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

Abstract

To provide an antibody binding to CDH6 and having internalization activity, an antibody-drug conjugate of the antibody and a drug having antitumor activity, a pharmaceutical product comprising the antibody-drug conjugate and having therapeutic effects on a tumor, a method for treating a tumor using the antibody, the antibody-drug conjugate or the pharmaceutical product, and the like.SOLUTION: The present invention provides an anti-CDH6 antibody having internalization activity, and an antibody-drug conjugate of the antibody and a drug having antitumor activity.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Cadherin is a glycoprotein present on the surface of the cell membrane that binds calcium. The N-terminal extracellular domains bind to each other in a neutron-dependent manner, and act as intercellular adhesion molecules. They also function as signaling molecules responsible for cell-cell interactions. Among these, the classical cadherins have five extracellular domains (ext cellular domain, EC domain), one transmembrane domain, and an intracellular domain Classic cadherins are single-pass transmembrane proteins consisting of two domains. Based on sequence homology, it is classified as a member of the type I family, represented by E-cadherin and N-cadherin. , classified into the Type II family.

[0003] Cadherin-6 (CDH6) is a member of the type II cadherin family. It is a single-pass transmembrane protein consisting of 790 amino acids, and its N-terminal end is The human CDH6 gene was first cloned in 1995. (Non-Patent Document 1), and accessions such as NM_004932 and NP_004923 (NCBI) It can be referenced by session number.

[0004] CDH6 is specifically expressed in the brain and kidney during development and is involved in the formation of central nervous system circuits ( Non-patent literature 2, Non-patent literature 3) and during nephron development in the kidney (Non-patent literature 4, Non-patent literature 5) In normal adult tissues, CDH6 expression is reported to play an important role in the renal and urinary It is localized to tubules and bile duct epithelial cells.

[0005] On the other hand, CDH6 expression is specifically elevated in tumor tissue in several types of adult cancer. It is known that CDH6 expression is associated with poor prognosis in human renal cell carcinoma, especially renal clear cell carcinoma. It has been reported that there is a correlation between these two and that they can be used as tumor markers (Non-patent Document 6, Non-patent Document High expression of CDH6 has also been reported in human ovarian cancer (Non-patent Document 8), and in human thyroid cancer It has also been reported that CDH6 is involved in epithelial-mesenchymal transition and metastasis of adenocarcinoma (Non-Patent Document 9). , CDH6 has also been reported to be expressed in human cholangiocarcinoma and human small cell lung cancer. (Non-patent literature 12, 13).

[0006] Cancer is a leading cause of death, and its incidence is expected to increase with the aging of the population. However, the treatment needs are still not fully met. Due to their low activity, they can cause side effects due to their toxicity not only to tumor cells but also to normal cells. The problem is that the drug's effects cannot be fully achieved because an insufficient amount of drug is administered. For this reason, in recent years, the molecular and cellular characteristics that show characteristic mutations and high expression in cancer cells have been investigated. The development of more selective molecular targeted drugs and antibody drugs that target specific molecules involved in cancer development is needed. It is being carried out.

[0007] Antibodies are highly stable in the blood and bind specifically to target antigens, which is expected to reduce side effects. Many antibody drugs have been developed against molecules that are highly expressed on the surface of cancer cells. Antibody-drug conjugates (ADCs) are a type of technology that utilizes the antigen-specific binding ability of antibodies. Antibody-Drug Conjugates (ADCs) are examples of ADCs. It binds to antigens expressed on the surface of cancer cells and internalizes the antigens into the cells through this binding. ADC is an antibody bound to a drug with cytotoxicity. ADC is a drug that efficiently targets cancer cells. By delivering drugs efficiently, the drugs accumulate in cancer cells and kill them. It is expected that ADCs will be effective (Non-Patent Document 10, Patent Documents 1 and 2). Adcetris (commercial name: ADCETRIS) is a D30 monoclonal antibody conjugated to monomethyl auristatin E. (brentuximab vedotin) for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma It has also been approved as a drug by combining emtansine with an anti-HER2 monoclonal antibody. Kadcyla™ (trastuzumab emtansine) is effective in treating HER2-positive patients with progression or recurrence It is used to treat breast cancer.

[0008] The characteristics of target antigens suitable for ADC as antitumor drugs are that they are specifically and highly expressed on the surface of cancer cells. It is expressed in normal cells, but is expressed at low levels or not at all in normal cells. It can be internalized in cells. It is an antigen. Furthermore, the key point of antibodies suitable for ADC is that they are not secreted from the cell surface. Its features include specific binding to target antigens and high internalization ability. The internalization ability of antibodies depends on the properties of both the target antigen and the antibody. , predicting antigen binding sites suitable for internalization from the target molecular structure, and determining antibody binding strength and physical properties. It is difficult to easily predict which antibodies have high internalization ability based on the above information. Obtaining antibodies with high internalization ability against antigens will enable the development of highly effective ADCs. This has become an important issue in the field (Non-Patent Document 11).

