicates that it is bound to La, and the wavy line indicates that it is bound to the sugar chain or remodeled sugar chain of Ab, as described in any one of [7] to

[14] .

[16] L belongs to the following group: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGPL-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 2 -OC(=O)-GGVA-NH-B-CH2-OC(=O)-, and -Z 3 Selected from -CH2-OC(=O)-GGVA-NH-B-CH2-OC(=O)-, Here, Z 1 The structural formula is as follows: [ka] Show, Z 2 The structural formula is as follows: [ka] Show, Z 3 The structural formula is as follows: [ka] Show, Here, Z 1 , Z 2 and Z 3 In the structural formula, the asterisk indicates a bond with La, the wavy line indicates a bond with the sugar chain or remodeled sugar chain of Ab, and, B is an antibody-drug conjugate representing a 1,4-phenyl group, as described in any one of [7] to

[15] .

[17] L belongs to the following group: -Z 1-C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 Selected from -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, Here, B is a 1,4-phenyl group, and Z 1 The following is the structural formula: [ka] Show, Here, Z 1 In the structural formula, the asterisk is Z 1 The antibody-drug conjugate described in

[16] shows that it is bound to an adjacent C(=O), and the wavy line indicates that it is bound to the sugar chain or remodeled sugar chain of Ab.

[18] L is represented as -Lb-La-Lp-NH-B-CH2-O(C=O)-*, In the formula, the asterisk indicates that it is bound to D. B represents a 1,4-phenyl group, Lp indicates -GGVA- or -VA. La is -(CH2)n 9 -C(=O)-, or -(CH2CH2)n10 -C(=O)-NH-(CH2CH2O)n 11 -CH2CH2-C(=O)- is shown as n 9 is an integer from 2 to 7, n 10 is an integer from 1 to 3, n 11 is an integer from 6 to 10, indicating Lb is -(Succinimid-3-yl-N)-, an antibody-drug conjugate described in any one of [1] to [6].

[19] L is the following group: -(Succinimid-3-yl-N)-(CH2)5-C(=O)-VA-NH-B-CH2-OC(=O)-, -(Succinimid-3-yl-N)-(CH2)5-C(=O)-GGVA-NH-B-CH2-OC(=O)-, and -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- is shown as any one selected from Here, B is a 1,4-phenyl group, an antibody-drug conjugate described in

[18] .

[20] The antibody is IgG, an antibody-drug conjugate described in any one of [1] to

[19] .

[21] The antibody is IgG1, IgG2 or IgG4, an antibody-drug conjugate described in

[20] .

[22] The antibody binds to tumor cells and is characterized by being taken up and internalized into tumor cells, an antibody-drug conjugate described in any one of [1] to

[21] . ​​​​​ The N297-glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2 or a mixture thereof, or N297-(Fuc)SG having the structure represented by the following formula, and is the antibody-drug conjugate described in

[24] or

[25] :

Chemical formula

Chemical formula

Chemical formula

[27] n 5 An antibody-drug conjugate as described in

[26] , wherein is an integer between 2 and 5.

[28] The following formula: [ka] (In the formula, m 2 This indicates 1 or 2. L is a linker that connects the N297 glycan chain of Ab to D, and belongs to the following group: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- It is one of the linkers selected from the following: Here, B is a 1,4-phenyl group, and Z 1 The following is the structural formula: [ka] Show, Here, Z shown above 1 In the structural formula, the asterisk is Z 1 The wavy line indicates that it is bonded to the adjacent C(=O), and the wavy line indicates that it is bonded to the N297 sugar chain of Ab. Ab represents an IgG antibody or a functional fragment of said antibody. The N297 glycan of Ab is one of the following: N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, or N297-(Fuc)SG, having the structure shown in the following formula. [ka] [ka] [ka] In the above formula, the wavy line indicates that the antibody is bound to Asn297. L(PEG) in the N297 sugar chain is -NH-CH2CH2-(O-CH2CH2)n 5 -* indicates Here, n 5 The value is an integer between 2 and 5, and the leftmost amino group is amide-bonded to the carboxylic acid at position 2 of the non-reducing end of the sialic acid on the 1-3 chain side and / or 1-6 chain side of the β-Man branched chain of the N297 sugar chain, and the asterisk indicates Z in the linker L. 1 This indicates that it is bonded to the nitrogen atom at position 1 or 3 on the triazole ring. D is one of the following groups: [ka] Here, the asterisk in the formula indicates that it is coupled with L. An antibody-drug conjugate as described in any one of the following

[25] -

[28] .

[30] Next group: [ka] [ka] [ka] [ka] Selected from, In each of the structural formulas shown above, m 2 This indicates that it is either 1 or 2. Ab indicates that it is an IgG antibody or a functional fragment thereof. The N297 glycan of Ab is one of the following: N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, or N297-(Fuc)SG, having the structure shown in the following formula. [ka] [ka] [ka] In the formula, the wavy line indicates that the antibody is bound to Asn297. The L(PEG) in the N297 sugar chain is -NH-CH2CH2-(O-CH2CH2)3-*, Here, the leftmost amino group is amide-bonded to the carboxylic acid at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain of the N297 glycan, either on the 1-3 chain side or / or the 1-6 chain side, and the asterisk indicates a bond to the nitrogen atom at position 1 or 3 on the triazole ring in each structural formula, representing an antibody-drug conjugate.

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

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

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

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

[30] or

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

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

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

[34] An antibody or functional fragment of the antibody described in any one of

[30] to

[33] , comprising the heavy chain variable region and the light chain variable region described in (a) or (b) below: (a) A heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 21, and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 19, (b) A heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 25, and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 23.

[35] An 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 (a) to (e) below, and a light chain variable region consisting of an amino acid sequence selected from the group consisting of (f) to (k): (a) Amino acid sequence described in Sequence ID No. 54, (b) Amino acid sequence described in Sequence ID No. 58, (c) Amino acid sequence described in Sequence ID No. 62, (d) Amino acid sequences having at least 95% homology to the sequences of framework regions other than each CDR sequence in the sequences of (a) to (c), (e) Amino acid sequences in which one or more amino acids are deleted, substituted, or added in the framework region of each CDR sequence in the sequences of (a) to (c), (f) Amino acid sequence described in Sequence ID No. 38, (g) Amino acid sequence described in Sequence ID No. 42, (h) Amino acid sequence described in Sequence ID No. 46, (i) Amino acid sequence described in Sequence ID No. 50, In sequences (j)(f)~(i), amino acid sequences having at least 95% homology to the sequences of framework regions other than each CDR sequence, and An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the framework region of each CDR sequence in the sequences (k)(f)~(i).

[36] The antibody or 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 (a) to (e) below: (a) A heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO. 54 and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO. 38, (b) Heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO. 58 and light chain variable region consisting of the amino acid sequence described in SEQ ID NO. 42, (c) Heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO. 54 and light chain variable region consisting of the amino acid sequence described in SEQ ID NO. 46, (d) A light chain variable region consisting of a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 58 and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 50, and (e) A heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 62 and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 46.

[37] The antibody described in any one of

[30] to

[36] , or a functional fragment of the antibody, wherein the antibody is a chimeric antibody.

[38] The antibody described in any one of

[30] to

[36] , or a functional fragment of the antibody, wherein the antibody is a humanized antibody.

[39] The antibody comprising the heavy chain constant region of human IgG1, human IgG2, or human IgG4, or the antibody according to any one of

[30] to

[38] or a functional fragment of the antibody.

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

[38] or

[39] , comprising a heavy chain and a light chain selected from the group consisting of (a) to (e) below: (a) A heavy chain consisting of the amino acid sequence described in amino acid numbers 20-471 of SEQ ID NO. 52 and a light chain (H1L1) consisting of the amino acid sequence described in amino acid numbers 21-234 of SEQ ID NO. 36, (b) A heavy chain consisting of the amino acid sequence described in amino acid numbers 20-471 of SEQ ID NO: 56 and a light chain (H2L2) consisting of the amino acid sequence described in amino acid numbers 21-234 of SEQ ID NO: 40, (c) A heavy chain consisting of the amino acid sequence described in amino acid numbers 20-471 of SEQ ID NO. 52 and a light chain (H1L3) consisting of the amino acid sequence described in amino acid numbers 21-234 of SEQ ID NO. 44, (d) A heavy chain consisting of the amino acid sequence described in amino acid numbers 20-471 of SEQ ID NO: 56 and a light chain (H2L4) consisting of the amino acid sequence described in amino acid numbers 21-234 of SEQ ID NO: 48, and (e) A heavy chain consisting of the amino acid sequence described in amino acid numbers 20-471 of SEQ ID NO: 60 and a light chain (H3L3) consisting of the amino acid sequence described in amino acid numbers 21-234 of SEQ ID NO: 44. An antibody or functional fragment of the antibody described in

[30] or

[31] that binds to a site on an antigen recognized by any one of the antibodies described in

[41] ,

[32] to

[36] and

[40] . An antibody or functional fragment of the antibody described in

[30] or

[31] that competes with any one of the antibodies described in

[42] ,

[32] to

[36] and

[40] for binding to CLDN6 and / or CLDN9. A polynucleotide encoding an antibody as described in any one of the items

[43] ,

[30] , or

[42] .

[44] Polynucleotides as described in

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

[45] ,

[43] , or

[44] . Host cells transformed with the expression vectors described in

[46]

[45] .

[47] Host cells described in

[46] are eukaryotic cells.

[48] ​​Host cells as described in

[47] , in which the host cell is an animal cell. A method for producing an antibody or a functional fragment of the antibody according to

[30] to

[42] , characterized by comprising the steps of culturing a host cell according to any one of

[49] ,

[46] to

[48] , and collecting a target antibody from the culture obtained in the said step. An antibody or a functional fragment of the antibody, characterized by being obtained by the manufacturing method described in

[50]

[49] .

[51] An 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, aspartic acid isomerization, methionine oxidation, 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 both 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) A step of culturing host cells described in any one of

[46] to

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

[56] The method for producing the antibody according to

[55] , further comprising the step of purifying the reaction solution from step ii) by purifying it with a hydroxyapatite column to obtain the (Fucα1,6)GlcNAc antibody.

[57] The following steps: i) A step of producing a glycosylation remodeling antibody by the method described in

[55] or

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

[29] , comprising the step of reacting a drug linker (manufacturing intermediate) having DBCO with an azide group in the glycan of a glycan remodeling antibody produced in step i). A glycosylation remodeling antibody characterized by being obtained by the manufacturing method described in

[58] ,

[55] , or

[56] . An antibody-drug conjugate characterized by being obtained by the manufacturing method described in

[59] and

[57] .

[60] Next group: [ka] [ka] [ka] , or, [ka] Selected from, In each of the structural formulas shown above, m 2 This indicates that it is either 1 or 2. Ab indicates that it is an antibody or a functional fragment of said antibody or an anti-HER2 antibody as described in any one of

[30] -

[42] ,

[50] -

[54] , and

[58] . The N297 glycan of Ab is one of the following: N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, or N297-(Fuc)SG, having the structure shown in the following formula. [ka] [ka] [ka] In the formula, the wavy line indicates that the antibody is bound to Asn297. L(PEG) indicates -NH-CH2CH2-(O-CH2CH2)3-*, where the leftmost amino group is amide-bonded to the carboxylic acid at position 2 of the non-reducing sialic acid at the 1-3 and / or 1-6 branched end of the β-Man branch of the N297 sugar chain, and the asterisk indicates bond to the nitrogen atom at position 1 or 3 on the triazole ring in each structural formula, representing an antibody-drug conjugate.

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

[29] ,

[59] -

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

[62] The following formula [ka] (In the formula, l represents an integer from 2 to 8, E represents a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated hetero ring with a spiro bond that may be substituted with 1-4 halogen atoms. R 9 and R 10 Each of these 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 The following (i) to (iii) are selected: (i)R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded. R 13This 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 This represents a hydrogen atom. R 12 and R 13 These combine to form a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated heterocycle or CH2=, or (iii)R 11 and R 12 Together, they form a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from group 9. R 13 This indicates a single bond. R 14 and R 15 Both indicate a hydrogen atom, or R 14 and R 15 Together, they show that it is an imine bond (C=N). R 16 and R 17 This refers to (a) or (b) below, (a)R 16 and R 17 Together, they form an imine bond (N=C). (b)R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)-* In the formula, the asterisk represents R 16 This indicates that it is bonded to an adjacent nitrogen atom. B' indicates a phenyl group or a heteroaryl group. Lp' indicates a linker consisting of an amino acid sequence that can be cleaved in target cells. La' belongs to the following group: -C(=O)-(CH2CH2)n 6 -C(=O)-, -C(=O)-(CH2CH2)n 6 -C(=O)-NH-(CH2CH2)n 7 -C(=O)-, -C(=O)-(CH2CH2)n 6-C(=O)-NH-(CH2CH2O)n 7 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 6 -NH-C(=O)-(CH2CH2O)n 7 -CH2CH2-C(=O)-, -(CH2)n 8 -OC(=O)-, -(CH2)n 12 -C(=O)- and -(CH2CH2)n 13 -C(=O)-NH-(CH2CH2O)n 14 -CH2CH2-C(=O)- Show one of the following to be selected: Here, in the formula, n 6 n is an integer between 1 and 3. 7 n is an integer from 1 to 5. 8 n is an integer between 0 and 2. 12 n is an integer between 2 and 7. 13 n is an integer between 1 and 3. 14 This represents an integer between 6 and 10. J represents one of the following: [ka] Here, we show that in the structural formula of J shown above, the asterisk is bonded to La'. R 17 This indicates that it is a hydroxyl group or a C1-C3 alkoxy group. V and W are, independently, an oxygen atom, a nitrogen atom, or a sulfur atom. Group 7 consists of a) C1-C6 alkoxy groups which may be substituted with 1-3 halogen atoms. b) C1-C6 alkyl groups which may be substituted with one or more halogen atoms, a hydroxyl group, or any one selected from -OCOR', -NR'R'', -C(=NR')-NR''R''' and -NHC(=NR')-NR''R'''. c) Halogen atom, d) C3-C5 cycloalkoxy groups, e) C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R''', h)-NHC(=NR')-NR''R''', i) -NHCOR', or j) Showing a hydroxyl group, Here, 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 C1-C6 alkoxy group which may be substituted with 1-3 halogen atoms. Compounds represented by, salts thereof, or hydrates thereof.

[63] E represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 9 and R 10 However, each independently exhibits a C1-C3 alkoxy group, R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded. R 13 However, this indicates that it is 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 is a) C1-C3 alkoxy groups which may be substituted with 1-3 halogen atoms. b) C1-C3 alkyl groups which may be substituted with one or more halogen atoms, hydroxyl groups, -OCOR'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''. c) C3-C5 cycloalkoxy group, d)-C(=NR')-NR''R''', e)-NHC(=NR')-NR''R''', or f) Showing a hydroxyl group, Here, R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, and, Group 11 is a compound described in

[62] , a salt thereof, or a hydrate thereof, representing a halogen atom, a hydroxyl group, or a C1-C3 alkoxy group.

[64] E represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 9 and R 10 However, each independently exhibits a C1-C3 alkoxy group, R 11 However, it shows a hydrogen atom. R 12 and R 13 However, R 12 and R 13 Together with the bonded carbon atoms, it forms a 3-5 membered saturated hydrocarbon ring or CH2=, and, The compounds described in

[62] , their salts, or their hydrates, wherein V and W are oxygen atoms.

[65] E represents a 3-5 member saturated hydrocarbon ring of a spiro bond which may be substituted with 1-2 halogen atoms, R 9 and R 10 However, each independently exhibits a C1-C3 alkoxy group, R 11 , R 12 and R 13 However, R 11 The carbon atoms to which it is bonded, and R 12 and R 13 Together with the bonded carbon atom, it forms 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 C1-C3 alkoxy group which may be substituted with 1 to 3 halogen atoms, the compounds described in

[62] , salts thereof, or hydrates thereof.

[66] A compound according to any one of

[62] to

[65] , a salt thereof, or a hydrate thereof, wherein B' is one selected from 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.

[67] The compounds described in

[66] , salts thereof, or hydrates thereof, wherein B' is a 1,4-phenyl group.

[68] Lp' is the following group: A compound described in any one of

[62] to

[67] , a salt thereof, or a hydrate thereof, which is an amino acid residue selected from -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, and -GGPL-.

[69] La' is the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, -(CH2)5-C(=O)-, and -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)- A compound, a salt thereof, or a hydrate thereof, selected from any one of

[62] to

[68] .

[70] R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)-* As shown, In the formula, B' is a 1,4-phenyl group. Lp' indicates one of the following groups: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-, La' indicates one of the following groups: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, -(CH2)5-C(=O)- and -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)- ,and, J represents one of the following: [ka] Here, in the structural formula of J, the asterisk indicates that it is bonded to La'. The compound, salt thereof, or hydrate thereof, as described in any one of

[62] to

[69] .

[71] R 16 However, the following groups: J 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-B'-CH2-OC(=O)-, J 1-C(=O)-CH2CH2-C(=O)-GGFG-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGPL-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 2 -OC(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 3 -CH2-OC(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 4 -(CH2)5-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 4 -(CH2)5-C(=O)-VA-NH-B'-CH2-OC(=O)-, and J 4 -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)- Selected from, Here, J 1 , J 2 , J 3 and J 4 The structural formula is as follows: [ka] Show, Here, J 1 , J 2 , J 3 and J 4 In the structural formula, the asterisk indicates that it is bonded to an adjacent group, and also, B' is a 1,4-phenyl group, and is one of the compounds listed in any one of

[62] to

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

[72] R 16 However, the following groups: J 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 4 -(CH2)5-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, and, J 4 -(CH2)5-C(=O)-VA-NH-B'-CH2-OC(=O)- Selected from, Here, B' is a 1,4-phenyl group, J1 , J 4 The structural formula is as follows: [ka] Show, Here, J 1 and J 4 In the structural formula, the asterisk indicates a bond with an adjacent group. The compound, salt thereof, or hydrate thereof, as described in any one of

[62] to

[71] .

[73] The following formula: [ka] Any one of the compounds indicated by, a salt thereof, or a hydrate thereof.

[74] The following formula: [ka] Any one of the compounds indicated by, a salt thereof, or a hydrate thereof.

[75] D is given by the following equation: [ka] An antibody-drug conjugate as described in any one of the following [1]-

[29] and

[59] -

[61] .

[76] The following formula: [ka] A compound, a salt thereof, or a hydrate thereof, as shown in any one of

[62] to

[72] and

[74] .

[77] D is given by the following equation: [ka] An antibody-drug conjugate as described in any one of the following [1]-

[29] and

[59] -

[61] .

[78] The following formula: [ka] A compound, a salt thereof, or a hydrate thereof, as shown in any one of

[62] to

[72] and

[74] . A pharmaceutical composition characterized by comprising an antibody-drug conjugate, a salt thereof, or a hydrate thereof, as described in any one of

[79] [1]~

[29] ,

[59] ~

[61] ,

[75] and

[77] ; an antibody or a functional fragment of said antibody, as described in any one of

[30] ~

[42] ,

[50] ~

[54] and

[58] ; and any compound, a salt thereof, or a hydrate thereof, as described in any one of

[62] ~

[74] ,

[76] and

[78] .

[80] A pharmaceutical composition according to any one of

[79] , characterized in that it is an antitumor drug.

[81] The pharmaceutical composition according to

[80] , characterized in that the tumor is a tumor expressing CLDN6 and / or CLDN9.

[82] The pharmaceutical composition according to

[80] or

[81] , characterized in that the tumor is ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor), lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, testicular cancer, placental choriocarcinoma, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, or esophageal cancer. A method for treating a tumor, characterized by administering to an individual an antibody-drug conjugate, a salt thereof, or a hydrate thereof, as described in any one of

[83] [1]~

[29] ,

[59] ~

[61] ,

[75] and

[77] , an antibody or a functional fragment of said antibody, as described in any one of

[30] ~

[42] ,

[50] ~

[54] and

[58] , and a compound, a salt thereof, or a hydrate thereof, as described in any one of

[62] ~

[74] ,

[76] and

[78] .

[84] A method for treating a tumor according to

[83] , characterized in that the tumor is a tumor expressing CLDN6 and / or CLDN9.

[85] The treatment method according to

[83] or

[84] , characterized in that the tumor is ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor), lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, testicular cancer, placental choriocarcinoma, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, or esophageal cancer. A method for treating a tumor, characterized by administering to an individual simultaneously, separately, or sequentially an antibody-drug conjugate, a salt thereof, or a hydrate thereof, an antibody or a functional fragment of said antibody, as described in any one of

[86] [1]~

[29] ,

[59] ~

[61] ,

[75] and

[77] , a pharmaceutical composition comprising at least one selected from a compound, a salt thereof, or a hydrate thereof, as described in any one of

[62] ~

[74] ,

[76] and

[78] , and at least one antitumor drug.

