Anti- CDH6 antibodies and Anti- CDH6 antibody-drug conjugates

Abstract

To provide antibodies specifically binding to CDH6 and having high internalizing activity.SOLUTION: Provided are antibodies having internalizing activity of binding to CDH6, antibody-drug conjugates of the antibodies and drugs having anti-tumor activity, pharmaceutical products having therapeutic effects on tumors using the antibody-drug conjugates, and methods for treating tumors using the antibodies, the antibody-drug conjugates, or the pharmaceutical products. The present invention provides anti- CDH6 antibodies having internalizing activity, antibody-drug conjugates of the antibodies and drugs having anti-tumor activity, and pharmaceutical products and methods for treating tumors using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-CDH6 antibody that binds to CDH6 and has internalization activity, a method for producing the anti-CDH6 antibody, an antibody-drug conjugate comprising the antibody, an antitumor agent comprising the antibody-drug conjugate, and the like. [Background technology]

[0002] Cadherins are glycoproteins present on the cell membrane surface. They function as cell-cell adhesion molecules and signaling molecules involved in cell-cell interactions by binding together their N-terminal extracellular domains in a calcium ion-dependent manner. Within the cadherin superfamily, the classical cadherin group is a single-pass transmembrane protein consisting of five extracellular domains (EC domains), one transmembrane region, and an intracellular domain. Based on amino acid sequence homology, classical cadherins are classified into the type I family, represented by E-cadherin and N-cadherin, and the type II family.

[0003] Cadherin-6 (CDH6) is a single-pass transmembrane protein consisting of 790 amino acids and classified as a type II cadherin family member, with the N-terminus extracellular and the C-terminus intracellular. The human CDH6 gene was first cloned in 1995 (Non-Patent Document 1) and can be accessed by accession numbers such as NM_004932 and NP_004923 (NCBI).

[0004] CDH6 is specifically expressed in the brain and kidney during development, and has been reported to play an important role in the formation of central nervous system circuits (Non-Patent Documents 2 and 3) and in the development of kidney nephrons (Non-Patent Documents 4 and 5). In normal adult tissues, CDH6 expression is limited to renal tubules, bile duct epithelial cells, etc.

[0005] On the other hand, it is known that CDH6 expression is specifically elevated in tumor sites in several types of adult cancers. It has been reported that CDH6 expression correlates with poor prognosis in human renal cell carcinoma, particularly renal clear cell carcinoma, and that it may be used as a tumor marker (Non-Patent Documents 6 and 7). High CDH6 expression has also been reported in human ovarian cancer (Non-Patent Document 8), and CDH6 has been reported to be involved in epithelial-mesenchymal transition and metastasis in human thyroid cancer (Non-Patent Document 9). CDH6 expression has also been reported in human bile duct carcinoma and human small cell lung carcinoma (Non-Patent Documents 12 and 13).

[0006] Cancer is a leading cause of death, and its incidence is expected to increase with the aging of the population, yet there is still an insufficient need for treatment. Conventional chemotherapy drugs have side effects due to their low selectivity, which can cause damage to not only tumor cells but also normal cells, and the inability to administer sufficient amounts of drug can result in insufficient efficacy. For this reason, in recent years, more selective molecular targeted drugs and antibody drugs have been developed that target molecules that exhibit characteristic mutations or high expression in cancer cells, or specific molecules involved in the carcinogenesis of cells.

[0007] Antibodies are highly stable in the blood and bind specifically to target antigens, which is expected to reduce side effects. Numerous antibody drugs targeting molecules highly expressed on the surface of cancer cells have been developed. One technology utilizing the antigen-specific binding ability of antibodies is antibody-drug conjugates (ADCs). ADCs are antibodies that bind to antigens expressed on the surface of cancer cells and internalize the antigens into the cells through this binding, and are coupled to cytotoxic drugs. ADCs are expected to efficiently deliver drugs to cancer cells, thereby accumulating the drug within the cells and killing them (Non-Patent Document 10, Patent Documents 1 and 2). For example, Adcetris™ (brentuximab vedotin), an anti-CD30 monoclonal antibody conjugated to monomethyl auristatin E, has been approved as a treatment for Hodgkin's lymphoma and anaplastic large cell lymphoma. Additionally, Kadcyla™ (trastuzumab emtansine), an anti-HER2 monoclonal antibody conjugated to emtansine, is used to treat HER2-positive advanced or recurrent breast cancer.

[0008] Characteristics of target antigens suitable for ADCs as antitumor drugs include high and specific expression on the surface of cancer cells and low or no expression on normal cells, the ability to be internalized within the cells, and the lack of secretion of the antigen from the cell surface. Important characteristics of antibodies suitable for ADCs include high internalization capacity in addition to specific binding to the target antigen. The internalization capacity of an antibody depends on the properties of both the target antigen and the antibody. It is difficult to predict an antigen-binding site suitable for internalization from the molecular structure of the target, or to easily predict an antibody with high internalization capacity from the binding strength or physical properties of the antibody. Therefore, obtaining antibodies with high internalization capacity for target antigens is an important challenge in developing highly effective ADCs (Non-Patent Document 11).

[0009] A known ADC targeting CDH6 is one in which DM4 is conjugated to an anti-CDH6 antibody that specifically binds to the EC domain 5 (EC5) of CDH6 (Patent Document 3). [Advanced Technology Documents] [Chartered documents]

[0010]

Patent Document 1

Patent document 2

Patent Document 3

Non-licensed literature

[0011] [Non-licensed document 1] Shimoyama Y,et al.,Cancer Research,2206-2211,55,May 15,1995 [Non-licensed document 2] Inoue T,et al.,Developmental Biology,183-194,1997 [Non-licensed document 3] Osterhout JA,et al.,Neuron,632-639,71,Aug 25,2011

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

[0012] An object of the present invention is to provide an antibody that specifically binds to CDH6 and has high internalization activity, an antibody-drug conjugate containing the antibody and having high anti-tumor activity, a pharmaceutical product that uses the antibody-drug conjugate and has a therapeutic effect against tumors, and a method for treating tumors using the antibody, antibody-drug conjugate, or pharmaceutical product. [Means for solving the problem]

[0013] The present inventors conducted extensive research to achieve the above-mentioned object and surprisingly found that an antibody that specifically binds to extracellular domain 3 (herein also referred to as EC3) of CDH6 has extremely high internalization activity in cells expressing CDH6 and is useful as an ADC antibody. Furthermore, the inventors found that an anti-CDH6 antibody-drug conjugate in which an intracellularly toxic drug is bound to the anti-CDH6 antibody via a linker with a specific structure exhibits stronger antitumor activity than conventional CDH6-drug conjugates.

[0014] The present invention includes the following inventions: [1] An antibody or a functional fragment of the antibody that specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and has the ability to be internalized into cells; [2] The following (1) to (5) for binding to the amino acid sequence set forth in SEQ ID NO: 4: (1) An antibody having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 53 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 56. (2) An antibody having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 69. (3) An antibody having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 73. (4) An antibody having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 73; and (5) An antibody having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 77. the antibody or functional fragment thereof according to [1], which has competitive inhibitory activity with at least one of the antibodies selected from the group consisting of: [3] (1) to (4) below: (1) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 24, (3) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34, and (4) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 44, and CDRL1, CDRL2, and CDRL3 selected from the group consisting of: (5) to (9) below: (5) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, (6) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 27, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 29, (7) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, (8) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and (9) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of: [1] The antibody or functional fragment of the antibody according to any one of [1] and [2]. [4] (1) to (5) below: (1) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 27, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 29, (3) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 39; (4) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 44; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 49; (5) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and CDRH1, CDRH2 and CDRH3 selected from the group consisting of: The antibody or functional fragment of the antibody according to any one of [1] to [3]; [5] The antibody or functional fragment of any one of [1] to [4] above, which is humanized; [6] (1) to (4) below: (1) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63; (2) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 67; (3) an amino acid sequence having at least 95% sequence identity to the sequences of the framework regions other than each CDR sequence in the amino acid sequences of (1) to (2); and (4) An amino acid sequence in which one or several amino acids are deleted, substituted, or added in the framework region sequences other than each CDR sequence in the amino acid sequences of (1) to (3). and a light chain variable region selected from the group consisting of: (5) to (9) below: (5) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 71; (6) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 75; (7) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 79; (8) An amino acid sequence having at least 95% sequence identity to the sequences of the framework regions other than each CDR sequence in the amino acid sequences of (5) to (7), and (9) An amino acid sequence in which one or several amino acids are deleted, substituted, or added in the framework region sequences other than each CDR sequence in the amino acid sequences of (5) to (8). a heavy chain variable region selected from the group consisting of:

[0023] The antibody or functional fragment of the antibody according to any one of [1] to [5], [7] (1) to (4) below: (1) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 71; (2) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 75; (3) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 67 and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 75, or (4) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 79; a light chain variable region and a heavy chain variable region of any one of

[0023] The antibody or functional fragment of the antibody according to any one of [1] to [6], [8] (1) to (4) below: (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 69; (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73; (3) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73, or (4) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 77; the antibody or functional fragment thereof according to any one of [1] to [7], [9] The antibody or functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 69;

[10] The antibody or functional fragment of [8], which has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73;

[11] The antibody or functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73;

[12] The antibody or functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 77;

[13] A functional fragment of the antibody according to any one of [1] to

[12] , wherein the functional fragment is selected from the group consisting of Fab, F(ab')2, Fab', and Fv;

[14] A polynucleotide encoding the antibody or a functional fragment of the antibody according to any one of [1] to

[13] ;

[15] (1) to (5) below: (1) A polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; (2) a polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 24; and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 27, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 29; (3) a polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 39; (4) A polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 44, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 49; and (5) A polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; The polynucleotide according to

[14] , comprising any one of the polynucleotides selected from the group consisting of:

[16] The polynucleotide according to

[14] or

[15] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 69;

[17] The polynucleotide according to

[14] or

[15] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73;

[18] The polynucleotide according to

[14] or

[15] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73;

[19] The polynucleotide according to

[14] or

[15] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 77;

[20] An expression vector containing the polynucleotide according to any one of

[14] to

[19] .

[21] A host cell transformed with the expression vector according to

[20] ;

[22] The host cell according to

[21] , wherein the host cell is a eukaryotic cell;

[23] A method for producing an antibody or a functional fragment of the antibody, comprising the steps of culturing the host cell according to

[21] or

[22] , and collecting the antibody of interest or a functional fragment of the antibody from the culture obtained in the step;

[24] The antibody or functional fragment of the antibody according to any one of [1] to

[13] , wherein the heavy chain or light chain has undergone 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 to the N-terminus, amidation of a proline residue, pyroglutamic oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus;

[25] The antibody according to

[24] , wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain;

[26] The antibody according to

[25] , wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains;

[27] The antibody according to any one of

[24] to

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

[28] The antibody or functional fragment of the antibody according to any one of the items [1] to

[13] and

[24] to

[27] , wherein glycosylation is regulated to enhance antibody-dependent cellular cytotoxicity;

[29] An antibody-drug conjugate comprising the antibody or functional fragment of the antibody according to any one of the group consisting of [1] to

[13] and

[24] to

[28] , to which a drug is bound;

[30] The antibody-drug conjugate according to

[29] , wherein the drug is an antitumor compound;

[31] The antitumor compound has the following formula:

[0015] [ka]

[0016] the antibody-drug conjugate according to

[30] , which is an antitumor compound represented by the formula:

[32] The antibody and the drug are represented by the following formulas (a) to (f): (a)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, The antibody-drug conjugate according to any one of

[29] to

[31] , wherein the antibody-drug conjugate is linked via a linker having a structure selected from the group consisting of: (Here, the antibody is bound at the terminus of -(Succinimid-3-yl-N). The antitumor compound is bound to the carbonyl group of the -CH2CH2CH2-C(=O)- moiety of (a), (b), (e), or (f), the CH2-O-CH2-C(=O)- moiety of (c), or the CH2CH2-O-CH2-C(=O)- moiety of (d), using the nitrogen atom of the amino group at position 1 as the binding site. In the above formula, GGFG represents an amino acid sequence connected by peptide bonds consisting of glycine-glycine-phenylalanine-glycine. -(Succinimid-3-yl-N)- has the following formula:

[0017] [ka]

[0018] The structure is represented by the formula:

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

[29] to

[32] , wherein the linker is represented by any formula selected from the group consisting of the following (c), (d), and (e): (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

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

[29] to

[33] , wherein the linker is represented by the following formula (c) or (e): (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

[35] The following formula:

[0019] [ka]

[0020] The antibody-drug conjugate according to any one of

[29] to

[34] , having a structure represented by the following formula: Here, AB represents an antibody or a functional fragment of the antibody, and n represents the average number of drug-linker structures bound to the antibody per antibody. The antibody and the linker are bound via a sulfhydryl group derived from the antibody;

[36] The following formula:

[0021] [ka]

[0022] The antibody-drug conjugate according to any one of

[29] to

[34] , having a structure represented by the following formula: Here, AB represents an antibody or a functional fragment of the antibody, and n represents the average number of drug-linker structures bound to the antibody per antibody. The antibody and the linker are bound via a sulfhydryl group derived from the antibody;

[37] The antibody is one of the following (1) to (4): (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 69; (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73; (3) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73, or (4) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 77; the antibody-drug conjugate according to any one of

[29] to

[36] , which is an antibody or a functional fragment of the antibody comprising the light chain and the heavy chain of any one selected from the group consisting of:

[38] The antibody-drug conjugate according to

[37] , wherein the antibody is an antibody or a functional fragment of the antibody, comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 69;

[39] The antibody-drug conjugate according to

[37] , wherein the antibody is an antibody or a functional fragment of the antibody, comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 77;

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

[29] to

[39] , wherein the heavy chain or light chain has undergone 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 to the N-terminus, amidation of a proline residue, pyroglutamic oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus;

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

[29] to

[40] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 1 to 10;

[42] The antibody-drug conjugate according to

[41] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 2 to 8;

[43] The antibody-drug conjugate according to

[42] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 5 to 8;

[44] The antibody-drug conjugate according to

[43] , wherein the average number of the selected drug-linker structures bound per antibody is 7 to 8;

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

[29] to

[44] , a salt thereof, or a hydrate thereof;

[46] The pharmaceutical composition according to

[45] , which is an antitumor drug;

[47] The pharmaceutical composition according to

[46] , wherein the tumor is a tumor that expresses CDH6;

[48] ​​The pharmaceutical composition according to

[46] or

[47] , wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma;

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

[29] to

[44] , a salt thereof, or a hydrate thereof;

[50] The method of treatment according to

[49] , wherein the tumor is a tumor expressing CDH6;

[51] The method of treatment according to

[49] or

[50] , wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma;

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

[29] to

[44] , a salt thereof, or a hydrate thereof, and at least one antitumor drug;

[53] A method for producing an antibody-drug conjugate, comprising a step of reacting the antibody or functional fragment thereof according to any one of the items [1] to

[13] and

[24] to

[28] , or the antibody or functional fragment thereof obtained by the production method according to

[23] , with a drug-linker intermediate compound; or

[54] A method for producing an antibody-drug conjugate, comprising the steps of: culturing the host cell according to

[21] or

[22] ; collecting an antibody of interest or a functional fragment of the antibody from the culture obtained in the step; and reacting the antibody or the functional fragment of the antibody obtained in the step with a drug-linker intermediate compound. [Effects of the Invention]