[0009] ADCs targeting CDH6 are specific to the EC domain 5 (EC5) of CDH6. An ADC in which DM4 is conjugated to an anti-CDH6 antibody that binds to CDH6 is known (Patent Document 3). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2014 / 057687 [Patent Document 2] US2016 / 0297890 [Patent Document 3] WO2016 / 024195 [Non-patent literature]

[0011] [Non-Patent Document 1] Shimoyama Y,et al.,Cancer Research,2206-2211,55,May 15,1995 [Non-patent document 2] Inoue T,et al.,Developmental Biology,183-194,1997 [Non-patent document 3] Osterhout JA,et al.,Neuron,632-639,71,Aug 25,2011 [Non-patent document 4] Cho EA,et al.,Development,803-812,125,1998 [Non-Patent Document 5] Mah SP,et al.,Developmental Biology,38-53,223,2000 [Non-patent document 6] Paul R,et al.,Cancer Research,2741-2748,July 1,57,1997 [Non-Patent Document 7] Shimazui T,et al.,Cancer,963-968,101(5),Sep.1,2004 [Non-patent document 8] Koebel M,et al.,PLoS Medicine,1749-1760,5(12),e232,Dec.2008 [Non-Patent Document 9] Gugnoni M,et al.,Oncogene,667-677,36,2017 [Non-Patent Document 10] Polakis P.,Pharmacological Reviews,3-19,68,2016 [Non-Patent Document 11] Peters C, et al.,Bioscience Reports,1-20,35,2015 [Non-Patent Document 12] Goeppert B,et al.,Epigenetics,780-790,11(11),2016 [Non-Patent Document 13] Yokoi S, et al.,American Journal of Pathology,207-216,161,1,2002 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide an antibody that specifically binds to CDH6 and has high internalization activity, Antibody-drug conjugate having high antitumor activity containing an antibody, and the antibody-drug conjugate and pharmaceuticals having therapeutic effects against tumors using antibodies, antibodies, and antibody-drug conjugates. The present invention provides a method for treating tumors using lectins or pharmaceuticals. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to achieve the above object, and have surprisingly found that CDH An antibody that specifically binds to the extracellular domain 3 (herein also referred to as EC3) of C6 is It has extremely high internalization activity in DH6-expressing cells and is useful as an ADC antibody. Furthermore, we found that the anti-CDH6 antibody can be linked to a specific structure of a drug that exerts intracellular toxicity. The anti-CDH6 antibody-drug conjugates linked via the linker of this structure are It was found to exhibit stronger antitumor activity than the six-drug conjugate.

[0014] The present invention includes the following inventions: [1] Internalization that specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and is taken up into cells an antibody or a functional fragment of said antibody; [2] The following (1) to (5) for binding to the amino acid sequence set forth in SEQ ID NO: 4: (1) a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 53 and SEQ ID NO: an antibody having a heavy chain consisting of an amino acid sequence set forth in positions 20 to 471 of SEQ ID NO:56; (2) a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and SEQ ID NO: an antibody having a heavy chain consisting of an amino acid sequence set forth in positions 20 to 471 of SEQ ID NO:69; (3) a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and SEQ ID NO: an antibody having a heavy chain consisting of an amino acid sequence set forth in positions 20 to 471 of B.V.73; (4) a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 65 and SEQ ID NO: An antibody having a heavy chain consisting of an amino acid sequence set forth in positions 20 to 471 of 73; and (5) a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 and SEQ ID NO: an antibody having a heavy chain consisting of an amino acid sequence set forth in positions 20 to 471 of B.V.77;

[0033] The antibody has a competitive inhibitory activity with at least one of the antibodies selected from the group consisting of the antibody or a functional fragment of the antibody according to [1]; [3] (1) to (4) below: (1) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14 3. (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22 and the amino acid sequence set forth in SEQ ID NO: 23 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24 3. (3) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32 and the amino acid sequence set forth in SEQ ID NO: 33 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 35. 3, and (4) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42 and the amino acid sequence set forth in SEQ ID NO: 43 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 44 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 45. 3. CDRL1, CDRL2 and CDRL3 according to any one of the groups consisting of: ,and, (5) to (9) below: (5) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ ID NO: 18 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 19 and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 20. 3. (6) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 27 and the amino acid sequence set forth in SEQ ID NO: 28 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 29 and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 30. 3. (7) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37 and the amino acid sequence set forth in SEQ ID NO: 38 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39 and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 39. 3. (8) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47 and the amino acid sequence set forth in SEQ ID NO: 48 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 49 and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 49. 3, and (9) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ ID NO: 60 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 19 and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 20. 3. CDRH1, CDRH2 and CDRH3 according to any one of the groups consisting of: Including, [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 and the amino acid sequence set forth in SEQ ID NO: 13 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 19 RH3, (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22 and the amino acid sequence set forth in SEQ ID NO: 23 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 28 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 29 RH3, (3) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32 and the amino acid sequence set forth in SEQ ID NO: 33 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 35. 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 38 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39 RH3, (4) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42 and the amino acid sequence set forth in SEQ ID NO: 43 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 44 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 45. 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 47, and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 48 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 49 RH3, and (5) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 60 CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 19 RH3, CDRL1, CDRL2 and CDRL3 selected from the group consisting of: , CDRH2 and CDRH3, The antibody or functional fragment of the antibody according to any one of [1] to [3]; [5] The antibody according to any one of [1] to [4], which is humanized, or the function of the antibody sexual fragment; [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) In the amino acid sequences of (1) to (2), the amino acid sequence of the framework region other than each CDR sequence is an amino acid sequence having at least 95% sequence identity to the sequence, and (4) In the amino acid sequences of (1) to (3), the amino acid sequence of the framework region other than each CDR sequence is An amino acid sequence in which one or several amino acids have been deleted, substituted, or added 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) In the amino acid sequences of (5) to (7), the amino acid sequence of the framework region other than each CDR sequence is an amino acid sequence having at least 95% sequence identity to the sequence, and (9) In the amino acid sequences of (5) to (8), a sequence of a framework region other than each CDR sequence is An amino acid sequence in which one or several amino acids have been deleted, substituted, or added 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 the amino acid sequence set forth in SEQ ID NO: 71 a heavy chain variable region consisting of the amino acid sequence (2) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63 and the amino acid sequence set forth in SEQ ID NO: 75 a heavy chain variable region consisting of the amino acid sequence (3) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 67 and the amino acid sequence set forth in SEQ ID NO: 75 a heavy chain variable region consisting of the amino acid sequence (4) a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 63 and the amino acid sequence set forth in SEQ ID NO: 79 a heavy chain variable region consisting of the amino acid sequence 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 light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 69 a heavy chain consisting of amino acid sequences 20 to 471; (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 73 a heavy chain consisting of amino acid sequences 20 to 471; (3) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 73 a heavy chain consisting of the amino acid sequence of positions 20 to 471, or (4) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 77 a heavy chain consisting of amino acid sequences 20 to 471;