[87] Compounds exhibiting proton NMR with peak positions substantially similar to those listed in Table 1 or Table 2. [Effects of the Invention]

[0008] The novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugate provided by the present invention is useful as an antitumor agent because it possesses excellent antitumor activity and safety. Furthermore, the PBD derivative of the present invention has antitumor activity and is useful as a drug in the conjugate. Moreover, the antibody of the present invention recognizes or binds to tumor cells and is therefore useful as an antibody in the conjugate. [Brief explanation of the drawing]

[0009] [Figure 1]This diagram schematically represents the drug-conjugate (molecule of (I)) of the present invention. (a) represents drug D, (b) represents linker L, (c) represents N3-L(PEG)-, and (d) represents the N297 glycan (where the white oval represents NeuAc(Sia), the white hexagon represents Man, the filled hexagon represents GlcNAc, the white rhombus represents Gal, and the white inverted triangle represents Fuc). In (b) and (c), the azide group (black teardrop shape) of (c) reacts with the alkyne structure in the spacer (white semicircle) of (b) to form a triazole ring and bind together. The Y-shape represents antibody Ab. Furthermore, in this schematic diagram, the N297 glycan is conveniently represented as N297-(Fuc)MSG, and a configuration is shown in which sialic acid with an azide group attached to a PEG linker (N3-L(PEG)-) is present only on one branch of each N297 glycan, while the other branch does not have sialic acid at its non-reducing end. However, by adopting N297-(Fuc)SG, a configuration in which sialic acid with an azide group attached to a PEG linker is present at the non-reducing ends of both branched ends is also possible. This representation method applies throughout this specification unless otherwise specified. [Figure 2] This is a schematic diagram showing the structures of the (Fucα1,6)GlcNAc antibody (molecule (II) in Figure 2A) and the MSG-type glycan remodeling antibody (molecule (III) in Figure 2B), which are intermediates in the production of the drug-conjugate of the present invention. In both figures, the Y-shape represents antibody Ab, as in Figure 1. In Figure 2A, (e) shows the N297 glycan consisting only of GlcNAc linked by α-glycosidic bonds at positions 1 and 6 of Fuc. In Figure 2B, (d) shows the N297 glycan as in Figure 1, and (f) shows the structure of the PEG linker portion having an azide group, with the azide group at the end being used for binding to linker L. The binding mode of the PEG linker having an azide group is the same as described in Figure 1. [Figure 3]This is a schematic diagram of the process for producing MSG-type glycan remodeling antibodies from antibodies produced in animal cells. The molecules (II) and (III) in the diagram represent (Fucα1,6)GlcNAc-antibody and MSG-type glycan remodeling antibody, respectively, as in Figure 2. The molecule (IV) is an antibody produced in animal cells, a mixture of molecules with heterogeneous N297 glycans. Figure 3A shows the process of producing a homogeneous (Fucα1,6)GlcNAc-antibody (II) by treating the heterogeneous N297 glycans of (IV) with a hydrolytic enzyme such as EndoS. Figure 3B shows the process of producing the MSG-type glycan remodeling antibody (III) by transferring the glycans of an MSG-type glycan donor molecule to the GlcNAc of the N297 glycan of antibody (II) using a glycosyltransferase such as the 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. In the MSG-type N297 glycan remodeling antibody produced, the sialic acid at the non-reducing end is similarly modified, as explained in Figure 2B. In Figure 3B, MSG is shown as the donor molecule for convenience, but by using SG(10) as the glycan donor, the remodeling antibody in (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] This study demonstrates the efficacy of anti-HER2 antibody-drug conjugates ADC26, ADC19, and ADC54 on NCI-N87 cells, a human gastric cancer cell line, transplanted subcutaneously. [Figure 5] This demonstrates the efficacy of the anti-HER2 antibody-drug conjugate ADC49, trastuzumab, and anti-LPS antibody-drug conjugate ADC53 on NCI-N87 cells, a human gastric cancer cell line, transplanted subcutaneously. [Figure 6] This study demonstrates the effects of anti-HER2 antibody-drug conjugate ADC49, anti-LPS antibody-drug conjugate ADC53, or trastuzumab-tesirine (Reference Example 1) on subcutaneously transplanted human breast cancer cell line KPL-4. [Figure 7]This study demonstrates the effects of the anti-HER2 antibody-drug conjugate ADC49 or trastuzumab-tesirine (Reference Example 1) on JIMT-1 cells, a human breast cancer cell line transplanted subcutaneously. [Figure 8] This study demonstrates the effects of the anti-CLDN6 antibody-drug conjugate ADC40, or the anti-CLDN6 antibody (H1L1)-Tesirine (Reference Example 1), on OV-90 cells, a human ovarian cancer cell line transplanted subcutaneously. [Figure 9] This study demonstrates the effects of the anti-CLDN6 antibody-drug conjugate ADC40, or the anti-CLDN6 antibody (H1L1)-Tesirine (Reference Example 1), on NIH:OVCAR-3 cells, a human ovarian cancer cell line transplanted subcutaneously. [Figure 10] This study demonstrates the efficacy of antibody-drug conjugates, either anti-TROP2 antibody-drug conjugate ADC50 or anti-LPS antibody-drug conjugate ADC53, against FaDu cells, a human head and neck cancer cell line, transplanted subcutaneously. [Figure 11] The full-length amino acid sequence (SEQ ID NO: 1) and the full-length cDNA sequence (SEQ ID NO: 2) of human CLDN6 are shown. [Figure 12] The full-length amino acid sequence (SEQ ID NO: 3) and the full-length cDNA sequence (SEQ ID NO: 4) of human CLDN9 are shown. [Figure 13] The amino acid sequences of CDRL1-3 of the B1 antibody light chain (SEQ ID NOs. 5-7) are shown. [Figure 14] The amino acid sequence of CDRL3 in the humanized B1 antibody light chain L4 (SEQ ID NO: 8) is shown. [Figure 15] The amino acid sequences of CDRH1-3 in the B1 antibody heavy chain (SEQ ID NOs. 9-11) are shown. [Figure 16] The amino acid sequences of CDRL1-3 of the C7 antibody light chain (SEQ ID NOs. 12-14) are shown. [Figure 17] The amino acid sequences of CDRH1-3 in the C7 antibody heavy chain (SEQ ID NOs. 15-17) are shown. [Figure 18] The nucleotide sequence of the cDNA encoding the variable region of the B1 antibody light chain (SEQ ID NO: 18) and the amino acid sequence of the variable region of the B1 antibody light chain (SEQ ID NO: 19) are shown. Underlined characters in the amino acid sequence indicate the CDR sequence. [Figure 19] The nucleotide sequence of the cDNA encoding the variable region of the B1 antibody heavy chain (SEQ ID NO: 20) and the amino acid sequence of the variable region of the B1 antibody heavy chain (SEQ ID NO: 21) are shown. Underlined characters in the amino acid sequence indicate the CDR sequence. [Figure 20] The nucleotide sequence of the cDNA encoding the variable region of the C7 antibody light chain (SEQ ID NO: 22) and the amino acid sequence of the variable region of the C7 antibody light chain (SEQ ID NO: 23) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 21] The nucleotide sequence of the cDNA encoding the variable region of the C7 antibody heavy chain (SEQ ID NO: 24) and the amino acid sequence of the variable region of the C7 antibody heavy chain (SEQ ID NO: 25) are shown. Underlined characters in the amino acid sequence indicate the CDR sequence. [Figure 22] The amino acid sequence of the chB1 light chain (SEQ ID NO: 28) and the DNA fragment containing the DNA sequence encoding the chB1 light chain (SEQ ID NO: 29) are shown. Underlined characters in the amino acid sequence indicate the CDR sequence. [Figure 23] The amino acid sequence of the variable region of the chB1 light chain (SEQ ID NO: 30) and the nucleotide sequence encoding the chB1 light chain variable region (SEQ ID NO: 31) are shown. Underlined amino acid sequences indicate 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. Underlined elements in the amino acid sequence indicate the CDR sequence. [Figure 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. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 26] The amino acid sequence of the humanized antibody light chain hL1 (SEQ ID NO: 36) and the nucleotide sequence encoding the humanized antibody light chain hL1 (SEQ ID NO: 37) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [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. Underlined parts in the amino acid sequence indicate the CDR sequence. [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. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 29] The amino acid sequence of the variable region of the humanized antibody light chain hL2 (SEQ ID NO: 42) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL2 (SEQ ID NO: 43) are shown. [Figure 30] The amino acid sequence of the humanized antibody light chain hL3 (SEQ ID NO: 44) and the nucleotide sequence encoding the humanized antibody light chain hL3 (SEQ ID NO: 45) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 31] The amino acid sequence of the variable region of the humanized antibody light chain hL3 (SEQ ID NO: 46) and the nucleotide sequence encoding the variable region of the humanized antibody light chain hL3 (SEQ ID NO: 47) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 32] The amino acid sequence of the humanized antibody light chain hL4 (SEQ ID NO: 48) and the nucleotide sequence encoding the humanized antibody light chain hL4 (SEQ ID NO: 49) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 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. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 34] The amino acid sequence of the humanized antibody heavy chain hH1 (SEQ ID NO: 52) and the nucleotide sequence encoding the humanized antibody heavy chain hH1 (SEQ ID NO: 53) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 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. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 36] The amino acid sequence of the humanized antibody heavy chain hH2 (SEQ ID NO: 56) and the nucleotide sequence encoding the humanized antibody heavy chain hH2 (SEQ ID NO: 57) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 37] The amino acid sequence of the variable region of the humanized antibody heavy chain hH2 (SEQ ID NO: 58) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH2 (SEQ ID NO: 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. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 39] The amino acid sequence of the variable region of the humanized antibody heavy chain hH3 (SEQ ID NO: 62) and the nucleotide sequence encoding the variable region of the humanized antibody heavy chain hH3 (SEQ ID NO: 63) are shown. Underlined parts in the amino acid sequence indicate the CDR sequence. [Figure 40] Flow cytometry analysis of B1 and C7 antibodies demonstrates their binding affinity to human CLDN6 and its family molecules CLDN3, CLDN4, and CLDN9. [Figure 41] This demonstrates the antibody internalization activity of B1 and C7 antibodies by Mab-ZAP. [Figure 42] The binding activity of humanized anti-CLDN6 antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 to CLDN6 and its family molecules is demonstrated by flow cytometry. [Figure 43] The amino acid sequences of the trastuzumab light chain (SEQ ID NO: 64) and heavy chain (SEQ ID NO: 65) are shown. [Figure 44] The amino acid sequences of the light chain (SEQ ID NO: 73) and heavy chain (SEQ ID NO: 75) of the Trastuzumab mutant are shown. [Figure 45]This figure shows a comparison of the amino acid sequences of chB1_H, the heavy chain of the human chimeric anti-CLDN6 antibody chB1, and the humanized antibody heavy chains hH1, hH2, and hH3. "·" indicates an amino acid residue identical to that of chB1_H, and the locations where amino acid residues are listed indicate substituted amino acid residues. [Figure 46] This figure shows a comparison of the amino acid sequences of chB1_L, the light chain of the human chimeric anti-CLDN6 antibody chB1, and the humanized antibody light chains hL1, hL2, hL3, and hL4. "·" indicates an amino acid residue identical to chB1_L, and where amino acid residues are listed, they indicate substituted amino acid residues. [Figure 47] This study demonstrates the effects of the anti-HER2 antibody-drug conjugates ADC49 and ADC55 on subcutaneously transplanted human breast cancer cell line KPL-4. [Figure 48] This study demonstrates the effect of the anti-HER2 antibody-drug conjugate ADC55 on JIMT-1 cells, a human breast cancer cell line transplanted subcutaneously. [Figure 49] This study demonstrates the effects of anti-HER2 antibody-drug conjugates ADC49 and ADC55, and anti-LPS antibody-drug conjugate ADC53 on subcutaneously transplanted human pancreatic cancer cell line CFPAC-1. [Modes for carrying out the invention]

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

[0011] In the present invention, examples of "halogen atoms" include fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, and the like.

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

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

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

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

[0016] In the present invention, "C3-C5 cycloalkoxy group" means a cycloalkoxy group having a saturated cyclic hydrocarbon group with 3 to 5 carbon atoms. Examples of "C3-C5 cycloalkoxy groups" include cyclopropoxy groups, cyclobutoxy groups, and cyclopentyloxy groups.

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

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

[0019] In the present invention, "heteroaryl group" refers to groups such as thienyl group, pyrrolyl group, pyrazolyl group, triazolyl group, oxazolyl group, oxadiazolyl group, thiazolyl group, pyridyl group, pyrimidyl group, pyridazyl group, pyrazinyl group, quinolyl group, quinoxalyl group, benzothiophenyl group, benzimidazolyl group, benzotriazolyl group, and benzofuranyl group.

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

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

[0022] [Antibody-drug conjugates] The antibody-drug conjugate of the present invention is represented by the following formula. [ka] m1 represents the number of drug-binding molecules per antibody in the antibody-drug conjugate, Ab represents the antibody or a functional fragment of the antibody, L represents the linker connecting Ab and D, and D represents the drug. <Drugs> The drug D bound to the antibody-drug conjugate of the present invention is described below. Drug D of the present invention is preferably an antitumor compound. In this antitumor compound, part or all of the linker is cleaved within tumor cells, releasing the antitumor compound portion and exhibiting its antitumor effect. If the linker is cleaved at the drug binding site, the antitumor compound is released in its original structure, and its original antitumor effect is exerted. The antitumor compound in the antibody-drug conjugate of the present invention is of general formula (V)

[0023] [ka] This is a pyrrolobenzodiazepine derivative (PBD derivative) represented by [formula]. It is explained below. The asterisk indicates that it is bound to linker L.

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

[0025] A represents a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated heterocycle with a spirobond, preferably a 3-5 member saturated hydrocarbon ring (cyclopropane, cyclobutane, cyclopentane), more preferably cyclopropane, cyclobutane, and most preferably cyclopropane. The 3-5 member saturated hydrocarbon ring of the spiro bond may be substituted with 1-4 halogen atoms, preferably with 1 or 2 fluorine atoms (e.g., 2,2-difluorocyclopropane).

[0026] R 1 , R 2 Each of these 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'', preferably a C1-C6 alkoxy group, a C1-C6 alkyl group, or a hydroxyl group, more preferably a C1-C3 alkoxy group, and most preferably a methoxy group.

[0027] R 3 , R 4 and R 5 This refers to (i) to (iii) below. (i)R 3 and R 4 When these groups come together and form a double bond with the carbon atom to which each group is bonded, as shown below, [ka] R5 This 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 preferably an aryl group which may have one or more substituents selected from group 1.

[0028] R 5 The "aryl group" in "an 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 "an 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] The above R 5 Examples of substituents on the aryl group or heteroaryl group are listed below as a) to j). a) C1-C6 alkoxy groups which may be substituted with 1-3 halogen atoms, b) A C1-C6 alkyl group which may be substituted with one of the following: 1-3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''. c) Halogen atom, d) C3-C5 cycloalkoxy groups, e) C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R''', h)-NHC(=NR')-NR''R''', i)-NHCOR' j) Hydroxyl group Here, in b), f) to i), R', R'', and R'''' each independently represent a hydrogen atom and a C1-C6 alkyl group, and preferably each independently represents a hydrogen atom or a C1-C3 alkyl group.

[0031] a) to j) are preferably as follows: a) A C1-C3 alkoxy group which may be substituted with 1-3 halogen atoms, More preferably, the group is a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, or a trifluoromethoxy group; even more preferably, it is a methoxy group, an ethoxy group, or a trifluoromethoxy group; and most preferably, it is a methoxy group. b) A C1-C3 alkyl group which may be substituted with 1-3 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 which may be substituted with 1-3 halogen atoms, a hydroxyl group, or any of the following 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, even more 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, -CH2OCOMe, -CH2-NHC(=NH)-NH2, or -CH2-NHC(=NMe)-NH2. c) A halogen atom, preferably a fluorine atom or a chlorine atom. d) A C3-C5 cycloalkoxy group, or more preferably a cyclopropoxy group. e) A C1-C3 alkylthio group, or more preferably a methylthio group or an ethylthio group. f) -NR'R'', where R' and R'' are independently a hydrogen atom or a C1-C3 alkyl group, more preferably -NH2, -NHMe, -NMe2, -NHEt, or -NEt2. 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)-NH2 and -C(=NMe)-NH2. 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、 It is -NHC(=NMe)-NH2. i) -NHCOR', where R' is a hydrogen atom or a C1-C3 alkyl group, more preferably -NHCOMe 、 -NHCOEt, j) It is a hydroxyl group.

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

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

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

[0035] R 5 If the group is a phenyl group, more preferably it is 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-hydroxymethylphenyl group, a 4-acetoxymethylphenyl group, or a 4-carbamimidoamidemethylphenyl group. Even more preferably it is a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxy-phenyl group, a 4-hydroxymethylphenyl group, a 4-acetoxymethylphenyl group, a 4-carbamimidoamidemethylphenyl group, or a 4-trifluoromethylphenyl group. R 5 If the group 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 substituents are preferably a), b), d), g), h), j), more preferably a), b).

[0037] R 5 If the group is a heteroaryl group, the substituent can be in any position, but if it is a 3-pyridyl group, it is preferably at position 6 or / and 5, if it is a 2-pyridyl group, it is preferably at position 5 or / and 4, or at position 5 or / and 6, and if it is a 4-pyridyl group, it is preferably at position 2 or / and 6.

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

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

[0040] R 5 In the phrase "a C1-C6 alkyl group having one or more substituents selected from group 2", the "C1-C6 alkyl group" is preferably a C1-C3 alkyl group, and more preferably a methyl group or an ethyl group.

[0041] R 5 The substituents of the "C1-C6 alkyl group having one or more substituents selected from group 2" are a halogen atom, a hydroxyl group, or a C1-C6 alkoxy group (preferably a C1-C3 alkoxy group), more preferably a hydroxyl group, a methoxy group, or an ethoxy group, and more preferably a hydroxyl group.

[0042] (ii)R 3 However, when referring to a hydrogen atom, R 4 and R 5 R 4 and R 5 Together with the bonded carbon atom, it forms a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, or forms CH2=, as shown below. [ka] , or, [ka] The aforementioned 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, and preferably with 1 or 2 fluorine atoms. R 4 and R 5 These groups combine to form a preferably 3-5 membered saturated hydrocarbon ring or CH2=, more preferably cyclopropane, cyclobutane, or CH2= (exomethylene group), and even more preferably cyclopropane. R 4 and R 5When they come together to form a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated heterocycle, it is preferable that A is the same 3-5 member saturated hydrocarbon ring or 3-5 member saturated heterocycle. More preferably, A is a 3-5 member saturated hydrocarbon ring, and R 4 and R 5 Together, they form a 3-5 member saturated hydrocarbon ring, more preferably A is a cyclopropane ring, and R 4 and R 5 They come together to form a cyclopropane ring.

[0043] (iii)R 3 , R 4 and R 5 However, R 3 The carbon atoms to which it is bonded, and R 4 and R 5 Together with the bonded carbon atom, it forms a benzene ring or a 6-membered heterocycle which may have one or more substituents selected from group 3. The following equation is R 3 and R 4 This shows a case where these elements come together to form a benzene ring which may have one or more substituents. [ka] The substituents on the benzene ring or heterocycle may be in any position, and if there are multiple substituents, they may be the same or different.

[0044] The substituents on the benzene ring or heterocycle are a halogen atom, a C1-C6 alkyl group or C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms, preferably a halogen atom, a C1-C3 alkyl group or 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 six-membered heterocycle which may have one or more substituents" is preferably an unsubstituted benzene ring.

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

[0047] R 6 , R 7 Both indicate a hydrogen atom, or R 6 and R 7 These two atoms combine to form an imine bond (C=N).

[0048] R 8 This is a hydroxyl group or a C1-C3 alkoxy group, preferably a hydroxyl group, a methoxy group, and more preferably a hydroxyl group. 8 This may also be a bisulfite adduct (OSO3M (where M is a metal cation)). R 8 Because it is bonded to a chiral carbon atom, it has the stereoconfiguration shown in the substructure (Va) or (Vb) below. The dashed line indicates a bond with Y in general formula (V), and the asterisk indicates a bond with L. [ka]

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

[0050] Drug D of the present invention is preferably one compound selected from the following group. [ka] , [ka] [ka]

[0051] <Linker structure> The linker structure for binding an antitumor drug to an antibody in the antibody-drug conjugate of the present invention will be described. The linker L is given by the following formula: -Lb-La-Lp-NH-B-CH2-O(C=O)-* This is shown. The asterisk indicates that the drug D is bound to the nitrogen atom at the N10' position, and Lb indicates a spacer that connects the sugar chain or remodeled sugar chain of La to Ab, or a spacer that connects the side chain of an amino acid residue (e.g., cysteine, lysine, etc.) of antibody Ab to La.

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

[0053] Lp represents a linker consisting of an amino acid sequence that can be cleaved in vivo or in target cells. Lp is cleaved, for example, by the action of enzymes such as esterases and peptidases. Lp is a peptide residue composed of 2 to 7 (preferably 2 to 4) amino acids. That is, it is composed of oligopeptide residues in which 2 to 7 amino acids are linked by peptide bonds. Lp is bonded to the carbonyl group of La in Lb-La- at its N-terminus and forms an amide bond with the amino group (-NH-) of the -NH-B-CH2-O(C=O)- portion of the linker at its C-terminus. The bond between the C-terminus of Lp and the -NH- is cleaved by enzymes such as esterase.

[0054] The amino acids that make up Lp are not particularly limited, but for example, they are L- or D-amino acids, and preferably L-amino acids. In addition to α-amino acids, they may also be amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid, and may even be unnatural amino acids such as N-methylated amino acids.

[0055] The amino acid sequence of Lp is not particularly limited, but possible 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). Of these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline (Cit) are preferred. These amino acids may be duplicated, and the amino acid sequence may contain arbitrarily selected amino acids. Furthermore, the pattern of drug release can be controlled depending on the type of amino acid.

[0056] As a specific example of linker Lp, -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- One could list these: Here, '(D-)V' represents D-valine, '(D-)P' represents D-proline, and '(D-)D' represents D-aspartic acid.

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

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

[0059] Lb indicates either i) a spacer that connects La to the glycan or remodeled glycan of antibody Ab, or ii) a spacer that connects La to the side chain of an amino acid residue (e.g., cysteine, lysine, etc.) of antibody Ab. If Lb is i), then La indicates one of the following selected groups. -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -NH-C(=O)-(CH2CH2O)n 3 -CH2CH2-C(=O)-, -(CH2)n 4 -OC(=O)- Here, in the formula, n 2 n is an integer from 1 to 3 (preferably 1 or 2), n 3 n is an integer from 1 to 5 (preferably an integer from 2 to 4, more preferably 2 or 4), n 4 This represents an integer between 0 and 2 (preferably 0 or 1).

[0060] If Lb is i), then La preferably represents one selected from the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)- -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, and -OC(=O)- La is more preferably -C(=O)-CH2CH2-C(=O)- or -C(=O)-(CH2CH2)2-C(=O)-.

[0061] The spacer for Lb is not particularly limited, but for example, the spacer shown in the following equation can be used. [ka] [ka] [ka] In the structural formulas of Lb shown above, the asterisk represents -(C=O) or -(CH2)n at the left end of La. 4 The wavy line indicates that it is bound to Ab's sugar chain or a remodeled sugar chain. In the structural formulas of each of the Lb(Lb-1, Lb-2, or Lb-3) shown above, the triazole ring moiety formed by the click reaction between the azide group and DBCO has geometric isomerism, and one of these two types of structures, or a mixture thereof, exists in one Lb. In one molecule of the antibody-drug conjugate of the present invention, m 1 There are several '-LD's, m 1 Each Lb (Lb-1, Lb-2, or Lb-3) in L of each individual '-LD' contains either one or both of these two types of structures.

[0062] If Lb is i), then L is preferably represented by -Lb-La-Lp-NH-B-CH2-O(C=O)-*, B is a 1,4-phenyl group, Lp indicates one of the following groups: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- La indicates one of the following groups: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)- Lb represents one of the structural formulas of Lb shown above.

[0063] If Lb is i), then L is more preferably one selected from the following group. -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B-CH2-OC(=O)-, -Z 1-C(=O)-CH2CH2-C(=O)-GGPL-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 2 -OC(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 3 -CH2-OC(=O)-GGVA-NH-B-CH2-OC(=O)- Here, Z 1 The structural formula of Lb shown above is: [ka] This indicates Z 2 The structural formula of Lb shown above is: [ka] This indicates Z 3 The structural formula of Lb above is shown below: [ka] This indicates that B is a 1,4-phenyl group.