[0023] The anti-CDH6 antibodies of the present invention are characterized by specifically recognizing the EC domain 3 (EC3) of CDH6 and possessing high internalization activity. Anti-CDH6 antibody-drug conjugates, in which an intracellularly toxic drug is bound to the anti-CDH6 antibody of the present invention via a linker of a specific structure, are expected to achieve excellent antitumor effects and safety when administered to patients with cancer cells expressing CDH6. In other words, the anti-CDH6 antibody-drug conjugates of the present invention are useful as antitumor agents. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the results of flow cytometry analysis of the binding of four rat anti-CDH6 monoclonal antibodies (clone numbers rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or hCDH6-transfected 293T cells. The horizontal axis represents FITC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents the cell count. [Figure 2-1] Figure 2-1 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or the negative control antibody Rat IgG2b to control cells or full-length hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the number of cells. [Figure 2-2] Figure 2-2 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or EC1-deficient hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 2-3] Figure 2-3 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or EC2-deficient hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 2-4]Figure 2-4 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or EC3-deficient hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 2-5] Figure 2-5 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or EC4-deficient hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 2-6] Figure 2-6 shows the binding of four rat anti-CDH6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control to control cells or EC5-deficient hCDH6-transfected 293 cells. The horizontal axis shows FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 3] Figure 3 shows the results of flow cytometry analysis of CDH6 expression on the cell membrane surface of four human tumor cell lines (NIH human ovarian tumor cell lines: OVCAR-3, PA-1, and ES-2, and human renal cell tumor cell line 786-O). The horizontal axis represents FITC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents the number of cells. [Figure 4] Figure 4 shows graphs evaluating the internalization activity of four rat anti-CDH6 antibodies (rG019, rG055, rG056, and rG061) or a rat IgG control in NIH:OVCAR-3 and 786-O cells using the anti-rat IgG reagent Rat-ZAP conjugated with a toxin (saporin) that inhibits protein synthesis, or the toxin-free Goat Anti-Rat IgG, Fc(gamma) Fragment Specific as a negative control. The vertical axis of the graph represents ATP activity (RLU). Below each graph, the cell viability (%) is shown, calculated as the relative viability, with the number of viable cells in wells containing the negative control instead of Rat-ZAP set at 100%. [Figure 5]5 shows the binding of the human chimeric anti-CDH6 antibody chG019 to human CDH6 and monkey CDH6. The horizontal axis shows the antibody concentration, and the vertical axis shows the amount of binding as mean fluorescent intensity. [Figure 6-1] Figures 6-1 and 6-2 show the binding affinity of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02) or the negative control antibody human IgG1 to human CDH6, monkey CDH6, mouse CDH6, and rat CDH6. The horizontal axis represents antibody concentration, and the vertical axis represents the amount of binding as mean fluorescent intensity. [Figure 6-2] Figures 6-1 and 6-2 show the binding affinity of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02) or the negative control antibody human IgG1 to human CDH6, monkey CDH6, mouse CDH6, and rat CDH6. The horizontal axis represents antibody concentration, and the vertical axis represents the amount of binding as mean fluorescent intensity. [Figure 7-1] Figure 7-1 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control antibody hIgG1 to control cells or full-length hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell number. [Figure 7-2] Figure 7-2 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control antibody hIgG1 to control cells or EC1-deficient hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 7-3]Figure 7-3 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control antibody hIgG1 to control cells or EC2-deficient hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell number. [Figure 7-4] Figure 7-4 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control antibody hIgG1 to control cells or EC3-deficient hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell count. [Figure 7-5] Figure 7-5 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control hIgG1 to control cells or EC4-deficient hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell number. [Figure 7-6] Figure 7-6 shows the binding of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), anti-CDH6 antibody NOV0712, or negative control hIgG1 to control cells or EC5-deficient hCDH6-transfected 293α cells. The horizontal axis shows APC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis shows the cell number. [Figure 8] Figure 8 shows the results of flow cytometry examining the expression of human CDH6 in the 786-O / hCDH6 stable-expressing cell line and the parent cell line 786-O. The horizontal axis represents the fluorescence intensity of Alexa Fluor 647, which indicates the amount of antibody binding, and the vertical axis represents the cell number. [Figure 9]9 shows a binding competition assay between four unlabeled humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), the anti-CDH6 antibody NOV0712, or a negative control hIgG1, using (a) labeled NOV0712 or (b) labeled H01L02. The horizontal axis shows the final concentration of unlabeled antibody upon addition, and the vertical axis shows the amount of binding in terms of mean fluorescent intensity. [Figure 10-1] Figure 10-1 shows graphs evaluating the internalization activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), the anti-CDH6 antibody NOV0712, and a negative control antibody in NIH:OVCAR-3 cells using the anti-human IgG reagent Hum-ZAP conjugated with a protein synthesis-inhibiting toxin (saporin) or the negative control F(ab')2 Fragment Goat Anti-human IgG, Fc(gamma) Fragment Specific, which is not conjugated with a toxin. The vertical axis of the graph represents ATP activity (RLU). Below each graph, cell viability (%) is shown, calculated as the relative viability, with the number of viable cells in wells containing the negative control instead of Hum-ZAP set at 100%. [Figure 10-2] Figure 10-2 shows graphs evaluating the internalization activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), the anti-CDH6 antibody NOV0712, and a negative control antibody in 786-O cells using Hum-ZAP, an anti-human IgG reagent conjugated with a protein synthesis-inhibiting toxin (saporin), or the negative control F(ab')2 Fragment Goat Anti-human IgG, Fc(gamma) Fragment Specific, which is not conjugated with a toxin. The vertical axis of the graph represents ATP activity (RLU). Below each graph, the cell viability (%) is shown, calculated as the relative viability, with the number of viable cells in wells containing the negative control instead of Hum-ZAP set at 100%. [Figure 10-3]Figure 10-3 shows graphs evaluating the internalization activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03, and H04L02), the anti-CDH6 antibody NOV0712, and a negative control antibody in PA-1 cells using Hum-ZAP, an anti-human IgG reagent conjugated with a protein synthesis-inhibiting toxin (saporin), or the negative control F(ab')2 Fragment Goat Anti-human IgG, Fc(gamma) Fragment Specific, which is not conjugated with a toxin. The vertical axis of the graph represents ATP activity (RLU). Below each graph, cell viability (%) is shown, calculated as the relative viability, with the number of viable cells in wells containing the negative control instead of Hum-ZAP set at 100%. [Figure 11] 11 shows the in vitro cytostatic activity of four humanized hG019-drug conjugates (H01L02-DXd, H02L02-DXd, H02L03-DXd, and H04L02-DXd) or NOV0712-DM4 against PA-1 cells. The horizontal axis represents the antibody-drug conjugate concentration, and the vertical axis represents cell viability (%). [Figure 12] Figure 12 shows the in vivo antitumor effects of four humanized hG019-drug conjugates (H01L02-DXd, H02L02-DXd, H02L03-DXd, and H04L02-DXd) or NOV0712-DM4. The effects were evaluated using an animal model in which the CDH6-positive human renal cell carcinoma cell line 786-O was transplanted into immunodeficient mice. The horizontal axis indicates the number of days, the vertical axis indicates tumor volume, and the error bars indicate standard error (SE). [Figure 13] Figure 13 shows the in vivo antitumor effects of humanized hG019-drug conjugates H01L02-DXd, NOV0712-DM4, and NOV0712-DXd. The effects were evaluated using an animal model in which the CDH6-positive human ovarian tumor cell line PA-1 was transplanted into immunodeficient mice. The horizontal axis indicates the number of days, the vertical axis indicates tumor volume, and the error bars indicate SE values. [Figure 14]Figure 14 shows the in vivo antitumor effects of humanized hG019-drug conjugates H01L02-DXd and NOV0712-DM4. The effects were evaluated using an animal model in which the CDH6-positive human ovarian tumor cell line NIH:OVCAR-3 was transplanted into immunodeficient mice. The horizontal axis indicates the number of days, the vertical axis indicates tumor volume, and the error bars indicate SE values. [Figure 15] Figure 15 shows the in vivo antitumor effects of humanized hG019-drug conjugates H01L02-DXd and NOV0712-DM4. The effects were evaluated using an animal model in which the CDH6-positive human renal cell carcinoma cell line 786-O was transplanted into immunodeficient mice. The horizontal axis indicates the number of days, the vertical axis indicates tumor volume, and the error bars indicate SE values. [Figure 16] Figure 16 shows the in vivo antitumor effects of humanized hG019-drug conjugates H01L02-DXd and NOV0712-DM4. The effects were evaluated using an animal model in which the CDH6-negative human ovarian tumor cell line ES-2 was transplanted into immunodeficient mice. The horizontal axis indicates the number of days, the vertical axis indicates tumor volume, and the error bars indicate SE values. DETAILED DESCRIPTION OF THE INVENTION

[0025] Preferred embodiments for carrying out the present invention will now be described with reference to the drawings. Note that the embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention should not be construed as being narrow.

[0026] In this specification, the terms "cancer" and "tumor" are used interchangeably.

[0027] As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA and cRNA.

[0028] As used herein, the terms "polynucleotide" and "nucleotide" are used interchangeably with "nucleic acid" and include DNA, RNA, probes, oligonucleotides, and primers. As used herein, "polynucleotide" and "nucleotide" can be used interchangeably unless otherwise specified.

[0029] As used herein, the terms "polypeptide" and "protein" may be used interchangeably.

[0030] As used herein, the term "cells" includes cells within an animal body and cultured cells.

[0031] As used herein, "CDH6" may be used interchangeably with CDH6 protein. As used herein, human CDH6 may be referred to as "hCDH6."

[0032] As used herein, the term "cytotoxic activity" refers to the induction of pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0033] As used herein, "exerting toxicity within a cell" means exhibiting toxicity within a cell in some form, and refers to not only direct damage but also any effect on the structure, function, or metabolism of a cell, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, reduction in the activity of various enzymes, or inhibition of the action of cell growth factors.

[0034] As used herein, the term "functional antibody fragment," also referred to as "antigen-binding fragment of antibody," refers to a partial fragment of an antibody that retains antigen-binding activity, and includes Fab, F(ab'), Fv, scFv, diabody, linear antibody, and multispecific antibody fragments formed from antibody fragments. Antigen-binding fragments of antibodies also include Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab') under reducing conditions. However, they are not limited to these molecules, as long as they retain the ability to bind to an antigen. These antigen-binding fragments also include proteins produced in suitable host cells using genetically engineered antibody genes, as well as full-length antibody protein molecules treated with appropriate enzymes.

[0035] As used herein, "epitope" refers to a partial peptide or partial three-dimensional structure of CDH6 to which a specific anti-CDH6 antibody binds. Epitopes, which are partial peptides of CDH6, can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of the antigen are prepared. These partial structures can be prepared using known oligonucleotide synthesis techniques. For example, a series of polypeptides sequentially shortened to appropriate lengths from the C-terminus or N-terminus of CDH6 are prepared using recombinant DNA techniques well known to those skilled in the art. The reactivity of antibodies against these polypeptides is then examined to determine the approximate recognition site. After this, shorter peptides are synthesized and their reactivity with these peptides is examined to determine the epitope. Furthermore, if an antibody that binds to a membrane protein consisting of multiple extracellular domains has an epitope consisting of a three-dimensional structure composed of multiple domains, the domain to which it binds can be determined by modifying the amino acid sequence of a specific extracellular domain to alter the three-dimensional structure. The epitope, which is the partial three-dimensional structure of an antigen to which a specific antibody binds, can also be determined by identifying the amino acid residues of the antigen adjacent to the antibody using X-ray structural analysis.

[0036] As used herein, "binding to the same epitope" refers to an antibody that binds to a common epitope. If a second antibody binds to a partial peptide or partial three-dimensional structure bound by a first antibody, it can be determined that the first and second antibodies bind to the same epitope. Alternatively, by confirming that the second antibody competes with the binding of the first antibody to the antigen (i.e., that the second antibody interferes with the binding of the first antibody to the antigen), it can be determined that the first and second antibodies bind to the same epitope, even if the specific sequence or structure of the epitope has not been determined. As used herein, "binding to the same epitope" refers to a case in which the first and second antibodies are determined to bind to a common epitope by one or both of the determination methods. If the first and second antibodies bind to the same epitope and the first antibody has a special effect such as antitumor activity or internalization activity, it is expected that the second antibody will also have a similar activity.

[0037] As used herein, "CDR" refers to complementarity determining region (CDR). It is known that the heavy and light chains of an antibody molecule each contain three CDRs. CDRs, also known as hypervariable regions, are located within the variable regions of the heavy and light chains of an antibody and are particularly highly variable in their primary structure. They are separated into three regions in the primary structure of the heavy and light chain polypeptide chains. As used herein, the CDRs of an antibody are represented as CDRH1, CDRH2, and CDRH3 for the heavy chain CDRs, from the amino-terminal end of the heavy chain amino acid sequence, and as CDRL1, CDRL2, and CDRL3 for the light chain CDRs, from the amino-terminal end of the light chain amino acid sequence. These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0038] As used herein, "hybridizing under stringent conditions" refers to hybridizing at 68°C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (Clontech), or hybridizing using a DNA-immobilized filter at 68°C in the presence of 0.7 to 1.0 M NaCl, followed by washing at 68°C using a 0.1 to 2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate), allowing identification, or hybridizing under equivalent conditions.

[0039] In this specification, "one to several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0040] 1.CDH6 Cadherins are glycoproteins present on the cell membrane surface that function as cell-cell adhesion molecules and signaling molecules responsible for cell-cell interactions by binding together their N-terminal extracellular domains in a calcium ion-dependent manner. Of the cadherin superfamily, the group of molecules classified as classic cadherins are single-pass transmembrane proteins consisting of five extracellular domains (EC domains), one transmembrane region, and an intracellular domain.

[0041] CDH6 (Cadherin-6) is a single-pass transmembrane protein consisting of 790 amino acids and classified as a type II cadherin family member, with the N-terminus extracellular and the C-terminus intracellular. The human CDH6 gene was first cloned in 1995 (Non-Patent Document 1) and can be accessed by accession numbers such as NM_004932 and NP_004923 (NCBI).

[0042] The CDH6 protein used in the present invention can be directly purified from CDH6-expressing cells of humans or non-human mammals (rat, mouse, monkey, etc.), or prepared from a cell membrane fraction of such cells. It can also be obtained by synthesizing CDH6 in vitro or by producing it in host cells through genetic engineering. Specifically, genetic engineering can involve incorporating CDH6 cDNA into an expression vector and then synthesizing it in a solution containing enzymes, substrates, and energy sources necessary for transcription and translation, or by expressing CDH6 by transforming other prokaryotic or eukaryotic host cells. Alternatively, the CDH6 protein can be obtained from the above-described genetically engineered CDH6-expressing cells or cell lines expressing CDH6. Alternatively, CDH6 can be expressed in the body of an immunized animal by directly administering an expression vector incorporating CDH6 cDNA to the animal.

[0043] Furthermore, CDH6 also includes proteins that have the amino acid sequence of the above CDH6 in which one or several amino acids have been substituted, deleted, and / or added, and that have biological activity equivalent to that of the protein.

[0044] The human CDH6 protein has the amino acid sequence set forth in SEQ ID NO: 1. The extracellular region of the human CDH6 protein is composed of extracellular domain 1 (also referred to herein as EC1) comprising the amino acid sequence of positions 54 to 159 of the amino acid sequence set forth in SEQ ID NO: 1; extracellular domain 2 (also referred to herein as EC2) comprising the amino acid sequence of positions 160 to 268 of the amino acid sequence set forth in SEQ ID NO: 1; extracellular domain 3 (also referred to herein as EC3) comprising the amino acid sequence of positions 269 to 383 of the amino acid sequence set forth in SEQ ID NO: 1; extracellular domain 4 (also referred to herein as EC4) comprising the amino acid sequence of positions 384 to 486 of the amino acid sequence set forth in SEQ ID NO: 1; and extracellular domain 5 (also referred to herein as EC5) comprising the amino acid sequence of positions 487 to 608 of the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequences of EC1 to EC5 are set forth in SEQ ID NOs: 2 to 6, respectively (Table 1).

[0045] 2. Preparation of Anti-CDH6 Antibody An example of an anti-CDH6 antibody of the present invention is an anti-CDH6 antibody that recognizes an amino acid sequence including the amino acid sequence set forth in SEQ ID NO: 4 and has internalization activity. An example of an anti-CDH6 antibody of the present invention is an anti-CDH6 antibody that specifically recognizes an amino acid sequence including the amino acid sequence set forth in SEQ ID NO: 4 and has internalization activity. An example of an anti-CDH6 antibody of the present invention is an anti-CDH6 antibody that recognizes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4 and has internalization activity. An example of an anti-CDH6 antibody of the present invention is an anti-CDH6 antibody that specifically recognizes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4 and has internalization activity. An antibody that "specifically recognizes an amino acid sequence including the amino acid sequence set forth in SEQ ID NO: 4" or "specifically recognizes the EC3 domain" means that the antibody recognizes or binds to the EC3 domain of CDH6 more strongly than other extracellular domains of CDH6.

[0046] The anti-CDH6 antibodies of the present invention may be derived from any species, preferably human, monkey, rat, mouse, or rabbit. When derived from a species other than human, they are preferably chimerized or humanized using well-known techniques. The antibodies of the present invention may be polyclonal or monoclonal, preferably monoclonal.

[0047] The anti-CDH6 antibodies of the present invention are antibodies that can target tumor cells, i.e., they have the properties of being able to recognize tumor cells, being able to bind to tumor cells, and / or being taken up and internalized within tumor cells, etc. Therefore, the anti-CDH6 antibodies of the present invention can be linked to compounds having anti-tumor activity via a linker to form antibody-drug conjugates.

[0048] Antibody binding to tumor cells can be confirmed using flow cytometry. Antibody uptake into tumor cells can be confirmed using (1) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to visualize the antibody uptake by the cell under a fluorescent microscope (Cell Death and Differentiation, 2008, 15, 751-761), (2) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to measure the amount of fluorescence uptake by the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon intracellular uptake, suppressing cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant complex protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin.

[0049] As used herein, "high internalization ability" means that the viability of CDH6-expressing cells administered with the antibody and a saporin-labeled anti-rat IgG antibody (expressed as a relative rate, with the cell viability when no antibody is added being 100%) is preferably 70% or less, more preferably 60% or less.

[0050] Since an antibody-drug conjugate is bound to a compound that exerts an antitumor effect, it is preferable, but not essential, that the antibody itself have an antitumor effect. For the purpose of specifically and / or selectively exerting the cytotoxicity of an antitumor compound in tumor cells, it is important and preferable that the antibody has the property of being internalized and transported into tumor cells.

[0051] Anti-CDH6 antibodies can be obtained by immunizing animals with a polypeptide antigen and collecting and purifying the antibodies produced in vivo using methods commonly used in this field. Preferably, CDH6 that retains its three-dimensional structure is used as the antigen. One such method is DNA immunization.

[0052] The origin of the antigen is not limited to humans, and animals can also be immunized with antigens derived from animals other than humans, such as mice and rats. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to the heterologous antigen with human antigens.

[0053] Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, 495-497; Kennet, R. ed., Monoclonal Antibodies, 365-367, Plenum Press, NY (1980)), and a monoclonal antibody can be obtained.

[0054] A specific method for obtaining antibodies against CDH6 will be described below.

[0055] (1) Antigen preparation Antigens can be obtained by genetically engineering a gene encoding an antigen protein to produce it in a host cell. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0056] Alternatively, antibodies can be obtained without using an antigen protein by incorporating the cDNA of the antigen protein into an expression vector and administering it to an animal to be immunized, thereby expressing the antigen protein in the body of the animal and producing antibodies against the antigen protein.

[0057] (2) Production of anti-CDH6 monoclonal antibody The anti-CDH6 antibody used in the present invention is not particularly limited, and for example, an antibody specified by the amino acid sequence shown in the sequence listing of the present application can be preferably used. The anti-CDH6 antibody used in the present invention preferably has the following properties: (1) An antibody characterized by the following properties: (a) Specific binding to CDH6 (b) It has the ability to be internalized into CDH6-expressing cells by binding to CDH6. (2) The antibody according to (1) above, wherein CDH6 is human CDH6. (3) It specifically recognizes the EC3 of human CDH6 and has internalization activity. The method for obtaining an antibody against CDH6 of the present invention is not particularly limited as long as an anti-CDH6 antibody can be obtained, but it is preferable to use CDH6 that retains its higher-order structure as the antigen.

[0058] One example of a preferred method for obtaining antibodies is DNA immunization. DNA immunization involves genetically introducing an antigen-expressing plasmid into an animal such as a mouse or rat, causing the antigen to be expressed within the animal, thereby inducing immunity against the antigen. Gene transfer methods include direct intramuscular injection of the plasmid, intravenous injection of transfection reagents such as liposomes or polyethyleneimine, viral vector techniques, injection of gold particles with attached plasmids using a gene gun, and hydrodynamic techniques, in which a large amount of plasmid solution is rapidly injected intravenously. Regarding gene transfer by intramuscular injection of an expression plasmid, a technique known as in vivo electroporation, in which the plasmid is injected intramuscularly and then electroporated at the same site, is known to improve expression levels (Aihara H, Miyazaki J. Nat Biotechnol. 1998 Sep;16(9):867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. Proc Natl Acad Sci USA. 1999 Apr 13;96(8):4262-7.). This technique can be further improved by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug;8(16):1264-70). Hybridomas can also be produced by known methods, for example, using the Hybrimune Hybridoma Production System (Cyto Pulse Sciences).