[0023] The antibody or functional fragment thereof according to any one of [1] to [7], Piece; [9] A light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 69 [8] The antibody according to [8], which has a heavy chain consisting of an amino acid sequence of amino acids 20 to 471. Functional fragments of;

[10] A light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 73 [8] The antibody according to [8], having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of Functional fragments of the body;

[11] A light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 65 and SEQ ID NO: 73 [8] The antibody according to [8], having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of Functional fragments of the body;

[12] A light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 77 [8] The antibody according to [8], having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of Functional fragments of the body;

[13] The functional fragment is selected from the group consisting of Fab, F(ab')2, Fab', and Fv. a functional fragment of the antibody according to any one of [1] to

[12] ;

[14] A method for producing a composition comprising coating the antibody or a functional fragment of the antibody according to any one of [1] to

[13] . a polynucleotide that encodes

[15] (1) to (5) below: (1) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14 a polynucleotide encoding a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and and a heavy chain variable region encoding CDRH3 having the amino acid sequence set forth in SEQ ID NO: 19. a polynucleotide (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 22 and the amino acid sequence set forth in SEQ ID NO: 23 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24 a polynucleotide encoding a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 28, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 29, and and a heavy chain variable region encoding CDRH3 having the amino acid sequence set forth in SEQ ID NO: 29. a polynucleotide (3) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32 and the amino acid sequence set forth in SEQ ID NO: 33 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 35. a polynucleotide encoding a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 38, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and and a heavy chain variable region encoding CDRH3 having the amino acid sequence set forth in SEQ ID NO: 39. a polynucleotide (4) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 42 and the amino acid sequence set forth in SEQ ID NO: 43 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 44 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 45. a polynucleotide encoding a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 47; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 48, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 49, and and a heavy chain variable region encoding CDRH3 having the amino acid sequence set forth in SEQ ID NO: 49. a polynucleotide comprising the sequence (5) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13 CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14 a polynucleotide encoding a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17; CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 61, and and a heavy chain variable region encoding CDRH3 having the amino acid sequence set forth in SEQ ID NO: 19. a polynucleotide

[14] The polynucleotide according to any one of the following: polynucleotides;

[16] A polynucleotide encoding a light chain consisting of the amino acid sequence from positions 21 to 233 of SEQ ID NO: 61. A heavy chain encoding the nucleotide sequence of amino acids 20 to 471 of SEQ ID NO: 69. The polynucleotide according to

[14] or

[15] , which comprises a polynucleotide

[17] A polynucleotide encoding a light chain consisting of the amino acid sequence from positions 21 to 233 of SEQ ID NO: 61. A heavy chain encoding the nucleotide sequence of amino acids 20 to 471 of SEQ ID NO: 73. The polynucleotide according to

[14] or

[15] , which comprises a polynucleotide

[18] A polynucleotide encoding a light chain consisting of the amino acid sequence from positions 21 to 233 of SEQ ID NO: 65. A heavy chain encoding the nucleotide sequence of amino acids 20 to 471 of SEQ ID NO: 73. The polynucleotide according to

[14] or

[15] , which comprises a polynucleotide

[19] A polynucleotide encoding a light chain consisting of the amino acid sequence from positions 21 to 233 of SEQ ID NO: 61. A heavy chain encoding the nucleotide sequence and amino acid sequence 20 to 471 of SEQ ID NO: 77 The polynucleotide according to

[14] or

[15] , which comprises a polynucleotide

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

[14] to

[19] . Kutar;

[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 process for culturing the host cell according to

[21] or

[22] , and a method for culturing the host cell obtained by the process. and recovering the antibody of interest or a functional fragment of the antibody from the culture. a method for producing the antibody or a functional fragment of the antibody;

[24] The heavy or light chain may undergo N-linked glycosylation, O-linked glycosylation, or N-terminal processing. cleavage, C-terminal processing, deamidation, isomerization of aspartic acid, and methionine Oxidation, addition of methionine residues to the N-terminus, amidation of proline residues, addition of glutamine or Pyroglutamate oxidation of the N-terminal glutamic acid and one or two carboxyl-terminal [1] to