[0064] L is most preferably one of the following: -Z 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1-C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- Here, B is a 1,4-phenyl group, and Z 1 The structural formula of Lb above is shown below: [ka] That is the case.

[0065] In the case of Lb in ii), if the amino acid residue is a cysteine ​​residue, the spacer for Lb is not particularly limited, but for example, -(Succinimid-3-yl-N)- is one example. 『-(Succinimid-3-yl-N)-』 is expressed as follows:

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

[0067] If Lb is ii), then L is represented as -Lb-La-Lp-NH-B-CH2-O(C=O)-*, B is a 1,4-phenyl group, Lp represents one of the following: -GGVA-, -GG-(D-)VA-, -VA-, or -GGFG-. La is -(CH2)n 9 -C(=O)-, or -(CH2CH2)n 10 -C(=O)-NH-(CH2CH2O)n 11 -CH2CH2-C(=O)- n 9 n is an integer from 2 to 7 (preferably an integer from 2 to 5, more preferably 2 or 5), n 10 n is an integer between 1 and 3 (preferably 1), n 11 This represents an integer between 6 and 10 (preferably 8), Lb represents -(Succinimid-3-yl-N)-.

[0068] If Lb is ii), then L is preferably one of the following: -(Succinimid-3-yl-N)-(CH2)5-C(=O)-VA-NH-B-CH2-OC(=O)-, -(Succinimid-3-yl-N)-(CH2)5-C(=O)-GGVA-NH-B-CH2-OC(=O)-, or, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- Here, B is a 1,4-phenyl group.

[0069] It is believed that the antibody-drug conjugate of the present invention, after most of it has migrated into tumor cells, releases the drug D portion (hereinafter referred to as the released drug) when the linker portion (e.g., Lp) is cleaved by an enzyme or the like and activated, thereby exhibiting antitumor activity. Therefore, it is preferable that the antibody-drug conjugate of the present invention be stable outside tumor cells. <Free drugs and manufacturing intermediates> The intermediate and free drug of the antibody-drug conjugate of the present invention are given by the following formula:

[0070] [ka] This is shown below.

[0071] The free drug of the present invention is generated after it has been transferred into tumor cells and the linker L portion of the antibody-drug conjugate has been cleaved. For example, drugs 1 to 16 of Examples 45 to 54 and 150 to 152 are examples of this drug. The antibody-drug conjugate of the present invention is prepared using this manufacturing intermediate.

[0072] The free drug in the antibody-drug conjugate of the present invention is (a)R 16 , R 17 R 16 and R 17 This is the case where they come together to form an imine bond (N=C). The manufacturing intermediate in the antibody-drug conjugate of the present invention is (b)R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)-* This is the case shown by [the symbol]. Therefore, in the formula, l and n 1 , E and the aforementioned A, R 9 and R 1 , R 10 and R 2 , R 11 and R 3 , R 12 and R 4 , R 13 and R 5 , R 14 and R 6 , R 15 and 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 are synonymous.

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

[0074] E represents a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated heterocycle with a spirobond, preferably a 3-5 member saturated hydrocarbon ring (cyclopropane, cyclobutane, cyclopentane), more preferably cyclopropane or cyclobutane, and most preferably cyclopropane. The 3-5 member saturated hydrocarbon ring of the spiro bond may be substituted with 1-4 halogen atoms, preferably with 1 or 2 fluorine atoms (e.g., 2,2-difluorocyclopropane).

[0075] R 9 , R 10 Each of these 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'', preferably a C1-C6 alkoxy group, a C1-C6 alkyl group, or a hydroxyl group, more preferably a C1-C3 alkoxy group, and most preferably a methoxy group.

[0076] R 11 , R 12 and R 13 This refers to (i) to (iii) below. (i)R 11 and R 12 When they come together and form a double bond with the carbon atom to which each group is bonded, R 13 This 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, preferably an aryl group which may have one or more substituents selected from group 7.

[0077] R 13 The "aryl group" in "an aryl group or heteroaryl group having one or more substituents selected from group 7" is preferably a phenyl group or a naphthyl group, and more preferably a phenyl group.

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

[0079] The above R 13 Examples of substituents on the aryl group or heteroaryl group are listed below as a) to j). a) C1-C6 alkoxy groups which may be substituted with 1-3 halogen atoms, b) A C1-C6 alkyl group which may be substituted with one of the following: 1-3 halogen atoms, a hydroxyl group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''. c) Halogen atom, d) C3-C5 cycloalkoxy groups, e) C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R''', h)-NHC(=NR')-NR''R''', i)-NHCOR', j) Hydroxyl group Here, in b), f) to i), R', R'', and R'''' each independently represent a hydrogen atom and a C1-C6 alkyl group, and preferably each independently represents a hydrogen atom or a C1-C3 alkyl group.

[0080] a) to j) are preferably as follows: a) A C1-C3 alkoxy group which may be substituted with 1-3 halogen atoms, More preferably, the group is a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, or a trifluoromethoxy group. More preferably are methoxy groups, ethoxy groups, and trifluoromethoxy groups, with the most preferred being methoxy groups. b) A C1-C3 alkyl group which may be substituted with 1-3 halogen atoms, a hydroxyl group, or any of the following selected from -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, the C1-C3 alkyl group may be substituted with 1-3 halogen atoms, a hydroxyl group, -OCOR', -C(=NR')-NR''R''', or -NHC(=NR')-NR''R''', where R', R'', and R''' are independently a hydrogen atom and a methyl group, respectively. More 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, -CH2OCOMe, -CH2-NHC(=NH)-NH 2、 It is -CH2-NHC(=NMe)-NH2. c) A halogen atom, preferably a fluorine atom or a chlorine atom. d) A C3-C5 cycloalkoxy group, or more preferably a cyclopropoxy group. e) A C1-C3 alkylthio group, or more preferably a methylthio group or an ethylthio group. f) -NR'R'', where R' and R'' are independently a hydrogen atom or a C1-C3 alkyl group. More preferably, these are -NH2, -NHMe, -NMe2, -NHEt, and -NEt2. g)-C(=NR')-NR''R''', where R', R'', and R'''' are each independently a hydrogen atom or a C1-C3 alkyl group. More specifically, -C(=NH)-NH2 and -C(=NMe)-NH2. h)-NHC(=NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group. Rather, -NHC(=NH)-NH 2、 It is -NHC(=NMe)-NH2. i) -NHCOR' R' is a hydrogen atom or a C1-C3 alkyl group. fukai, -NHCOMe 、 -NHCOEt, j) It is a hydroxyl group.

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

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

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

[0084] R 13If the group is a phenyl group, more preferably it is 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-hydroxymethylphenyl group, a 4-acetoxymethylphenyl group, or a 4-carbamimidomethylphenyl group. More preferably, the group is a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxyphenyl group, a 4-hydroxymethylphenyl group, a 4-acetoxymethylphenyl group, a 4-carbamimidomethylphenyl group, or a 4-trifluoromethylphenyl group. R 13 If the group 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 substituents are preferably a), b), d), g), h), j), more preferably a), b). R 13 If the group is a heteroaryl group, the substituent can be in any position, but if it is a 3-pyridyl group, it is preferably at position 6 or / and 5, if it is a 2-pyridyl group, it is preferably at position 5 or / and 4, or at position 5 or / and 6, and if it is a 4-pyridyl group, it is preferably at position 2 or / and 6. R 13 When the substituent is a heteroaryl group, there may be multiple substituents, but preferably there is one or two, and preferably one. R 13 If the group is a pyridyl group, it is preferably a 6-methoxy-3-pyridyl group or a 6-methyl-3-pyridyl group. R 13 When the group is a 3-thienyl group or a 6-quinoxalyl group, it is preferably unsubstituted.

[0086] R 13In the phrase "a C1-C6 alkyl group having one or more substituents selected from group 8", the "C1-C6 alkyl group" is preferably a C1-C3 alkyl group, and more preferably a methyl group or an ethyl group. R 13 The substituents of the "C1-C6 alkyl group having one or more substituents selected from group 8" are a halogen atom, a hydroxyl group, or a C1-C6 alkoxy group (preferably a C1-C3 alkoxy group), more preferably a hydroxyl group, a methoxy group, or an ethoxy group, and more preferably a hydroxyl group.

[0087] (ii)R 11 However, when referring to a hydrogen atom, R 12 and R 13 R 12 and R 13 Together with the bonded carbon atom, it forms a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, or forms a CH2=. The aforementioned 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, and preferably with 1 or 2 fluorine atoms. R 12 and R 13 These groups combine to form a preferably 3-5 membered saturated hydrocarbon ring or CH2=, more preferably cyclopropane, cyclobutane, or CH2= (exomethylene group), and even more preferably cyclopropane. R 12 and R 13 When they come together to form a 3-5 member saturated hydrocarbon ring or a 3-5 member saturated heterocycle, it is preferable that E is the same 3-5 member saturated hydrocarbon ring or saturated heterocycle as R. More preferably, E is a 3-5 member saturated hydrocarbon ring, and R is the same 3-5 member saturated hydrocarbon ring as E. 12 and R 13 Together they form a 3-5 member saturated hydrocarbon ring, more preferably E is a cyclopropane ring, and R 12 and R 13 They come together to form a cyclopropane ring.

[0088] (iii)R 11 , R 12 and R 13 However, R 11 The carbon atoms to which it is bonded, and R 12 and R 13 Together with the bonded carbon atom, it forms a benzene ring or a six-membered heterocycle which may have one or more substituents selected from group 9. The substituents on the benzene ring or heterocycle may be in any position, and if there are multiple substituents, they may be the same or different. The substituents on the benzene ring or heterocycle are a halogen atom, a C1-C6 alkyl group or C1-C6 alkoxy group which may be substituted with 1 to 3 halogen atoms, preferably a halogen atom, a C1-C3 alkyl group or 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 six-membered heterocycle which may have one or more substituents" is preferably an unsubstituted benzene ring.

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

[0090] R 14 , R 15 Both indicate a hydrogen atom, or R 14 and R 15 These two atoms combine to form an imine bond (C=N).

[0091] V and W are, independently, an oxygen atom, a nitrogen atom, and a sulfur atom, and are preferably oxygen atoms.

[0092] R 16 , R 17 teeth, (a)R 16 , R 17 R 16 and R 17They come together to form an imine bond (N=C), or (b)R 16 This represents J-La'-Lp'-NH-B'-CH2-O(C=O)-*, and R 17 This is represented by a hydroxyl group or a C1-C3 alkoxy group.

[0093] (b)R above 16 However, in the case of J-La'-Lp'-NH-B'-CH2-O(C=O)-*, the asterisk in the formula indicates that it is bonded to the N10' position of the pyrrolobenzodiazepine ring shown in the above formula.

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

[0095] Lp' represents a linker consisting of an amino acid sequence that can be cleaved in vivo or in target cells. Lp is cleaved by enzymes such as esterases and peptidases.

[0096] As a concrete example of linker Lp', -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-, -GGFGGGF- can be listed. Here, '(D-)V' represents D-valine, '(D-)P' represents D-proline, and '(D-)D' represents D-aspartic acid.

[0097] The linker Lp' is preferably as follows: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- More preferably, -GGVA-, -GGVCit-, and -VA- can be mentioned.

[0098] La' represents one of the following groups: -C(=O)-(CH2CH2)n 6 -C(=O)-, -C(=O)-(CH2CH2)n 6 -C(=O)-NH-(CH2CH2)n 7 -C(=O)-, -C(=O)-(CH2CH2)n 6 -C(=O)-NH-(CH2CH2O)n 7 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 6 -NH-C(=O)-(CH2CH2O)n 7 -CH2CH2-C(=O)-, -(CH2)n 8 -OC(=O)-, -(CH2)n 12 -C(=O)- and -(CH2CH2)n 13 -C(=O)-NH-(CH2CH2O)n 14 -CH2CH2-C(=O)- Here, in the formula, n 6 n is an integer from 1 to 3 (preferably 1 or 2), n 7 n is an integer from 1 to 5 (preferably an integer from 2 to 4, more preferably 2 or 4), n 8 n is an integer between 0 and 2 (preferably 0 or 1), n 12 n is an integer from 2 to 7 (preferably an integer from 2 to 5, more preferably 2 or 5), n 13 n is an integer between 1 and 3 (preferably 1), n 14 This represents an integer between 6 and 10 (preferably 8).

[0099] La' preferably represents one selected from the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, -(CH2)2-C(=O)-, -(CH2)5-C(=O)-, and -CH2CH2-C(=O)-NH -(CH2CH2O)8-CH2CH2-C(=O)-

[0100] La' is more precisely -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, or -(CH2)5-C(=O)-.

[0101] J is not particularly limited as 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, but examples include the compound shown in the following formula. [ka]

[0102] In each of the structural formulas of J shown above, the asterisk is -(C=O) or -(CH2)n at the left end of La'. 8 This indicates that it is bound to [something].

[0103] Alternatively, J may be a compound or halogen atom that binds to the side chain of an amino acid residue (e.g., cysteine, lysine, etc.) of antibody Ab, for example, a maleidyl group represented by the following formula. [ka] In the maleimidyl group shown above, the asterisk is the -(CH2)n at the left end of La'. 12 , or -(CH2CH2)n 13 This indicates that it is bound to [something].

[0104] R 16This is preferably represented by J-La'-Lp'-NH-B'-CH2-O(C=O)-*, B' is a 1,4-phenyl group, Lp' indicates one of the following groups: -GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-, -GGPL- La' indicates one of the following groups: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -OC(=O)-, -CH2-OC(=O)-, -(CH2)5-C(=O)- and -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)- J represents one of the following structural formulas: [ka] Here, we show that in the structural formula of J, the asterisk is bonded to La'.

[0105] R 16 More preferably, it is one selected from the following group. J 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-GGVK-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-GGPL-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B’-CH2-OC(=O)-、 J 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B’-CH2-OC(=O)-、 J 2 -OC(=O)-GGVA-NH-B’-CH2-OC(=O)-、J 3 -CH2-OC(=O)-GGVA-NH-B’-CH2-OC(=O)-、 J<* 4 -(CH2)5-C(=O)-GGVA-NH-B’-CH2-OC(=O)-、 J 4 -(CH2)5-C(=O)-VA-NH-B’-CH2-OC(=O)-、及び J 4-CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)- Here, J 1 , J 2 , J 3 , J 4 The structural formula shown below is: [ka] Here, J 1 , J 2 , J 3 and J 4 In the structural formula, the asterisk is J 1 , J 2 , J 3 Or J 4 This indicates that it is bonded to an adjacent group. B' is a 1,4-phenyl group.

[0106] R 16 Most preferably, it is one of the following: J 1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1-C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 4 -(CH2)5-C(=O)-VA-NH-B'-CH2-OC(=O)- Here, B' is a 1,4-phenyl group, J 1 , J 4 The structural formula of J above is shown below: [ka] Here, J 1 and J 4 In the structural formula, the asterisk is J 1 Or J 4 This indicates that it is bonded to an adjacent group.

[0107] R 17 This is a hydroxyl group or a C1-C3 alkoxy group, preferably a hydroxyl group or a methoxy group. R 17 This may also be a bisulfite adduct (OSO3M (where M is a metal cation)). R 17 Because it is bonded to a chiral carbon atom, it has the stereoconfiguration shown in the substructure (VIa) or (VIb) below. The dashed 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 one compound selected from the following group. [ka]

[0109] This free drug may be released within tumor cells with a portion of linker L bound to it, but even in this state, it is an excellent drug that exhibits superior antitumor effects. After the free drug migrates to tumor cells, it is further oxidized to R 16 , R 17 Although dehydrogenation may occur, it still exhibits excellent antitumor effects even in this state.

[0110] The manufacturing intermediate is preferably one compound selected from the following group. [ka] [ka]

[0111] The manufacturing intermediate is preferably one compound selected from the following group. [ka] [ka] [ka] [ka]

[0112] <Antibody> In this invention, the terms "cancer" and "tumor" are used interchangeably. In the present invention, "gene" means a nucleotide or nucleotide sequence containing a nucleotide sequence that codes for the amino acids of a protein, or its complementary chain. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, RNA, etc., which are nucleotide sequences containing a nucleotide sequence that codes for the amino acids of a protein or their complementary chains, are included in the meaning of "gene". Examples of the "CLDN6 gene" of the present invention include DNA, mRNA, cDNA, cRNA, etc., containing a nucleotide sequence that codes for the amino acid sequence of the CLDN6 protein. In this invention, "nucleotide," "polynucleotide," or "nucleotide sequence" and "nucleic acid" are synonymous, and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc., are also included in the meaning of "nucleotide" or "nucleotide sequence." In this invention, the terms "polypeptide," "peptide," and "protein" are used interchangeably. In this invention, "CLDN6" is used interchangeably with "CLDN6 protein".

[0113] In this invention, "cells" include cells from an animal organism and cultured cells. In this invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, and is not limited to direct trauma, but includes causing any structural or functional damage to cells, such as DNA breakage, base dimer formation, chromosome breakage, damage to the cell division apparatus, and a decrease in the activity of various enzymes.

[0114] In the present invention, "functional fragment of an antibody" is also called "antigen-binding fragment of an antibody" and 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 antibodies, and polyspecific antibodies formed from antibody fragments. Furthermore, 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 antibody antigen-binding fragments. However, the invention is not limited to these molecules as long as they have the ability to bind to an antigen. In addition, these antigen-binding fragments include not only full-length antibody proteins treated with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes. The functional fragment of the present invention includes a functional fragment that retains aspartic acid (Asn297) and surrounding amino acids that are modified by well-conserved N-linked glycans in the Fc region of the IgG heavy chain, and that has the ability to bind to an antigen.

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

[0116] In this invention, "CDR" refers to the complementarity determining region. It is known that antibody molecules have three CDRs each in their heavy and light chains. CDRs, also called hypervariable regions, are sites within the variable regions of the antibody's heavy and light chains that exhibit particularly high variability in their primary structure, and are separated into three locations on the primary structure of the polypeptide chains of the heavy and light chains. In this specification, the CDRs of the antibody are denoted 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 denoted as CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These sites are in close proximity to each other in terms of 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 at 68°C in the presence of 0.7-1.0 M NaCl using a DNA-immobilized filter, followed by washing at 68°C with a 0.1-2 times concentration SSC solution (1x concentration SSC consists of 150 mM NaCl and 15 mM sodium citrate) to identify the DNA, or hybridizing under conditions equivalent thereto. In this invention, "1 to several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0118] In the present invention, antibodies that recognize or bind to CLDN6 or CLDN6 and CLDN9 may be referred to as "anti-CLDN6 antibody" and "anti-CLDN6 / CLDN9 antibody," 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 antibody."

[0119] The antibody used in the antibody-drug conjugate of the present invention refers to an immunoglobulin, which is a molecule containing an antigen-binding site that binds immunospecifically to an antigen. The antibody of the present invention may be any of the classes IgG, IgE, IgM, IgD, IgA, and IgY, but IgG is preferred. Furthermore, any of the subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 may be used, but IgG1, IgG2, and IgG4 are preferred. When using IgG1 or IgG4, the effector function can be adjusted by substituting some of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, WO94 / 29351). The antibodies may originate from any species, but preferably from humans, rats, mice, and rabbits. If they originate from a species other than humans, it is preferable to chimerize or humanize them using well-known techniques. The antibodies of the present invention may be polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, as well as chimeric antibodies, humanized antibodies, human antibodies, functional fragments thereof, or modified versions thereof.

[0120] The antibody of the present invention is preferably an antibody that can target tumor cells. That is, since a drug having antitumor activity is conjugated via a linker, it is preferable that the antibody has one or more properties: the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be taken up and internalized within tumor cells, and the ability to damage tumor cells. The binding affinity of antibodies to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay that visualizes antibodies taken up into cells using a fluorescence microscope with a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay that measures the amount of fluorescence when taken up into cells 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) the Mab-ZAP assay (Bio Techniques 28:162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody and suppresses cell proliferation by releasing a toxin upon uptake into cells. Recombinant complex proteins of the catalytic domain of diphtheria toxin and protein G can also be used as immunotoxins. In the present invention, "high internalization ability" means that the viability of target antigen-expressing cells (e.g., CLDN6-expressing cells) to which the antibody and saporin-labeled anti-mouse or rat IgG antibody have been added (expressed as a relative rate with the cell viability without antibody added being 100%) is preferably 70% or less, more preferably 60% or less.

[0121] The antibody-drug conjugate of the present invention has a compound that exerts an antitumor effect attached to it; therefore, it is preferable, but not essential, that the antibody itself has an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxic effect of the antitumor compound in tumor cells, it is important and preferable that the antibody has the property of internalizing and migrating into tumor cells. From the viewpoint of exerting an antitumor effect, the property of the antibody internalizing and migrating into tumor cells is important and preferable because it allows the drug to specifically and selectively damage tumor cells. The antitumor activity of an antibody refers to its cytotoxic activity and anti-cellular effect on tumor cells. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems. Examples of such antibodies include antibodies against tumor-associated antigens, such as anti-CLDN6 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-DLL3 (Delta-like protein 3) antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-FGFR2 antibody (e.g., WO201315206), anti-G250 antibody, anti-MUC1 antibody (e.g., WO2011012309), anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-EGFR antibody, and anti-DR5 antibody, but are not limited to these. Preferably, the antibodies of the present invention are anti-CLDN6 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-CD98 antibody, and anti-TROP2 antibody, and more preferably anti-CLDN6 antibody and anti-HER2 antibody (e.g., Trastuzumab, Trastuzumab variant).

[0122] The antibodies of the present invention can be obtained by immunizing animals with antigenic polypeptides using methods commonly practiced in this art, and then collecting and purifying the antibodies produced in vivo. The antigen is not limited to human origin; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between the obtained antibodies that bind to the heterologous antigen and the human antigen. Furthermore, monoclonal antibodies can be obtained by establishing hybridomas and fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (e.g., Nature (1975) 256, pp. 495-497, Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)) (see below). Antigens can be obtained by genetically modifying host cells to produce the gene that codes for the antigen protein.

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

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

[0125] The anti-CLDN6 antibody used in the present invention is described below. The embodiments described below are merely examples of typical embodiments of the present invention and should not be interpreted as narrowing the scope of the invention.