[0059] Specific examples of obtaining monoclonal antibodies include the following. (a) CDH6 cDNA can be inserted into an expression vector (e.g., pcDNA3.1: Thermo Fisher Scientific) and then the vector can be directly administered to an animal to be immunized (e.g., a rat or mouse) by electroporation, a gene gun, or other methods, thereby expressing CDH6 in the animal, thereby inducing an immune response. The vector can be administered by electroporation or other methods once or multiple times, preferably multiple times, if necessary to increase the antibody titer. (b) collecting tissues (e.g., lymph nodes) containing antibody-producing cells from the animal in which the immune response has been induced; (c) preparation of myeloma cells (hereinafter referred to as "myeloma") (e.g., mouse myeloma SP2 / 0-ag14 cells); (d) cell fusion between antibody-producing cells and myelomas; (e) Selection of hybridomas producing the desired antibody; (f) division into single-cell clones (cloning); (g) the cultivation of hybridomas or the rearing of animals implanted with hybridomas for the large-scale production of monoclonal antibodies, as the case may be; and / or (h) Examination of the physiological activity (internalization activity) and binding specificity of the monoclonal antibody thus produced, or assay of its properties as a labeling reagent.

[0060] The antibody titer measurement method used here includes, for example, flow cytometry or Cell-ELISA, but is not limited to these methods.

[0061] Examples of hybridoma strains established in this manner include anti-CDH6 antibody-producing hybridomas rG019, rG055, rG056, and rG061. In this specification, the antibody produced by the anti-CDH6 antibody-producing hybridoma rG019 will be referred to as the "rG019 antibody" or simply "rG019," the antibody produced by the hybridoma rG055 will be referred to as the "rG055 antibody" or simply "rG055," the antibody produced by the hybridoma rG056 will be referred to as the "rG056 antibody" or simply "rG056," and the antibody produced by the hybridoma rG061 will be referred to as the "rG061 antibody" or simply "rG061."

[0062] The light chain variable region of the rG019 antibody consists of the amino acid sequence set forth in SEQ ID NO: 10. The amino acid sequence of the light chain variable region of the rG019 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 11. The light chain variable region of the rG019 antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14. The heavy chain variable region of the rG019 antibody consists of the amino acid sequence set forth in SEQ ID NO: 15. The amino acid sequence of the heavy chain variable region of the rG019 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 16. The heavy chain variable region of the rG019 antibody has CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19. The sequence of the rG019 antibody is shown in Table 1.

[0063] The light chain variable region of the rG055 antibody consists of the amino acid sequence set forth in SEQ ID NO:20. The amino acid sequence of the light chain variable region of the rG055 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO:21. The light chain variable region of the rG055 antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO:22, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:23, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:24. The heavy chain variable region of the rG055 antibody consists of the amino acid sequence set forth in SEQ ID NO:25. The amino acid sequence of the heavy chain variable region of the rG055 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO:26. The heavy chain variable region of the rG055 antibody has CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO:27, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO:28, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO:29. The sequence of the rG055 antibody is shown in Table 1.

[0064] The light chain variable region of the rG056 antibody consists of the amino acid sequence set forth in SEQ ID NO: 30. The amino acid sequence of the light chain variable region of the rG056 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 31. The light chain variable region of the rG056 antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34. The heavy chain variable region of the rG056 antibody consists of the amino acid sequence set forth in SEQ ID NO: 35. The amino acid sequence of the heavy chain variable region of the rG056 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 36. The heavy chain variable region of the rG056 antibody has CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 39. The sequence of the rG056 antibody is shown in Table 1.

[0065] The light chain variable region of the rG061 antibody consists of the amino acid sequence set forth in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of the rG061 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 41. The light chain variable region of the rG061 antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 44. The heavy chain variable region of the rG061 antibody consists of the amino acid sequence set forth in SEQ ID NO: 45. The amino acid sequence of the heavy chain variable region of the rG061 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 46. The heavy chain variable region of the rG061 antibody has CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 48, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 49. The sequence of the rG061 antibody is shown in Table 1.

[0066] Furthermore, even when steps "2. Production of anti-CDH6 antibodies" (a) to (h) are repeated to obtain a separate, independent monoclonal antibody, or when a separate monoclonal antibody is obtained by another method, it is possible to obtain an antibody with internalization activity equivalent to that of the rG019, rG055, rG056, or rG061 antibody. Examples of such antibodies include antibodies that bind to the same epitope as the rG019, rG055, rG056, or rG061 antibodies. If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure to which the rG019, rG055, rG056, or rG061 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the rG019, rG055, rG056, or rG061 antibody. Furthermore, by confirming that the monoclonal antibody competes with the binding of rG019, rG055, rG056, or rG061 antibodies to CDH6 (i.e., that the monoclonal antibody interferes with the binding of rG019, rG055, rG056, or rG061 antibodies to CDH6), it can be determined that the monoclonal antibody binds to the same epitope as an anti-CDH6 antibody, even if the specific sequence or structure of the epitope has not been determined. If it is confirmed that the epitope is the same, it is strongly expected that the monoclonal antibody will have antigen-binding ability, biological activity, and / or internalization activity equivalent to those of the rG019, rG055, rG056, or rG061 antibodies.

[0067] (3) Other antibodies The antibodies of the present invention include not only the above-mentioned monoclonal antibodies against CDH6, but also genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, or human antibodies. These antibodies can be produced using known methods.

[0068] Chimeric antibodies include antibodies in which the variable and constant regions are heterologous, such as chimeric antibodies in which the variable regions of a mouse- or rat-derived antibody are joined to the constant regions of a human-derived antibody (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0069] Examples of chimeric antibodies derived from rat anti-human CDH6 antibodies include antibodies consisting of a light chain comprising each of the light chain variable regions of a rat anti-human CDH6 antibody described herein (e.g., rG019 antibody, rG055 antibody, rG056 antibody, or rG061 antibody) and a human-derived constant region, and a heavy chain comprising each of the heavy chain variable regions and a human-derived constant region.

[0070] Other examples of chimeric antibodies derived from rat anti-human CDH6 antibodies include antibodies consisting of a light chain comprising a light chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in each light chain variable region of a rat anti-human CDH6 antibody described herein (e.g., rG019 antibody, rG055 antibody, rG056 antibody, or rG061 antibody) has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in each heavy chain variable region has been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0071] Other examples of chimeric antibodies derived from rat anti-human CDH6 antibodies include antibodies consisting of a light chain comprising a light chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in each light chain variable region of a rat anti-human CDH6 antibody described herein (e.g., rG019 antibody, rG055 antibody, rG056 antibody, or rG061 antibody) have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in each heavy chain variable region have been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0072] Examples of chimeric antibodies derived from the rG019 antibody include antibodies consisting of a light chain containing a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 and a heavy chain containing a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and the antibody may have a constant region derived from any human.

[0073] Other examples of chimeric antibodies derived from the rG019 antibody include antibodies comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 in which one to several, one to three, one to two, and preferably one amino acid residue has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 15 in which one to several, one to three, one to two, and preferably one amino acid residue has been substituted with another amino acid residue, and the antibody may have a constant region derived from any human.

[0074] Other examples of chimeric antibodies derived from the rG019 antibody include antibodies consisting of a light chain comprising a light chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15 have been substituted with another amino acid residue, and the antibody may have a constant region derived from any human.

[0075] Another example of a chimeric antibody derived from the rG019 antibody is an antibody consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58, and the antibody may have a constant region of any human origin. The amino acid sequence set forth in SEQ ID NO: 58 is a sequence in which the cysteine ​​residue in CDRH2 in the amino acid sequence set forth in SEQ ID NO: 15 is substituted with a proline residue.

[0076] A specific example of a chimeric antibody derived from the rG019 antibody is an antibody consisting of a light chain consisting of the full-length light chain amino acid sequence shown in SEQ ID NO: 53 and a heavy chain consisting of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 56. This chimeric anti-human CDH6 antibody is referred to herein as a "chimeric G019 antibody," "chG019 antibody," or "chG019." The full-length light chain amino acid sequence of the chG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 54, and the full-length heavy chain amino acid sequence of the chG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 57.

[0077] The amino acid sequence of the light chain variable region of the chG019 antibody is identical to that of the rG019 antibody and consists of the amino acid sequence set forth in SEQ ID NO: 10. The light chain of the chG019 antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, which are identical to the CDRL1, CDRL2, and CDRL3 of the light chain of rG019, respectively. The amino acid sequence of the light chain variable region of the chG019 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 55.

[0078] The heavy chain variable region amino acid sequence of the chG019 antibody consists of the amino acid sequence set forth in SEQ ID NO: 58. The heavy chain of the chG019 antibody has a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19. The amino acid sequence set forth in SEQ ID NO: 58 is a sequence in which the cysteine ​​residue in CDRH2 of the amino acid sequence set forth in SEQ ID NO: 15 is substituted with a proline residue. The CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60 is a sequence in which the cysteine ​​residue in the rG019 CDRH2 set forth in SEQ ID NO: 18 is substituted with a proline residue. The heavy chain variable region amino acid sequence of the chG019 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 59.

[0079] The sequence of the chG019 antibody is shown in Table 1.

[0080] Examples of chimeric antibodies derived from the rat anti-human CDH6 antibody rG055 include chimeric antibodies consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 20 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 25, and the antibody may have a constant region derived from any human.

[0081] Examples of chimeric antibodies derived from the rat anti-human CDH6 antibody rG056 include chimeric antibodies consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 30 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 35, and the antibody may have a constant region derived from any human.

[0082] Examples of chimeric antibodies derived from the rat anti-human CDH6 antibody rG061 include chimeric antibodies consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 45, and the antibody may have a constant region derived from any human.

[0083] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) has been incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies in which not only the CDR sequence but also some framework amino acid residues have been grafted onto a human antibody by CDR grafting (WO 90 / 07861), and antibodies in which the amino acid sequence of some CDRs has been modified while maintaining the antigen-binding ability.

[0084] As used herein, a humanized antibody derived from the rG019, rG055, rG056, rG061, or chG019 antibody is not limited to a specific humanized antibody as long as it retains all six CDR sequences unique to the rG019, rG055, rG056, rG061, or chG019 antibody and has internalization activity. As long as the humanized antibody retains internalization activity, the amino acid sequences of some of the CDRs may also be modified.

[0085] Examples of humanized antibodies of the chG019 antibody include any combination of light chains comprising a light chain variable region selected from the group consisting of: (1) the amino acid sequence set forth in SEQ ID NO: 63 or 67; (2) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (1) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (3) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (1) above; and heavy chains comprising a heavy chain variable region selected from the group consisting of: (4) the amino acid sequence set forth in SEQ ID NO: 71, 75, or 79; (5) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (4) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (6) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (4) above.

[0086] Furthermore, antibodies in which one of the heavy or light chains is humanized and the other is the light or heavy chain of a rat antibody or a chimeric antibody can also be used. Examples of such antibodies include any combination of light chains comprising a light chain variable region selected from the group consisting of: (1) the amino acid sequence set forth in SEQ ID NO: 63 or 67; (2) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (1) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (3) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (1) above; and heavy chains comprising a heavy chain variable region selected from the group consisting of: (4) the amino acid sequence set forth in SEQ ID NO: 15, 25, 35, 45, or 58; (5) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (4) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (6) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (4) above. Other examples include any combination of light chains comprising a light chain variable region selected from the group consisting of: (1) the amino acid sequence set forth in SEQ ID NO: 10, 20, 30, or 40; (2) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (1) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (3) the amino acid sequence of (1) above, in which one or several amino acids have been deleted, substituted, or added; and heavy chains comprising a heavy chain variable region selected from the group consisting of: (4) the amino acid sequence set forth in SEQ ID NO: 71, 75, or 79; (5) an amino acid sequence having at least 95% or more identity to the amino acid sequence of (4) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence); and (6) the amino acid sequence of (4) above, in which one or several amino acids have been deleted, substituted, or added.

[0087] Conservative amino acid substitutions are preferred for amino acid substitutions herein. Conservative amino acid substitutions are substitutions that occur within amino acid groups that are related in their side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Such amino acid substitutions are preferably made to the extent that they do not impair the properties of the substance having the original amino acid sequence.

[0088] Antibodies with suitable combinations of the above-mentioned light chain and heavy chain include antibodies consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 63 (herein also referred to as the hL02 light chain variable region amino acid sequence) or a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 67 (herein also referred to as the hL03 light chain variable region amino acid sequence), and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 71 (herein also referred to as the hH01 heavy chain variable region amino acid sequence), a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 75 (herein also referred to as the hH02 heavy chain variable region amino acid sequence), or a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 79 (herein also referred to as the hH04 heavy chain variable region amino acid sequence). Preferred examples include an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 71; an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 75; an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 79; an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 67 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 71; an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 67 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 75; or an antibody consisting of a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 67 and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 79.More preferred examples include an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 71; an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 75; an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 79; or an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 67 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 75.

[0089] Other examples of antibodies with suitable combinations of light chains and heavy chains include antibodies comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 (also referred to herein as the full-length hL02 light chain amino acid sequence), or a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 (also referred to herein as the full-length hL03 light chain amino acid sequence), and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69 (also referred to herein as the full-length hH01 heavy chain amino acid sequence), a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73 (also referred to herein as the full-length hH02 heavy chain amino acid sequence), or a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77 (also referred to herein as the full-length hH04 heavy chain amino acid sequence). Preferred examples include an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69; an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73; and an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77. an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69; an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73; or an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77.More preferred examples include an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69 (also referred to herein as "H01L02 antibody" or "H01L02"); an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73 (also referred to herein as "H02L02 antibody" or "H02L02"); an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77 (also referred to herein as the "H04L02 antibody" or "H04L02"); or an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73 (also referred to herein as the "H02L03 antibody" or "H02L03"). The sequences of the H01L02 antibody, H02L02 antibody, H02L03 antibody, and H04L02 antibody are shown in Table 1.

[0090] By combining sequences that show high identity with the above heavy chain and light chain amino acid sequences, it is possible to select antibodies with biological activity equivalent to each of the above antibodies. Such identity is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the heavy chain or light chain amino acid sequences, it is also possible to select antibodies with biological activity equivalent to each of the above antibodies.

[0091] Identity between two amino acid sequences can be determined by aligning the sequences using the default parameters of ClustalW version 2 (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG (2007), "Clustal W and Clustal X version 2.0", Bioinformatics. 23(21):2947-2948).

[0092] In the full-length amino acid sequence of the hL02 light chain shown in SEQ ID NO: 61, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region, and the amino acid sequence consisting of amino acid residues 129 to 233 is a constant region. In the full-length nucleotide sequence of the hL02 light chain shown in SEQ ID NO: 62, the nucleotide sequence consisting of nucleotides 1 to 60 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 61 to 384 encodes a variable region, and the nucleotide sequence consisting of nucleotides 385 to 699 encodes a constant region.

[0093] In the full-length amino acid sequence of the hL03 light chain shown in SEQ ID NO: 65, the amino acid sequence of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence of amino acid residues 21 to 128 is a variable region, and the amino acid sequence of amino acid residues 129 to 233 is a constant region. In the full-length nucleotide sequence of the hL03 light chain shown in SEQ ID NO: 66, the nucleotide sequence of nucleotides 1 to 60 encodes a signal sequence, the nucleotide sequence of nucleotides 61 to 384 encodes a variable region, and the nucleotide sequence of nucleotides 385 to 699 encodes a constant region.

[0094] In the full-length amino acid sequence of the hH01 heavy chain shown in SEQ ID NO: 69, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is a variable region, and the amino acid sequence consisting of amino acid residues 142 to 471 is a constant region. In the full-length nucleotide sequence of the hH01 heavy chain shown in SEQ ID NO: 70, the nucleotide sequence consisting of nucleotides 1 to 57 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 58 to 423 encodes a variable region, and the nucleotide sequence consisting of nucleotides 424 to 1413 encodes a constant region.

[0095] In the full-length amino acid sequence of the hH02 heavy chain shown in SEQ ID NO: 73, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 141 is a variable region, and the amino acid sequence consisting of amino acid residues 142 to 471 is a constant region. In the full-length nucleotide sequence of the hH02 heavy chain shown in SEQ ID NO: 74, the nucleotide sequence consisting of nucleotides 1 to 57 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 58 to 423 encodes a variable region, and the nucleotide sequence consisting of nucleotides 424 to 1413 encodes a constant region.

[0096] In the full-length amino acid sequence of the hH04 heavy chain shown in SEQ ID NO: 77, the amino acid sequence of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence of amino acid residues 20 to 141 is a variable region, and the amino acid sequence of amino acid residues 142 to 471 is a constant region. In the full-length nucleotide sequence of the hL04 heavy chain shown in SEQ ID NO: 78, the nucleotide sequence of nucleotides 1 to 57 is a signal sequence, the nucleotide sequence of nucleotides 58 to 423 encodes the variable region, and the nucleotide sequence of nucleotides 424 to 1413 encodes the constant region.

[0097] [Table 1-1]

[0098] Table 1-2

[0099] Table 1-3

[0100] Table 1-4

[0101] Table 1-5

[0102] Table 1-6

[0103] Table 1-7

[0104] Table 1-8

[0105] Table 1-9

[0106] Table 1-10

[0107] Table 1-11

[0108] [Table 1-12]

[0109] [Table 1-13]

[0110] [Table 1-14]

[0111] [Table 1-15]

[0112] (In this specification, Tables 1-1 to 1-15 may be collectively referred to as Table 1.) Further examples of the antibodies of the present invention include human antibodies that bind to CDH6. An anti-CDH6 human antibody refers to a human antibody that has only the gene sequence of an antibody derived from a human chromosome. Anti-CDH6 human antibodies can be obtained by a method using human antibody-producing mice carrying human chromosomal fragments containing the heavy and light chain genes of human antibodies (Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999.; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

[0113] Specifically, such human antibody-producing mice can be produced by generating knockout animals and transgenic animals as genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci have been destroyed and instead the human immunoglobulin heavy and light chain gene loci have been introduced via a yeast artificial chromosome (YAC) vector or the like, and by crossbreeding these animals.

[0114] Alternatively, eukaryotic cells can be transformed using recombinant DNA technology with cDNA encoding each of the heavy and light chains of such a human antibody, preferably with a vector containing the cDNA, and the transformed cells that produce the recombinant human monoclonal antibody can be cultured to obtain the antibody from the culture supernatant.

[0115] Here, as the host, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, and myeloma cells can be used.

[0116] Methods for obtaining phage-display-derived human antibodies selected from a human antibody library are also known (see, for example, Wormstone, I. et al., Investigative Ophthalmology & Visual Science. (2002) 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109(3), pp. 427-431).

[0117] For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105-1116) can be used, in which the variable region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv), and phages that bind to the antigen are selected.

[0118] By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.