[13] , which have undergone one or more modifications selected from the group consisting of amino acid deletions. the antibody or a functional fragment of the antibody according to any one of the preceding claims;

[25] The antibody according to

[0024] , wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain;

[26] The antibody according to

[0025] , 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

[0026] , wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated;

[28] Glycosylation is regulated to enhance antibody-dependent cellular cytotoxicity.[1]

[13] and

[24] to

[27] . is a functional fragment of the antibody;

[29] Any one selected from the group consisting of [1] to

[13] and

[24] to

[28] An antibody-drug conjugate in which a drug is bound to the antibody or functional fragment of the antibody according to any one of the preceding items. to;

[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)-GGF G-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-CH2 CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH- CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C (=O)-GGFG-NH-CH2CH2CH2-C(=O)-,

[29] ~

[31] The antibody-drug conjugate according to any one of claims 1 to 31. (wherein the antibody binds at the end of -(Succinimid-3-yl-N) The antitumor compounds are (a), (b), and (c) with the nitrogen atom of the amino group at position 1 as the binding site. e) or (f), the -CH2CH2CH2-C(=O)- moiety of (c), the CH2-O-CH 2-C(=O)- portion or the CH2CH2-O-CH2-C(=O)- portion of (d) In the above formula, GGFG is glycine-glycine-phenylalanine. The amino acid sequence is shown, connected by peptide bonds consisting of lysines. -(Succinimid-3-yl-N)- has the following formula:

[0017] [ka]

[0018] The structure is represented by the formula: Attaches to a methylene group in the linker structure;

[33] The linker is any one selected from the group consisting of (c), (d), and (e) below. The antibody-drug conjugate according to any one of

[29] to

[32] , represented by the formula: (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-CH2 CH2CH2-C(=O)-;

[34] The linker is any one of

[29] to

[33] represented by the following formula (c) or (e): The antibody-drug conjugate of claim 1, (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-CH2 CH2CH2-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: DUGATE: Here, AB represents an antibody or a functional fragment of the antibody. n represents a drug-phosphoric acid bonded to the antibody. The figure shows the average number of bonds per antibody in the linker structure. bonded via a phenyl group;

[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: DUGATE: Here, AB represents an antibody or a functional fragment of the antibody. n represents a drug-phosphoric acid bonded to the antibody. The figure shows the average number of bonds per antibody in the linker structure. bonded via a phenyl group;

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

[29] to

[36] , which is a functional fragment. to;

[38] The antibody has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a sequence Antibody containing a heavy chain consisting of the amino acid sequence of 20 to 471 of No. 69 or function of said antibody the antibody-drug conjugate according to

[37] , which is a functional fragment;

[39] The antibody has a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 61 and a sequence Antibody containing a heavy chain consisting of the amino acid sequence 20 to 471 of No. 77 or function of said antibody the antibody-drug conjugate according to

[37] , which is a functional fragment;

[40] The heavy or light chain may undergo N-linked glycosylation, O-linked glycosylation, or N-terminal processing. cleavage, C-terminal processing, deamidation, isomerization of aspartic acid, and methionine Oxidation, addition of methionine residues to the N-terminus, amidation of proline residues, addition of glutamine or Pyroglutamation of N-terminal glutamic acid and one or two amino acids at the carboxyl terminus

[29] ~

[39] the antibody-drug conjugate according to any one of the preceding claims;

[41] The average number of conjugates per antibody for one selected drug-linker structure is 1–10. The antibody-drug conjugate according to any one of

[29] to

[40] , wherein the range is

[42] The average number of linkages per antibody of one selected drug-linker structure is 2-8.

[41] an antibody-drug conjugate according to the present invention;

[43] The average number of linkages per antibody of one selected drug-linker structure is 5-8.

[42] An antibody-drug conjugate according to the present invention;

[44] The average number of conjugations per antibody for each selected drug-linker structure was 7–8. an antibody-drug conjugate according to

[43] ;

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

[29] to

[44] , or 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. composition;

[48] ​​The tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, Thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' The pharmaceutical composition according to

[46] or

[47] , characterized in that the compound is a tumor or neuroblastoma. ;

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

[29] to

[44] , or a salt thereof or a hydrate thereof to an individual. treatment method;

[50] The method according to

[49] , wherein the tumor expresses CDH6. treatment method;

[51] The tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, Thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' the method for treating a tumor according to

[49] or

[50] , characterized in that the tumor is a thyroid tumor or a neuroblastoma;

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

[29] to

[44] , or a salt thereof or a hydrate thereof, and a pharmaceutical composition comprising at least one selected from the group consisting of A method for treating tumors, characterized by administering two antitumor drugs simultaneously, separately or sequentially to an individual. treatment method;

[53] Any one selected from the group consisting of [1] to

[13] and

[24] to

[28]

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

[23] , or an antibody obtained by the production method according to

[23] . reacting the antibody or a functional fragment of the antibody with a drug-linker intermediate compound. a method for producing an antibody-drug conjugate, the method comprising:

[54] A step of culturing the host cell according to

[21] or

[22] , and the culture medium obtained in the step a step of collecting the antibody of interest or a functional fragment of the antibody from the culture; and The method includes a step of reacting an antibody or a functional fragment of the antibody with a drug-linker intermediate compound. A method for producing an antibody-drug conjugate, characterized by: [Effects of the Invention]