[0126] 1. CLDN6 and CLDN9 CLDN6 is a 220-amino acid, four-transmembrane protein belonging to the Claudin family, with its N-terminus and C-terminus located within the cell. The amino acid and DNA sequences of human CLDN6 are publicly available in databases and can be accessed using accession numbers such as NP_067018 (Sequence ID 1 (Figure 11)) and NM_021195 (Sequence ID 2 (Figure 11) (both from NCBI)). Regarding the amino acid sequence of the human CLDN6 protein (hereinafter referred to as "CLDN6 amino acid sequence"), the extracellular region consists of an extracellular domain (EC1) consisting of amino acids 29-81 of sequence number 1 in the sequence listing, and an extracellular domain (EC2) consisting of amino acids 138-160. CLDN9 is a 217-amino acid, four-transmembrane protein belonging to the Claudin family, with its N-terminus and C-terminus located intracellularly. CLDN9 has high homology to CLDN6. The amino acid and DNA sequences of human CLDN9 are publicly available in databases and can be accessed using accession numbers such as NP_066192 (Sequence ID 3 (Figure 12)) and NM_020982 (Sequence ID 4 (Figure 12)) (both from NCBI).

[0127] 2. Anti-CLDN6 antibody An example of the anti-CLDN6 antibody of the present invention is an anti-CLDN6 antibody that recognizes a higher-order structure consisting of two extracellular regions of CLDN6, namely the amino acid sequences from 29 to 81 and the amino acid sequences from 138 to 160 from the N-terminus, as shown in Sequence ID No. 1 of the sequence listing, and that has internalization activity. The anti-CLDN6 antibody of the present invention is an antibody that can target tumor cells, that is, it possesses the properties of being able to recognize tumor cells, being able to bind to tumor cells, and being taken up and internalized within tumor cells. Therefore, the anti-CLDN6 antibody of the present invention and a compound having antitumor activity can be conjugated via a linker to form an antibody-drug conjugate. The anti-CLDN6 antibody of the present invention may also have antitumor activity.

[0128] Anti-CLDN6 antibodies can be obtained by immunizing animals with an antigenic polypeptide using methods commonly employed in this field, and then collecting and purifying the antibodies produced in vivo. Since CLDN6 is a four-transmembrane protein, proteins that retain their three-dimensional structure can be used as antigens, and cell-based immunization is one such method. Furthermore, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to the known method described above, thereby obtaining a monoclonal antibody.

[0129] The following describes in detail how to obtain antibodies against CLDN6. 1) Preparation of antigens CLDN6 can be used by purifying it directly from human tumor tissue or tumor cells, or by preparing the cell membrane fraction of such cells. Alternatively, CLDN6 can be obtained by synthesizing it in vitro (e.g., using a rapid translation system (RTS) from Roche Diagnostics) or by inducing its production in host cells through genetic engineering. Specifically, in genetic engineering, the antigen can be obtained by incorporating the CLDN6 cDNA into an expressionable vector and then synthesizing it in a solution containing the enzymes, substrates, and energy substances necessary for transcription and translation, or by transforming other prokaryotic or eukaryotic host cells to express CLDN6. Furthermore, CLDN6-expressing cells obtained through the aforementioned genetic engineering, or cell lines expressing CLDN6, can also be used as the CLDN6 protein. It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein, which is a link between the extracellular domain of the membrane protein CLDN6 and the constant domain of an antibody, in an appropriate host-vector system. It is also possible to use the transformed organism itself as an antigen, as described above. Furthermore, cell lines expressing CLDN6 can also be used as antigens. Examples of such cell lines include the human pancreatic cancer cell line NOR-P1, the human ovarian cancer cell lines NIH:OVCAR-3, OV-90, or OAW28, the human ovarian teratoma cell line PA-1, the human liver cancer cell line HuH-7, the human gestational choriocarcinoma cell line JEG-3, and the human pluripotent embryonic cancer cell line NTERA-2 clone D1, but are not limited to these cell lines as long as they express CLDN6. The CLDN9 protein used in this invention can be prepared and used in the same manner. 2) Production of anti-CLDN6 monoclonal antibodies The anti-CLDN6 antibody used in the present invention is not particularly limited, but for example, an antibody identified by the amino acid sequence shown in the sequence listing of this application can be suitably used. The anti-CLDN6 antibody used in the present invention is preferably one that has the following characteristics. (1) An antibody characterized by having the following characteristics (a) and (b); (a) Recognize or bind 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 this invention, "specific recognition," or "specific binding," means binding that is not nonspecific adsorption. A criterion for determining whether binding is specific or not can be, for example, the dissociation constant (hereinafter referred to as "KD"). The KD value of a preferred antibody of this invention for CLDN6 and / or CLDN9 is 1 × 10⁻⁶. -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 M or less. The binding of antigens and antibodies in this invention can be measured or determined by methods such as ELISA, RIA, and Surface Plasmon Resonance (SPR) analysis. The binding of antigens and antibodies expressed on the cell surface can be measured by methods such as flow saturation. (b) It has the activity to internalize CLDN6 and / or CLDN9-expressing cells by binding to CLDN6 and / or CLDN9. (2) The antibody described in (1) above, wherein CLDN6 and / or CLDN9 is human CLDN6 and / or human CLDN9.

[0130] The method for obtaining an antibody against CLDN6 according to the present invention typically involves the following steps, but is not limited thereto. (Method using hybridomas) (a) Purification of biomolecules to be used as antigens, or preparation of antigen-expressing cells, and administration of said biomolecules or antigen-expressing cells to animals. (b) From the aforementioned animals in which an immune response has been induced, collect tissue containing antibody-producing cells (e.g., lymph nodes), (c) Preparation of myeloma cells (hereinafter referred to as "myeloma") (for example, mouse myeloma SP2 / 0-ag14 cells), (d) Cell fusion between antibody-producing cells and myeloma, (e) Selection of hybridoma groups that produce the target antibody, (f) Divide into single-cell clones (cloning), (g) In some cases, culturing hybridomas for the mass production of monoclonal antibodies, or raising animals transplanted with hybridomas, (h) Examination of the physiological activity (internalization activity) and binding specificity of the monoclonal antibody produced in this manner, or testing of its properties as a labeling reagent. Examples of antibody titer measurement methods used here include, but are not limited to, flow cytometry or Cell-ELISA.

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

[0132] Furthermore, even if the monoclonal antibody is obtained separately and independently by repeating steps (a) to (h) of "Production of anti-CLDN6 antibody," or by obtaining the monoclonal antibody separately by other methods, it is possible to obtain an antibody with internalization activity equivalent to that of the B1 antibody or C7 antibody. An example of such an antibody is an antibody that binds to the same epitope as the B1 antibody or C7 antibody. If the newly produced monoclonal antibody binds to the partial peptide or partial three-dimensional structure to which the B1 antibody or C7 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the B1 antibody or C7 antibody. In addition, by confirming that the monoclonal antibody competes for the binding of the B1 antibody or C7 antibody to CLDN6 (i.e., the monoclonal antibody prevents the binding of the B1 antibody 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 epitope sequence or structure has not been determined. If the epitopes are confirmed to be identical, it is strongly expected that the monoclonal antibody will have antigen-binding ability, biological activity, and / or internalization activity equivalent to that of the B1 antibody or C7 antibody.

[0133] The antibodies of the present invention include not only monoclonal antibodies against CLDN6 as described above, but also genetically modified recombinant antibodies, such as chimeric antibodies, humanized antibodies, and human antibodies, which are artificially modified for purposes such as reducing heterologous antigenicity against humans. These antibodies can be manufactured using known methods. (1) Chimeric antibody Chimeric antibodies include antibodies in which the variable region and constant region are heterogeneous, such as chimeric antibodies in which the variable region of a mouse or rat-derived antibody is conjugated to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). The chimeric antibody derived from mouse anti-human CLDN6 antibody B1, as exemplified by the present invention, is an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 21 (Figure 19) and a light chain containing a light chain variable region shown in SEQ ID NO: 19 (Figure 18), and may have any human-derived constant region. 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"). The amino acid sequence of the chB1 antibody can be described as an antibody containing a heavy chain having an amino acid sequence consisting of amino acid residues 20 to 471 of sequence number 32 (Figure 24) in the sequence listing, and a light chain having an amino acid sequence consisting of amino acid residues 21 to 234 of sequence number 28 (Figure 22) in the sequence listing. In the heavy chain sequence shown in sequence number 32 (Figure 24) of the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 19 is the signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is the heavy chain variable region, and the amino acid sequence consisting of residues 142 to 471 is the heavy chain constant region. In addition, in the light chain sequence shown in sequence number 28 (Figure 22) of the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is the signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is the light chain variable region, and the amino acid sequence consisting of amino acid residues 128 to 234 is the light chain constant region. The amino acid sequences of the variable regions of the heavy and light chains of the chB1 antibody are listed in sequence number 34 (Figure 25) and sequence number 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 Sequence ID No. 33 (Figure 24) of the sequence listing. The nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in Sequence 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 Sequence ID No. 33 of the sequence listing encodes the variable region of the chB1 antibody heavy chain, and the nucleotide sequence consisting of nucleotides 424 to 1413 of the nucleotide sequence shown in Sequence ID No. 33 of the sequence listing encodes the constant region of the chB1 antibody heavy chain. The nucleotide sequence of the heavy chain variable region of the chB1 antibody is listed as sequence number 35 in the sequence listing (Figure 25). The light chain amino acid sequence of the chB1 antibody is encoded by the nucleotide sequence shown in sequence number 29 of the sequence listing (Figure 22). The nucleotide sequence consisting of nucleotides 26 to 85 of the nucleotide sequence shown in sequence number 29 of the sequence listing encodes the signal sequence of the chB1 antibody light chain, the nucleotide sequence consisting of nucleotides 86 to 406 of the nucleotide sequence shown in sequence number 29 of the sequence listing encodes the variable region of the chB1 antibody light chain, and the nucleotide sequence consisting of nucleotides 407 to 727 of the nucleotide sequence shown in sequence number 29 of the sequence listing encodes the constant region of the chB1 antibody light chain. The nucleotide sequence of the light chain variable region of the chB1 antibody is listed as sequence number 31 in the sequence listing (Figure 23). (2) Humanized antibodies Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) is incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies in which the CDR sequence and some amino acid residues of the framework are transplanted into a human antibody using the CDR transplantation method (WO90 / 07861), and antibodies in which the amino acid sequence of some CDRs is modified while maintaining the ability to bind to the antigen. However, as a humanized antibody derived from a B1 antibody or C1 antibody, it is not limited to a specific humanized antibody, as long as it retains all six CDR sequences of the B1 antibody or C1 antibody and has CLDN6 binding activity. Furthermore, humanized antibody variants in which the amino acid sequence of one to several (preferably one to two, more preferably one) CDRs has been modified are also not limited to a specific humanized antibody, as long as they recognize the CLDN6 protein or have CLDN6 protein binding activity. Examples of the anti-CLDN6 humanized antibody or functional fragment thereof of the present invention include: CDRH1 consists of the amino acid sequence shown in sequence number 9 of the sequence listing (Figure 15) or an amino acid sequence in which one to several amino acids (preferably one to two) of the amino acid sequence are substituted. CDRH2 consists of the amino acid sequence shown in sequence number 10 of the sequence listing (Figure 15) or an amino acid sequence in which one to several amino acids (preferably one to two) of said amino acid sequence are substituted, A heavy chain having a variable region containing CDRH3, which consists of the amino acid sequence shown in sequence number 11 of the sequence listing (Figure 15) or an amino acid sequence in which one to several amino acids (preferably one to two) of said amino acid sequence are substituted, and CDRL1 consists of the amino acid sequence shown in sequence number 5 of the sequence listing (Figure 13) or an amino acid sequence in which one to several amino acids (preferably one to two) of the said amino acid sequence are substituted. CDRL2 consists of the amino acid sequence shown in sequence number 6 of the sequence listing (Figure 13) or an amino acid sequence in which one to several amino acids (preferably one to two) of the amino acid sequence are substituted, The light chain includes a variable region containing CDRL3, which consists of the amino acid sequence shown in sequence number 7 of the sequence listing (Figure 13) or an amino acid sequence in which one to several amino acids (preferably one to two) of said amino acid sequence are substituted. Examples of the present invention include antibodies that recognize the CLDN6 protein or that retain the CLDN6 protein-binding activity of said antibody, or functional fragments of said antibody. Examples of amino acid substitutions of CDR in the above-mentioned anti-CLDN6 humanized antibody or its functional fragment include, preferably, one to several (preferably one to two) amino acid substitutions of CDRL3. An example is the CDRL3 shown in sequence number 8 of the sequence listing (Figure 14), in which amino acids 4 and 5 of sequence number 7 of the sequence listing are substituted.

[0134] Examples of the heavy chain variable regions of the humanized antibody having the above-mentioned CDRH include the amino acid sequences shown in sequence number 54 (Figure 35), sequence number 58 (Figure 37), and sequence number 62 (Figure 39) of the sequence listing. Examples of the light chain variable regions of the humanized antibody having the above-mentioned CDRL include the amino acid sequences shown in sequence number 38 (Figure 27), sequence number 42 (Figure 29), sequence number 46 (Figure 31), and sequence number 50 (Figure 33) of the sequence listing.

[0135] As a humanized antibody containing the above combination of heavy chain variable region and light chain variable region, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in sequence number 54 (Figure 35) of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in sequence number 38 (Figure 27) of the sequence listing, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in sequence number 58 (Figure 37) of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in sequence number 42 (Figure 29) of the sequence listing, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in sequence number 54 (Figure 35) of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in sequence number 46 (Figure 31) of the sequence listing, A humanized antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in sequence number 58 (Figure 37) of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in sequence number 50 (Figure 33) of the sequence listing, A suitable example of a humanized antibody is one that includes a heavy chain variable region consisting of the amino acid sequence shown in sequence number 62 (Figure 39) of the sequence listing, and a light chain variable region consisting of the amino acid sequence shown in sequence number 46 (Figure 31) of the sequence listing.

[0136] The full-length sequence of a humanized antibody comprising the above 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 at amino acid numbers 20-471 of sequence number 52 (Figure 34) in the sequence listing, and a light chain consisting of the amino acid sequence shown at amino acid numbers 21-234 of sequence number 36 (Figure 26) in the sequence listing, A humanized antibody (H2L2) comprising a heavy chain consisting of the amino acid sequence shown at amino acid numbers 20-471 of sequence number 56 (Figure 36) in the sequence listing, and a light chain consisting of the amino acid sequence shown at amino acid numbers 21-234 of sequence number 40 (Figure 28) in the sequence listing, A humanized antibody (H1L3) comprising a heavy chain consisting of the amino acid sequence shown at amino acid numbers 20-471 of sequence number 52 (Figure 34) in the sequence listing, and a light chain consisting of the amino acid sequence shown at amino acid numbers 21-234 of sequence number 44 (Figure 30) in the sequence listing, A humanized antibody (H2L4) comprising a heavy chain consisting of the amino acid sequence shown at amino acid numbers 20-471 of sequence number 56 (Figure 36) in the sequence listing, and a light chain consisting of the amino acid sequence shown at amino acid numbers 21-234 of sequence number 48 (Figure 32) in the sequence listing, or An example of a humanized antibody (H3L3) is one that includes a heavy chain consisting of the amino acid sequence shown at amino acid numbers 20-471 of sequence number 60 (Figure 38) in the sequence listing, and a light chain consisting of the amino acid sequence shown at amino acid numbers 21-234 of sequence number 44 (Figure 30) in the sequence listing. In addition, in the heavy chain amino acid sequences shown in sequence number 52 (Figure 34), 56 (Figure 36), or 60 (Figure 38) of the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 19 is the signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is the heavy chain variable region, and the amino acid sequence consisting of amino acid residues 142 to 471 is the heavy chain constant region. Furthermore, in the light chain amino acid sequences shown in sequence numbers 36 (Figure 26), 40 (Figure 28), 44 (Figure 30), or 48 (Figure 32) of the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is the signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is the light chain variable region, and the amino acid sequence consisting of amino acid residues 128 to 234 is the light chain constant region.

[0137] The base sequence encoding the heavy chain amino acid sequence of the above humanized antibody H1L1 is SEQ ID NO: 53 (Figure 34), and the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 37 (Figure 26). The base sequence encoding the heavy chain amino acid sequence of the humanized antibody H2L2 is SEQ ID NO: 57 (Figure 36), and 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), and 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), and the base sequence encoding the light chain amino acid sequence is SEQ ID NO: 49 (Figure 32). The polynucleotides encoding the heavy chain amino acid sequence of the humanized antibody H3L3 are shown in SEQ ID NO: 61 (Figure 38), and the polynucleotides encoding the light chain amino acid sequence are shown in SEQ ID NO: 45 (Figure 30). The base sequence encoding the amino acid sequence of the heavy chain variable region of the above humanized antibody H1L1 is SEQ ID NO: 55 (Figure 35), and the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 39 (Figure 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 (Figure 37), and the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 43 (Figure 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), and 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), and the base sequence encoding the amino acid sequence of the light chain variable region is SEQ ID NO: 51 (Figure 33). The polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the humanized antibody H3L3 is shown as SEQ ID NO: 63 (Figure 39), and the polynucleotide sequence encoding the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 47 (Figure 31). Furthermore, the nucleotide sequences consisting of nucleotides 1 to 57 of each nucleotide sequence shown in sequence numbers 53 (Figure 34), 57 (Figure 36), and 61 (Figure 38) of the sequence listing encode the signal sequence of the humanized antibody heavy chain, the nucleotide sequences consisting of nucleotides 58 to 423 encode the amino acid sequence of the variable region of the humanized antibody heavy chain, and the nucleotide sequences consisting of nucleotides from nucleotides 424 to 1413 encode the constant region of the antibody heavy chain. Furthermore, the nucleotide sequences consisting of nucleotides 1 to 60 of each nucleotide sequence shown in sequence numbers 37 (Figure 26), 41 (Figure 28), 45 (Figure 30), and 49 (Figure 32) of the sequence listing encode the signal sequence of the humanized antibody light chain, the nucleotide sequences consisting of nucleotides 61 to 381 encode the amino acid sequence of the variable region of the humanized antibody light chain, and the nucleotide sequences consisting of nucleotides 382 to 702 encode the constant region of the antibody light chain.

[0138] Antibodies comprising the above-mentioned combination of heavy chain variable region and light chain variable region, 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 above-mentioned combination of heavy chain and light chain, are also included in the antibodies of the present invention, as long as they have binding activity to CLDN6. Furthermore, antibodies comprising the above-mentioned combination of heavy chain variable region and light chain variable region, or antibodies having a CDR with the same amino acid sequence as the CDR of an antibody comprising the above-mentioned combination of heavy chain and light chain, and wherein the identity or homology of the amino acid sequence excluding the amino acid sequence of the antibody's CDR is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 99% or more, are also included in the antibodies of the present invention, as long as they have binding activity to CLDN6. Furthermore, 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, it is possible to select antibodies with biological activity equivalent to that of the antibodies described above. In this specification, conservative amino acid substitutions are preferred (WO2013154206). Conservative amino acid substitutions are substitutions that occur within a group of amino acids related to the amino acid side chain. Such amino acid substitutions should preferably be performed within a range that does not degrade the properties of the substance having the original amino acid sequence. The homology between two amino acid sequences can be determined by 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 via the internet at www.ncbi.nlm.nih.gov / blast. (3) Human antibodies The antibodies of the present invention can further include human antibodies that bind to CLDN6 and / or CLDN9. Anti-CLDN6 and / or CLDN9 human antibodies refer to human antibodies that have only the gene sequence of an antibody derived from a human chromosome. Anti-CLDN6 human antibodies can be obtained by a method using human antibody-producing mice that have human chromosome fragments containing the genes for the heavy and light chains of human antibodies (see Nature Genetics (1997) 16, pp. 133-143; Nucl. Acids Res. (1998) 26, pp. 3447-3448; Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73, Kluwer Academic Publishers, 1999; Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.). Specifically, these human antibody-producing mice can be created as genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci are disrupted and replaced with human immunoglobulin heavy and light chain gene loci via a yeast artificial chromosome (YAC) vector or the like. These can be produced by creating knockout animals and transgenic animals, and then crossbreeding these animals. Furthermore, by using genetic engineering technology to transform eukaryotic cells with cDNA encoding the heavy and light chains of such human antibodies, preferably a vector containing the cDNA, and culturing the transformed cells that produce the recombinant human monoclonal antibody, this antibody can also be obtained from the culture supernatant. Here, as the host, for example, eukaryotic cells, preferably CHO cells, or mammalian cells such as lymphocytes or myeloma can be used. Furthermore, methods for obtaining human antibodies derived from phage displays selected from human antibody libraries are also known (see 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, etc.). For example, a phage display method can be used to select phages that bind to an antigen by expressing the variable region of a human antibody as a single-chain antibody (scFv) on the phage surface (Nature Biotechnology (2005), 23, (9), pp. 1105-1116). By analyzing the genes of phages selected through antigen binding, it is possible to determine the DNA sequence encoding the variable region of human antibodies that bind to the antigen. Once the DNA sequence of the scFv that binds to the antigen is identified, an expression vector containing that sequence can be constructed and introduced into a suitable host to express it, thereby obtaining human antibodies (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu.Rev.Immunol(1994)12,p.433-455, Nature Biotechnology(2005)23(9),p.1105-1116).

[0139] The chimeric antibodies, humanized antibodies, or human antibodies obtained by the above methods can be evaluated for their binding affinity to antigens using known methods, and suitable antibodies can be selected. Another example of an indicator used when comparing the properties of antibodies is their stability. Differential scanning calorimetry (DSC) is a device that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. By measuring Tm values ​​using DSC and comparing these values, differences in thermal stability can be compared. It is known that the storage stability of antibodies shows a certain correlation with their thermal stability. (Pharmaceutical Development and Technology (2007) 12, pp. 265-273), suitable antibodies can be selected using thermal stability as an indicator. Other indicators for antibody selection include high yield in appropriate host cells and low aggregation in aqueous solution. For example, the 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 indicators described above to select the antibody most suitable for administration to humans.

[0140] The antibodies of the present invention also include 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 antibodies of the present invention also include modified antibodies. Such modified antibodies are those that have undergone chemical or biological modifications. Chemical modifications include the attachment of chemical moieties to the amino acid backbone, and chemical modifications of N-linked or O-linked carbohydrate chains. Biological modifications include those that have undergone post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation), and those that have had a methionine residue added to the N-terminus by expression using prokaryotic host cells. Furthermore, modified antibodies that have been labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescently labeled, or affinity-labeled antibodies, are also included in the meaning of such modified antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and for detection or isolation of antibodies or antigens. Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the glycosylation (glycosylation, defucoseation, etc.) of the antibodies of the present invention. Known techniques for regulating antibody glycosylation include WO1999 / 54342, WO2000 / 61739, and WO2002 / 31140, but are not limited to these. The antibodies of the present invention also include antibodies in which such glycosylation has been regulated. Such modifications may be made at any position on the antibody or its functional fragment, or at any desired position, and one or more identical or two or more different modifications may be made at one or more positions. In this invention, the term "modified antibody fragment" also includes "fragment of a modified antibody."