[0119] Once the DNA sequence of an antigen-binding scFv is identified, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression to obtain a human antibody (WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0120] If a newly created human antibody binds to a partial peptide or partial three-dimensional structure to which any one of the rat anti-human CDH6 antibodies, chimeric anti-human CDH6 antibodies, or humanized anti-human CDH6 antibodies described herein (e.g., rG019 antibody, rG055 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody) binds, it can be determined that the human antibody binds to the same epitope as the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody. Alternatively, the human antibody competes with the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody described herein (e.g., rG019 antibody, rG055 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody) for binding to CDH6 (e.g., the human antibody competes with the rG019 antibody, rG055 antibody, rG056 antibody, rG0 By confirming that the human antibody binds to the same epitope as the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody described herein, even if the specific sequence or structure of the epitope has not been determined, it can be determined that the human antibody binds to the same epitope as the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody described herein, even if the specific sequence or structure of the epitope has not been determined. Herein, if a newly created human antibody is determined to "bind to the same epitope" by at least one of these determination methods, it can be said that the newly created human antibody "binds to the same epitope" as the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody described herein. If it is confirmed that the epitope is identical, the human antibody is expected to have biological activity equivalent to that of a rat anti-human CDH6 antibody, a chimeric anti-human CDH6 antibody, or a humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG055 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody).

[0121] The chimeric, humanized, or human antibodies obtained by the above methods can be evaluated for their antigen-binding ability by known methods, and suitable antibodies can be selected.

[0122] Another example of an index used to compare antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument that can quickly and accurately measure the thermal denaturation midpoint (Tm), a good indicator of the relative structural stability of proteins. Differences in thermal stability can be compared by measuring Tm values ​​using DSC and comparing the values. It is known that the storage stability of antibodies correlates somewhat with their thermal stability (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273). Therefore, suitable antibodies can be selected using thermal stability as an index. Other indexes 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 have the highest thermal stability. Therefore, it is necessary to comprehensively evaluate the above-mentioned indexes to select the antibody most suitable for human administration.

[0123] The antibodies of the present invention also include modified antibodies. The term "modified antibodies" refers to antibodies of the present invention that have been chemically or biologically modified. Chemical modifications include attachment of a chemical moiety to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, and the like. Biological modifications include those that have undergone post-translational modifications (e.g., glycosylation to an N- or O-linkage, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, or pyroglutamation of N-terminal glutamine or N-terminal glutamic acid), and those that have undergone expression in prokaryotic host cells to add a methionine residue to the N-terminus. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens, among other purposes.

[0124] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating antibody sugar chain modification are known, including, but not limited to, those described in International Publication Nos. 1999 / 54342, 2000 / 61739, and 2002 / 31140. The antibodies of the present invention also include antibodies with modified sugar chain modifications.

[0125] When an antibody gene is isolated and then introduced into a suitable host to produce an antibody, a suitable combination of host and expression vector can be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence and a gene encoding the light chain sequence of an antibody described herein. When transforming a host cell, 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.

[0126] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y., Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), and FreeStyle 293F cells (Invitrogen).

[0127] When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis.

[0128] Antibodies can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro. The yield of the antibody during this culture may vary depending on the antibody sequence, and antibodies with equivalent binding activity can be selected based on the yield as an indicator to determine which antibodies are easy to produce as pharmaceuticals. Therefore, the antibodies of the present invention also include antibodies obtained by a method for producing the antibody, which method comprises the steps of culturing the transformed host cells and recovering the desired antibody or a functional fragment of the antibody from the culture obtained in this step.

[0129] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)). Similarly, it is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention also include antibodies that have undergone such modifications and functional fragments of such antibodies, including deletions in which one or two amino acids are deleted from 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 the antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody of the present invention are not limited to the above types. The two heavy chains constituting the antibody of the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions, but an example of a major component of the antibody of the present invention is one in which one amino acid residue is deleted at the carboxyl terminus of both of the two heavy chains.

[0130] The isotype of the antibody of the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG4.

[0131] The biological activities of an antibody generally include antigen-binding activity, the activity of binding to an antigen and thereby being internalized into cells expressing the antigen, the activity of neutralizing antigen activity, the activity of enhancing antigen activity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). The function of the antibody of the present invention is binding activity to CDH6, preferably the activity of being internalized into CDH6-expressing cells by binding to CDH6. Furthermore, the antibody of the present invention may have ADCC, CDC, and / or ADCP activity in addition to the cell-internalizing activity.

[0132] The obtained antibodies can be purified to homogeneity. Antibodies can be separated and purified using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, 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.

[0133] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.

[0134] These chromatographies can be carried out using liquid chromatography such as HPLC or FPLC.

[0135] Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0136] It is also possible to purify antibodies by using a carrier on which an antigen is immobilized, and taking advantage of their binding to the antigen.

[0137] 3. Anti-CDH6 Antibody-Drug Conjugates (1) Drugs The anti-CDH6 antibody obtained in "2. Production of anti-CDH6 antibody" above can be converted into an anti-CDH6 antibody-drug conjugate by binding a drug via a linker structure moiety. The drug is not particularly limited as long as it has a substituent or partial structure that can be bound to the linker structure. Anti-CDH6 antibody-drug conjugates can be used for various purposes depending on the drug to be bound. Examples of such drugs include substances with antitumor activity, substances effective against blood diseases, substances effective against autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

[0138] (1)-1 Antitumor compounds An example of using an antitumor compound as the compound bound to the anti-CDH6 antibody-drug conjugate of the present invention is described below. The antitumor compound is not particularly limited as long as it is a compound with antitumor effect and has a substituent or partial structure that can be bound to a linker structure. In the antitumor compound, the linker is partially or completely cleaved within tumor cells, releasing the antitumor compound moiety and thereby exerting its antitumor effect. When the linker is cleaved at the binding site with the drug, the antitumor compound is released in its original structure, thereby exerting its original antitumor effect.

[0139] The anti-CDH6 antibody obtained in the above "2. Production of anti-CDH6 antibody" can be converted into an anti-CDH6 antibody-drug conjugate by binding an antitumor compound via a linker structure moiety.

[0140] One example of an antitumor compound that can be used in the present invention is the camptothecin derivative exatecan ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione; the following formula:

[0141] [ka]

[0142] can be preferably used. This compound can be easily obtained by, for example, the method described in U.S. Patent Publication No. US2016 / 0297890 or other known methods, and the amino group at position 1 can be preferably used as a binding site to a linker structure. Furthermore, exatecan may be released within tumor cells with a portion of the linker still bound, but even in this state, it is a compound that exhibits excellent antitumor effects.

[0143] Since exatecan has a camptothecin structure, it is known that in an acidic aqueous medium (e.g., at about pH 3), the equilibrium shifts toward a structure in which the lactone ring is formed (closed ring isomer), whereas in a basic aqueous medium (e.g., at about pH 10), the equilibrium shifts toward a structure in which the lactone ring is opened (open ring isomer). Drug conjugates incorporating exatecan residues corresponding to such closed ring structures and open ring structures are expected to have equivalent antitumor effects, and it goes without saying that both are encompassed within the scope of the present invention.

[0144] Other antitumor compounds include, for example, antitumor compounds described in the literature (Pharmacological Reviews, 68, p3-19, 2016), and examples thereof include auristatins such as doxorubicin, calchemicin, dorastatin 10, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and pyrrolobenzodiazepines (Py Examples of antitumor agents include the dimeric benzodiazepine (C2H) dimer SG2000 (SJG-136), camptothecin derivatives SN-38 and duocarmycins such as CC-1065, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or its derivatives), and taxol or its derivatives.

[0145] In antibody-drug conjugates, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of small molecules, they are usually obtained as a mixture of drugs with different numbers of conjugated drugs. The number of drugs bound to one antibody molecule is specified and expressed as an average value, i.e., the average drug conjugation number. In the present invention, as a general rule, unless otherwise specified, i.e., except when referring to an antibody-drug conjugate with a specific drug conjugation number contained in a mixture of antibody-drug conjugates with different drug conjugation numbers, the average drug conjugation number is used. The number of exatecans bound to an antibody molecule can be controlled, and the average number of drugs bound per antibody can be about 1 to 10, preferably 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8, more preferably 5 to 8, even more preferably 7 to 8, and still more preferably 8. Those skilled in the art will be able to design a reaction for binding the required number of drugs to an antibody from the description of the Examples of the present application, and will be able to obtain an antibody-drug conjugate in which the number of exatecans bound is controlled.

[0146] (2) Linker structure The linker structure that binds a drug to an anti-CDH6 antibody in the anti-CDH6 antibody-drug conjugate of the present invention will be described.

[0147] In the antibody-drug conjugate of the present application, the linker structure connecting the anti-CDH6 antibody and the drug is not particularly limited as long as it can be used as an antibody-drug conjugate, and can be appropriately selected depending on the intended use. Examples of linker structures include those described in the publicly known literature (Pharmacol Rev 68:3 -19, January 2016, Protein Cell DOI 10.1007 / s13238-016-0323-0, etc., and more specific examples include VC (valine-citrulline), MC (maleimidocaproyl), SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate, SS (disulfide), SPDB (N-succinimidyl 4-(2-pyridyldithio)butyrate) Examples include 4-(2-pyridyldithio)butyrate, SS / hydrazone, hydrazone, and carbonate.

[0148] Other examples include the linker structure described in U.S. Patent Publication US2016 / 0297890 (for example, those described in paragraphs

[0260] to

[0289] ), and the following structure can be suitably used. Note that the left end of the structure shown below is the binding site with the antibody, and the right end is the binding site with the drug. Furthermore, GGFG in the following linker structure represents an amino acid sequence connected by a peptide bond consisting of glycine-glycine-phenylalanine-glycine (GGFG). -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0149] More preferably, the following can be mentioned: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Even more preferably, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Examples include:

[0150] The antibody binds to the end of -(Succinimid-3-yl-N) (for example, in "-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-", the end opposite to where (-CH2CH2CH2CH2CH2-) is bound (the left end)), and the antitumor compound binds to the end opposite to -(Succinimid-3-yl-N) (the right end in the above example, via the carbonyl group of CH2-O-CH2-C(=O)-). "-(Succinimid-3-yl-N)-" is a compound represented by the following formula:

[0151] [ka]

[0152] The structure is shown below. Position 3 of this partial structure is the binding site for the anti-CDH6 antibody. The binding to the antibody at position 3 is characterized by the formation of a thioether bond. The nitrogen atom at position 1 of this structural portion is bonded to the carbon atom of a methylene group present in the linker containing this structure.

[0153] In the antibody-drug conjugate of the present invention in which the drug is exatecan, the antibody is preferably bound to a drug-linker structural moiety having the following structure: The average number of these drug-linker structural moieties bound per antibody may be 1 to 10, preferably 2 to 8, more preferably 5 to 8, even more preferably 7 to 8, and even more preferably 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0154] More preferably, it is: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0155] More preferably, the following can be mentioned: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0156] Also, -(NH-DX) is represented by the following formula:

[0157] [ka]

[0158] This structure represents the group formed when one hydrogen atom is removed from the amino group at position 1 of exatecan.

[0159] (3) Method for producing antibody-drug conjugates There are no particular limitations on the antibodies that can be used in the antibody-drug conjugates of the present invention, as long as they are anti-CDH6 antibodies having internalization activity as described in the above section "2. Production of anti-CDH6 antibodies" and in the Examples, or functional fragments of such antibodies.

[0160] Next, a representative method for producing the antibody-drug conjugate of the present invention will be described. In the following, the compound numbers shown in each reaction scheme will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be referred to in the same manner.

[0161] (3)-1 Manufacturing method 1 Among the antibody-drug conjugates represented by the following formula (1), those in which the anti-CDH6 antibody and linker structure are linked via a thioether can be produced by reacting an anti-CDH6 antibody obtained by reducing the disulfide bonds to sulfhydryl groups with compound (2) available by a known method (for example, available by the method described in US2016 / 297890 (e.g., the method described in paragraphs

[0336] to

[0374] ). For example, it can be produced by the method described below.

[0162]

number

[0163] [wherein AB represents an antibody having a sulfhydryl group. where L 1teeth, -(Succinimid-3-yl-N)- L 1 ' represents a maleimidyl group represented by the following formula:

[0164] [ka]

[0165] -L 1 -L X has any of the structures shown in the following formulas: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0166] Among these, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0167] Furthermore, the following can be preferably mentioned: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0168] In addition, in the above reaction scheme, antibody-drug conjugate (1) is depicted as a structure in which one structural moiety from the drug to the linker terminal is bound to one antibody, but this is a description for convenience of explanation, and in reality, multiple structural moieties are often bound to one antibody molecule. This situation also applies to the following description of the production method.

[0169] That is, the antibody-drug conjugate (1) can be produced by reacting compound (2) obtainable by a known method (e.g., obtainable by the method described in US2016 / 297890 (e.g., the method described in paragraphs

[0336] to

[0374] )) with antibody (3a) having a sulfhydryl group.

[0170] Antibody (3a) having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples of methods include, but are not limited to, reacting Traut's reagent with the amino groups of an antibody; reacting N-succinimidyl S-acetylthioalkanoates with the amino groups of an antibody followed by hydroxylamine; reacting N-succinimidyl 3-(pyridyldithio)propionate with the amino groups of an antibody followed by a reducing agent; or reacting an antibody with a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the interchain disulfide bonds in the antibody to generate sulfhydryl groups.

[0171] Specifically, an antibody with partially or completely reduced intra-chain disulfides can be obtained by reacting the antibody with TCEP as a reducing agent in a buffer solution containing a chelating agent, in an amount of 0.3 to 3 molar equivalents per antibody inter-chain disulfide. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These can be used at a concentration of 1 mM to 20 mM. Examples of buffer solutions that can be used include sodium phosphate, sodium borate, and sodium acetate solutions. In a specific example, an antibody (3a) with partially or completely reduced sulfhydryl groups can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours.

[0172] Here, a reaction for adding a sulfhydryl group to the drug-linker moiety can be carried out, thereby linking the drug-linker moiety via a thioether bond.

[0173] Next, 2 to 20 molar equivalents of compound (2) can be used per antibody (3a) having a sulfhydryl group to produce antibody-drug conjugate (1) in which 2 to 8 drugs are bound per antibody. Specifically, a solution of compound (2) is added to a buffer containing antibody (3a) having a sulfhydryl group and the reaction is allowed to proceed. Examples of the buffer include sodium acetate, sodium phosphate, and sodium borate. The reaction is allowed to proceed at a pH of 5 to 9, preferably around pH 7. Examples of solvents that can be used to dissolve compound (2) include organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP). The organic solvent solution containing compound (2) is added to a buffer containing antibody (3a) having a sulfhydryl group at a concentration of 1 to 20% v / v and the reaction is allowed to proceed. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by deactivating the reactivity of unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC relative to the compound (2) used and incubating at room temperature for 10 to 30 minutes.

[0174] (4) Identification of antibody-drug conjugates The produced antibody-drug conjugate (1) can be concentrated, buffer exchanged, purified, and the antibody concentration and average number of drugs bound per antibody molecule measured by the following common procedures, allowing the antibody-drug conjugate (1) to be identified.

[0175] (4)-1 Common Procedure A: Concentration of Aqueous Solution of Antibody or Antibody-Drug Conjugate The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (50,000 MWCO, Millipore Corporation) container and concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000 to 3800 G for 5 to 20 minutes.

[0176] (4)-2 Common Procedure B: Antibody Concentration Measurement Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. The 280 nm extinction coefficient (1.3 mL mg) differs for each antibody. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used.

[0177] (4)-3 Common Procedure C: Buffer Exchange of Antibody A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 matrix was equilibrated with phosphate buffer (50 mM, pH 6.0) containing 50 mM sodium chloride and 2 mM EDTA (referred to herein as PBS 6.0 / EDTA) according to the manufacturer's instructions. 2.5 mL of antibody solution was loaded onto each NAP-25 column, and a 3.5 mL fraction was eluted with 3.5 mL of PBS 6.0 / EDTA. This fraction was concentrated using common procedure A, and the antibody concentration was measured using common procedure B. The antibody concentration was then adjusted to 20 mg / mL using PBS 6.0 / EDTA.

[0178] (4)-4 Common Procedure D: Purification of Antibody-Drug Conjugates A NAP-25 column was equilibrated with one of the commercially available acetate buffers (10 mM, pH 5.5; referred to herein as ABS) containing sorbitol (5%). The antibody-drug conjugate reaction solution (approximately 2.5 mL) was loaded onto the NAP-25 column and eluted with the manufacturer's specified volume of buffer to obtain an antibody fraction. This fraction was then loaded onto the NAP-25 column again and eluted with buffer. This gel filtration purification procedure was repeated two or three times to obtain an antibody-drug conjugate free of unbound drug linkers and small molecules (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide).

[0179] (4)-5 Common Procedure E: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates The bound drug concentration in an antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths, 280 nm and 370 nm, and then performing the following calculation.

[0180] Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in a system (additivity of absorbance), assuming that there is no change in the molar absorption coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Formula (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (2) where A 280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. 、 A370 indicates the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, and A A,280 indicates the absorbance of the antibody at 280 nm, and A A,370 indicates the absorbance of the antibody at 370 nm, and A D,280 denotes the absorbance of the conjugate precursor at 280 nm, and A D,370 denotes the absorbance of the conjugate precursor at 370 nm, and ε A,280 denotes the molar extinction coefficient of the antibody at 280 nm, and ε A,370 denotes the molar extinction coefficient of the antibody at 370 nm, and ε D,280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 is the molar extinction coefficient of the conjugate precursor at 370 nm, and C A indicates the antibody concentration in the antibody-drug conjugate, and C D indicates the drug concentration in the antibody-drug conjugate.

[0181] where ε A,280、 ε A,370、 ε D,280、 ε D,370 is a value prepared in advance (a calculated estimated value or an actual measured value obtained from UV measurement of the compound). For example, ε A,280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A,370 is usually zero. D,280 and ε D,370 The A of an antibody-drug conjugate solution can be obtained by measuring the absorbance of a solution in which the conjugate precursor used is dissolved at a certain molar concentration, using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). 280 and A 370 By measuring these values ​​and substituting them into equations (1) and (2) and solving the simultaneous equations, C A and C D Furthermore, C D C AThe average number of drugs bound per antibody can be calculated by dividing by this.

[0182] (4)-6 Common Procedure F: Measurement of the average number of drugs bound per antibody molecule in antibody-drug conjugates (2) The average number of drugs bound per antibody molecule in an antibody-drug conjugate can also be determined by high-performance liquid chromatography (HPLC) analysis using the following method, in addition to the aforementioned "(4)-5 Common Procedure E." The following describes a method for measuring the average number of drugs bound by HPLC when the antibody and the drug linker are disulfide-bonded. Those skilled in the art can refer to this method to appropriately measure the average number of drugs bound by HPLC depending on the type of bond between the antibody and the drug linker.