[0023] The anti-CDH6 antibody of the present invention specifically recognizes the EC domain 3 (EC3) of CDH6, The anti-CDH6 antibody of the present invention is characterized by its high internalization activity. Anti-CDH6 antibody-drug conjugates in which a drug exhibiting the activity is linked via a linker with a specific structure Gate has shown excellent anti-cancer activity when administered to patients with cancer cells expressing CDH6. It is expected that the anti-CDH6 antibody-drug combination of the present invention will achieve anti-tumor efficacy and safety. The conjugates 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 of the present invention will now be described with reference to the drawings. The embodiment described below is an example of a typical embodiment of the present invention. This will not narrow the scope of the present invention.

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

[0027] In this specification, the term "gene" includes not only DNA but also its mRNA, cDNA, and The cRNA is also included.

[0028] As used herein, the terms "polynucleotide" and "nucleotide" are used interchangeably with "nucleic acid." This includes DNA, RNA, probes, oligonucleotides, and primers. In the specification, the terms "polynucleotide" and "nucleotide" are used interchangeably unless otherwise specified. It can be used for various purposes.

[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. In this document, human CDH6 may be referred to as "hCDH6."

[0032] As used herein, "cytotoxic activity" refers to any activity that causes pathological changes in cells. This refers to damage that occurs not only directly but also in DNA breaks, the formation of base dimers, and chromosome damage. Damage to the structure and function of cells, including cleavage, damage to the cell division apparatus, and reduced activity of various enzymes This means causing

[0033] As used herein, the term "to exert toxicity within a cell" means to exhibit toxicity within a cell in some way. This refers to damage that occurs not only directly but also in DNA breaks, the formation of base dimers, and chromosome breaks. It causes various damages such as cell division, damage to the cell division apparatus, reduction in enzyme activity, and inhibition of the action of cell growth factors. It refers to the effects on the structure, function, and metabolism of cells.

[0034] As used herein, the term "functional fragment of an antibody" is also referred to as an "antigen-binding fragment of an antibody," and is a fragment that binds to an antigen. These fragments refer to partial fragments of antibodies that have the binding activity of Fab, F(ab')2, Fv, and s This includes cFv, diabody, linear antibody, and multispecific antibody formed from antibody fragments. In addition, Fab, a monovalent fragment of the variable region of an antibody, is obtained by treating F(ab')2 under reducing conditions. However, these fragments are not limited to the antigen-binding fragments of antibodies as long as they have the ability to bind to antigens. These antigen-binding fragments are not limited to those described above. In addition, the full-length antibody protein molecule can be cleaved by an appropriate enzyme. Not only those treated with natural antibodies, but also those genetically modified antibody genes can be used to infect suitable hosts. Proteins produced in cells are also included.

[0035] As used herein, the term "epitope" refers to a partial peptide of CDH6 to which a specific anti-CDH6 antibody binds. The epitope, which is a partial peptide of CDH6, is This 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. For example, a suitable peptide can be synthesized from the C-terminus or N-terminus of CDH6. A series of polypeptides of successively shorter lengths can be produced by recombinant DNA techniques well known to those skilled in the art. After preparing them, we examined the reactivity of the antibodies to them and determined the rough recognition site. By synthesizing shorter peptides and examining the reactivity with these peptides, It is also possible to determine the target of the antibody. If the antibody that binds to the epitope has a three-dimensional structure consisting of multiple domains, By modifying the amino acid sequence of the extracellular domain, the three-dimensional structure can be altered. The part of the antigen that a particular antibody binds to can be determined by the The three-dimensional structure of the epitope is determined by X-ray structural analysis, which identifies the amino acid residues of the antigen adjacent to the antibody. It can also be determined by specifying

[0036] As used herein, "binding to the same epitope" refers to antibodies that bind to a common epitope. The second antibody binds to the partial peptide or partial three-dimensional structure to which the first antibody binds. If the first and second antibodies bind to the same epitope, it can be determined that the first and second antibodies bind to the same epitope. Alternatively, a second antibody competes with the binding of the first antibody to the antigen (i.e., The specific epitope is identified by checking that the body prevents the first antibody from binding to the antigen. Even if the sequence or structure of the peptide has not been determined, the first antibody and the second antibody may have the same epitope. In the present specification, the term "binding to the same epitope" means The first antibody and the second antibody are found to have a common epitope by one or both of the determination methods. The first and second antibodies bind to the same epitope. and the first antibody has a specific effect such as antitumor activity or internalization activity, It is expected that the antibody will also have similar activity.

[0037] As used herein, "CDR" refers to a complementarity determining region (CDR). The heavy and light chains of an antibody molecule are called the "antibody determining region." It is known that each of these has three CDRs. These are also called the variable region of an antibody, and are located within the variable regions of the heavy and light chains of an antibody. Therefore, this is a site where the variation in the primary structure is particularly high, and the primary structure of the heavy and light polypeptide chains is In the present specification, the CDRs of an antibody are separated into three regions. The heavy chain CDRs are CDRH1, CDRH2, CDRH3, CDRH4, CDRH5, CDRH6, CDRH7, CDRH8, CDRH9, CDRH10, CDRH11, CDRH12, CDRH13, CDRH14, CDRH15, CDRH16, CDRH17, CDRH18, CDRH19 ... The light chain CDRs are represented as CDRL1, CDRH2, and CDRH3, respectively, from the amino-terminal end of the light chain amino acid sequence. These sites are designated CDRL2 and CDRL3. These sites are close to each other in the three-dimensional structure and bind This determines the specificity for the antigen.