[0142] When antibody genes are isolated and then introduced into a suitable host to produce antibodies, a suitable host and expression vector combination can be used. Specific examples of antibody genes include combinations of genes encoding the heavy chain sequence and light chain sequence of the antibody described herein. When transforming host cells, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or they can be inserted into separate expression vectors. When using eukaryotic cells as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, animal cells include mammalian cells, such as monkey cells (Cell (1981) 23, pp. 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, pp. 4126-4220), and FreeStyle 293F cells (Invitrogen). Examples of prokaryotic cells that can be used include E. coli and Bacillus subtilis. Antibodies can be obtained by introducing the target antibody gene into these cells by transformation and culturing the transformed cells in vitro. In this culture, the yield may differ depending on the antibody sequence, and it is possible to select antibodies that are easy to produce as pharmaceuticals from among antibodies with equivalent binding activity, using yield as an indicator. Therefore, the antibodies of the present invention also include antibodies obtained by a method for producing antibodies, which is characterized by including the steps of culturing the transformed host cells and collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step.

[0143] The antibody gene described above is preferably a polynucleotide containing one of the polynucleotides described in any one of (a) to (e) below. (a) A combination of a polynucleotide encoding the heavy chain amino acid sequence and a polynucleotide encoding the light chain amino acid sequence of one of the following humanized antibodies: B1 or C7 antibody, chB1 antibody, and one of the humanized antibodies H1L1, H2L2, H1L3, H2L4, or H3L3. (b) A combination of polynucleotides encoding the heavy chain amino acid sequence containing CDRH1-CDRH3 and the light chain amino acid sequence containing CDRL1-CDRL3 of one of the following antibodies: B1 or C7 antibody, chB1 antibody, and humanized antibody H1L1, H2L2, H1L3, H2L4, or H3L3. (c) A combination of a polynucleotide encoding a heavy chain amino acid sequence containing the amino acid sequence of the heavy chain variable region of a B1 or C7 antibody, a chB1 antibody, and one of the humanized antibodies H1L1, H2L2, H1L3, H2L4, or H3L3, and a polynucleotide encoding a light chain amino acid sequence containing the amino acid sequence of the light chain variable region. (d) A polynucleotide that hybridizes under stringent conditions with a polynucleotide complementary to any one of (a) to (c), and a polynucleotide encoding the amino acid sequence of an antibody that binds to CDLN6, and (e) A polynucleotide that codes for the amino acid sequence of a polypeptide, wherein 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 is substituted, deleted, added, or inserted in any one of the polynucleotides described in (a) to (c), and also codes for the amino acid sequence of an antibody that binds to CLDN6. The present invention includes nucleotides encoding the antibody of the present invention, a functional fragment thereof, or a modified version thereof, a recombinant vector into which the gene is inserted, and cells into which the gene or the vector is introduced. The present invention also includes a method for producing an antibody, a functional fragment thereof, or a modified thereof, comprising the steps of culturing the cells and recovering the antibody, a functional fragment thereof, or a modified thereof from the culture.

[0144] It is known that antibodies produced in mammalian cultured cells lose a lysine residue at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705:129-134 (1995)), and that two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain are also lost, with a proline residue newly located at the carboxyl terminus being amidated (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector function (such as complement activation or antibody-dependent cytotoxicity) of the antibody. Therefore, the antibody according to the present invention includes antibodies that have undergone such modifications and functional fragments of such antibodies, as well as deletions in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and amidated deletions (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody according to the present invention are not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be one of the full-length heavy chains and heavy chains selected from the group consisting of the above-mentioned deletions, or a combination of either two types. The quantity ratio of each deletion may be affected by the type of mammalian cultured cell that produces the antibody according to the present invention and the culture conditions, but a case in which one amino acid residue at the carboxyl terminal is deleted in both heavy chains can be cited as the main component of the antibody according to the present invention.

[0145] Examples of isotypes for the anti-CLDN6 antibody of the present invention include IgG (IgG1, IgG2, IgG3, IgG4), but IgG1, IgG2, or IgG4 are preferred. When IgG1 is used as the isotype for the antibody of the present invention, the effector function can be adjusted by substituting some 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), with IgG1 LALA being preferred. L234A and L235A represent substitutions of leucine to alanine at positions 234 and 235, respectively, as identified by the EU index (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85), and G237A represents a substitution of glycine to alanine at position 237, as identified by the EU index.

[0146] The biological activities of antibodies generally include antigen-binding activity, activity that internalizes an antigen into cells expressing that antigen by binding to it, activity that neutralizes the activity of an antigen, activity that enhances the activity of an antigen, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cell-mediated cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP). However, the function of the antibody according to the present invention is binding activity to CLDN6, and preferably, activity that internalizes an antigen into CLDN6-expressing cells by binding to CLDN6. Furthermore, the antibody according to the present invention may also possess ADCC activity, CDC activity, and / or ADCP activity in addition to intracellular internalization activity.

[0147] The obtained antibodies can be purified to a uniform degree. Antibodies can be separated and purified using the same separation and purification methods used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, and isoelectric focusing (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 are not limited to these methods. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. These chromatography processes can be performed using liquid chromatography such as HPLC or FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. Furthermore, it is possible to purify antibodies by utilizing their binding properties to antigens using a carrier on which the antigen is immobilized.

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

[0149] <Glycan remodeling> In recent years, methods have been reported for remodeling heterogeneous antibody glycoproteins using enzymatic reactions to uniformly introduce functional sugar chains (ACS Chemical Biology 2012, 7, 110, ACS Medicinal Chemistry Letters 2016, 7, 1005). Attempts have also been made to synthesize homogeneous ADCs by introducing drugs site-specifically using this sugar chain remodeling technology (Bioconjugate Chemistry 2015, 26, 2233, Angew.Chem.Int.Ed. 2016,55,2361-2367, US2016361436).

[0150] The present invention's glycan remodeling method first uses a hydrolytic enzyme to excise heterogeneous glycans attached to a protein (such as an antibody), leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereinafter referred to as the "acceptor"). Next, a separately prepared glycan of any desired type is prepared (hereinafter referred to as the "donor"), and this acceptor and donor are linked using a glycosyltransferase. This allows for the synthesis of a homogeneous glycoprotein with any desired glycan structure.

[0151] In this invention, "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-" and "MSG-". When these abbreviations are used in a structural formula, oxygen or nitrogen atoms belonging to glycosidic bonds with other structural units at the reducing end are not included in the abbreviation representing the glycan, unless otherwise specified.

[0152] In this invention, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described, for convenience, with the carbon atom bonded to the oxygen atom constituting the ring and directly bonded to the hydroxyl group (or oxygen atom belonging to the glycosidic bond) designated as position 1 (position 2 only in the case of sialic acid). The names of the example compounds are given based on the overall chemical structure, and this rule does not necessarily apply.

[0153] In the present invention, when a sugar chain is represented by a symbol (e.g., GLY, SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol shall include the carbon atom at the reducing end, and the N or O atoms belonging to the N- or O-glycosidic bond shall not be included in the symbol.

[0154] In the present invention, unless otherwise specified, the substructure of an amino acid linked to a sugar chain in its side chain shall be indicated by enclosing the side chain portion in parentheses, for example, as "(SG-)Asn".

[0155] The antibody-drug conjugate of the present invention is as follows: [ka] As shown, the antibody Ab or its functional fragment may be bound to L directly from the side chain of its amino acid residue (e.g., cysteine, lysine, etc.) or to L from the glycans of Ab or a remodeled glycan, preferably to L from the glycans of Ab or a remodeled glycan, and more preferably to L from the remodeled glycans of Ab.

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

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

[0158] IgG possesses a well-conserved N-linked glycan at the 297th asparagine residue in the Fc region of its heavy chain (hereinafter referred to as "Asn297 or N297"), 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 in Edelman et al. (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85), each amino acid is identified by its Eu number (Eu INDEX). For example, Asn297, to which an N-linked glycan is attached in the Fc region, corresponds to position 297 in the Eu number. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by representing it with its Eu number.

[0160] In the antibody-drug conjugate of the present invention, more preferably, the antibody or its functional fragment is bound to L from a glycan attached to the side chain of Asn297 (hereinafter referred to as "N297 glycan"), and even more preferably, the antibody or its functional fragment is bound to L from the N297 glycan, and the N297 glycan is a remodeled glycan. The following formula shows the case in which the antibody-drug conjugate of the present invention is bound to L from the N297 glycan of the antibody or its functional fragment. [ka] Antibodies that possess such remodeled glycans are called glycan remodeling antibodies.

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

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

[0163] The remodeled sugar chain 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 that the antibody is bound to Asn297. L(PEG) is -(CH2CH2-O)n 5 It shows -CH2CH2-NH-, indicating that the rightmost amino group is amide-bonded to the carboxylic acid at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, in particular linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 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 that the antibody is bound to Asn297. L(PEG) is -(CH2CH2-O)n 5 It shows -CH2CH2-NH-, indicating that the rightmost amino group is amide-bonded to the carboxylic acid at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-6 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, in particular linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 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 that the antibody is bound to Asn297. L(PEG) is -(CH2CH2-O)n 5The -CH2CH2-NH- symbol indicates that the rightmost amino group is amide-bonded to the carboxylic acid at position 2 of the sialic acid at both the 1-3 and 1-6 non-reducing ends of the β-Man branched chain of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring of linker L, particularly Lb in linker L. Here, n 5 is an integer between 2 and 10, preferably between 2 and 5.

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

[0168] In the antibody-drug conjugate of the present invention, if the N297 glycan of the antibody is N297-(Fuc)SG, then since the antibody is a dimer, the antibody-drug conjugate is a molecule to which four linkers L and four drug D are bound (as described above m 2 =2). For example, Example 72: ADC6 is the case where the N297 glycan is N297-(Fuc)SG.

[0169] The N297 glycan is preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, and more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2. In the antibody-drug conjugate of the present invention, if the N297 glycan of the antibody is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, a uniform 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) A step of culturing a host cell (e.g., an animal cell (CHO cell, etc.)) described in any one of the above

[46] to

[48] , and collecting the target antibody from the culture obtained, ii) A step to produce an antibody ((Fucα1,6)GlcNAc-antibody) in which the N297 glycan is (Fucα1,6)GlcNAc by treating the antibody obtained in step i) with a hydrolase (Figure 3A), Preferably, the reaction solution is further purified by a step including a hydroxyapatite column to purify the (Fucα1,6)GlcNAc antibody, and, iii)-1 or iii)-2, either one of the steps (Figure 3B). iii)-1 A step to synthesize a glycosylation remodeling antibody in which an azide group has been introduced to sialic acid by introducing a PEG linker (N3-L(PEG)) having an azide group at the carbonyl group of the carboxylic acid at position 2 of MSG(9) or SG(10), and reacting a glycosylation donor molecule with an oxazoline-treated reducing end with a (Fucα1,6)GlcNAc- antibody in the presence of a glycosyltransferase. iii)-2 A step to synthesize a glycan remodeling antibody in which an azide group is introduced into a PEG linker (N3-L(PEG)) having an azide group on the carbonyl group of the carboxylic acid at position 2 of the sialic acid of (MSG-)Asn or (SG-)Asn, which may have a protected or modified α-amino group, and after reacting with a hydrolase, the reducing end of the glycan donor molecule is oxazolinened, and the PEG linker is reacted with a (Fucα1,6)GlcNAc- antibody in the presence of a glycosyltransferase. Furthermore, glycosylation remodeling antibodies or functional fragments thereof obtained by such manufacturing methods, or modified versions thereof, are also included in the present invention.

[0171] The manufacturing intermediate for the antibody-drug conjugate described above 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 manufacturing intermediate with an MSG1, MSG2, or SG type glycan remodeling antibody or a functional fragment of the antibody, in which a PEG linker having an azide group is introduced to the sialic acid of the glycan obtained in steps i) to iii) above.

[0172] In the N297 glycan of the present invention, the fucose-added GlcNAc(Fucα1,6)GlcNAc) at the reducing end is preferably derived from an antibody produced in animal cells, and the glycan on the non-reducing end is preferably remodeled to have a glycan structure similar to that of the MSG(MSG1, MSG2) or SG described above. In both cases, the carboxylic acid bonded at the 2-position of the sialic acid at the non-reducing end is used to bind to L(PEG). Such glycan remodeling antibodies having MSG (MSG1, MSG2) or SG-type N297 glycans can be manufactured by the method shown in Figure 3, for example, according to the method described in WO2013 / 120066. When antibodies are produced as recombinant proteins using animal cells as a host according to known methods (step i above), the N297 glycan has a fucose-linked N-linked glycan structure as its basic structure, but is obtained as a mixture of antibodies or fragments of glycans having various structures with diverse modifications to the non-reducing end structure and constituent sugars (IV in Figure 3A). Antibodies produced in animal cells in this manner can be treated with hydrolytic enzymes such as EndoS, which hydrolyzes the glycosidic bond between GlcNAcβ1-4GlcNAc in the chitobiose structure at the reducing end, yielding an antibody molecule with a single glycan structure containing only (Fucα1,6)GlcNAc as the N297 glycan (referred to as "(Fucα1,6)GlcNAc-antibody," see Figure 2A) (Figure 3A) (step ii above).

[0173] As the enzyme used in the hydrolysis reaction of the N297 glycan, EndoS or a mutant enzyme that retains its hydrolytic activity can be used.

[0174] By using the (Fucα1,6)GlcNAc-antibody obtained by the above hydrolysis reaction as a glycan acceptor molecule and reacting it with MSG (MSG1, MSG2) or SG-type glycan donor molecules using a glycosyltransferase such as EndoS D233Q or EndoS D233Q / Q303L mutant (e.g., WO2017010559), an antibody having MSG (MSG1, MSG2) or SG-type N297 glycan with the above structure (see Figure 2B) (Figure 3B) (steps iii)-1, iii)-2).

[0175] Number of drug conjugates per antibody molecule in an antibody-drug conjugate (m) 2 If the value is 1, a glycan donor molecule having MSG, MSG1, or MSG2 as the glycan is used. Such glycans can be obtained by separating (MSG-)Asn1 or (MSG2-)Asn from commercially available monosialo-Asn free (1S2G / 1G2S-10NC-Asn, Glycotechnology Research Institute Co., Ltd., hereinafter referred to as "(MSG-)Asn") as a raw material according to the method described in Example 56, or they can be used as a mixture without separation.

[0176] Number of drug conjugates per antibody molecule in an antibody-drug conjugate (m) 2 If the value is 2, a glycosylation donor molecule having SG(10) as the sugar chain is used in this glycosylation reaction. Such an SG(10) sugar chain may be obtained, for example, from SGP by hydrolysis, or a commercially available SG(10) sugar chain such as disialooctasaccharide (Tokyo Chemical Industries, Ltd.) may be used.

[0177] The MSG (MSG1, MSG2) or SG-type sugar chain contained in the donor molecule has a PEG linker (N3-L(PEG)) with an azide group at the 2-position of its sialic acid. To introduce the PEG linker (N3-L(PEG)) with an azide group at the 2-position of the sialic acid, known reactions (condensation reactions, etc.) in the field of organic synthesis can be used with MSG (MSG(9)), MSG1 or MSG2, or disialooctasaccharide (SG(10)) and the PEG linker (N3-L(PEG)) with an azide group, namely N3-(CH2CH2-O)n5-CH2CH2-NH2 (where n5 is an integer from 2 to 10, preferably an integer from 2 to 5). That is, the carboxylic acid at the 2-position of the sialic acid and the amino group at the right end of N3-(CH2CH2-O)n5-CH2CH2-NH2 form an amide bond through a condensation reaction.

[0178] Furthermore, MSG (MSG1, MSG2) or SG-type glycans can also be obtained by introducing a PEG linker having an azide group (N3-(CH2CH2-O)n5-CH2CH2-NH2) into the carboxylic acid at the 2-position of the sialic acid of a raw material such as (MSG1-)Asn, (MSG2-)Asn, or (SG-)Asn (Glycotechnology Institute), which may have a protected or modified α-amino group, using a condensation reaction, and then reacting it with a hydrolytic enzyme such as EndoM or EndoRp (iii)-2 above). Examples of α-amino group protecting groups include, but are not limited to, acetyl (Ac) group, t-Butoxycarbonyl (Boc) group, benzoyl (Bz) group, benzyl (Bzl) group, Carbobenzoxy (Cbz) group, and 9-Fluorenylmethoxycarbonyl (Fmoc) group. The α-amino group protecting group is preferably an Fmoc group. Examples of modifying groups for the α-amino group include hydroxyacetyl groups or PEG structures that improve water solubility. (MSG1-)Asn, (MSG-2)Asn, or (SG-)Asn preferably have an α-amino group protected by the protecting group. If the α-amino group is protected by a protecting group (e.g., an Fmoc group), the protecting group can be removed as needed after introducing a PEG linker having an azide group and before treating with a hydrolase.

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

[0180] Various enzymes (glycosyltransferases) can be used in the glycosyltransfer reaction as long as they have the activity to transfer complex glycans to the N297 glycan. However, the preferred enzyme is EndoS D233Q, a modified version of EndoS in which the hydrolysis reaction is suppressed by substituting Asp at position 233 with Gln. Glycosyltransfer reactions using EndoS D233Q are described in WO2013 / 120066, etc. Alternatively, modified enzymes such as EndoS D233Q / Q303L (WO2017010559), which are further mutated versions of EndoS D233Q, may also be used.

[0181] The purification of antibodies after antibody glycosylation (glycosylation and glycosylation reactions) aims to separate them from the low-molecular-weight compounds and enzymes used in the reaction. Such purification is usually performed using gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc., but additional purification using a hydroxyapatite column may also be performed. In other words, the present invention provides a method for producing a drug-conjugate that includes a further purification step using a hydroxyapatite column in the purification step of the intermediate from the reaction solution after glycosylation of the antibody. According to reports on glycosylation (J. Am. Chem. Soc. 2012, 134, 12308-12318., Angew. Chem. Int. Ed. 2016, 55, 2361-2367), the reaction mixture treated with an antibody hydrolase is purified using a Protein A column (affinity chromatography column). However, this purification method does not completely remove the hydrolase (EndoS, etc.), and residual enzymes can affect the subsequent glycosylation reaction. Therefore, after investigating purification methods, we found that purifying the reaction mixture treated with an antibody hydrolase using a Protein A column followed by a hydroxyapatite column (CHT column, Bio-Rad Laboratories, Inc.) improved the reaction efficiency of the subsequent glycosylation reaction without the influence of residual enzymes.

[0182] The antibody-drug conjugate of the present invention is most preferably one 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 This shows that 1), Antibody Ab is an IgG antibody (preferably IgG1, IgG2, IgG4, more preferably IgG1) or a functional fragment thereof. The N297 glycan is one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, a mixture thereof, or N297-(Fuc)SG (preferably N297-(Fuc)MSG1). L(PEG) indicates -NH-CH2CH2-(O-CH2CH2)3-*, where the leftmost amino group is amide-bonded to the carboxylic acid at position 2 of the non-reducing sialic acid on the 1-3 and / or 1-6 side (preferably the 1-3 side) of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the triazole ring of Lb in the linker L. For convenience, as the most preferred antibody-drug conjugate above, one molecule of the conjugate contains 2 or 4 "-(N297 glycan)-LD" ("(N297 glycan)-(N1Lb)LD") in which the N297 glycan is bonded to the nitrogen atom at position 1 on the triazole ring of Lb in L.2 =1 or 2) or has 2 or 4 "-(N297 sugar chain)-LD" ("(N297 sugar chain)-(N3Lb)LD") bonded to the nitrogen atom at position 3 (m 2 =1 or 2) The structure is described, but one molecule of the conjugate contains '(N297 sugar chain)- (N1Lb)LD' (m 2 If = 1, then 1 item, m 2 If =2, then 1, 2, 3 items) and '(N297 sugar chain)- (N3Lb)LD' (m 2 If = 1, then 1 item, m 2 In the case of =2, this also includes antibody-drug conjugates that have both (3, 2, 1). That is, one molecule of the conjugate may contain only one of either '(N297 glycan)-(N1Lb)LD' or '(N297 glycan)-(N3Lb)LD', or both.

[0183] Furthermore, Ab is preferably an anti-CLDN6 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-DLL3 antibody, anti-FAP antibody, anti-CDH11 antibody, anti-A33 antibody, anti-CanAg antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD98 antibody, anti-TROP2 antibody, anti-CEA antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-FGFR2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-In These are tegrin antibody, PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody, anti-EGFR antibody, and anti-DR5 antibody; more preferably, anti-CLDN6 antibody, anti-CLDN6 / CLDN9 antibody, anti-HER2 antibody, anti-CD98 antibody, and anti-TROP2 antibody; even more preferably, the anti-CLDN6 antibody (e.g., Examples 106, 107, 108, 109) and anti-HER2 antibody (e.g., Trastuzumab, Trastuzumab variant).

[0184] The antibody-drug conjugates of the present invention and the anti-CLDN6 antibody or anti-HER2 antibody-drug conjugates of the present invention exhibit strong tumor activity (in vivo antitumor activity, in vitro anti-cellular activity), good pharmacokinetics and physical properties, and high safety, making them useful as pharmaceuticals.

[0185] The antibody-drug conjugates, free drugs, or their manufacturing intermediates of the present invention may contain stereoisomers or optical isomers derived from an asymmetric carbon atom, such as geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers. However, all of these isomers, optical isomers, and mixtures thereof are included in the present invention. The PBD derivatives (V) or (VI) of the present invention have an asymmetric carbon at the 11' position, and therefore optical isomers exist. In this specification, all of these isomers and mixtures thereof are represented by a single formula, i.e., the general formula (V) or (VI). Accordingly, (V) or (VI) includes all optical isomers and mixtures of optical isomers in any proportion. The absolute stereochemistry at the 11' position of (V) or (VI) can be determined by X-ray crystallography or NMR such as the Mosher method of the crystalline product or intermediate or its derivatives. In this case, the absolute stereochemistry may be determined using a crystalline product or intermediate derivatized with a reagent having an asymmetric center whose stereochemistry is known. Stereoisomers can be obtained by isolating the synthesized compound according to the present invention 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 affecting its efficacy and safety. The production of antibody-drug conjugates is carried out by defining reaction conditions, such as the amount of raw materials and reagents used, so that the number of drugs bound is constant. However, unlike the chemical reaction of low-molecular-weight compounds, it is usually obtained as a mixture with different numbers of drugs bound. The number of drugs bound to one antibody molecule can be specified as an average value, i.e., the average drug-to-antibody ratio (DAR). The number of pyrrolobenzodiazepine derivatives bound to antibody molecules is controllable, and it is possible to bind pyrrolobenzodiazepine derivatives in the range of 1 to 10 as the average drug-to-antibody ratio (DAR) per antibody, 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 from the remodeled sugar chain of the antibody, the number of drug conjugates per antibody molecule in the antibody-drug conjugate is m. 2 m is an integer of 1 or 2. If the sugar chain is an N297 sugar chain, and the sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, then m 2 The value is 1, and the 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 glycan is N297-(Fuc)SG, m 2 The value of is 2, and the 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, those skilled in the art can design a reaction to conjugate the required number of drugs to an antibody based on the descriptions in the examples of this application, and obtain an antibody with controlled conjugation of pyrrolobenzodiazepine derivatives.