[0183] F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates) The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to cleave the disulfide bond between the light and heavy chains of the antibody-drug conjugate. The resulting sample is used for HPLC analysis.

[0184] F-2.HPLC analysis The HPLC analysis is carried out under the following measurement conditions.

[0185] HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 x 50 mm, 1.7 μm, 130 Å; Waters, P / N 186002884) Column temperature: 80℃ Mobile phase A: 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol in water Mobile phase B: acetonitrile solution containing 0.075% TFA and 15% 2-propanol Gradient program: 14%-36% (0-15 min), 36%-80% (15-17 min), 80%-14% (17-17.01 min), 14% (17.01-25 min) Sample injection volume: 10 μL F-3. Data Analysis F-3-1: The light chain (L0) and heavy chain (H0) of an antibody to which no drug is bound are compared with the light chain (L) to which a drug is bound. i ) and heavy chain (heavy chain with i drugs bound: H i ) become more hydrophobic and their retention times increase in proportion to the number of bound drugs, and are therefore eluted in the order of, for example, L0, L1, H0, H1, H2, and H3. By comparing the retention times with L0 and H0, the detected peak can be assigned to any of L0, L1, H0, H1, H2, and H3. The number of bound drugs can be determined by those skilled in the art, but is preferably L0, L1, H0, H1, H2, and H3.

[0186] F-3-2 Because the drug linker has UV absorption, the peak area value is corrected according to the molar absorption coefficients of the light chain, heavy chain, and drug linker according to the number of drug linkers bound, using the following formula.

[0187]

number

[0188]

number

[0189] Here, the molar extinction coefficients (280 nm) of the light and heavy chains of each antibody can be estimated from the amino acid sequences of the light and heavy chains of each antibody using a known calculation method (Protein Science, 1995, vol. 4, pp. 2411-2423). In the case of H01L02, the molar extinction coefficient of the light chain was estimated to be 31,710, and the molar extinction coefficient of the heavy chain was estimated to be 79,990, based on the amino acid sequence. The molar extinction coefficient (280 nm) of the drug linker was determined by measuring the actual molar extinction coefficient (280 nm) of a compound prepared by reacting each drug linker with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether. The wavelength at which absorbance is measured can be determined appropriately by those skilled in the art, but is preferably the wavelength at which the antibody peak can be measured, more preferably 280 nm.

[0190] F-3-3 Calculate the ratio (%) of each chain peak area to the total corrected peak area according to the following formula.

[0191]

number

[0192] F-3-4 Calculate the average number of drugs bound per antibody molecule in the antibody-drug conjugate according to the following formula.

[0193] Average number of drug bindings = (L0 peak area ratio x 0 + L1 peak area ratio x 1 + H0 peak area ratio x 0 + H1 peak area ratio x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100 x 2 To ensure a certain amount of antibody-drug conjugate, multiple antibody-drug conjugates produced under similar conditions and with similar average drug numbers (e.g., about ±1) can be mixed to create a new lot. In this case, the average drug number will fall within the average drug number before mixing.

[0194] One specific example of an antibody-drug conjugate of the present invention is a conjugate of the following formula:

[0195] [ka]

[0196] or the following formula:

[0197] [ka]

[0198] Examples of the compound include those having the structure shown below.

[0199] Here, AB represents the anti-CDH6 antibody disclosed herein, which is conjugated to the conjugated linker via a sulfhydryl group derived from the antibody. Here, n is synonymous with the so-called Drug-to-Antibody Ratio (DAR) and represents the drug-antibody ratio per antibody. That is, it represents the number of drugs bound to one antibody molecule, and this is a numerical value specified and expressed as the average value, i.e., the average number of drugs bound. In the case of the antibody-drug conjugates of the present invention represented by [Chemical Formula 9] and [Chemical Formula 10], n may be 2 to 8, preferably 5 to 8, more preferably 7 to 8, and even more preferably 8, as measured by common procedure F.

[0200] An example of the antibody-drug conjugate of the present invention is an antibody-drug conjugate or a pharmacologically acceptable salt thereof, in which the antibody represented by AB in the structure represented by the above formula [Chemical Formula 9] or [Chemical Formula 10] comprises an antibody having a heavy chain and a light chain, or a functional fragment thereof, selected from the group consisting of the following (a) to (g): (a) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69; (b) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73; (c) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77; (d) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69; (e) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73; (f) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77; or (g) An antibody according to any one of (a) to (f), wherein the heavy or light chain contains one or more modifications selected from the group consisting of post-translational modifications typified by N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, addition of a methionine residue to the N-terminus, amidation of proline residues, pyroglutamic acid oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[0201] 4. Pharmaceuticals The anti-CDH6 antibodies of the present invention and functional fragments of the antibodies described in the above section "2. Production of anti-CDH6 antibodies" and in the Examples bind to CDH6 on the surface of tumor cells and have internalization activity, and therefore can be used alone or in combination with other drugs as pharmaceuticals for the treatment of cancers such as renal cell tumors and ovarian tumors, for example, renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma.

[0202] It can also be used to detect cells expressing CDH6.

[0203] Furthermore, the anti-CDH6 antibody of the present invention and functional fragments of said antibody have internalization activity and can therefore be used as antibodies for antibody-drug conjugates.

[0204] Among the anti-CDH6 antibody-drug conjugates of the present invention described in the above section "3. Anti-CDH6 antibody-drug conjugates" and in the Examples, those that use a drug having anti-tumor activity such as cytotoxic activity as the drug are conjugates of an anti-CDH6 antibody with internalization activity and / or a functional fragment of the antibody with a drug having anti-tumor activity such as cytotoxic activity, and because they exhibit anti-tumor activity against cancer cells that express CDH6, they can be used as pharmaceuticals, particularly as therapeutic and / or preventive agents for cancer.

[0205] The anti-CDH6 antibody-drug conjugates of the present invention may become hydrated by absorbing moisture or by adsorbing water when left in the air or when subjected to recrystallization or purification procedures. Such water-containing compounds or pharmacologically acceptable salts are also encompassed by the present invention.

[0206] When the anti-CDH6 antibody-drug conjugate of the present invention has a basic group such as an amino group, it can form a pharmacologically acceptable acid addition salt, if desired. Examples of such acid addition salts include hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0207] When the anti-CDH6 antibody-drug conjugate of the present invention has an acidic group such as a carboxy group, it can form a pharmacologically acceptable base addition salt, if desired. Examples of such base addition salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; 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.

[0208] The present invention also encompasses anti-CDH6 antibody-drug conjugates in which one or more atoms constituting the antibody-drug conjugate are substituted with an isotope of that atom. There are two types of isotopes: radioactive isotopes and stable isotopes. Examples of isotopes include hydrogen isotopes (H and H), carbon isotopes (C, C, and C), nitrogen isotopes (N and N), oxygen isotopes (O, O, and O), and fluorine isotope (F). Compositions containing isotope-labeled antibody-drug conjugates are useful, for example, as therapeutic agents, preventive agents, research reagents, assay reagents, diagnostic agents, in vivo imaging diagnostic agents, and the like. Isotopically labeled antibody-drug conjugates and mixtures of isotope-labeled antibody-drug conjugates in any proportion are also encompassed by the present invention. Isotopically labeled antibody-drug conjugates can be produced by methods known in the art, for example, by using isotope-labeled starting materials instead of the starting materials in the production methods of the present invention described below.

[0209] In vitro cytocidal activity can be measured, for example, by measuring cell proliferation inhibitory activity. For example, cancer cell lines overexpressing CDH6 are cultured, and anti-CDH6 antibody-drug conjugates are added to the culture system at various concentrations to measure the inhibitory activity against focus activity, colony formation, and spheroid growth. Here, for example, renal cell tumor- and ovarian tumor-derived cancer cell lines can be used to examine the cytostatic activity against renal cell tumors and ovarian tumors.

[0210] The therapeutic effect on cancer in vivo using experimental animals can be measured, for example, by administering an anti-CDH6 antibody-drug conjugate to nude mice transplanted with a tumor cell line that highly expresses CDH6, and then measuring changes in the cancer cells. Here, for example, by using an animal model in which cells derived from renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, or thyroid cancer are transplanted into immunodeficient mice, the therapeutic effect on renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, or thyroid cancer can be measured.

[0211] The type of cancer to which the anti-CDH6 antibody-drug conjugates of the present invention are applicable is not particularly limited as long as CDH6 is expressed in the cancer cells to be treated, and examples include renal cell carcinoma (e.g., renal clear cell carcinoma or papillary renal cell carcinoma), ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, and neuroblastoma, but is not limited to these as long as CDH6 is expressed. More preferred examples of cancer include renal cell carcinoma (e.g., renal clear cell carcinoma or papillary renal cell carcinoma) and ovarian cancer.

[0212] The anti-CDH6 antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0213] Substances used in pharmaceutical compositions containing the anti-CDH6 antibody-drug conjugates of the present invention can be appropriately selected and applied in terms of dosage amount and administration concentration from pharmaceutical additives and other substances commonly used in this field.

[0214] The anti-CDH6 antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, 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 and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation will correspond to the mode of administration.

[0215] Various delivery systems are known and can be used to administer the anti-CDH6 antibody-drug conjugates of the present invention. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, the antibody-drug conjugates are administered by infusion. Parenteral administration is a preferred route of administration.

[0216] In representative embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the medicament may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form (e.g., as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent). Where the medicament is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients may be mixed prior to administration.

[0217] The pharmaceutical compositions of the present invention may contain only the anti-CDH6 antibody-drug conjugate of the present application, or may contain the anti-CDH6 antibody-drug conjugate and at least one other cancer therapeutic agent. The anti-CDH6 antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. The other anti-cancer agents used for this purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or sequentially, or at different administration intervals. Examples of such cancer therapeutic agents include tyrosine kinase inhibitors such as imatinib, sunitinib, and regorafenib; CDK4 / 6 inhibitors such as palbociclib; HSP90 inhibitors such as TAS-116; MEK inhibitors such as MEK162; and immune checkpoint inhibitors such as nivolumab, pembrolizumab, and ipilimumab. However, any agent with antitumor activity may be used without limitation.

[0218] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0219] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the anti-CDH6 antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its medicinal effect at a smaller dose, the higher the affinity (lower the Kd value) of the antibody-drug conjugate for the antigen. Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, approximately 0.001 to 100 mg / kg can be administered once or multiple times at intervals of once every 1 to 180 days. Preferably, 0.1 to 50 mg / kg, more preferably 1 to 50 mg / kg, 1 to 30 mg / kg, 1 to 20 mg / kg, 1 to 15 mg / kg, 2 to 50 mg / kg, 2 to 30 mg / kg, 2 to 20 mg / kg, or 2 to 15 mg / kg may be administered multiple times at intervals of once every 1 to 4 weeks, preferably once every 2 to 3 weeks. [Example]

[0220] The present invention will be specifically described by the following examples, but the present invention is not limited thereto. Furthermore, these examples should not be construed as limiting in any sense. Unless otherwise specified, each genetic manipulation procedure in the following examples was performed according to the method described in "Molecular Cloning" (Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) or other experimental methods used by those skilled in the art. Alternatively, when using commercially available reagents or kits, the procedures were performed according to the instructions provided with the commercially available products. Reagents, solvents, and starting materials not specifically described herein are readily available from commercial sources.

[0221] Example 1: Obtaining rat anti-human CDH6 antibody with internalization activity 1)-1 Construction of human, mouse, rat, and cynomolgus monkey CDH6 expression vectors A cDNA expression vector (OriGene, RC217889) encoding human CDH6 protein (NP_004923) was incorporated into a mammalian expression vector according to methods known to those skilled in the art to generate the human CDH6 expression vector pcDNA3.1-hCDH6. The amino acid sequence of the human CDH6 ORF (open reading frame) is shown in SEQ ID NO: 1.

[0222] A cDNA expression vector (OriGene, MC221619) encoding mouse CDH6 protein (NP_031692) was inserted into a mammalian expression vector according to methods known to those skilled in the art to create the mouse CDH6 expression vectors pcDNA3.1-mCDH6 and p3xFLAG-CMV-9-mCDH6. The amino acid sequence of the mouse CDH6 ORF is shown in SEQ ID NO:7.

[0223] The human CDH6 expression vectors pcDNA3.1-rCDH6 and p3xFLAG-CMV-9-rCDH6 were constructed by incorporating the cDNA fragments of the cDNA expression vector (OriGene, RN211850) encoding the rat CDH6 protein (NP_037059) into mammalian expression vectors according to methods well known to those skilled in the art. The amino acid sequence of the rat CDH6 ORF is shown in SEQ ID NO:8.

[0224] The cDNA encoding cynomolgus monkey CDH6 protein was cloned using cDNA synthesized from cynomolgus monkey kidney total RNA as a template with primer 1 (5'-CACCATGAGAACTTACCGCTACTTCTTGCTGCTC-3') (SEQ ID NO: 85) and primer 2 (5'-TTAGGAGTCTTTGTCACTGTCCACTCCTCC-3') (SEQ ID NO: 86). The resulting sequence was confirmed to match the extracellular domain of cynomolgus monkey CDH6 (NCBI, XP_005556691.1). Furthermore, the full-length sequence was confirmed to match that of cynomolgus monkey CDH6 (EHH54180.1) registered at EMBL. The cynomolgus monkey CDH6 expression vector, pcDNA3.1-cynoCDH6, was constructed by incorporating the fragment into a mammalian expression vector using methods known to those skilled in the art. The amino acid sequence of the cynomolgus monkey CDH6 ORF is shown in SEQ ID NO: 9.

[0225] The resulting plasmid DNA was prepared in large quantities using the EndoFree Plasmid Giga Kit (QIAGEN).

[0226] 1)-2 Immunity Female WKY / Izm rats (Japan SLC) were used for immunization. First, the rats' lower legs were pretreated with Hyaluronidase (Sigma-Aldrich), and then the human CDH6 expression vector pcDNA3.1-hCDH6 prepared in Example 1)-1 was intramuscularly injected into the same areas. Next, in vivo electroporation was performed at the same areas using ECM830 (BTX) and a two-needle electrode. Similar in vivo electroporation was repeated approximately once every two weeks, after which the rat's lymph nodes or spleens were collected and used for hybridoma production.

[0227] 1)-3 Hybridoma production Lymph node cells or spleen cells were electrofused with mouse myeloma SP2 / 0-ag14 cells (ATCC, No. CRL-1 581) using an LF301 Cell Fusion Unit (BEX), then suspended in ClonaCell-HY Selection Medium D (StemCell Technologies), diluted, and cultured at 37°C and 5% CO2. Each hybridoma colony that emerged was recovered as a monoclone and suspended in ClonaCell-HY Selection Medium E (StemCell Technologies) and cultured at 37°C and 5% CO2. After adequate cell proliferation, frozen stocks of each hybridoma cell were prepared, and the resulting hybridoma culture supernatant was used for screening of anti-human CDH6 antibody-producing hybridomas.

[0228] 1)-4 Screening of antibody-producing hybridomas by Cell-ELISA method 1)-4-1 Preparation of antigen gene-expressing cells for Cell-ELISA 293α cells (a stable cell line derived from HEK293 expressing integrin αv and integrin β3) were cultured at 5x10 in DMEM medium containing 10% FBS. 5The cells were transfected with pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, or pcDNA3.1 (negative control) DNA using Lipofectamine 2000 (Thermo Fisher Scientific). 100 μL of each transfection was dispensed into a 96-well plate (Corning) and cultured in 10% FBS-containing DMEM medium at 37°C in 5% CO2 for 24 to 27 hours. The transfected cells were used in Cell-ELISA while still adherent.

[0229] 1)-4-2 Cell-ELISA After removing the culture supernatant from the expression vector-transfected 293α cells prepared in Example 1)-4-1, hybridoma culture supernatant was added to each of the pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, or pcDNA3.1-transfected 293α cells and allowed to stand for 1 hour at 4° C. The cells in the wells were washed once with 5% FBS-containing PBS(+), and then anti-rat IgG-peroxidase antibody produced in rabbit (SIGMA) diluted 500-fold with 5% FBS-containing PBS(+) was added and allowed to stand for 1 hour at 4° C. After washing the cells in the wells three times with 5% FBS-containing PBS(+), 100 μL of OPD coloring solution (0.05 M trisodium citrate, 0.1 M disodium hydrogen phosphate 12-water, pH 4.5, dissolved in o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H2O2 at 0.4 mg / mL and 0.6% (v / v), respectively) was added to each well. The coloring reaction was allowed to proceed with occasional stirring, and 100 μL of 1 M HCl was added to each well to stop the coloring reaction. The absorbance at 490 nm was then measured using a plate reader (ENVISION: PerkinElmer). Hybridomas producing culture supernatants with higher absorbance from 293α cells transfected with pcDNA3.1-hCDH6 and pcDNA3.1-cynoCDH6 expression vectors compared to control 293α cells transfected with pcDNA3.1 were selected as antibody-producing hybridomas that bind to human and cynomolgus monkey CDH6.

[0230] 1)-5 Screening of selective binding antibodies to cynomolgus monkey CDH6 by flow cytometry 1)-5-1 Preparation of antigen gene-expressing cells for flow cytometry analysis 5 × 10 293T cells 4 cells / cm 2 225cm 2The cells were seeded into flasks (Sumitomo Bakelite Co., Ltd.) and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% CO2. 293T cells were transfected with pcDNA3.1-cynoCDH6 or pcDNA3.1 (as a negative control) using Lipofectamine 2000 and further cultured overnight at 37°C and 5% CO2. 293T cells transfected with each vector were treated with TrypLE Express (Thermo Fisher Scientific), washed with DMEM containing 10% FBS, and then suspended in PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.

[0231] 1)-5-2 Flow cytometry analysis The binding specificity of antibodies produced by hybridomas producing antibodies that bind to human and cynomolgus monkey CDH6, selected by Cell-ELISA in Example 1)-4, to cynomolgus monkey CDH6 was further confirmed by flow cytometry. The suspension of transiently expressing 293T cells prepared in Example 1)-5-1 was centrifuged, the supernatant removed, and each cell was suspended in hybridoma culture supernatant and allowed to stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were suspended in Anti-Rat IgG FITC conjugate (SIGMA) diluted 500-fold with 5% FBS-containing PBS and allowed to stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were resuspended in 5% FBS-containing PBS containing 2 μg / mL 7-aminoactinomycin D (Molecular Probes) and detected using a flow cytometer (FC500: Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). After excluding 7-aminoactinomycin D-positive dead cells by gating, a histogram of the FITC fluorescence intensity of live cells was created. Hybridomas producing antibodies in which the histogram of pcDNA3.1-cynoCDH6-transfected 293T cells was shifted to the stronger fluorescence intensity side compared to the fluorescence intensity histogram of control pcDNA3.1-transfected 293T cells were selected as hybridomas producing antibodies that specifically bind to cynomolgus monkey CDH6 expressed on the cell membrane surface.