[0038] As used herein, the term "hybridize under stringent conditions" refers to the hybridization of a commercially available hybrid. ExpressHyb Hybridization Solution Hybridization was performed at 68°C in ion (Clontech) or by fixing the DNA. Hybridization using filters in the presence of 0.7-1.0 M NaCl at 68°C After this, add 0.1 to 2x SSC solution (1x SSC is 150 mM NaCl , 15 mM sodium citrate) at 68°C. This refers to hybridization under conditions that allow hybridization to occur between two or more DNA fragments or conditions equivalent thereto.

[0039] In the present specification, "1 to several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 It means 1, 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, and their N-terminal domains are linked in a calcium ion-dependent manner. The binding of the extracellular domains at the ends of the membranes allows them to function as cell-cell adhesion molecules and mediate cell-cell interactions. It functions as a signaling molecule that carries out the functions of the cadherin superfamily. Cadherins are a group of molecules that contain five extracellular domains (EC domains) and one extracellular domain. It is a single-pass transmembrane protein consisting of two transmembrane regions and an intracellular domain.

[0041] CDH6 (Cadherin-6) is classified as a member of the type II cadherin family. It is a single-pass transmembrane protein consisting of 790 amino acids, with the N-terminal end being extracellular and the C-terminal end being The human CDH6 gene was first cloned in 1995 (Non-Patent Document 1), NM_004932, NP_004923 (NCBI), etc. It is more accessible.

[0042] The CDH6 protein used in the present invention can be expressed in humans and non-human mammals (rats, mice, monkeys, etc.). It is used by directly purifying it from CDH6-expressing cells, or by using the cell membrane fraction of the cells. CDH6 can be prepared and used, or can be synthesized in vitro or can be obtained by genetically engineering host cells to produce them. Specifically, CDH6 cDNA was inserted into an expression vector, and then transcription and translation were performed. synthesis in a solution containing the enzymes, substrates and energy sources required for translation, or other prokaryotic or by transforming eukaryotic host cells to express CDH6. Therefore, the protein can be obtained. cells or cell lines expressing CDH6 as the CDH6 protein. Alternatively, an expression vector incorporating CDH6 cDNA can be directly introduced into the immunized animal. It is also possible to administer the antibody to the immunized animal to express CDH6 in the body of the immunized animal.

[0043] In addition, in the amino acid sequence of the CDH6, one or several amino acids are substituted, deleted, or and / or a protein consisting of an added amino acid sequence and having biological activity equivalent to that of the protein. Proteins are also included in CDH6.

[0044] The human CDH6 protein has the amino acid sequence set forth in SEQ ID NO: 1. Human CDH6 The extracellular region of the protein is amino acids 54 to 159 of the amino acid sequence set forth in SEQ ID NO: 1. Extracellular domain 1 (also referred to herein as EC1) having the amino acid sequence set forth in SEQ ID NO: 1 The extracellular domain 2 (Honmei) has the amino acid sequence of 160 to 268 of the amino acid sequence described above. (hereinafter also referred to as EC2) and the amino acid sequence from 269 to 383 of the amino acid sequence set forth in SEQ ID NO: 1. extracellular domain 3 (herein also referred to as EC3), having the amino acid sequence of SEQ ID NO: An extracellular domain having the amino acid sequence of positions 384 to 486 of the amino acid sequence described in No. 1 4 (also referred to as EC4 herein), and 487 of the amino acid sequence set forth in SEQ ID NO: 1. Extracellular domain 5 (also referred to as EC5 herein) having an amino acid sequence of amino acids 1 to 608 The amino acid sequences of EC1 to EC5 are 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 antibody comprising the amino acid sequence shown in SEQ ID NO:4. Examples of the anti-CDH6 antibodies of the present invention include those that recognize the sequence and have internalization activity. An example of an anti-CDH6 antibody is a CDH6 antibody that specifically binds to an amino acid sequence containing the amino acid sequence shown in SEQ ID NO: 4. Examples of the anti-CDH6 antibodies of the present invention include those that specifically recognize CDH6 and have internalization activity. An example of a CDH6 antibody is one that recognizes the amino acid sequence shown in SEQ ID NO: 4. The anti-CDH6 antibodies of the present invention can be exemplified by those that exhibit both the anti-CDH6 activity and the internalization activity. An example of an antibody is one that specifically recognizes the amino acid sequence shown in SEQ ID NO: 4. An example of an anti-CDH6 antibody is an antibody having the sequence "SEQ ID NO: 4" and an internalization activity. "specifically recognizes an amino acid sequence containing the amino acid sequence shown in "Specifically recognize" means that the antibody specifically recognizes the EC3 domain of CDH6 as a different domain from other extracellular domains of CDH6. It refers to the ability to recognize or bind strongly to a target protein compared to an antibody.