[0187] Furthermore, the antibody-drug conjugates, free drugs, or manufacturing intermediates of the present invention may absorb moisture when left in the air or recrystallized, resulting in adsorbed water or the formation of hydrates. Compounds and salts containing such water are also included in the present invention.

[0188] If the antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention has a basic group such as an amino group, it can optionally be made into a pharmaceutically acceptable salt. Examples of such salts include hydrohalides such as hydrochloride and hydroiodide; inorganic salts such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfons such as methanesulfonates, trifluoromethanesulfons, and ethanesulfons; allylsulfons such as benzenesulfons and p-toluenesulfons; organic salts such as formic acid, acetic acid, malic acid, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0189] When the antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention has an acidic group such as a carboxyl group, it is generally possible to form a base addition salt. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; and organic amine salts such as 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.

[0190] The antibody-drug conjugate, free drug, or manufacturing intermediate of the present invention may exist as a hydrate by absorbing moisture from the air, etc. The solvate of the present invention is not particularly limited as long as it is pharmaceutically acceptable, but specifically, hydrates, ethanol hydrates, 2-propanol hydrates, etc. are preferred. Furthermore, if a nitrogen atom is present in the antibody-drug conjugate, free drug, or manufacturing 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] Furthermore, the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. The antibody-drug conjugates, free drugs, or manufacturing intermediates of the present invention may also contain non-natural proportions of atomic isotopes in one or more of the atoms constituting them. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), Iodine-125( 125 I) or carbon-14 ( 14 Examples include C). Furthermore, the compounds of the present invention include, for example, tritium ( 3 H), Iodine-125( 125 I) or carbon-14 ( 14 The compounds can be radiolabeled with radioisotopes 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 agents. All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0192] [Manufacturing method] Next, a typical method for producing the antibody-drug conjugate and free drug or their intermediates according to the present invention will be described. In the following, the compound numbers shown in each reaction formula 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 similarly.

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

[0194] Each step of the following methods A through Q can be used to carry out the desired reaction using known organic chemical techniques. The solvent used in each step of the reaction described below, from Method A to Method Q, is not particularly limited as long as it does not inhibit the reaction, does not adversely affect the reaction, and dissolves the starting materials to some extent. In the reactions of each step of the following methods A through Q, the reaction temperature will vary depending on the solvent, starting materials, reagents, etc., and the reaction time will also vary depending on the solvent, starting materials, reagents, reaction temperature, etc. In the reactions of each step of Method A to Q described below, after the reaction is complete, each target compound is collected from the reaction mixture according to conventional methods. For example, the reaction mixture is neutralized as appropriate, and if insoluble matter is present, it is removed by filtration. Then, water and an immiscible organic solvent such as ethyl acetate are added to separate the organic layer containing the target compound. After washing with water, etc., it is dried with anhydrous magnesium sulfate, anhydrous sodium sulfate, etc., filtered, and the solvent is removed by distillation to obtain the target compound. The obtained target compound can be separated and purified by conventional methods, if necessary, such as recrystallization, reprecipitation, chromatography (for example, adsorption column chromatography using carriers such as silica gel, alumina, magnesium silicate-based Florisil, SO3H-silica (manufactured by Fuji Silycia); partition column chromatography using carriers such as Sephadex LH-20 (manufactured by Pharmacia), Amberlite XAD-11 (manufactured by Rohm & Haas), Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), etc., in appropriate combinations, and eluted with an appropriate eluent). For target compounds insoluble in solvent, the obtained crude solid product can be purified by washing with a solvent. The target compounds from each step can also be used directly in the next reaction without purification.

[0195] In the reactions of each step of the following methods A through Q, J, La', Lp', B', E, V, W, 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(Lp')' indicates the same meaning as above. (Lp')' indicates one of the dipeptide residues represented by -VA-, -FG-, -PI-, -VCit-, -VK-, -(D-)PI-, -PL-, -(D-)VA-, or -GF-. (R 13 )' is R 13 If there is a hydroxyl group or amino group on the substituent, a protecting group may be used, and if there is no protecting group, R 13 This indicates (R 17 )' is a state in which the hydroxyl group is protected by a protecting group such as tert-butyldimethylsilyloxy group or R 17 To indicate one of the following.

[0196] PRO 1 PRO 4 PRO 6 PRO 8 PRO 9 This indicates an amino group protecting group. Preferably, PRO 1 PRO 4 PRO 8 PRO 9 These include allyloxycarbonyl group, 2,2,2-trichloroethyloxycarbonyl group, trimethylsilylethoxymethoxy group, benzyloxycarbonyl group, or 9-fluorenylmethyloxycarbonyl group. Also, PRO 6 Preferably, the group is a 2-(trimethylsilyl)ethoxymethyl group, a methoxymethyl group, etc. Also, PRO 2 PRO 3 PRO 5 PRO 7 PRO 10 PRO 11 PRO 12 This indicates a protecting group for a hydroxyl group, phenol group, or carboxyl group used in the field of organic synthesis chemistry. Preferably, it is an acetyl group, a benzyl group, a tert-butyldimethylsilyl (TBDMS) group, a triisopropylsilyl group, or a tert-butyl group.

[0197] X 2This represents a leaving group used in the field of organic synthesis chemistry. Preferably, it is 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, preferably a methyl group, ethyl group, benzyl group, tert-butyl group, etc. R b The leaving group is one that forms an enol sulfonate, and preferably it is a trifluoromethanesulfonyl group. Furthermore, amino groups and hydroxyl groups not explicitly protected in methods A through Q may be protected using protecting groups as needed. Deprotection may also be performed as needed, and after protection, deprotection may be carried out and a different protecting group may be attached.

[0198] A method This manufacturing method is a method for producing compound (12a), 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) in a solvent (diethyl ether, tetrahydrofuran (THF), dichloromethane, ethanol, etc., or a mixture thereof) with a reducing agent (lithium aluminum hydride, diborane, lithium borohydride, sodium borohydride, borane-tetrahydrofuran complex, or sodium bis(2-methoxyethoxy)aluminum hydride, etc.) at a temperature from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 50°C. The amount of reducing agent used is 1 to an excess mole, preferably 1 to 5 moles, relative to compound (1a). If necessary, a Lewis acid (lithium chloride, calcium chloride, tin chloride, trifluoroborane ether complex, etc.) may be added. The reaction time is from 1 minute to 60 hours, preferably from 5 minutes to 24 hours.

[0200] A-2 process (2a) → (3a): Introduction of protecting group (tert-butyldimethylsilyl group, etc.) PRO 2 If the compound is a TBDMS group, the reaction is carried out by reacting compound (2a) with a silylation agent (tert-butyldimethylsilyl chloride, tert-butyldimethylsilyl trifluoromethanesulfonate, etc.) in a solvent (dichloromethane, acetonitrile, tetrahydrafuran, N,N-dimethylformamide (DMF), etc., or a mixture thereof) at a temperature of -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. For every mole of compound (2a), 1 to an excess mole of silylation agent, preferably 1 to 5 moles, is used, and 1 to an excess mole of base, preferably 1 to 5 moles, is used. 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 the group 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 carbon, etc.) from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. The reaction is usually carried out under a hydrogen atmosphere, but if necessary, cyclohexene, 1,4-cyclohexadiene, etc. may be used as a hydrogen donor. The reaction time is 10 minutes to 100 hours, preferably from 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 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, or 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride. The condensing agent is used in amounts of 0.3 to 5 moles, preferably 0.4 to 2 moles, of carboxylic acid (compound (A)) per mole of compound (4a), and 1 to an excess mole, preferably 1 to 5 moles, of the condensing agent. Additionally, a base (triethylamine, diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine, etc.) and an additive (1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, etc.) are added as needed. The base is used in amounts ranging from a catalytic amount to an excess mole, preferably 0.2 to 3 moles, per mole of compound (4a). The additive is used in amounts ranging from a catalytic amount to an excess, 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. Furthermore, when a carboxylic acid (compound (A)) is converted to an acid halide through a condensation reaction, 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 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 every mole of compound (4a), 0.3 to 5 moles, preferably 0.4 to 2 moles, of the acid halide are used, and the amount of base is from a catalytic amount to an excess mole, preferably 0.2 to 5 moles. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. To prepare an acid halogen compound of a carboxylic acid (compound (A)), the carboxylic acid (compound (A)) is treated with oxalyl chloride, thionyl chloride, etc., in a solvent (benzene, toluene, dichloromethane, dichloroethane, etc., or a mixture thereof) at a temperature 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, etc., is added. For carboxylic acid (compound (A)), 1 to an excess mole, preferably 1 to 10 moles, of oxalyl chloride or thionyl chloride is used. 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-carbon, nickel, etc.) from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. The reaction is usually carried out under a hydrogen atmosphere, but if necessary, cyclohexene, 1,4-cyclohexadiene, hydrazine, etc. may be used as a hydrogen donor. The reaction time is from 10 minutes to 72 hours, preferably from 30 minutes to 24 hours. Furthermore, the reduction of the nitro group can also be carried out under the following conditions. Compound (5a) is reacted 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 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. For every mole of compound (5a), 1 to an excess mole of reducing agent is used, preferably 1 to 100 moles, and 1 to an excess mole of acid is added. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0204] A-6 Step (6a) → (7a): Carbamate reaction Compound (6a) is reacted with triphosgene (isocyanating agent) in a solvent (THF, dichloromethane, DMF, etc., or a mixture thereof) at a temperature from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C, to generate an isocyanate intermediate in situ, and then treated with an alcohol represented by general formula (B). If necessary, a base (triethylamine, diisopropylethylamine, sodium carbonate, sodium hydroxide, etc.) is added. For every mole of compound (6a), 0.3 to an excess mole of triphosgene (isocyanating agent), preferably 0.35 to 3 moles, is used, 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 alcohol (B) is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The alcohol (B) used in this process can be produced according to the L method described later.

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

[0206] A-8 Step (8a) → (9a): Oxidation reaction Compound (8a) is reacted with an oxidizing agent (chlorosulfonium salt, Dessmartin reagent, tetrabutylammonium lutenate, pyridinium chlorochromate, nitroxyl radical oxidation catalyst, etc.) in a solvent (acetone, dichloromethane, pyridine, etc., or a mixture thereof) at a temperature 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 additive (tetrabutylammonium bromide, potassium bromide, etc.) are added. The oxidizing agent is used in an amount of 0.005 to an excess mole, preferably 0.005 to 10 moles, per mole of compound (8a). Alternatively, 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 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

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

[0208] A-10 Step (10a) → (11a): Deprotection reaction PRO 3 When the compound is a triisopropylsilyl group, it is produced by treating compound (10a) with lithium acetate in a solvent (DMF, water, etc., or a mixture thereof) 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 Compound (11a) is prepared by reacting it 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 from -20°C to the boiling point of the solvent used in the reaction, preferably from 0°C to the boiling point. A base (potassium carbonate, cesium carbonate, etc.) may be added as needed. For every mole of compound (11a), 1 mole to an excess mole of alkylating agent, preferably 1 to 10 moles, and 0.4 to an excess mole of base, preferably 0.5 to 5 moles, are used. The reaction time is from 1 minute to 60 hours, preferably from 5 minutes to 24 hours.

[0210] B method This manufacturing method involves compound (1), R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded, R 14 , R 15 This is a method for producing compound (10b), an intermediate necessary for producing a compound in which hydrogen is present. [ka]

[0211] B-1 step (1b) → (2b): Deprotection group reaction PRO 7If the group is a triisopropylsilyl group, it is manufactured according to step A-10 of Method A. PRO 7 If the group is a benzyl group, it is manufactured according to step A-3 of Method A.

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

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

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

[0215] B-5 Step (5b) → (6b): Enol sulfonylation reaction R b When the compound is a trifluoromethanesulfonyl group, the reaction is carried out by reacting compound (5b) with trifluoromethanesulfonic anhydride, etc., in a solvent (such as dichloromethane) at a temperature from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 30°C. A base (such as 2,6-lutidine) is added as needed. The amount of trifluoromethanesulfonic anhydride used is 1 mole to an excess mole, preferably 1 mole to 5 moles, per mole of compound (5b). The amount of base used is 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) Compound (6b) is reacted in a solvent (ethanol, toluene, 1,4-dioxane, DMF, tetrahydrafuran, water, etc., or a mixture thereof) at a temperature from 0°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 120°C, in the presence of a transition metal catalyst (tetrakis(triphenylphosphine)palladium, dichlorobis(benzonitrile)palladium(II), etc.) and an organoboron compound (4-methoxyphenylboronic acid, etc.). If necessary, a base (sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, 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, per mole of compound (6b), the organoboron compound is used in an amount of 1 mole to an excess mole, preferably 1 to 10 moles, the base in an amount of 1 to 5 moles, and the additive in an amount of 0.1 to 5 moles. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0217] B-7 Step (7b) → (8b): Reduction reaction For example, PRO 6 If the compound is a 2-(trimethylsilyl)ethoxymethyl group, the compound (7b) is treated with a reducing agent (lithium borohydride, sodium borohydride, etc.) in a solvent (diethyl ether, THF, dichloromethane, ethanol, etc., or a mixture thereof) at a temperature from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 50°C. The amount of reducing agent used is 1 to an excess mole, preferably 1 to 30 moles, 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 mixture thereof) and stirring. The amount of silica gel used is 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 the imino group The reaction is carried out by treating compound (8b) in a solvent (THF, dichloromethane, N,N-dimethylformamide, etc., or a mixture thereof) with a reducing agent (sodium borohydride, boron cyanohydride, sodium triacetoxyborohydride, 2-picolineborane, pyridineborane, etc.) at a temperature from -78°C to the boiling point of the solvent used in the reaction, preferably from -78°C to 50°C. The amount of reducing agent used is 1 to an excess mole, preferably 1 to 5 moles, relative to compound (8b). The reaction time is from 1 minute to 60 hours, preferably from 5 minutes to 24 hours.

[0219] B-9 process (9b) → (10b): Introduction of protecting group PRO 8 If the compound is an allyloxycarbonyl group, (9b) is reacted with allyl chloroformate, diallyl dicarbonate, etc. in a solvent (benzene, toluene, pyridine, diethyl ether, dichloromethane, THF, 1,4-dioxane, water, etc., or a mixture thereof) at a temperature 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, potassium carbonate, sodium hydroxide, etc.) is added. For every mole of compound (9b), 1 mole to an excess mole of allyl chloroformate is used, preferably from 1 mole to 10 moles, and 1 mole to an excess amount of base is used, preferably from 1 to 10 moles. The reaction time is 10 minutes to 72 hours, preferably from 10 minutes to 48 hours. PRO 8 If it is a 2'2'2'-trichloroethoxycarbonyl group, Compound (9b) is reacted with 2,2,2-trichloroethyl chloroformate, etc., in a solvent (benzene, toluene, pyridine, diethyl ether, dichloromethane, THF, 1,4-dioxane, water, etc., or a mixture thereof) at a temperature 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, etc.) is added. For 1 mole of compound (9b), 1 mole to an excess mole of 2,2,2-trichloroethyl chloroformate is used, preferably from 1 mole to 10 moles, and 1 mole to an excess amount of base is used, preferably from 1 to 10 moles. The reaction time is 10 minutes to 72 hours, preferably from 30 minutes to 48 hours.

[0220] C method This manufacturing method involves compound (1), R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded, R 14 , R 15 This is a method for producing compound (14c), an intermediate necessary for producing a compound in which hydrogen is present. Compound (10b) can also be produced by this method. [ka]

[0221] C-1 process (1c) → (2c): Introduction of protecting group PRO 5If the compound 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 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. For every mole of compound (1c), 1 mole to an excess mole of acetylating agent, preferably 1 mole to 20 moles, and a catalytic amount to an excess mole of base, preferably 0.1 to 20 moles, is used. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours. PRO 5 If it is a TBDMS group, it is manufactured according to Method A, Step A-2.

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

[0223] C-6 step (6c)→(7c): Deprotection group reaction PRO 9 When the compound is a 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 a temperature from -20°C to the boiling point of the solvent, preferably from 0°C to 40°C. The amount of metal reagent used is 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 the compound is an allyloxycarbonyl group, the reaction is carried out in a solvent (dichloromethane, DMF, THF, etc., or a mixture thereof) 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 (such as tetrakis(triphenylphosphine)palladium) and an allyl group scavenger (such as pyrrolidine, morpholine, or barbituric acid). The amount of palladium catalyst used is 0.005 to 1 mole, preferably 0.005 to 0.5 moles, per mole of compound (6c). The amount of allyl group scavenger used is 1 mole to an excess mole, preferably 1 to 10 moles. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

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

[0225] D-1 process ~ D-6 process, D-9 process, D-10 process Process D-1 is manufactured according to process B-6 of method B, process D-2 according to process A-5 of method A, process D-3 according to process B-9 of method B, process D-4 according to process A-7 of method A, process D-5 according to process A-8 of method A, process D-6 according to process C-6 of method C, process D-9 according to process B-8 of method B, and process D-10 according to process B-9 of method B.

[0226] D-7 process and D-8 process Compound (7d) can also be produced by following steps D-7, which is similar to step B-6 of Method B, and step D-8, which is similar to step B-7 of Method B.

[0227] E method Method E is a method for 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 process involves the production of compound (1e) by a coupling reaction between compound (11a) produced by method A and compound (10b) produced by method B. Compound (11a) is produced by coupling compound (10b) with compound (10b) in a solvent (THF, DMF, N,N-dimethylacetamide, or a mixture thereof) at a temperature from -20°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C, in the presence of a base (potassium carbonate, cesium carbonate, etc.). Compound (10b) is used in an amount of 1 mole to an excess mole, preferably 0.7 to 1.5 moles, per 0.5 moles of compound (11a). The amount of base used is 1 to 5 moles. 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 performing a coupling reaction with compound (12a) produced by method A and compound (14c) produced by method C, similar to step E-1.

[0230] E-3 process Process E-3 is manufactured according to process A-7 of Method A.

[0231] E-4 process This process involves using compound (2e) in PRO 4 and PRO 8 If the protecting group is the same, this is a step to produce compound (4e) by carrying out a deprotection reaction similar to step C-6 of the C method.

[0232] PRO of compound (2e) 4 and PRO 8 If the protecting groups are different, compound (4e) can also be produced by carrying out the deprotection reaction stepwise through steps E-5 and E-6.

[0233] Processes E-5 and E-6 are manufactured according to Method C, Process C-6.

[0234] F method Compound (4e) can also be produced from the intermediate compound (10f) of this synthesis method. This method describes how to produce compound (10f) and compound (4e). [ka]

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

[0236] F-11 process PRO 10 and (R 17 If both protecting groups of the hydroxyl groups in )' are TBDMS groups, the product is manufactured according to step A-7 of Method A.

[0237] F-12 process This step involves the protecting group PRO with compound (9f). 4 and PRO 9 If the same, this is a step to produce compound (10f) by carrying out a deprotection group reaction, similar to step C-6 of the C method.

[0238] F-13 and F-14 processes PRO of compound (9f) 4 and PRO 9 If the protecting groups are different, compound (10f) can also be produced by a stepwise deprotection reaction via steps F-13 and F-14. Steps F-13 and F-14 are produced according to step C-6 of the C method.

[0239] G method This manufacturing method involves, among compound (1), R 11 R represents a hydrogen atom, 12 and R 13They come together to form a spiro ring, R 14 , R 15 This is a method for producing an intermediate compound (11g) that exhibits a hydrogen atom. [ka]

[0240] G-1~G-2 process, G-5~G-11 process, Process G-1 is manufactured according to Method A's A-4 process, process G-2 according to Method A's A-5 process, process G-5 according to Method A's A-11 process, process G-6 according to Method B's B-7 process, process G-7 according to Method B's B-8 process, process G-8 according to Method B's B-9 process, process G-9 according to Method E's E-1 process, process G-10 according to Method A's A-7 process, and process G-11 according to Method C's C-6 process.

[0241] Step G-3: Introduction of protecting group The compound (2g) is reacted with a chloromethoxy ether reagent (2-(chloromethoxy)ethyltrimethylsilane, chloromethyl methyl ether, benzylchloromethyl ether, etc.) in a solvent (THF, DMF, dioxane, etc., or a mixture thereof) at a temperature 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, hexamethyldisilazane lithium) is added. The reagent is used in amounts of 1 to an excess mole, preferably 1 to 5 moles, per mole of compound (2g). The base is used in amounts of 1 to an excess mole, 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 If the group is a benzyl group, it is manufactured according to step A-3 of Method A. PRO 7 If the group is a triisopropylsilyl group, it is manufactured according to step A-10 of Method A.

[0243] H method This manufacturing method involves, among compound (1), R 11R represents a hydrogen atom, 12 and R 13 They come together to form a spiro ring, R 14 , R 15 This is a method for producing an intermediate compound (9h) for manufacturing a compound with an imine bond (C=N). In this manufacturing method, R 12 and R 13 The spiro ring formed is synonymous with E, so it is also represented as E. [ka]

[0244] H-1 process ~ H-10 process Process H-1 is manufactured according to process A-4 of method A, process H-2 according to process A-1 of method A, process H-3 according to process C-1 of method C, process H-4 according to process A-4 of method A, process H-5 according to process A-5 of method A, process H-6 according to process B-9 of method B, process H-7 according to process A-6 of method A, process H-8 according to process B-3 of method B, process H-9 according to process A-8 of method A, and process H-10 according to process C-6 of method C.

[0245] Method I This manufacturing method involves compound (1), R 11 and R 12 Together they form a benzene ring, R 13 It is a single bond, R 14 , R 15 This is a method for producing compound (11i), an intermediate for producing a compound in which two substances combine to form an imine. [ka]

[0246] I-1 process ~ I-11 process Process I-1 is manufactured according to process A-4 of method A, process I-2 according to process A-5 of method A, process I-3 according to process B-9 of method B, process I-4 according to process A-7 of method A, process I-5 according to process A-8 of method A, process I-6 according to process A-2 of method A, process I-7 according to process A-10 of method A, process I-8 according to process A-11 of method A, process I-9 according to process E-1 of method E, process I-10 according to process A-7 of method A, and process I-11 according to process C-6 of method C.