[0232] 1)-6 Determination of the isotype of rat monoclonal antibodies Among the rat anti-CDH6 antibody-producing hybridomas selected in Example 1)-5, clones rG019, rG055, rG056, and rG061 were selected, which were suggested to bind strongly and specifically to human and monkey CDH6, and the isotype of each antibody was identified. The heavy chain subclass and light chain type of the antibodies were determined using a RAT MONOCLONAL ANTIBODY ISOTYPING TEST KIT (DS Pharma Biomedical). As a result, it was confirmed that the subclass of all four clones, rG019, rG055, rG056, and rG061, was IgG2b and the isotype was κ chain.

[0233] 1)-7 Preparation of rat anti-CDH6 antibody 1)-7-1 Preparation of culture supernatant Rat anti-human CDH6 monoclonal antibody was purified from hybridoma culture supernatant. First, rat anti-CDH6 monoclonal antibody-producing hybridomas were grown to sufficient numbers in ClonaCell-HY Selection Medium E (StemCell Technologies). The medium was then replaced with Hybridoma SFM (Thermo Fisher Scientific) supplemented with 20% Ultra Low IgG FBS (Thermo Fisher Scientific) and cultured for 4–5 days. The culture supernatant was collected and passed through a 0.8 μm filter, followed by a 0.2 μm filter to remove insoluble matter.

[0234] 1)-7-2 Purification of rat anti-CDH6 antibody Antibodies (rat anti-CDH6 antibodies (rG019, rG055, rG056, rG061)) were purified from the culture supernatant of the hybridoma prepared in Example 1)-7-1 by Protein G affinity chromatography. The antibodies were adsorbed onto a Protein G column (GE Healthcare Biosciences), washed with PBS, and then eluted with 0.1 M glycine / hydrochloric acid (pH 2.7). The eluate was adjusted to pH 7.0-7.5 with 1 M Tris-HCl (pH 9.0). The buffer was then replaced with HBS or (25 mM histidine / 5% sorbitol, pH 6.0) using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF30K, Sartorius) and the antibody was concentrated to a concentration of 1 mg / mL. Finally, the purified sample was filtered through a Minisart-Plus filter (Sartorius).

[0235] Example 2: In vitro evaluation of rat anti-CDH6 antibodies 2)-1 Evaluation of binding ability of rat anti-CDH6 antibody by flow cytometry The human CDH6 binding activity of the rat anti-CDH6 antibodies prepared in Example 1)-7 was evaluated by flow cytometry. 293T cells (ATCC) were transiently transfected with pcDNA3.1-hCDH6 prepared in Example 1)-1 using Lipofectamine 2000 (Thermo Fisher Scientific) and cultured overnight at 37°C and 5% CO2 to prepare a cell suspension. The transfected 293T cell suspension was centrifuged, the supernatant was removed, and the cells were suspended in one of four rat anti-CDH6 monoclonal antibodies (clone numbers rG019, rG055, rG056, and rG061) prepared in Example 1)-7 or a rat IgG control (R&D Systems) at a final concentration of 10 ng / mL. The cells were then incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were suspended in anti-rat IgG (whole molecule)-FITC antibody produced in rabbit (SIGMA) diluted 50-fold in 5% FBS-containing PBS and incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were detected using a flow cytometer (FC500: Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figure 1. In the histograms in Figure 1, the horizontal axis represents FITC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents cell number. The shaded histogram represents the results using negative control 293T cells not transfected with hCDH6, while the open solid line histogram represents the results using hCDH6-transfected 293T cells. The fluorescence intensity was enhanced by antibody binding to hCDH6 on the cell surface. The rat IgG control did not bind to any of the cells. As a result, it was confirmed that the four rat anti-CDH6 monoclonal antibodies produced bound to pcDNA3.1-hCDH6-transfected 293T cells.

[0236] 2)-2 Analysis of CDH6 binding sites of rat anti-CDH6 antibodies by flow cytometry 2)-2-1 Construction of human CDH6 domain deletion expression vectors The full-length extracellular sequence of human CDH6 contains five extracellular domains: EC1 (SEQ ID NO: 2), EC2 (SEQ ID NO: 3), EC3 (SEQ ID NO: 4), EC4 (SEQ ID NO: 5), and EC5 (SEQ ID NO: 6). Genes expressing full-length human CDH6 with one deletion from each of the five EC domains were synthesized by GeneArt, and these genes were then inserted into the mammalian expression vector p3xFLAG-CMV-9 (SIGMA-ALDRICH) according to methods known to those skilled in the art, to prepare expression vectors for each domain deletion, with EC1 to EC5 deleted, respectively.

[0237] 2)-2-2 Epitope analysis of rat anti-CDH6 antibodies using domain-deleted variants by flow cytometry The binding epitopes of rat anti-human CDH6 antibodies were identified by flow cytometry analysis using 293α cell lines transfected with each EC domain deletion vector. The 293α cell line, a cell line stably transfected with integrin αv and integrin β3 expression vectors into HEK293 cells, was transiently transfected with each domain deletion expression vector prepared in Example 2)-2-1 and pcDNA3.1-hCDH6 expressing full-length human CDH6 using Lipofectamine 2000 (Thermo Fisher Scientific). The cells were then cultured overnight at 37°C in 5% CO2, after which a cell suspension was prepared. The transfected 293α cell suspension was centrifuged and the supernatant removed. Four rat anti-CDH6 monoclonal antibodies (clone numbers rG019, rG055, rG056, and rG061) prepared in Example 1)-7 or a rat IgG control (R&D Systems) were added to a final concentration of 20 nM, and the cells were suspended and incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, an anti-rat IgG (whole molecule)-FITC antibody produced in rabbit (SIGMA) diluted 50-fold with 5% FBS-containing PBS was added and suspended, and the cells were incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, detection was performed using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figures 2-1 to 2-6. In the histograms in Figures 2-1 to 2-6, the horizontal axis represents FITC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents the number of cells. The shaded histograms represent the results using non-transfected negative control 293α cells, while the open solid histograms represent the results using full-length hCDH6 or 293 cells lacking each EC domain. When the antibody binds to full-length hCDH6 or each EC domain deletion on the cell surface, the fluorescence intensity increases. The rat IgG control does not bind to any of the transfected cells. The four rat anti-CDH6 monoclonal antibodies we generated bind to full-length hCDH6, EC1 deletion, EC2 deletion, EC4 deletion, and EC5 deletion, but not to the EC3 deletion.These results demonstrated that the four rat anti-CDH6 monoclonal antibodies specifically bound to the EC3 epitope of hCDH6.

[0238] 2)-3 Internalization activity of rat anti-CDH6 antibody 2)-3-1 Confirmation of CDH6 expression in human tumor cell lines To select CDH6-positive human tumor cell lines for antibody evaluation, CDH6 expression information was searched for in public databases, and CDH6 expression on the cell membrane surface was assessed by flow cytometry. Human ovarian tumor cell lines NIH: OVCAR-3, PA-1, and ES-2, and human renal cell tumor cell line 786-O (all obtained from ATCC) were cultured at 37°C in 5% CO2, and then cell suspensions were prepared. After centrifugation and removal of the supernatant, cells were resuspended in a commercial anti-human CDH6 antibody (MABU2715, R&D Systems) or a negative control mouse IgG1 (BD Pharmingen) at a final concentration of 50 μg / mL and incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, cells were resuspended in a 50-fold diluted F(ab')2 fragment of FITC-conjugated goat anti-mouse immunoglobulins (Dako) in 5% FBS-containing PBS and incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, detection was performed using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figure 3. In the histograms in Figure 3, the horizontal axis represents FITC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents cell number. The shaded histograms represent staining with the negative control mIgG1, while the open solid histograms represent staining with an anti-human CDH6 antibody. The enhanced fluorescence intensity was due to antibody binding to hCDH6 on the cell surface. The mIgG1 control did not bind to any of the cells. These results demonstrated that the NIH:OVCAR-3, PA-1, and 786-O cell lines endogenously express CDH6 on their cell surfaces. In contrast, the ES-2 cell line did not express CDH6 at all.

[0239] 2)-3-2 Evaluation of the internalization activity of rat anti-CDH6 antibody The internalization activity of rat anti-CDH6 antibodies was evaluated using the Rat-ZAP (ADVANCED TARGETING SYSTEMS) anti-rat IgG reagent conjugated with a toxin (saporin) that inhibits protein synthesis. Specifically, the human CDH6-positive ovarian tumor cell line NIH:OVCAR-3 (ATCC) was cultured at 4 x 10 3 The human CDH6-positive renal cell carcinoma cell line 786-O (ATCC) was seeded at 1x10 cells / well in a 96-well plate and cultured overnight at 37°C in 5% CO2. 3 Cells were seeded at 1000 cells / well in a 96-well plate and cultured overnight. The next day, rat anti-CDH6 antibody (final concentration: 1 nM) or rat IgG2b antibody (R&D Systems) was added as a negative control. Furthermore, Rat-ZAP (final concentration: 0.5 nM) or non-toxin-conjugated Goat Anti-Rat IgG, Fc(gamma) Fragment Specific (JACKSON IMMUNORESEARCH) (final concentration: 0.5 nM) was added as a negative control, and the cells were cultured for 3 days at 37°C under 5% CO2. The number of viable cells was determined using CellTiter-Glo. TM ATP activity (RLU) was measured using a Luminescent Cell Viability Assay (Promega). This assay demonstrated that Rat-ZAP was internalized into cells dependent on the internalization activity of the rat anti-CDH6 antibody, resulting in the release of saporin, which inhibits protein synthesis, and thus inhibits cell proliferation. The inhibitory effect of the anti-CDH6 antibody on cell proliferation was expressed as relative viability, with the number of viable cells in wells containing a negative control instead of Rat-ZAP taken as 100%. Figure 4 shows a graph and a table of cell viability. These results demonstrate that the rat anti-CDH6 antibody binds to CDH6 and induces its internalization.

[0240] Example 3: Determination of the nucleotide sequence of cDNA encoding the variable region of rat anti-CDH6 antibody 3)-1 Amplification and sequencing of gene fragments of the rG019 heavy and light chain variable regions 3)-1-1 Preparation of total RNA from G019 To amplify cDNA containing the variable region of rG019, total RNA was prepared from G019 using TRIzol Reagent (Ambion).

[0241] 3)-1-2 Amplification of cDNA containing the heavy chain variable region of rG019 by 5'-RACE PCR and determination of the nucleotide sequence Amplification of cDNA containing the heavy chain variable region was carried out using approximately 1 μg of the total RNA prepared in Example 3)-1-1 and the SMARTer RACE cDNA Amplification Kit (Clontech). Primers used to amplify the cDNA variable region of the heavy chain gene of rG019 by PCR were UPM (Universal Primer A Mix: included in the SMARTer RACE cDNA Amplification Kit) and primers designed from the sequence of a known rat heavy chain constant region.

[0242] The cDNA containing the heavy chain variable region amplified by 5'-RACE PCR was cloned into a plasmid, and then the nucleotide sequence of the heavy chain variable region cDNA was analyzed by sequencing.

[0243] The determined nucleotide sequence of the cDNA encoding the variable region of the heavy chain of rG019 is shown in SEQ ID NO:16, and the amino acid sequence is shown in SEQ ID NO:15.

[0244] 3)-1-3 Amplification of cDNA containing the light chain variable region of rG019 by 5'-RACE PCR and determination of the nucleotide sequence The same method as in Example 3)-1-2 was used, except that UPM (Universal Primer A Mix, included in the SMARTer RACE cDNA Amplification Kit) and primers designed from the known rat light chain constant region sequences were used as primers for PCR amplification of the cDNA variable region of the rG019 light chain gene.

[0245] The determined nucleotide sequence of the cDNA encoding the variable region of the light chain of rG019 is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:10.

[0246] 3)-2 Amplification and sequencing of gene fragments of the heavy and light chain variable regions of rG055 The sequence was determined in the same manner as in Example 3)-1.

[0247] The determined nucleotide sequence of the cDNA encoding the heavy chain variable region of rG055 is shown in SEQ ID NO: 26, and the amino acid sequence is shown in SEQ ID NO: 25. The nucleotide sequence of the cDNA encoding the light chain variable region is shown in SEQ ID NO: 21, and the amino acid sequence is shown in SEQ ID NO: 20.

[0248] 3)-3 Amplification and sequencing of gene fragments of the heavy and light chain variable regions of rG056 The sequence was determined in the same manner as in Example 3)-1.

[0249] The determined nucleotide sequence of the cDNA encoding the heavy chain variable region of rG056 is shown in SEQ ID NO: 36, and the amino acid sequence is shown in SEQ ID NO: 35. The nucleotide sequence of the cDNA encoding the light chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 30.

[0250] 3)-4 Amplification and sequencing of gene fragments of the heavy and light chain variable regions of rG061 The sequence was determined in the same manner as in Example 3)-1.

[0251] The determined nucleotide sequence of the cDNA encoding the heavy chain variable region of rG061 is shown in SEQ ID NO: 46, and the amino acid sequence is shown in SEQ ID NO: 45. The nucleotide sequence of the cDNA encoding the light chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence is shown in SEQ ID NO: 40.

[0252] Example 4: Preparation of human chimeric anti-CDH6 antibody chG019 4)-1 Construction of expression vector for human chimeric anti-CDH6 antibody chG019 4)-1-1 Construction of chimeric and humanized light chain expression vector pCMA-LK Plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) was digested with the restriction enzymes XbaI and PmeI to obtain a fragment of approximately 5.4 kb. This fragment was ligated to a DNA fragment containing the DNA sequence encoding the human light chain signal sequence and human κ chain constant region shown in SEQ ID NO: 50 using an In-Fusion Advantage PCR Cloning Kit (Clontech) to produce pcDNA3.3 / LK.

[0253] pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.

[0254] 4)-1-2 Construction of chimeric and humanized IgG1-type heavy chain expression vector pCMA-G1 pCMA-LK was digested with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region. This DNA fragment was ligated to a DNA fragment containing the DNA sequence encoding the human heavy chain signal sequence and human IgG1 constant region shown in SEQ ID NO: 51 using an In-Fusion Advantage PCR cloning kit (Clontech) to construct pCMA-G1.

[0255] 4)-1-3 Construction of chG019 heavy chain expression vector A DNA fragment represented by nucleotides 36 to 440 of the nucleotide sequence of the chG019 heavy chain shown in SEQ ID NO: 57 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), pCMA-G1 was cleaved with the restriction enzyme BlpI and the synthesized DNA fragment was inserted into the site, thereby constructing a chG019 heavy chain expression vector. Note that the chG019 heavy chain had a sequence in which cysteine ​​in the CDR was substituted with proline to prevent unexpected disulfide bonds.

[0256] 4)-1-4 Construction of chG019 light chain expression vector A DNA fragment containing the DNA sequence encoding the chG019 light chain shown in SEQ ID NO: 52 was synthesized (GENEART). Using an In-Fusion HD PCR Cloning Kit (Clontech), the synthesized DNA fragment was ligated to a DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region, thereby constructing a chG019 light chain expression vector.

[0257] 4)-2 Production and purification of human chimeric anti-CDH6 antibody chG019 4)-2-1 Production of chG019 FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the manufacturer's instructions. 1.2 × 10 cells were cultured in the logarithmic growth phase. 9 FreeStyle 293F cells (Invitrogen) were seeded in a 3L Fernbach Erlenmeyer Flask (Corning) and diluted with FreeStyle 293 expression medium (Invitrogen) to a concentration of 2.0 × 10 6The cells were cultured at a concentration of 1000 cells / ml. 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector, and 1.8 mg of polyethyleneimine (Polyscience #24765) were added to 40 ml of Opti-Pro SFM medium (Invitrogen) and gently mixed. The mixture was then left for 5 minutes before being added to FreeStyle 293F cells. After incubation at 90 rpm in an incubator at 37°C and 8% CO2 for 4 hours, 600 ml of EX-CELL VPRO medium (SAFC Biosciences), 18 ml of GlutaMAX I (GIBCO), and 30 ml of Yeastolate Ultrafiltrate (GIBCO) were added. The cells were then cultured at 90 rpm in an incubator at 37°C and 8% CO2 for 7 days. The resulting culture supernatant was filtered through a disposable capsule filter (Advantec #CCS-045-E1H).

[0258] 4)-2-2 Purification of chG019 The culture supernatant obtained in Example 4)-2-1 was purified by a single-step process of rProtein A affinity chromatography. The culture supernatant was applied to a column packed with MabSelectSuRe (GE Healthcare Biosciences) equilibrated with PBS, and the column was washed with at least two column volumes of PBS. The antibody-containing fraction was then eluted with 2 M arginine hydrochloride solution (pH 4.0), and the antibody-containing fraction was collected. The fraction was then buffer-exchanged to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 5 mg / ml or higher. Finally, the purified sample was filtered through a Minisart-Plus filter (Sartorius).

[0259] 4)-3 Binding evaluation of the human chimeric anti-CDH6 antibody chG019 The CDH6 binding activity of the human chimeric anti-CDH6 antibody chG019 purified in Example 4)-2 was confirmed by flow cytometry. 293α cells were transiently transfected with pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, or pcDNA3.1 prepared in Example 1)-1 using Lipofectamine 2000. After overnight culture at 37°C and 5% CO2, cell suspensions were prepared. The cells were added with chG019 and incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were suspended in PE-labeled F(ab')2 Fragment anti-human IgG, Fcγ antibody (JACKSON IMMUNORESEARCH) diluted 500-fold with 5% FBS-containing PBS. The cells were then incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were resuspended in 5% FBS-containing PBS and subjected to detection using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). As shown in Figure 5, chG019 did not bind to the negative control pcDNA3.1-transfected 293T cells, but bound to pcDNA3.1-hCDH6 and pcDNA3.1-cynoCDH6-transfected 293T cells in an antibody concentration-dependent manner. In Figure 5, the horizontal axis represents antibody concentration, and the vertical axis represents the amount of binding (Mean Fluorescent Intensity). These results demonstrate that chG019 specifically binds to human and cynomolgus monkey CDH6 with nearly equivalent binding activity.

[0260] [Example 5: Production of humanized anti-CDH6 antibody] 5)-1 Design of humanized anti-CDH6 antibodies 5)-1-1 Molecular modeling of the variable region of chG019 Molecular modeling of the variable regions of chG019 was performed using a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The structure (PDB ID: 2I9L) registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)), which has high sequence identity to the variable regions of the heavy and light chains of chG019, was used as a template, and the commercially available protein 3D structure analysis program BioLuminate (Schrodinger) was used.