[0046] The anti-CDH6 antibody of the present invention may be derived from any species, but is preferably derived from humans or monkeys. Examples of the cells derived from a species other than humans include rats, mice, and rabbits. Preferably, the antibody of the present invention is chimerized or humanized using a polyclonal antibody. The antibody may be either a human or a monoclonal antibody, although monoclonal antibodies are preferred.

[0047] The anti-CDH6 antibody of the present invention is an antibody that can target tumor cells, i.e., it can recognize tumor cells. the ability to recognize, bind to, and / or be internalized by tumor cells Therefore, the anti-CDH6 antibody of the present invention and a compound having antitumor activity are The compounds can be linked via a linker to form an antibody-drug conjugate.

[0048] The binding of the antibody to tumor cells can be confirmed using flow cytometry. Antibody uptake into the cell can be achieved by (1) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody. Cell Death and Immunoglobulin Assay (CIM) Differentiation,2008,15,751-761), (2) Treatment anti- The amount of fluorescence taken up into the cells is measured using a secondary antibody (fluorescently labeled) that binds to the antibody. Sei(Molecular Biology of the Cell Vol.15, 5268-5282, December 2004) or (3) an antibody that binds to a therapeutic antibody. When notoxins are taken up into cells, toxins are released and cell proliferation is suppressed. Mab-ZAP assay (BioTechniques 28:162-165, As immunotoxins, diphtheria Recombinant complex protein of the catalytic domain of atoxin and protein G is also available. It is Noh.

[0049] The term "high internalization ability" used herein means that the antibody and the saporin-labeled anti-rat IgG antibody Viability of administered CDH6-expressing cells (relative rate with cell viability without antibody set to 100%) (represented by the formula (I)) is preferably 70% or less, more preferably 60% or less. do.

[0050] Antibody-drug conjugates are compounds that exert antitumor effects, so antibodies It is preferable, but not essential, that the compound itself has an antitumor effect. For the purpose of exerting toxicity specifically and / or selectively in tumor cells, antibodies are internalized. It is important and preferable that the compound has the property of translocating into tumor cells.

[0051] The anti-CDH6 antibody can be prepared by isolating the antigenic polypeptide using a method commonly used in the art. It can be obtained by immunizing an animal with the antibody and collecting and purifying the antibodies produced in the body. However, it is preferable to use CDH6 that maintains its three-dimensional structure as an antigen. An example of such a method is DNA immunization.

[0052] The origin of the antigen is not limited to humans, and antigens derived from animals other than humans, such as mice and rats, can also be used. Animals can also be immunized, in which case the resulting antibodies that bind to the foreign antigen and the human Antibodies applicable to human diseases can be selected by testing cross-reactivity with antigens.

[0053] Also, known methods (e.g., Kohler and Milstein, Nature e(1975) 256, 495-497, Kennet, R. ed., Monoclon. al Antibodies, 365-367, Plenum Press, NY( 1980)), antibody-producing cells that produce antibodies against antigens and myeloma cells are By fusion, hybridomas can be established and monoclonal antibodies can be obtained. do.

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

[0055] (1) Antigen preparation Antigens are produced by genetically engineering host cells to encode genes for antigenic proteins. Specifically, a vector capable of expressing an antigen gene is prepared. This can then be introduced into host cells to express the gene, and the expressed antigen can be purified. Immunize animals with genetically engineered antigen-expressing cells or cell lines expressing the antigen. Antibodies can also be obtained by using the method.

[0056] Alternatively, the cDNA of the antigen protein may be inserted into an expression vector without using the antigen protein. The antigen protein is expressed in the body of the immunized animal, and the antigen protein is expressed in the body of the immunized animal. Antibodies can also be obtained by raising antibodies against the substance.

[0057] (2) Production of anti-CDH6 monoclonal antibody The anti-CDH6 antibody used in the present invention is not particularly limited, and may be, for example, Antibodies specified by the amino acid sequence can be preferably used in the present invention. The anti-CDH6 antibody to be used 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 as far as an anti-CDH6 antibody can be obtained. Therefore, although not particularly limited, it is preferable to use CDH6 that retains its higher-order structure as an antigen. stomach.

[0058] A preferred example of a method for obtaining antibodies is DNA immunization. The method involves introducing an antigen-expressing plasmid into an individual animal, such as a mouse or rat, and then expressing the antigen individually. This is a method of inducing immunity against antigens by expressing them in the body. Methods include direct injection of the plasmid into the muscle, and the use of liposomes and polyethyleneimine. The methods of intravenous injection of transfection reagents, viral vectors, and plasmid-attached transfection A method in which gold particles containing plasmids are injected into the blood using a gene gun. A large amount of plasmid solution is rapidly injected intravenously. Hydrodynamic methods include intramuscular injection of expression plasmids. Regarding the gene transfer method, a method to improve the expression level is to inject the plasmid intramuscularly and then inject the elec- tromycin into the same site. A technique called in vivo electroporation The technique is known (Aihara H, Miyazaki J. Nat Biotech nol.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, Scherma n D.Proc Natl Acad Sci USA.1999 Apr 13;9 6(8):4262-7.) This method involves purifying the muscle with hyaluronidase before intramuscular injection of the plasmid. Treatment with α-glucan further increases the expression level (McMahon JM1, Signo ri E,Wells KE,Fazio VM,Wells DJ.Gene The r.2001 Aug;8(16):1264-70). Also, the production of hybridomas This can be done by known methods, for example, Hybridoma Production System (Cyto Pulse Sciences) It can also be done using