[0247] J method This manufacturing method involves compound (1), R 12 and R 13 Together they form CH2= and R 11 is hydrogen, R 14 , R 15 This is a method for producing compound (12j), an intermediate of a compound that forms an imine when combined with other components. [ka]

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

[0249] J-2 step: Protecting group introduction PRO 7 If the compound (2a) 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 a temperature of -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. For every mole of compound (2a), 1 to an excess mole of silylation agent, preferably 1 to 3 moles, and 1 to an excess mole of base, preferably 1 to 5 moles, are used. 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 according to process A-5 of method A, process J-4 according to process B-9 of method B, process J-5 according to process A-7 of method A, process J-6 according to process A-8 of method A, process J-7 according to process A-2 of method A, process J-8 according to process A-10 of method A, process J-9 according to process E-1 of method E, process J-10 according to process A-7 of method A, and process J-11 according to process C-6 of method C.

[0251] K method The K method is used for compound (1), where R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded, R 13 R is a hydroxymethyl group, 14 , R 15 This is a method for producing compound (7k), an intermediate necessary for manufacturing a compound that forms an imine when combined with other components. [ka]

[0252] K-1 process This step involves a carbonylation reaction with compound (6b) to produce compound (1k).

[0253] K-2 process This process involves producing compound (2k) by performing an aldehyde-selective reduction reaction on compound (1k).

[0254] K-3 process ~ K-7 process Process K-3 is manufactured according to Method A, Process A-2; Process K-4 according to Method B, Process B-7; Process K-5 according to Method E, Process E-1; Process K-6 according to Method A, Process A-7; and Process K-7 according to Method C, Process C-6.

[0255] L method The L method is a typical method for producing compound (B). [ka]

[0256] Peptide residues represented by the general formula (Lp')' can be produced by amino acid condensation reactions. PRO 4 It protects the N-terminus at the peptide residue (Lp')', PRO 12 It protects the C-terminus.

[0257] L-1 process The L-1 process is manufactured according to the B-9 process of Method B.

[0258] L-2 step: Deprotection reaction PRO 12 If the compound is a tert-butyl group, the reaction is carried out by reacting 2 liters of the compound with an acid (trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, acetic acid, etc.) in a solvent (such as dichloromethane) 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 mole per mole of 2 liters of the compound. The reaction time is 10 minutes to 72 hours, preferably 30 minutes to 24 hours.

[0259] L-3 process~L-4 process Process L-3 is manufactured according to Method B, Process B-9, and process L-4 is manufactured according to Method A, Process A-4.

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

[0261] M method The M method is a method for producing compound (2). Compound (2) shown in this manufacturing method is the R of the manufacturing intermediate of the present invention among compound (1). 16 This is equivalent to J-La'-Lp'-NH-B'-CH2-O(C=O)-*. [ka]

[0262] The compound (1m) shown in the above manufacturing method represents compounds (4e), (4f), (11g), (9h), (11i), (12j), (7k), and (8k) produced by methods E through K.

[0263] The (PBD)' shown in the above manufacturing method is [ka] Show, PBD [ka] This indicates. (PBD)' is R in PBD 13 The substituent above (such as a hydroxyl group) may be protected by a protecting group, and if there is no protecting group, it is equivalent to PBD (R 13 =(R 13 )') is. (Lp')” t The peptide residue denoted by -(Lp')' may have a functional group (such as an amino group) on the side chain of the amino acid residue denoted by Lp' protected by a protecting group, and if the protecting group is unsubstituted, it is equivalent to Lp'. Here, (Lp')' represents two amino acid sequences as shown below, and the side chain may be protected if it has a functional group (amino group, hydroxyl group). -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 may be protected if a functional group (amino group, hydroxyl group) is present in the side chain. -GG-, -EGG-, -DG-, -(D-)DG-, -EG-, -GGF-, -SG-, -KG-, -DGG-, -GGF-, -DDGG-, -KDGG-, or -GGFG-

[0264] (La')' indicates one of the following groups. -C(=O)-(CH2CH2)n 6 -C(=O), -C(=O)-(CH2CH2)n6 -NH-C(=O)-(CH2CH2O)n 7 -CH2CH2-C(=O)-, -(CH2)n 8 -OC(=O)-, -(CH2)n 12 -C(=O)-, and, -(CH2CH2)n 13 -C(=O)-NH-(CH2CH2O)n 14 -CH2CH2-C(=O)-

[0265] (La')'' represents one of the following groups. -NH-(CH2CH2)n 7 -C(=O)-, or -NH-(CH2CH2O)n 7 -CH2-C(=O)-, s or t each independently represents either 0 or 1. For example, in the M-1 process below, s and t are 0; in the M-3 process, s is 1 and t is 0; and in the M-5 process, s is 0 and t is 1.

[0266] (La')'-(La')'' s This is synonymous with La'. (Lp')'' t -(Lp')' becomes Lp' when a protecting group is present, but is synonymous with Lp' when there is no protecting group. In the above manufacturing method, Lx represents a hydrogen atom or a leaving group (such as hydroxysuccinimide). In the above manufacturing method, 1m, 9m, 10m, 11m, and PBD or (PBD)' in compound (2) indicate that the asterisk (N10' position) is bonded to the rightmost C(=O)- of -OC(=O)-.

[0267] M-1 process This process involves a condensation reaction between compound (1m) and compound (2m), which are produced by methods E through K, to produce compound (11m). If Lx=H and compound (2m) is a carboxylic acid, compound (2m) can be produced according to step A-4 of method A. When Lx is a leaving group (such as hydroxysuccinimide or p-nitrophenoxy group), 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 a temperature from -30°C to the boiling point of the solvent used in the reaction, preferably from 0°C to 50°C. For every mole of compound (1m), 0.9 to an excess mole, preferably 0.9 to 2 moles, of compound (2m) is used. In addition, a base (such as triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine, or diazabicycloundecene) is added as needed. For every mole of compound (1m), 1 mole to an excess amount, preferably 1 to 5 moles, of base is used. 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 manufactured according to process M-1, process M-3 according to method A A-4, process M-4 according to process M-1, process M-5 according to method A A-4, and process M-8 according to method A A-4.

[0269] M-6 process This process involves a condensation reaction with compound (6m) to produce an active ester intermediate (7m). The compound (6m) is reacted with hydroxysuccinimide, etc., in a solvent (benzene, toluene, diethyl ether, dichloromethane, THF, DMF, etc., or a mixture thereof) at a temperature 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 or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The amount of condensing agent used is 1 to an excess mole, preferably 1 to 5 moles, per mole of compound (6m). The amount of hydroxysuccinimide used is 1 to an excess mole, 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 process involves a condensation reaction between compound (1m) and compound (7m) in the same manner as in the M-1 process to produce compound (11m).

[0271] M-9 process This process involves a deprotection reaction with compound (9m) to produce compound (10m). 4 When the compound is a 9-fluorenylmethyloxycarbonyl group, the reaction is carried out by reacting the 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 from -20°C to the boiling point of the solvent, preferably from 0°C to 40°C. For every mole of compound (9m), 1 to an excess mole of base is used, preferably from 1 to 10 moles. The reaction time is from 1 minute to 72 hours, preferably from 5 minutes to 24 hours.

[0272] M-10 process This process involves a condensation reaction between compound (10m) and compound (2m) or (4m) in the same manner as in step A-4 of Method A, to produce compound (11m).

[0273] M-11 process This process involves compound (11m) with (Lp')” t If -(Lp')' or PBD' has a protecting group, this is a step to produce compound (2) by deprotection. It is produced according to step B-3 of Method B and step C-6 of Method C. Furthermore, if (Lp')' or PBD' does not have a protecting group, step M-11 is omitted, in which case compound (11m) is synonymous with (2).

[0274] N method The N method uses the free drugs shown in (1), R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded, R 14 , R 15 is hydrogen, R 16 and R 17This describes a method for synthesizing compounds that combine to form an imine bond. [ka]

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

[0276] (R 13 )'=R 13 In this case, the manufacturing process follows steps N-9 and N-10 below.

[0277] N-9 process Process N-9 is manufactured according to process A-7 of Method A.

[0278] N-10 process PRO 4 and PRO 8 If the protecting group is the same, it is manufactured according to the E-4 process of the E method. 4 and PRO 8 If the protecting group is different, it is manufactured according to steps E-5 and E-6 of Method E.

[0279] (R 13 If )' has a protecting group, it is manufactured according to the following N-11 and N-12 steps.

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

[0281] N-12 process PRO 4 and PRO 8 If the same protecting group is used, it is manufactured according to steps E-3 and E-4 of the E method. 4 and PRO 8If the protecting group is different, it is manufactured according to steps E-3, E-5 and E-6 of Method E.

[0282] O method Method O is the method in which, of the free drugs shown in (1), R 11 and R 12 Together, they form a double bond with the carbon atom to which each group is bonded, R 14 and R 15 and R 16 and R 17 This is a method for producing a compound (6o) with an imine bond (C=N) by combining these two components. [ka]

[0283] O-1 process~O-6 process Process O-1 is manufactured according to Method E-1, process O-2 according to Method B-3, process O-3 according to Method A-8, process O-4 according to Method B-5, process O-5 according to Method B-6, and process O-6 according to Method B-7.

[0284] P method This manufacturing method uses (1) of the free agents, R 11 R represents a hydrogen atom, 12 and R 13 They come together to form a spiro ring, R 14 and R 15 and R 16 and R 17 This is a method for producing compounds with an imine bond (C=N) by combining the following. In this production method, the starting material compound (4h) is represented by R 12 and R 13 The spiro ring formed is synonymous with E, so it is also represented as E. [ka]

[0285] P-1 process~P-4 process Process P-1 is manufactured according to process B-9 of method B, process P-2 according to process B-3 of method B, process P-3 according to process A-8 of method A, and process P-4 according to process C-6 of method C.

[0286] Q method This manufacturing method uses (1) of the free agents, R 11 , R 14 and R 15 R represents a hydrogen atom, 12 and R 13 They come together to form a spiro ring, and R 16 and R 17 This is a method for producing compounds with an imine bond (C=N) by combining these elements. [ka]

[0287] Q-1 process~Q-6 process Process Q-1 is manufactured according to Method A's A-1 process, process Q-2 according to Method A's A-8 process, process Q-3 according to Method A's A-5 process, process Q-4 according to Method E's E-1 process, process Q-5 according to Method A's A-7 process, and process Q-6 according to Method C's C-6 process.

[0288] In the above, the amino group and hydroxyl group protecting groups that may be protected refer to protecting groups that can be cleaved by chemical methods such as hydrolysis, hydrolysis, electrolysis, and photolysis, and represent protecting groups commonly used in organic synthesis (see, for example, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc. (1999)). In the above, the "protecting groups" for hydroxyl groups that may be protected (e.g., alkylcarbonyl groups, silyl groups, or aralkyl groups), the "protecting groups" for carboxyl groups that may be protected (e.g., C1-C6 alkyl groups or aralkyl groups), and the "protecting groups" for amino groups that may be protected (e.g., alkoxycarbonyl groups) are not particularly limited as long as they are protecting groups for hydroxyl, carboxyl, and amino groups used in the field of organic synthesis chemistry. Steps requiring protection and deprotection are carried out according to known methods (for example, those described in "Protective Groups in Organic Synthesis" (Theodora W. Greene and Peter GMWuts, 1999, published by Wiley-Interscience Publications)).

[0289] R method: Antibody preparation Glycan remodeling antibodies can be manufactured by the method shown in Figure 3, for example, according to the method described in WO2013 / 120066. [ka]

[0290] R-1 step: Hydrolysis of the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing end chitobiose structure. This process involves preparing a glycosylation-cleaved antibody by using a known enzymatic reaction to cleave the N-linked glycan (N297-linked glycan) that binds to the aspartic acid at amino acid position 297 of the antibody's amino acid sequence. The target antibody (20 mg / ml) is placed in a buffer solution (such as 50 mM phosphate buffer solution) at a temperature of 0°C to 40°C and hydrolytic enzymes such as EndoS enzyme are used to hydrolyze the glycosidic bond between GlcNAcβ1 and 4GlcNAc at the reducing end chitobiose structure. The reaction time is 10 minutes to 72 hours, preferably 1 to 6 hours. For every 100 mg of antibody, 0.1 to 10 mg, preferably 0.1 to 3 mg, of wild-type EndoS enzyme is used. After the reaction is complete, (Fucα1,6)GlcNAc antibody, in which the sugar chain between GlcNAcβ1 and 4GlcNAc has been hydrolyzed, can be produced by affinity chromatography purification and / or hydroxyapatite column purification as described later.

[0291] R-2 process: Glycan transfer reaction This process involves using an enzymatic reaction to attach MSG (MSG1, MSG2) or SG-type glycan oxazoline (hereinafter referred to as "azide glycan oxazoline") having an azide group-containing PEG linker to the (Fucα1,6)GlcNAc antibody described above, in order to produce a glycan remodeling antibody. The glycosylation reaction is carried out by reacting the above-mentioned glycosylation-cleaving antibody with an azide glycan oxazoline in a buffer solution (such as phosphate buffer solution) at a temperature of 0°C to 40°C in the presence of a catalytic amount of glycosyltransferase such as EndoS (D233Q / Q303L). The reaction time is 10 minutes to 72 hours, preferably 1 to 6 hours. For every 100 mg of antibody, 1 to 10 mg, preferably 1 to 3 mg, of the EndoS enzyme (D233Q / Q303L) is used, and 2 to an excess equivalent, preferably 2 to 20 equivalents, of the azide glycan oxazoline is used. After the reaction is complete, the purified glycosylation remodeling antibody can be obtained by performing affinity chromatography purification and hydroxyapatite column purification. Azide glycan oxazolines can be prepared according to the methods described in Examples 55-57. N3-(CH2CH2-O)n5-CH2CH2-NH2, a PEG linker (N3-L(PEG)) containing an azide group, can be introduced into MSG (MSG1, MSG2) or disialooctasaccharide (Tokyo Chemical Industries, Ltd.) using reactions known in the field of organic synthesis (such as condensation reactions). Specifically, the carboxylic acid at the 2-position of the sialic acid and the amino group at the right end of N3-(CH2CH2-O)n5-CH2CH2-NH2 form an amide bond through a condensation reaction. When using a condensation reaction, the condensing agent may be N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), carbonyldiimidazole (CDI), 2-(2H-benzotriazole-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (BOP), 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), etc., and the solvent used in the reaction may be dichloromethane, DMF, THF, ethyl acetate, etc., or a mixture thereof, but is not particularly limited. The reaction temperature is usually in the range of -20°C to 100°C or up to the boiling point of the solvent, but preferably in the range of -5°C to 50°C. Additionally, organic bases such as triethylamine, diisopropylethylamine, N-methylmorpholine, or 4-dimethylaminopyridine, or inorganic bases such as potassium carbonate, sodium carbonate, potassium bicarbonate, or sodium bicarbonate may be added as needed. Furthermore, reaction accelerators such as 1-droxybenzotriazole and N-hydroxysuccinimide may be added. MSG, MSG1, or MSG2 can be obtained by hydrolyzing the aforementioned (MSG-)Asn or separated and purified (MSG1-)Asn or (MSG2-)Asn (Example 56) with a hydrolytic enzyme such as EndoM. Oxazolination can be prepared from MSG (MSG1, MSG2) or GlcNAc at the reducing end of SG-type glycans according to known papers (J. Org Chem., 2009, 74 (5), 2210-2212. Helv. Chim. Acta, 2012, 95, 1928-1936.).

[0292] In the preparation of the above-described glycosylation remodeling antibody, the concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be carried out according to the following common operations A to C. Common procedure A: Concentration of antibody aqueous solution Antibodies or antibody-drug conjugate solutions were placed in Amicon Ultra containers (30,000 to 50,000 MWCO, Millipore Co.), and the antibodies and antibody-drug conjugate solutions described later were concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000G to 4000G for 5 to 20 minutes. Common procedure B: Measurement of antibody concentration Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's specified method. At that time, different 280nm extinction coefficients (1.3 mL mg) were used for each antibody. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used. Common operation C: Antibody buffer exchange The antibody aqueous solution was concentrated using common procedure A by adding a buffer solution (phosphate-buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.). 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 manufacturing method involves conjugating the aforementioned glycan remodeling antibody and manufacturing intermediate (2) via a SPAAC (strain-promoted alkyne azide cycloaddition: J. AM. CHEM. SOC. 2004, 126, 15046-15047) reaction to produce an antibody-drug conjugate. In the formula, Ab represents the glycan remodeling antibody. [ka] The SPAAC reaction proceeds when a buffer solution of antibody Ab (such as sodium acetate solution, sodium phosphate solution, sodium borate solution, or a mixture thereof) is mixed with a solution of compound (2) dissolved in a suitable solvent (such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP), propylene glycol (PG), or a mixture thereof). The amount of compound (2) per mole of antibody is 2 moles to an excess mole, preferably 1 mole to 30 moles, and the ratio of the organic solvent to the antibody buffer is preferably 1 to 200% v / v. The reaction temperature is 0°C to 37°C, preferably 10°C to 25°C, and the reaction time is 1 to 150 hours, preferably 6 to 100 hours. The pH during the reaction is preferably 5 to 9.

[0294] Antibody-drug conjugates can be identified by performing buffer exchange, purification, measurement of antibody concentration and average number of drug conjugates per antibody molecule using the common operations A to C described above and common operations D to F described below.

[0295] Common operation D: Purification of antibody-drug conjugates The NAP-25 column was equilibrated with one of the following buffers: a commercially available acetate buffer containing 5% sorbitol (10 mM, pH 5.5; referred to as ABS herein). Approximately 1.5–2.5 mL of the antibody-drug conjugate reaction aqueous solution was placed on this NAP-25 column, and the antibody fraction was separated by elution with the manufacturer's specified amount of buffer. This separated fraction was then placed back on the NAP-25 column, and the gel filtration purification procedure was repeated 2–3 times to obtain the antibody-drug conjugate, free from unbound drug linkers, dimethyl sulfoxide, and propylene glycol. The concentration of the antibody-drug conjugate solution was adjusted as needed using common procedures A and C. Common procedure E: Measurement of antibody concentration in antibody-drug conjugates The concentration of the conjugated drug in an antibody-drug conjugate can be calculated using the Lambert-Beer law shown below. Equation (I) using Lambert-Beer's law is shown below.

number

number

number

number

[0296] Common procedure F: Measurement of the average number of drug molecules bound per antibody molecule in antibody-drug conjugates. The average number of drug molecules 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)] Mix an antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). Incubate the mixture at 37°C for 30 minutes to cleave the disulfide bond between the light and heavy chains of the antibody-drug conjugate. Use the resulting sample for HPLC analysis. [F-2.HPLC analysis] HPLC analysis will be performed under the following measurement conditions. HPLC System: Agilent 1290 HPLC System (Agilent Technologies) Detector: UV absorbance meter (measurement wavelengths: 280 nm, 329 nm) Column: BEH Phenyl (2.1 × 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 acetonitrile solution 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: 5 μL

[0297] [F-3. Data Analysis] [F-3-1] Compared to the light chain (L0) and heavy chain (H0) of an antibody that is not bound to a drug, the drug-bound light chain (L1) and heavy chain (H1), with one drug bound, with two drugs bound, and with three drugs bound, with the retention time of the heavy chain (H3) increases proportionally to the number of drugs bound, resulting in elution in the order of L0, L1, H0, H1, H2, H3. The order of L1 and H0 may vary, but the drug-unbound H0 does not have the characteristic 329nm wavelength absorption of the drug. Therefore, L1 and H0 can be distinguished by checking the 329nm wavelength absorption. By comparing the retention times of L0 and H0, the detection peak can be assigned to one of L0, L1, H0, H1, H2, or H3. [F-3-2] Because drug linkers have UV absorption, the peak area value is corrected according to the following formula using the molar extinction coefficients of the L chain, H chain, and drug linker, depending on the number of drug linkers.

number

number

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

[0299] The types of cancer 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.), kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain tumor, head and neck cancer, and their metastatic forms. However, the invention is not limited to these cancer cells, as long as the cancer cells being 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, but is more preferably to humans.

[0301] The substances used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention can be appropriately selected and applied from pharmaceutical additives and others commonly used in this field, in terms of dosage and concentration.

[0302] The antibody-drug conjugate of the present invention may be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically comprises one or more pharmaceutical carriers (e.g., sterile liquids (e.g., water and oil (oils of petroleum, animal, plant, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)))). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and aqueous glycerol solutions, may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition may also optionally contain trace amounts of wetting or emulsifying agents or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Their formulations correspond to the mode of administration.

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

[0304] In typical embodiments, the pharmaceutical composition is formulated according to a standard procedure as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the pharmacopoeia may also contain a solubilizer and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the components are supplied either separately (e.g., as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the activator) or mixed together in a unit dosage form. If the pharmacopoeia is to be administered by infusion, it may be administered in an infusion bottle containing, for example, sterile pharmaceutical-grade water or saline. If the pharmacopoeia is to be administered by injection, ampoules of sterile water or saline for injection may be provided, for example, so that the components can be mixed before administration.

[0305] The pharmaceutical composition of the present invention may consist solely of the antibody-drug conjugate of this application, or it may consist of the antibody-drug conjugate and at least one other cancer treatment agent. The antibody-drug conjugate of the present invention may also be administered together with other cancer treatment agents to enhance its anticancer effect. Other anticancer agents used for this purpose may be administered to the individual simultaneously with, separately from, or consecutively with the antibody-drug conjugate, or with varying administration intervals. Examples of such cancer treatment agents include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, or the drugs listed in pamphlet WO2003 / 038043, as well as LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), etc., but the term is not limited to drugs that have antitumor activity.

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

[0307] The composition and concentration of the pharmaceutical composition vary depending on the administration method, but the antibody-drug conjugate contained in the pharmaceutical composition of the present invention exhibits therapeutic effects even at small doses if its affinity for the antigen is high (low Kd value), i.e., the dissociation constant (Kd value) for the antigen is high. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can also be set based on the affinity between the antibody-drug conjugate and the antigen. When administering the antibody-drug conjugate of the present invention to humans, for example, approximately 0.001 to 100 mg / kg can be administered once or multiple times at intervals of 1 to 180 days.

[0308] Furthermore, the antibody or functional fragment of the antibody of the present invention can also be used as a pharmaceutical. In this case, the above-mentioned description of "antibody-drug conjugate" in relation to "pharmaceutical" can be appropriately replaced with a description of "antibody or functional fragment of the antibody."