[0261] 5)-1-2 Design of the amino acid sequence for humanized hG019 chG019 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The consensus sequences for human gamma chain subgroup 1 and kappa chain subgroup 1 defined by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) were selected as the heavy and light chain acceptors, respectively, because they share high identity with the framework regions of chG019. The donor residues to be transferred onto the acceptors were selected by analyzing three-dimensional models, using criteria such as those provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

[0262] 5)-2 Humanization of chG019 heavy chain The three designed heavy chains were designated hH01, hH02, and hH04. The full-length amino acid sequence of the heavy chain of hH01 is set forth in SEQ ID NO:69. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:69 is set forth in SEQ ID NO:70. The full-length amino acid sequence of the heavy chain of hH02 is set forth in SEQ ID NO:73. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:73 is set forth in SEQ ID NO:74. The full-length amino acid sequence of the heavy chain of hH04 is set forth in SEQ ID NO:77. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:77 is set forth in SEQ ID NO:78.

[0263] 5)-3 Humanization of chG019 light chain The two designed light chains were designated hL02 and hL03. The full-length amino acid sequence of the light chain of hL02 is set forth in SEQ ID NO:61. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:61 is set forth in SEQ ID NO:62. The full-length amino acid sequence of the light chain of hL03 is set forth in SEQ ID NO:65. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:65 is set forth in SEQ ID NO:66.

[0264] 5)-4 Design of humanized hG019 by combining heavy and light chains An antibody consisting of hH01 and hL02 is referred to as the "H01L02 antibody" or "H01L02." An antibody consisting of hH02 and hL02 is referred to as the "H02L02 antibody" or "H02L02." An antibody consisting of hH02 and hL03 is referred to as the "H02L03 antibody" or "H02L03." An antibody consisting of hH04 and hL02 is referred to as the "H04L02 antibody" or "H04L02."

[0265] 5)-5 Expression of humanized anti-CDH6 antibody 5)-5-1 Construction of humanized hG019 heavy chain expression vector 5)-5-1-1 Construction of humanized hG019-H01 type heavy chain expression vector A DNA fragment represented by nucleotides 36 to 440 of the nucleotide sequence of the humanized hG019-H01 type heavy chain shown in SEQ ID NO: 70 was synthesized (GENEART). A humanized hG019-H01 type heavy chain expression vector was constructed in the same manner as in Example 4)-1-3.

[0266] 5)-5-1-2 Construction of humanized hG019-H02 type heavy chain expression vector A DNA fragment represented by nucleotides 36 to 440 of the nucleotide sequence of the humanized hG019-H02 type heavy chain shown in SEQ ID NO: 74 was synthesized (GENEART). A humanized hG019-H02 type heavy chain expression vector was constructed in the same manner as in Example 4)-1-3.

[0267] 5)-5-1-3 Construction of humanized hG019-H04 type heavy chain expression vector A DNA fragment represented by nucleotides 36 to 440 of the nucleotide sequence of the humanized hG019-H04 type heavy chain shown in SEQ ID NO: 78 was synthesized (GENEART). A humanized hG019-H04 type heavy chain expression vector was constructed in the same manner as in Example 4)-1-3.

[0268] 5)-5-2 Construction of humanized hG019 light chain expression vector 5)-5-2-1 Construction of humanized hG019-L02 type light chain expression vector A DNA fragment containing a DNA sequence encoding the variable region of the humanized hG019-L02-type light chain represented by nucleotides 37 to 399 of the nucleotide sequence of the humanized hG019-L02-type light chain represented by SEQ ID NO: 62 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), pCMA-LK was cleaved with the restriction enzyme BsiWI and the synthesized DNA fragment was inserted into the site, thereby constructing a humanized hG019-L02-type light chain expression vector.

[0269] 5)-5-2-2 Construction of humanized hG019-L03 type light chain expression vector A DNA fragment containing a DNA sequence encoding the variable region of the humanized hG019-L03 type light chain represented by nucleotides 37 to 399 of the nucleotide sequence of the humanized hG019-L03 type light chain represented by SEQ ID NO: 66 was synthesized (GENEART). A humanized hG019-L03 type light chain expression vector was constructed in the same manner as in Example 5)-5-2-1.

[0270] 5)-5-3 Preparation of humanized hG019 5)-5-3-1 Production of H01L02, H02L02, H02L03, and H04L02 They were produced in the same manner as in Example 4)-2-1. H01L02, H02L02, H02L03, and H04L02 were produced by combining the heavy chains and light chains shown in Example 5)-4.

[0271] 5)-5-3-2 Two-step purification of H01L02, H02L02, H02L03, and H04L02 The culture supernatant obtained in Example 5)-5-3-1 was purified in two steps: rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a column (GE Healthcare Bioscience) packed with MabSelectSuRe equilibrated with PBS, and the column was washed with at least two column volumes of PBS. The antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was subjected to buffer exchange with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), diluted five-fold with 5 mM sodium phosphate / 50 mM MES / pH 7.0 buffer, and then applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0 buffer. A linear gradient elution with sodium chloride was performed, and the antibody-containing fractions were collected. The fractions were then dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) to exchange the buffer for HBSor (25 mM histidine / 5% sorbitol, pH 6.0). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 20 mg / ml. Finally, the purified sample was filtered through a Minisart-Plus filter (Sartorius).

[0272] [Reference Example 1: Production of anti-CDH6 antibody NOV0712] The anti-CDH6 antibody NOV0712 used in the examples was produced with reference to the amino acid sequences of the full-length light chain and full-length heavy chain of NOV0712 described in WO 2016 / 024195 (sequence numbers 235 and 234, respectively, in WO 2016 / 024195).

[0273] Reference example 1)-1 Anti-CDH6 antibody NOV0712 Reference Example 1)-1-1 Construction of heavy chain expression vector for anti-CDH6 antibody NOV0712 A DNA fragment containing the variable region of the heavy chain of NOV0712 represented by nucleotides 36 to 428 of the nucleotide sequence of the heavy chain of NOV0712 represented by SEQ ID NO: 84 was synthesized (GENEART). A heavy chain expression vector for NOV0712 was constructed in the same manner as in Example 4)-1-3. The amino acid sequence of the heavy chain of NOV0712 expressed by the heavy chain expression vector for NOV0712 is represented by SEQ ID NO: 83. In the amino acid sequence represented by SEQ ID NO: 83, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence.

[0274] Reference Example 1)-1-2 Construction of light chain expression vector for anti-CDH6 antibody NOV0712 A DNA fragment containing a DNA sequence encoding the variable region of the light chain of NOV0712 represented by nucleotides 37 to 405 of the nucleotide sequence of the light chain of NOV0712 represented by SEQ ID NO: 82 was synthesized (GENEART). A light chain expression vector for NOV0712 was constructed in the same manner as in Example 5)-5-2-1. The amino acid sequence of the light chain of NOV0712 expressed by the light chain expression vector for NOV0712 is represented by SEQ ID NO: 81. In the amino acid sequence represented by SEQ ID NO: 81, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence.

[0275] Reference Example 1)-2 Preparation of anti-CDH6 antibody NOV0712 Reference Example 1)-2-1 Production of anti-CDH6 antibody NOV0712 NOV0712 was produced in the same manner as in Example 4)-2-1.

[0276] Reference Example 1)-2-2 One-step purification of anti-CDH6 antibody NOV0712 The anti-CDH6 antibody NOV0712 was purified from the culture supernatant obtained in Reference Example 1)-2-1 in the same manner as in Example 4)-2-2 (antibody concentration: 5 mg / L in HBS or).

[0277] Example 6: In vitro evaluation of humanized hG019 and NOV0712 6)-1 Binding evaluation of humanized hG019 6)-1-1 Human CDH6 antigen binding ability of humanized hG019 The dissociation constant between the antibody and antigen (Recombinant Human CDH6 Fc His chimera, R&D Systems) was measured using a Biacore T200 (GE Healthcare Biosciences) by the capture method, in which the antigen was captured as a ligand by an immobilized anti-His antibody and the antibody was measured as an analyte. An anti-histidine antibody (His capture kit, GE Healthcare Biosciences) was covalently bound to a sensor chip CM5 (GE Healthcare Biosciences) at approximately 1000 RU using the amine coupling method. A reference cell was also immobilized in the same manner. HBS-P+ (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20) supplemented with 1 mM CaCl2 was used as the running buffer. Antigen was applied to a chip with immobilized anti-histidine antibody for 60 seconds, followed by the addition of a dilution series of antibody solutions (0.391-100 nM) at a flow rate of 30 μl / min for 300 seconds. The dissociation phase was then monitored for 600 seconds. A regenerating solution, glycine solution (pH 1.5) supplemented with 5 M MgCl2, was added twice for 30 seconds at a flow rate of 10 μl / min. Data were analyzed using the Steady State Affinity model in the analysis software (BIAevaluation software, version 4.1), and the dissociation constant (KD) was calculated. The results are shown in Table 2.

[0278] [Table 2]

[0279] 6)-1-2 Binding to human, monkey, mouse, and rat CDH6 293α cells were transiently transfected with pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, p3xFLAG-CMV-9-mCDH6, and p3xFLAG-CMV-9-rCDH6 prepared in Example 1)-1 using Lipofectamine 2000. After overnight culture at 37°C and 5% CO2, a cell suspension was prepared. Non-transfected 293α cells were used as a negative control. The 293α cell suspension prepared above was centrifuged, and the supernatant was removed. The cells were then suspended in one of four humanized hG019 antibodies (clone numbers H01L02, H02L02, H02L03, and H04L02) prepared in Example 5)-5-3 or a human IgG1 control (Calbiochem). The cells were then incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, anti-human Fcg PE goat F(ab') (Jackson Laboratory) diluted 500-fold in 5% FBS-containing PBS was added and suspended, and the cells were incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, detection was performed using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). In Figures 6-1 and 6-2, the horizontal axis represents antibody concentration, and the vertical axis represents binding amount (Mean Fluorescent Intensity). As shown in Figures 6-1 and 6-2, the negative control human IgG1 control did not bind to any of the CDH6-transfected cells. Four humanized hG019 antibodies (clone numbers H01L02, H02L02, H02L03, and H04L02) bound to human and cynomolgus monkey CDH6 but not to mouse or rat CDH6. None of the antibodies bind to cells transfected with the empty vector pcDNA3.1 (a negative control). Meanwhile, WO 2016 / 024195 shows that the NOV0712 antibody exhibits binding activity to human, cynomolgus monkey, mouse, and rat CDH6. These results demonstrate that the four humanized hG019 antibodies obtained herein are anti-CDH6 antibodies that exhibit binding properties different from those of the NOV0712 antibody.

[0280] 6)-2 Analysis of the CDH6 binding sites of humanized hG019 and NOV0712 6)-2-1 Epitope analysis using domain-deleted antibodies The domain-deleted expression vectors prepared in Example 2)-2-1 and pcDNA3.1-hCDH6 expressing full-length hCDH6 were transiently transfected using Lipofectamine 2000 (Thermo Fisher Scientific) and cultured overnight at 37°C in 5% CO2 to prepare a cell suspension. The transfected 293α cell suspension was centrifuged, the supernatant was removed, and the cells were suspended in one of four humanized hG019 antibodies (clone numbers H01L02, H02L02, H02L03, and H04L02) prepared in Example 5)-5-3, the anti-CDH6 antibody NOV0712 prepared in Reference Example 1, or human IgG1 (Calbiochem) as a negative control. The cells were then incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, APC-anti-human IgG goat F(ab')2 (Jackson Laboratory) diluted 500-fold in 5% FBS-containing PBS was added and suspended, and the cells were incubated at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, detection was performed using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figures 7-1 to 7-6. In the histograms in Figures 7-1 to 7-6, the horizontal axis represents the APC fluorescence intensity, which indicates the amount of antibody binding, and the vertical axis represents the cell count. The shaded histograms represent the results using non-transfected negative control 293α cells, while the open solid histograms represent the results using full-length hCDH6 or EC domain-deleted 293α cells. Fluorescence intensity increased when the antibody bound to full-length hCDH6 or each EC domain-deleted hCDH6 on the cell surface. The human IgG1 control did not bind to either transfected cell line. Four humanized hG019 antibodies (clone numbers: H01L02, H02L02, H02L03, and H04L02) bind to full-length hCDH6, EC1-deleted hCDH6, EC2-deleted hCDH6, EC4-deleted hCDH6, and EC5-deleted hCDH6, but not to EC3-deleted hCDH6. This indicates that the four humanized hG019 antibodies specifically bind to the EC3 epitope of hCDH6.On the other hand, the anti-CDH6 antibody NOV0712 binds to full-length hCDH6, EC1 deletion, EC2 deletion, EC3 deletion, and EC4 deletion, but not to the EC5 deletion. In other words, the anti-CDH6 antibody NOV0712 was shown to specifically bind to the EC5 epitope of hCDH6, which is consistent with the epitope information for NOV0712 described in WO 2016 / 024195. These results demonstrate that NOV0712 and the four humanized hG019 antibodies obtained herein are anti-CDH6 antibodies exhibiting different properties.

[0281] 6)-2-2 Antibody binding competition assay 6)-2-2-1 Creation of 786-O / hCDH6 stable expressing cell line The 786-O / hCDH6 stable cell line was generated by infecting 786-O cells (ATCC) with a recombinant retrovirus expressing full-length human CDH6. The human CDH6-expressing retroviral vector (pQCXIN-hCDH6) was generated by incorporating a cDNA expression vector (OriGene RC217889) encoding the human CDH6 protein (NP_004923) into the retroviral vector pQCXIN (CLONTECH) according to methods known to those skilled in the art. pQCXIN-hCDH6 was transiently transfected into RetroPack PT67 (CLONTECH) retroviral packaging cells using FuGene HD (Promega). After 48 hours, the culture supernatant containing the recombinant retrovirus was collected and added to a 786-O cell culture system to infect the cells. Three days after infection, infected cells were cultured at 37°C in 5% CO2 in medium supplemented with G418 (Gibco) at a final concentration of 50 mg / mL, and drug selection was performed to establish the cell line 786-O / hCDH6, which stably expresses human CDH6. High expression of human CDH6 in the stable expression line was confirmed by flow cytometry as in Example 2)-3-1 (Figure 8). Goat anti-mouse IgG1 secondary antibody Alexa Fluor 647 (Thermo Fisher Scientific), diluted 500-fold with 5% FBS-containing PBS, was used as the detection antibody. The results are shown in Figure 8. In the histogram in Figure 8, the horizontal axis represents the fluorescence intensity of Alexa Fluor 647, which indicates the amount of antibody binding, and the vertical axis represents the cell number. The shaded histogram represents cells stained with the negative control mIgG1, and the open solid line histogram represents cells stained with the anti-human CDH6 antibody. This shows that the antibody binding to hCDH6 on the cell surface resulted in increased fluorescence intensity. The mIgG1 control did not bind to either cell. These results demonstrate that the 786-O / hCDH6 stable expressing cell line expresses higher levels of human CDH6 than the parent 786-O cells.

[0282] 6)-2-2-2 Binding competition assay using labeled H01L02 and labeled NOV0712 Labeled H01L02 and labeled NOV0712 were prepared using the Alexa Fluor 488 Monoclonal Antibody Labeling Kit (Thermo Fisher). The cell suspension of the 786-O / hCDH6-stably expressing cell line prepared in 7)-2-2-1 was centrifuged, the supernatant was removed, and labeled NOV0712 or labeled H01L02 was added to a final concentration of 5 nM. Four types of humanized hG019 antibodies (clone numbers: H01L02, H02L02, H02L03, and H04L02) prepared in Example 5)-5-3, the anti-CDH6 antibody NOV0712 prepared in Reference Example 1, or human IgG1 (Calbiochem) as a negative control were added at the final concentrations shown on the horizontal axis in Figure 9, the suspension was suspended, and the mixture was allowed to stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, detection was performed using a flow cytometer (Canto II: BD Biosciences). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figure 9. The horizontal axis indicates the final concentration of unlabeled antibody at the time of addition, and the vertical axis indicates the amount of binding as mean fluorescent intensity. When unlabeled NOV0712 was added to cells treated with labeled NOV0712, the unlabeled antibody was replaced in a concentration-dependent manner, resulting in a decrease in the amount of binding of the labeled antibody, as they share the same epitope and compete for binding. On the other hand, when four humanized hG019 antibodies or human IgG1 as a negative control were added to cells treated with labeled NOV0712, there was no change in the amount of binding of the labeled antibody, indicating that these antibodies have different epitopes and do not compete for binding. Similarly, when four unlabeled humanized hG019 antibodies were added to cells to which labeled H01L02 had been added, they had the same epitope and competed for binding, resulting in a concentration-dependent displacement of the unlabeled antibody and a decrease in the amount of binding of the labeled antibody. On the other hand, when NOV0712 or human IgG1 (a negative control) was added to cells to which labeled H01L02 had been added, there was no change in the amount of binding of the labeled antibody, indicating that these antibodies have different epitopes and do not compete for binding.

[0283] 6)-3 Evaluation of internalization activity of humanized hG019 and NOV0712 The internalization activity of humanized hG019 and NOV0712 was evaluated using the anti-human IgG reagent Hum-ZAP (ADVANCED TARGETING SYSTEMS), which is conjugated with a toxin (saporin) that inhibits protein synthesis. The human CDH6-positive ovarian tumor cell line NIH:OVCAR-3 (ATCC) was cultured at 4 x 10 3 The human CDH6-positive renal cell carcinoma cell line 786-O (ATCC) was seeded at 1x10 cells / well in a 96-well plate and cultured overnight at 37°C in 5% CO2. 3 Human CDH6-positive ovarian tumor cell line PA-1 (ATCC) was seeded at 1x10 cells / well in a 96-well plate and cultured overnight. 3 The cells were seeded onto a 96-well plate at 1000 cells / well and cultured overnight at 37°C in 5% CO2. The next day, anti-CDH6 antibody (final concentration: 1 nM) or a human IgG1 antibody (Calbiochem) was added as a negative control. Hum-ZAP (final concentration: 0.5 nM) or a non-toxin-conjugated F(ab')2 Fragment Goat Anti-human IgG, Fc(gamma) Fragment Specific (JACKSON IMMUNORESEARCH) (final concentration: 0.5 nM) was then added as a negative control, and the cells were cultured for 3 days at 37°C in 5% CO2. The number of viable cells was determined using CellTiter-Glo. TMATP activity (RLU) was measured using a luminescent cell viability assay. In this assay, the internalization activity of the humanized anti-CDH6 antibody was dependent on the intracellular uptake of Hum-ZAP, which then releases saporin, a protein synthesis inhibitor, into the cells, suppressing cell proliferation. The cell proliferation inhibitory effect of the anti-CDH6 antibody was expressed as relative viability, with the number of viable cells in wells containing a negative control instead of Hum-ZAP being set at 100%. Figures 10-1 to 10-3 show graphs and cell viability tables. In this experiment, antibodies with strong internalization activity were expected to exhibit low cell viability. The results showed that NOV0712 exhibited significantly higher internalization activity than NOV0712, with predicted internalization rates of approximately 50-75% in all three cell lines. Based on the mechanism of action of ADCs, antibodies with high internalization activity are considered more suitable for ADC antibody development.