[0309] Furthermore, the free drug of the present invention (novel PBD derivative compound), its salt, or hydrate thereof can also be used as a pharmaceutical. In this case, the above-mentioned description of "antibody-drug conjugate" in relation to <pharmaceutical> can be appropriately replaced with a description of "free drug (novel PBD derivative compound), its salt, or hydrate thereof." [Examples]

[0310] The present invention will be specifically illustrated by the following examples, but the present invention is not limited thereto. Furthermore, these examples are not intended to be restrictive in any sense. Also, reagents, solvents, and starting materials not specifically mentioned herein are readily available from commercially available sources.

[0311] Example 1: Trastuzumab-Tesirine Step 1: Conjugation of antibody and drug linker To a 5 mM ethylenediamine tetraacetate-phosphate buffered saline solution (pH 6.5) (9.91 mg / mL, 0.70 mL) of trastuzumab (Reference Example 3), dipotassium phosphate aqueous solution (1.0 M, 0.0112 mL) and tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (10 mM, 0.0086 mL) were added at 20°C for 60 minutes and then at room temperature for 30 minutes. To the reaction solution, a dimethylacetamide solution (0.0415 mL) of tesirine (0.36 mg), synthesized according to the literature (Med. Chem. Lett. 2016, 7, 983-987), was added and the reaction was allowed to proceed at room temperature for 1 hour. To the reaction solution, N-acetylcysteine ​​aqueous solution (100 mM, 0.0024 mL) was added and the reaction was stopped after 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. Characteristic evaluation: The following characteristic values ​​were obtained using common operations E and F. Antibody concentration: 1.40 mg / mL, Antibody yield: 4.90 mg (71%), Average number of drug bindings 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 ethylenediamine tetraacetate-phosphate buffered saline solution (pH 6.5) containing anti-CLDN6(H1L1) antibody (9.87 mg / mL, 0.45 mL) was mixed with a 1.0 M dipotassium phosphate aqueous solution (0.0072 mL) and a 10 mM tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (0.0041 mL) at 20°C and reacted for 90 minutes at 20°C. To the reaction solution, a 0.0277 mL solution of N,N-dimethylacetamide containing tesurine (0.15 mg), synthesized according to the literature (Med. Chem. Lett. 2016, 7, 983-987), was added and reacted for 1 hour at 20°C. To the reaction solution, a 100 mM N-acetylcysteine ​​aqueous solution (0.001 mL) was added and reacted for 30 minutes to stop the reaction. Purification procedure: The above solution was purified using common procedure D to obtain 3.5 mL of a solution containing the target compound. Characteristic evaluation: The following characteristic values ​​were obtained using common operations E and F. Antibody concentration: 1.56 mg / mL, Antibody yield: 3.90 mg (88%), Average number of drug bindings per antibody molecule (n): 2.1

[0313] Reference example 3: Anti-HER2 antibody Trastuzumab The anti-HER2 antibody was prepared by referring to US5821337. The amino acid sequences of the light and heavy chains of trastuzumab are shown in SEQ ID NOs. 64 and 65.

[0314] Reference example 4: Anti-LPS antibody h#1G5-H1L1 The anti-LPS antibody was prepared by referring to WO2015 / 046505. The amino acid sequences of the light and heavy chains of h#1G5-H1L1 are shown in SEQ ID NOs. 66 and 67.

[0315] Reference example 5: Anti-TROP2 antibody hRS7 Anti-TROP2 antibodies were prepared by referring to WO2003 / 074566 and WO2015 / 098099 (Reference Example 1). The amino acid sequences of the light and heavy chains of hRS7 are shown in SEQ ID NOs. 68 and 69.

[0316] Reference example 6: Anti-CD98 antibody hM23-H1L1 The anti-CD98 antibody was prepared by referring to WO2015 / 146132. The amino acid sequences of the light and heavy chains of hM23-H1L1 are shown in SEQ ID NOs. 70 and 71.

[0317] [Synthesis of manufacturing intermediates] Example 1: Intermediate 1 [ka] Step 1: Benzyl (6S)-6-(hydroxymethyl)-5-azaspiro[2,4]heptane-5-carboxylate To a solution of 5-benzyl 6-methyl(6S)-5-azaspiro[2.4]heptane-5,6-dicarboxylate (104 mmol, WO2012087596) in THF (500 mL), lithium borohydride (4.30 g, 178 mmol) was added in small amounts at 0°C. After stirring at 0°C for 30 minutes, the mixture was stirred at room temperature for 2 hours. Water (180 mL) and 2 N hydrochloric acid (186 mL) were added at 0°C, and the mixture was removed by distillation under reduced pressure. The resulting residue was extracted four times with ethyl acetate, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After distillation under reduced pressure, the resulting residue (27.9 g, 90%) was used directly 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 obtained in step 1 above (27.9 g, 107 mmol) 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 saturated citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and then removed by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 50:50 (v / v)] to obtain the target product (32.5 g, 81%). 1 H-NMR(CDCl3)δ:7.39-7.34(5H,m),5.23-5.11(2H,m),4.10-3.48(4H,m),3.16-3.14(1H,m),2 .15-2.04(1H,m),1.81-1.77(1H,m),0.91-0.88(9H,m),0.65-0.55(4H,m),0.08-0.01(6H,m). 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 obtained in step 2 above (32.5 g, 86.5 mmol) 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 removed by reduced pressure distillation to obtain the target product (21.3 g, quantitative). 1 H-NMR(CDCl3)δ:3.79-3.77(1H,m),3.71-3.69(1H,m),3.65-3.60(1H,m),3.01-2.98 (2H,m),1.81-1.71(2H,m),0.90(9H,s),0.65-0.57(4H,m),0.08(3H,s),0.07(3H,s). 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 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) were dissolved in dichloromethane (500 mL), to which 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, the compound obtained in step 3 above (34.1 g, 141 mmol) and triethylamine (29.4 mL, 212 mmol) were slowly added dropwise in dichloromethane (100 mL) at -60 °C. After stirring the reaction solution overnight at room temperature, saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the reaction mixture was extracted with chloroform. The organic layer was washed with water and saturated brine and dried over anhydrous magnesium sulfate. Ethyl acetate and diethyl ether were added to the residue obtained by vacuum distillation, and the solid components were removed by filtration. The filtrate was then distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 25:75 (v / v)] to obtain the target product (55.0 g, 66%). 1 H-NMR(CDCl3)δ:7.72-7.66(1H,m),6.80-6.73(1H,m),4.53-4.49(1H,m),4.04-3.95(1H,m),3.91-3.8 8(3H,m),3.59-3.54(1H,m),3.36-3.25(0.5H,m),3.01-2.96(1.5H,m),2.24-2.20(0.3H,m),2.09-2.05 (0.7H,m),2.00-1.97(0.7H,m),1.69-1.67(0.3H,m),1.32-1.24(3H,m),1.12-1.05(18H,m),0.93-0.91 (6H,m),0.79-0.77(3H,m),0.71-0.62(2H,m),0.57-0.40(2H,m),0.12-0.10(4H,m),0.11-0.15(2H,m). 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 obtained in step 4 above (55.0 g, 92.8 mmol) 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 starting material had disappeared, the reaction mixture was filtered, and the filtrate was removed by reduced pressure distillation. The resulting target product (52.2 g, 100%) was used directly in the next reaction. 1 H-NMR(CDCl3)δ:6.71(1H,s),6.25(1H,s),4.55-4.28(2H,m),3.97(1H,m),3.75-3.62(3H,m),3.70(3H,s),3.09-3.07(1H,m),2 .24-2.19(1H,m),1.81-1.68(1H,m),1.27-1.22(3H,m),1.09-1.05(18H,m),0.90(9H,s),0.65-0.46(4H,m),0.07-0.03(6H,m). 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 obtained in step 5 above (18.6 g, 33.0 mmol) 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 were consumed, water was added to the reaction mixture, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 40:60 (v / v)] to obtain the target product (23.5 g, 74%). 1 H-NMR(CDCl3)δ:8.99(1H,m),8.58(1H,s),7.80(1H,s),7.55-7.53(2H,m),7.34-7.32(2H,m),6.77-6.75(2H,m),5.9 4-5.87(1H,m),5.40-5.38(1H,m),5.33-5.29(1H,m),5.23-5.21(1H,m),5.13(1H,m),5.10(2H,m),4.69-4.64(1H,m) ,4.62-4.52(2H,m),4.06-4.03(1H,m),3.98(1H,m),3.76-3.65(6H,m),3.04(1H,m),2.28-2.26(1H,m),2.18-2.13(1 H,m),1.46(3H,m),1.32-1.25(3H,m),1.11-1.09(18H,m),0.99-0.84(15H,m),0.65-0.40(4H,m),0.08-0.00(6H,m). 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 To a solution of the compound obtained in step 6 above (23.5 g, 24.3 mmol) in THF (50 mL), methanol (50 mL), and water (44 mL), acetic acid (200 mL) was added at room temperature. The reaction mixture was stirred at room temperature. After the starting material was removed, the reaction mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 0:100 (v / v)] to obtain the target product (18.0 g, 87%). 1 H-NMR(CDCl3)δ:8.64-8.62(1H,m),8.50(1H,m),7.69(1H,m),7.55-7.53(2H,m),7.34-7.32(2H,m),6.79-6.75 (3H,m),5.91-5.89(1H,m),5.39(1H,m),5.32-5.29(1H,m),5.23-5.21(1H,m),4.68-4.54(4H,m),4.31(1H,m),4 .06-4.04(1H,m),3.81-3.79(3H,m),3.76(3H,s),3.63-3.61(1H,m),3.13-3.11(1H,m),2.16-2.13(1H,m),1.8 7-1.81(2H,m),1.46-1.43(3H,m),1.30-1.24(3H,m),1.12-1.08(18H,m),0.98-0.91(6H,m),0.63-0.45(4H,m). 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 To a solution of dimethyl sulfoxide (3.75 mL, 52.8 mmol) in dichloromethane (300 mL), oxalyl chloride (2.17 mL, 25.3 mmol) was slowly added dropwise at -78°C under a nitrogen atmosphere. After addition, the reaction mixture was stirred at -78°C. To the reaction mixture, a solution of the compound obtained in step 7 (18.0 g, 21.1 mmol) in dichloromethane (50.0 mL) was slowly added dropwise. Triethylamine (14.6 mL, 105 mmol) was added to the reaction solution at -78°C. After addition, the refrigerant bath was removed and the temperature was slowly raised to room temperature. After the starting materials had disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with chloroform (200 mL). The organic layer was washed with water and saturated brine and dried over anhydrous magnesium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 0:60 (v / v)] to obtain the target product (16.5 g, 92%). 1H-NMR(CDCl3)δ:8.51-8.36(1H,m),7.54-7.38(2H,m),7.22-7.07(3H,m),6.73-6.64(1H,m),5.94-5.8 7(2H,m),5.33-5.22(3H,m),5.09(1H,m),4.97(1H,m),4.64-4.58(4H,m),4.02-4.00(1H,m),3.86-3.83 (3H,m),3.75-3.70(1H,m),3.61-3.54(2H,m),3.38-3.29(1H,m),2.40(1H,m),2.16-2.14(1H,m),1.74- 1.71(1H,m),1.44(3H,m),1.18-1.16(3H,m),1.05-1.00(18H,m),0.97-0.92(6H,m),0.72-0.60(4H,m). 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-alanineamide To a solution of the compound obtained in step 8 above (12.0 g, 14.1 mmol) and 2,6-lutidine (6.58 mL, 56.5 mmol) in dichloromethane (200 mL), tert-butyldimethylsilyl trifluoromethylsulfonate (9.73 mL, 42.3 mmol) was slowly added dropwise at 0°C under a nitrogen atmosphere. After stirring for 10 minutes under ice cooling, the ice bath was removed and the mixture was stirred at room temperature. After the starting materials had disappeared, water was added to the reaction mixture, and the reaction mixture was extracted with chloroform. The organic layer was washed with water and saturated brine and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 25:75 (v / v)] to obtain the target product (8.12 g, 60%). 1 H-NMR(CDCl3)δ:8.67-8.45(1H,m),7.50-7.44(2H,m),7.19(1H,s),7.13(2H,m),6.95(2H,m),6.62-6.57(2H,m), 6.01(1H,m),5.95-5.86(1H,m),5.33-5.13(3H,m),4.82(1H,m),4.65-4.54(3H,m),4.03-4.01(1H,m),3.84-3.82 (3H,m),3.73-3.66(1H,m),3.50-3.48(1H,m),3.27(1H,m),2.37-2.33(1H,m),2.19-2.13(1H,m),1.54-1.43(3H, m),1.22-1.13(3H,m),1.10-1.00(18H,m),0.97-0.91(6H,m),0.81(9H,s),0.76-0.59(4H,m),0.19--0.09(6H,m). 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 To a solution of the compound obtained in step 9 above (8.12 g, 8.42 mmol) in N,N-dimethylformamide (90 mL) and water (2 mL), lithium acetate (0.611 g, 9.26 mmol) was added and the mixture was stirred at room temperature. After the starting material had disappeared, water was added to the reaction mixture and extracted with ethyl acetate. The organic layer was washed with water and saturated brine and dried over anhydrous sodium sulfate. After filtration, the residue obtained by distillation under reduced pressure was purified by silica gel column chromatography [hexane:ethyl acetate = 100:0 (v / v) ~ 0:100 (v / v)] to obtain the target product (5.48 g, 81%). 1 H-NMR(CDCl3)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 obtained in step 10 above (2.40 g, 2.97 mmol) was reacted in the same manner as in step 1 of Example 4 to obtain the target product (2.73 g, 99%). 1 H-NMR(DMSO-D6)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]azosin-5(6H)-yl)-4-oxobutanoyl]glycylglycine To a mixture of glycylglycine (0.328 g, 2.49 mmol), N,N-diisopropylethylamine (0.433 mL, 2.49 mmol), and N,N-dimethylformamide (20 mL), 1-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidine-2,5-dione (1.00 g, 2.49 mmol, Click Chemistry Tools) and water (10 mL) were added at room temperature, and the mixture was stirred overnight at room temperature. The residue was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform: CMW = 100:0 (v / v) ~ 0:100 (v / v)] to obtain the target product (0.930 g, 89%). CMW refers to a partition organic layer with a chloroform:methanol:water ratio of 7:3:1 (v / v / v). 1 H-NMR(DMSO-D6)δ:12.58(1H,s),8.14-8.12(1H,m),8.08-8.07(1H,m),7.69-7.68(1H,m),7.62-7.61(1H,m),7.53-7.45(3H,m),7.40-7.29( 3H,m),5.05-5.01(1H,m),3.73-3.72(2H,m),3.66-3.60(3H,m),2.66- 2.60(1H,m),2.33-2.24(1H,m),2.08-2.04(1H,m),1.81-1.77(1H,m). MS(APCI,ESI)m / z:420[(M+H) + ].

[0329] Step 2: 2,5-Dioxopyrrolidine-1-yl N-[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]glycylglycinate To a solution of the compound obtained in step 1 above (0.612 g, 1.46 mmol) and N-hydroxysucciimide (0.168 g, 1.459 mmol) in dichloromethane (6 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.420 g, 2.19 mmol) was added and the mixture was stirred at room temperature for 21 hours. The mixture was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform: CMW = 100:0 (v / v) ~ 0:100 (v / v)] to obtain the target product (0.375 g, 50%). CMW refers to a partition organic layer with a chloroform:methanol:water ratio of 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) was dissolved in THF (150mL) and ethanol (150mL). Under a nitrogen atmosphere, 5% palladium carbon (54% water, 10.0g) was added, and the reaction solution was stirred under a hydrogen atmosphere at room temperature for three days. Chloroform was added to the reaction solution, and after filtration with Celite, the filtrate was removed by reduced pressure distillation. 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%). 1H-NMR(CDCl3)δ:7.36(1H,s),7.25(1H,s),6.01(1H,s),5.45-5.43(1H,m) ,4.69-4.67(1H,m),4.60-4.55(1H,m),4.23-4.21(1H,m),3.96(3H,s),3.7 6-3.68(2H,m),3.63-3.61(1H,m),3.56-3.53(1H,m),2.88-2.83(1H,m),2. 03-2.00(1H,m),1.00-0.98(2H,m),0.87(9H,s),0.10(6H,s),0.02(9H,s). MS (APCI, ESI) m / z: 523 (M+H) +

[0331] Step 2: (2R,11aS)-8-[(5-bromopentyl)oxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione To a solution of the compound obtained in step 1 above (10.8 g, 20.7 mmol) in N,N-dimethylformamide (30 mL), 1,5-dibromopentane (23.8 g, 103 mmol) and potassium carbonate (3.43 g, 24.8 mmol) were added at room temperature. After stirring at room temperature for 3 hours, water was added to the reaction solution and extracted with ethyl acetate. The resulting organic layer was washed with saturated brine, dried over sodium sulfate, and then removed by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 90:10 (v / v) ~ 50:50 (v / v)] to obtain the target product (14.5 g, quantitative). 1H-NMR(CDCl3)δ:7.34(1H,s),7.21(1H,s),5.52-5.49(1H,m),4.63-4.62(1H,m),4.58 -4.55(1H,m),4.24-4.22(1H,m),4.07-4.04(2H,m),3.92(3H,s),3.82-3.64(3H,m),3 .56-3.53(1H,m),3.45-3.43(2H,m),2.86-2.84(1H,m),2.04-2.00(1H,m),1.97-1.87 (4H,m),1.66-1.62(2H,m),1.01-0.98(2H,m),0.87(9H,s),0.10(6H,s),0.04(9H,s). MS(APCI,ESI)m / z:673[ 81 Br,(M+H) + ],671[ 79 Br,(M+H) + ].

[0332] Step 3: (2R,11aS)-8-[(5-bromopentyl)oxy]-2-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione To a THF (40 mL) solution of the compound (21.5 mmol) obtained in step 2 above, a 1 mol / L tetrabutylammonium fluoride THF solution (28.0 mL, 28.0 mmol) was added at 0°C. After stirring at room temperature for 30 minutes, water was added to the reaction solution, and it was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine. After drying with sodium sulfate, the residue was removed by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography [chloroform:methanol = 97.5:2.5 (v / v) ~ 92.5:7.5 (v / v)] to obtain the target product (11.3 g, 94%). 1H-NMR(CDCl3)δ:7.34(1H,s),7.21(1H,s),5.53-5.50(1H,m),4.69-4.64( 2H,m),4.32-4.30(1H,m),4.10-4.00(2H,m),3.91(3H,s),3.88-3.75(2H,m ),3.73-3.64(2H,m),3.45-3.44(2H,m),2.99-2.96(1H,m),2.15-2.09(1H ,m),1.99-1.85(5H,m),1.68-1.62(2H,m),1.01-0.95(2H,m),0.04(9H,s). MS(APCI,ESI)m / z:559[ 81 Br,(M+H) + ],557[ 79 Br,(M+H) + ].

[0333] Step 4: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2,5,11(3H,10H,11aH)-trione The compound obtained in step 3 above (11.3g, 20.2 mmol), tetrabutylammonium bromide (0.325g, 1.01 mmol), and potassium bromide (0.240g, 2.02 mmol) were dissolved in saturated sodium bicarbonate aqueous solution (60 mL) and dichloromethane (60 mL). nor-AZADO (0.0279g, 0.202 mmol) and sodium hypochlorite pentahydrate (2.03g, 27.2 mmol) were added at 0°C, and the mixture was stirred at 0°C for 30 minutes. Since some raw material remained, sodium hypochlorite pentahydrate (1.00g, 13.4 mmol) was added at 0°C, and the mixture was stirred at 0°C for 15 minutes. Furthermore, sodium hypochlorite pentahydrate (0.300g, 4.03 mmol) was added at 0°C, and the mixture was stirred at 0°C for 15 minutes, and the disappearance of the raw material was confirmed by TLC. An aqueous solution of sodium thiosulfate was added to the reaction solution, extracted with chloroform, and the resulting organic layer was dried over sodium sulfate. The residue was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 75:25 (v / v) ~ 40:60 (v / v)] to obtain the target product (9.74 g, 87%). 1 H-NMR(CDCl3)δ:7.33(1H,s),7.24(1H,s),5.56-5.53(1H,m),4.71-4.69(1H,m) ,4.66-4.63(1H,m),4.27-4.22(1H,m),4.12-4.02(2H,m),3.93-3.88(4H,m),3.8 2-3.75(1H,m),3.69-3.67(1H,m),3.61-3.56(1H,m),3.46-3.44(2H,m),2.82-2. 77(1H,m),1.97-1.89(4H,m),1.68-1.64(2H,m),1.05-0.93(2H,m),0.04(9H,s). MS(APCI,ESI)m / z:557[ 81 Br,(M+H) + ],555[ 79 Br,(M+H) + ].

[0334] Step 5: (11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl trifluoromethanesulfonate To a solution of the compound obtained in step 4 above (9.74 g, 17.5 mmol) in dichloromethane (160 mL), 2,6-lutidine (8.17 mL, 70.1 mmol) was added at -40°C and the mixture was stirred at -40°C for 10 minutes. Trifluoromethanesulfonic anhydride (8.85 mL, 52.6 mmol) was added to the reaction solution at -40°C and the mixture was stirred at -40°C for 30 minutes. A 10% aqueous citric acid solution was added to the reaction solution and extracted with chloroform, and the resulting organic layer was dried over sodium sulfate. The residue was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 95:5 (v / v) ~ 70:35 (v / v)], followed by purification by NH2 silica gel chromatography [hexane:ethyl acetate = 95:5 (v / v) ~ 65:35 (v / v)] to obtain the target product (7.10 g, 59%). 1 H-NMR(CDCl3)δ:7.32(1H,s),7.24(1H,s),7.15-7.14(1H,m),5.56-5.53(1 H,m),4.70-4.68(1H,m),4.66-4.63(1H,m),4...

Claims

1. An antibody or antigen-binding fragment of the antibody that binds to CLDN6 and / or CLDN9, comprising a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 9, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 10, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 11, and a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 5, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 6, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO:

7.

2. An antibody or antigen-binding fragment of the antibody that binds to CLDN6 and / or CLDN9, comprising a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 54 and a light chain variable region consisting of the amino acid sequence described in SEQ ID NO:

38.

3. An antibody or antigen-binding fragment of the antibody according to claim 1 or 2, comprising one or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, 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.

4. An antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 3, wherein one or more amino acid residues are deleted at the carboxyl terminus of the heavy chain.

5. The antibody or antigen-binding fragment of the antibody according to claim 4, wherein one amino acid residue is deleted at the carboxyl terminus of both of the two heavy chains.

6. The antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 5, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

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