[0284] Example 7: Preparation of humanized hG019-drug conjugates 7)-1 Preparation of antibody-drug conjugates H01L02-DXd Step 1: Antibody-drug conjugate (1)

[0285] [ka]

[0286] Antibody reduction: H01L02 prepared in Example 5 was reduced to 1.53 mL / min as the 280 nm extinction coefficient by the common procedure B described in Production Method 1. -1 cm -1Using 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.231 mL; 6.0 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0855 mL) were added to this solution (5.7 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bonds of the antibody.

[0287] Conjugation of antibody and drug linker: The above solution was incubated for 10 minutes at 15° C. Next, a 10 mM dimethyl sulfoxide solution (0.386 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15° C. for 1 hour to allow binding of the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

[0288] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H01L02-ADC."

[0289] Characterization: Common procedure E(ε D,280 =5440 、 ε D,370 =21240) to obtain the following characteristic values. Antibody concentration: 2.26 mg / mL, antibody yield: 42.9 mg (76%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 5.9; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.7.

[0290] 7)-2 Preparation of antibody-drug conjugates H02L02-DXd Step 1: Antibody-drug conjugate (2)

[0291] [ka]

[0292] Antibody reduction: H02L02 prepared in Example 5 was reduced to 1.51 mL / min as the 280 nm extinction coefficient by the common procedure B described in Production Method 1. -1 cm -1 Using 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.234 mL; 6.0 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0855 mL) were added to this solution (5.7 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bonds of the antibody.

[0293] Conjugation of antibody and drug linker: The above solution was incubated for 10 minutes at 15° C. Next, a 10 mM dimethyl sulfoxide solution (0.389 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15° C. for 1 hour to allow binding of the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0350 mL; 9 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

[0294] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H02L02-ADC."

[0295] Characterization: Common procedure E(ε D,280 =5440 、 ε D,370 =21240) to obtain the following characteristic values. Antibody concentration: 2.61 mg / mL, antibody yield: 49.6 mg (87%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 5.9; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.6.

[0296] 7)-3 Preparation of antibody-drug conjugates H02L03-DXd Step 1: Antibody-drug conjugate (3)

[0297] [ka]

[0298] Antibody reduction: H02L03 prepared in Example 5 was reduced to 1.53 mL / min as the 280 nm extinction coefficient by the common procedure B described in Production Method 1. -1 cm -1 Using 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.270 mL; 7.0 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0855 mL) were added to this solution (5.7 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bonds of the antibody.

[0299] Conjugation of antibody and drug linker: The above solution was incubated for 10 minutes at 15° C. Next, a 10 mM dimethyl sulfoxide solution (0.386 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15° C. for 1 hour to allow binding of the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

[0300] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H01L02-ADC."

[0301] Characterization: Common procedure E(εD,280 =5440 、 ε D,370 =21240) to obtain the following characteristic values. Antibody concentration: 2.71 mg / mL, antibody yield: 51.4 mg (91%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 5.7; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.6.

[0302] 7)-4 Preparation of antibody-drug conjugates H04L02-DXd Step 1: Antibody-drug conjugate (4)

[0303] [ka]

[0304] Antibody reduction: H04L02 prepared in Example 5 was reduced to 1.53 mL / min as the 280 nm extinction coefficient by the common procedure B described in Production Method 1. -1 cm -1 Using 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.232 mL; 6.0 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0855 mL) were added to this solution (5.7 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bonds of the antibody.

[0305] Conjugation of antibody and drug linker: The above solution was incubated for 10 minutes at 15° C. Next, a 10 mM dimethyl sulfoxide solution (0.386 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15° C. for 1 hour to allow binding of the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

[0306] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H04L02-ADC."

[0307] Characterization: Common procedure E(ε D,280 =5440 、 ε D,370 =21240) to obtain the following characteristic values. Antibody concentration: 2.56 mg / mL, antibody yield: 48.7 mg (87%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 5.8; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.6.

[0308] [Reference Example 2: Preparation of NOV0712-drug conjugate] Reference Example 2)-1 Preparation of antibody-drug conjugate NOV0712-DM4 Antibody-drug conjugates (5) Conjugation of antibody and drug linker: NOV0712 prepared in Reference Example 1 was conjugated to a drug linker in a concentration of 1.51 mL / min as calculated at 280 nm using the common procedure B described in Production Method 1. -1 cm -1 Using 20 mM HEPES 8.1 (LIFE TECHNOLOGIES HEPES, 1 M Buffer Solution (20 mL) was adjusted to pH 8.1 with 1 M sodium hydroxide and then made up to 1 L with distilled water) and C, the concentration was adjusted to 9.7 mg / mL, and the solution was incubated at 20°C for 10 minutes. Next, a DMA solution of 10 mM 1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid (described in WO2016 / 024195; 5.2 equivalents per antibody molecule), a DMA solution of 10 mM N2-deacetyl-deacetyl-N2-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4; 0.366 mL; 6.8 equivalents per antibody molecule), and 0.243 mL of DMA were added and incubated at 20°C for 16 hours to conjugate the drug linker to the antibody. Next, 1 M aqueous acetic acid was added to adjust the pH to 5.0, and the mixture was stirred at room temperature for another 20 minutes to quench the drug linker reaction.

[0309] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 28 mL of a solution containing the title antibody-drug conjugate "NOV0712-DM4."

[0310] Characterization: Common procedure E(ε A,280 =200500 、 ε A,252 =76295, ε D,280 =43170 、 and ε D,252 =23224) to obtain the following characteristic values. Antibody concentration: 2.58 mg / mL, antibody yield: 72.2 mg (93%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 3.0.

[0311] Reference Example 2)-2 Preparation of antibody-drug conjugate NOV0712-DXd Step 1: Antibody-drug conjugate (6)

[0312] [ka]

[0313] Antibody reduction: NOV0712 prepared in Reference Example 1 was reduced to 1.51 mL / min as absorbance coefficient at 280 nm by the common procedure B described in Production Method 1. -1 cm -1 The antibody was diluted to 9.26 mg / mL in PBS 6.0 / EDTA using ATP (prepared with ATP) and C. To this solution (6.6 mL) was added a 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.254 mL; 6.0 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0990 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bonds of the antibody.

[0314] Conjugation of antibody and drug linker: The above solution was incubated for 10 minutes at 15° C. Next, a 10 mM dimethyl sulfoxide solution (0.381 mL; 9 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15° C. for 1 hour to allow binding of the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0381 mL; 9 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

[0315] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 23.5 mL of a solution containing the title antibody-drug conjugate "NOV0712-ADC."

[0316] Characterization: Common procedure E(ε D,280 =5440 、 ε D,370 =21240) to obtain the following characteristic values. Antibody concentration: 2.26 mg / mL, antibody yield: 56.4 mg (92%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 6.4; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.8.

[0317] [Reference Example 3: Preparation of H01L02-DM4] Reference Example 3)-1 Preparation of antibody-drug conjugate H01L02-DM4 Antibody-drug conjugates (7) Conjugation of antibody and drug linker: H01L02 prepared in Example 5 was subjected to common procedure B described in Production Method 1 (1.53 mL mg as 280 nm extinction coefficient). -1 cm -1Using 20 mM HEPES 8.1 (LIFE TECHNOLOGIES HEPES, 1 M Buffer Solution (20 mL) was adjusted to pH 8.1 with 1 M sodium hydroxide and then made up to 1 L with distilled water) and the concentration was adjusted to 9.8 mg / mL using 20 mM HEPES 8.1 (LIFE TECHNOLOGIES HEPES, 1 M Buffer Solution (20 mL) was adjusted to pH 8.1 with 1 M sodium hydroxide and made up to 1 L with distilled water), and the solution was incubated at 20°C for 10 minutes. Next, a DMA solution of 10 mM 1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid (described in WO2016 / 024195; 11.5 equivalents per antibody molecule) and a DMA solution of 10 mM N2-deacetyl-N2-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) (0.082 mL; 15.1 equivalents per antibody molecule) were added and incubated at 20°C for 18 hours to allow the drug linker to bind to the antibody. Next, 1 M aqueous acetic acid was added to adjust the pH to 5.0, and the mixture was stirred at room temperature for 20 minutes to quench the drug linker reaction.

[0318] Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 3.5 mL of a solution containing the title antibody-drug conjugate "H01L02-DM4."

[0319] Characterization: Using the common procedure E described in Preparation Method 1 (using εA,280=223400, εA,252=85646, εD,280=4317, and εD,252=23224), the following property values ​​were obtained. Antibody concentration: 1.97 mg / mL, antibody yield: 6.90 mg (88%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 3.6.

[0320] Example 8: In vitro activity evaluation of antibody-drug conjugates 8)-1 In vitro cytostatic activity of antibody-drug conjugates against CDH6-positive human tumor cell lines CDH6-positive human ovarian tumor cell line PA-1 was cultured at 2 × 10 in MEM medium containing 10% FBS. 3The cells were seeded onto a 96-well plate at 100 μL / well and cultured overnight at 37°C in 5% CO2. The following day, four types of humanized hG019-drug conjugates (clone names: H01L02-DXd, H02L02-DXd, H02L03-DXd, and H04L02-DXd) prepared in Example 7 or the NOV0712-drug conjugate (NOV0712-DM4) prepared in Reference Example 2 were added to a final concentration of 0.0001 (nM) to 100 (nM). After 4 days of culture, the viable cells were counted using CellTiter-Glo. TM ATP was measured using a Luminescent Cell Viability Assay (Promega). Figure 11 shows the concentration-dependent cell growth inhibitory activity of each antibody-drug conjugate. The results show that the four humanized hG019-drug conjugates exhibited tumor cell growth inhibitory activity at lower concentrations than the NOV0712-drug conjugate, demonstrating their high antitumor activity.

[0321] Example 9: In vivo antitumor effect of antibody-drug conjugates The antitumor efficacy of antibody-drug conjugates was evaluated using an animal model in which CDH6-positive human tumor cell lines were transplanted into immunodeficient mice. Four- to five-week-old BALB / c nude mice (CAnN.Cg-Foxnl[nu] / CrlCrlj[Foxnlnu / Foxnlnu], Charles River, Japan) and SCID mice (CB17 / Icr-Prkdc[scid] / CrlCrlj, Charles River, Japan) were acclimated to specific-pathogen-free (SPF) conditions for at least 3 days before use. Mice were fed sterilized solid diet (FR-2, Funabashi Farms Co., Ltd.) and sterilized tap water (prepared with 5-15 ppm sodium hypochlorite solution). The major and minor diameters of the transplanted tumors were measured twice weekly using electronic digital calipers (CD-15CX, Mitutoyo Corp.), and tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = 1 / 2 x major axis (mm) x [minor axis (mm)] 2 All antibody-drug conjugates were diluted with ABS buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5) (NACALAI) and administered via the tail vein at the dose indicated in each example. A control group (vehicle group) was also administered ABS buffer in the same manner. Six mice per group were used in the experiment.

[0322] 9)-1 Antitumor effect (1) The CDH6-positive human renal cell carcinoma cell line 786-O (ATCC), whose CDH6 expression was confirmed in Example 2)-3-1, was suspended in Matrigel (Corning) and 5 × 10 6 The cells were subcutaneously transplanted into the right flank of male SCID mice (Day 0), and the mice were randomly assigned to groups on Day 18. On the day of group assignment, four types of antibody-drug conjugates prepared in Example 7 (clone names: H01L02-DXd, H02L02-DXd, H02L03-DXd, and H04L02-DXd) or NOV0712-DM4 prepared in Reference Example 2 were administered into the tail vein at a dose of 3 mg / kg. The results are shown in Figure 12. The horizontal axis indicates the number of days, the vertical axis indicates the tumor volume, and the error bars indicate SE values.

[0323] NOV0712-DM4 did not exhibit a significant antitumor effect in this tumor model. After administration of all four antibody-drug conjugates prepared in Example 7, tumor volume decreased, significant tumor regression was observed, and the tumor regression effect persisted for 24 days after administration (Figure 12).

[0324] 9)-2 Antitumor effect (2) The CDH6-positive human ovarian tumor cell line PA-1 (ATCC), whose CDH6 expression was confirmed in Example 2)-3-1, was suspended in Matrigel (Corning) and 8.5 × 10 6The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 11. On the day of group assignment, the antibody-drug conjugate H01L02-DXd prepared in Example 7 or NOV0712-DM4 or NOV0712-DXd prepared in Reference Example 2 was administered into the tail vein at doses of 1 and 3 mg / kg. The results are shown in Figure 13. The horizontal axis indicates the number of days, the vertical axis indicates the tumor volume, and the error bars indicate SE values.

[0325] NOV0712-DM4 did not exhibit antitumor effects in this tumor model at either the 1 or 3 mg / kg dose. In contrast, H01L02-DXd significantly reduced tumor volume and caused tumor regression at both the 1 and 3 mg / kg doses (Figure 13). Furthermore, when the efficacy of samples conjugated with the same drug DXd to the H01L02 antibody or NOV0712 antibody obtained herein was compared, H01L02-DXd exhibited stronger antitumor effects than NOV0712-DXd at both the 1 and 3 mg / kg doses. Thus, the H01L02 antibody of the present invention was shown to be a superior antibody-drug conjugate as an antitumor agent compared to the NOV0712 antibody (Figure 13).

[0326] 9)-3 Antitumor effect (3) The CDH6-positive human ovarian tumor cell line NIH:OVCAR-3 (ATCC), whose CDH6 expression was confirmed in Example 2)-3-1, was suspended in Matrigel (Corning) and diluted to 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 22. On the day of grouping, the antibody-drug conjugate H01L02-DXd prepared in Example 7 or NOV0712-DM4 prepared in Reference Example 2 was administered into the tail vein at doses of 1 and 3 mg / kg. The results are shown in Figure 14. The horizontal axis indicates the number of days, the vertical axis indicates the tumor volume, and the error bars indicate SE values.

[0327] NOV0712-DM4 showed no antitumor effect at 1 mg / kg, but showed an antitumor effect at 3 mg / kg, but tumor regrowth was observed starting 2 weeks after administration. On the other hand, H01L02-DXd significantly suppressed tumor volume increase at both 1 and 3 mg / kg doses, and the tumor growth inhibitory effect at 3 mg / kg was sustained for a long period of 31 days after administration (Figure 14).

[0328] Similarly, using PA-1 cells, the tumor growth inhibitory effect of NOV0712-DM4 prepared in Reference Example 2 or H01L02-DM4 prepared in Reference Example 3 was evaluated. H01L02-DM4 reduced tumor volume more than NOV0712-DM4, and the H01L02 antibody of the present invention was a more superior antibody than the NOV0712 antibody as an antibody-drug conjugate used as an antitumor agent.

[0329] 9)-4 Antitumor effect (4) The CDH6-positive human renal cell carcinoma cell line 786-O (ATCC), whose CDH6 expression was confirmed in Example 2)-3-1, was suspended in Matrigel (Corning) and 5 × 10 6 The cells were subcutaneously transplanted into the right flank of male SCID mice (Day 0), and the mice were randomly assigned to groups on Day 20. On the day of group assignment, the antibody-drug conjugate H01L02-DXd prepared in Example 7 or NOV0712-DM4 prepared in Reference Example 2 was administered into the tail vein at doses of 1 and 3 mg / kg. The results are shown in Figure 15. The horizontal axis indicates the number of days, the vertical axis indicates the tumor volume, and the error bars indicate SE values.

[0330] In this tumor model, NOV0712-DM4 did not show a significant antitumor effect at either the 1 or 3 mg / kg dose, whereas H01L02-DXd reduced tumor volume at either the 1 or 3 mg / kg dose, with significant tumor regression observed at 3 mg / kg, which persisted for 20 days after administration (Figure 15).

[0331] 9)-5 Antitumor effect (5) The CDH6-negative human ovarian tumor cell line ES-2 (ATCC), which was confirmed not to express CDH6 in Example 2)-3-1, was suspended in physiological saline and diluted to 1 × 10 6 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 7. On the day of group assignment, the antibody-drug conjugate H01L02-DXd prepared in Example 7 or NOV0712-DM4 prepared in Reference Example 2 was administered into the tail vein at doses of 1 and 3 mg / kg. The results are shown in Figure 16. The horizontal axis indicates the number of days, the vertical axis indicates the tumor volume, and the error bars indicate SE values.

[0332] In this tumor model, which does not express CDH6, H01L02-DXd and NOV0712-DM4 showed no antitumor effect at any dose. These results indicate that the antitumor effect of the antibody-drug conjugate in the CDH6-positive tumor model shown in Examples 9)-1, 9)-2, 9)-3, and 9)-4 is dependent on CDH6 expression in tumor cells, and that it is a selective and safe antitumor agent that exhibits an antitumor effect specifically against CDH6-positive tumors without causing cytotoxicity in CDH6-negative normal tissues (Figure 16). [Industrial Applicability]

[0333] The present invention provides an anti-CDH6 antibody having internalization activity and an antibody-drug conjugate comprising the antibody. The antibody-drug conjugate can be used as a therapeutic agent for cancer, etc.

Claims

1. A polynucleotide encoding an antibody or an antigen-binding fragment of the antibody, The antibody or antigen-binding fragment of the antibody is specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and has the ability to be internalized into cells; A light chain variable region consisting of an amino acid sequence having 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 63 and a heavy chain variable region consisting of an amino acid sequence having 95% or more identity to the amino acid sequence set forth in SEQ ID NO: 75, The polynucleotide.

2. A polynucleotide encoding an antibody or an antigen-binding fragment of the antibody, The antibody or antigen-binding fragment of the antibody is specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and has the ability to be internalized into cells; A light chain consisting of an amino acid sequence having 95% or more identity to the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61, and a heavy chain consisting of an amino acid sequence having 95% or more identity to the amino acid sequence of positions 20 to 471 of SEQ ID NO: 73, The polynucleotide.

3. 3. The polynucleotide of claim 1, wherein the antigen-binding fragment of the antibody is an antigen-binding fragment selected from the group consisting of Fab, F(ab')2, Fab', and Fv.

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