[0059] Specific examples of obtaining monoclonal antibodies include the following. (a) CDH6 cDNA was transfected into an expression vector (e.g., pcDNA3.1: Thermo Scientific) Fisher Scientific) for electroporation and gene gun applications. The vector is then directly administered to the immunized animal (e.g., rat or mouse) by the method described above. By expressing CDH6 in animals, an immune response can be induced. Administering vectors by electroporation etc. can increase antibody titers. If necessary, the treatment may be carried out once or multiple times, preferably multiple times; (b) Extract tissues containing antibody-producing cells (e.g., lymph nodes) from the above-mentioned animals in which an immune response has been induced. ) is collected; (c) myeloma cells (hereinafter referred to as "myeloma") (e.g., mouse myeloma SP2 / 0 -ag14 cells); (d) cell fusion between antibody-producing cells and myelomas; (e) Selection of hybridomas producing the desired antibody; (f) division into single-cell clones (cloning); (g) In some cases, culturing hybridomas for the large-scale production of monoclonal antibodies. Cultivation or rearing of animals implanted with hybridomas; and / or (h) The physiological activity (internalization activity) of the monoclonal antibody thus produced, and Examination of the binding specificity of or testing of properties as a labeling reagent.

[0060] The antibody titer used herein can be measured by, for example, flow cytometry or Cel Examples of the method include, but are not limited to, 1-ELISA.

[0061] An example of a hybridoma strain established in this way is the anti-CDH6 antibody-producing hybridoma. Examples of the rhodomas include rG019, rG055, rG056 and rG061. In the present specification, the antibody produced by the anti-CDH6 antibody-producing hybridoma rG019 is The antibody is referred to as "rG019 antibody" or simply "rG019," and the hybridoma rG055 The antibody produced by the hybridoma is referred to as "rG055 antibody" or simply "rG055." The antibody produced by rG056 is referred to as "rG056 antibody" or simply "rG056." The antibody produced by hybridoma rG061 is referred to as "rG061 antibody" or simply as "rG061 "

[0062] The light chain variable region of the rG019 antibody consists of the amino acid sequence shown in SEQ ID NO: 10. The amino acid sequence of the light chain variable region of the rG019 antibody is the nucleotide sequence shown in SEQ ID NO: 11. The light chain variable region of the rG019 antibody is encoded by the amino acid sequence shown in SEQ ID NO: 12. CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 13, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 15. The amino acid sequence of the chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO: 16. The heavy chain variable region of the rG019 antibody is a CD4 fragment consisting of the amino acid sequence shown in SEQ ID NO: 17. CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 and CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 19 The rG019 antibody has a CDRH3 consisting of the amino acid sequence shown in Table 1.

[0063] The light chain variable region of the rG055 antibody consists of the amino acid sequence shown in SEQ ID NO: 20. The amino acid sequence of the light chain variable region of the rG055 antibody is the nucleotide sequence shown in SEQ ID NO: 21. The light chain variable region of the rG055 antibody is encoded by the amino acid sequence shown in SEQ ID NO:22. CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 24. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 25. The amino acid sequence of the chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO: 26. The heavy chain variable region of the rG055 antibody is a CD4V nucleotide sequence consisting of the amino acid sequence shown in SEQ ID NO: 27. CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 28 and CDRH2 consisting of the amino acid sequence shown 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 shown in SEQ ID NO: 30. The amino acid sequence of the light chain variable region of the rG056 antibody is the nucleotide sequence shown in SEQ ID NO: 31. The light chain variable region of the rG056 antibody is encoded by the amino acid sequence shown in SEQ ID NO: 32. CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 33, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 34. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 35. The amino acid sequence of the chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO: 36. The heavy chain variable region of the rG056 antibody is a CD4V nucleotide sequence consisting of the amino acid sequence shown in SEQ ID NO: 37. CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 38 and CDRH2 consisting of the amino acid sequence shown 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 shown in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of the rG061 antibody is the nucleotide sequence shown in SEQ ID NO:41. The light chain variable region of the rG061 antibody is encoded by the amino acid sequence shown in SEQ ID NO:42. CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 43, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 44, and and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45. The amino acid sequence of the chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO: 46. The heavy chain variable region of the rG061 antibody is a CD4 fragment consisting of the amino acid sequence shown in SEQ ID NO: 47. CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 48 and CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 49 The sequence of the rG061 antibody is shown in Table 1.

[0066] Furthermore, the steps (a) to (h) of "2. Production of anti-CDH6 antibody" are repeated to separately If a monoclonal antibody is obtained independently or by other methods, Even if the antibody is obtained, the rG019 antibody, rG055 antibody, rG056 antibody or r It is possible to obtain an antibody that has an internalization activity equivalent to that of antibody G061. Examples of antibodies include rG019 antibody, rG055 antibody, rG056 antibody, and rG061 antibody. Examples of antibodies that bind to the same epitope as the human antibody include newly created monoclonal antibodies. The clonal antibody is an rG019 antibody, an rG055 antibody, an rG056 antibody, or an rG061 antibody. If the monoclonal antibody binds to the corresponding partial peptide or partial conformation, it is determined that the antibody is rG01. Binds to the same epitope as antibody rG059, antibody rG055, antibody rG056, or antibody rG061 Furthermore, it can be determined that the rG019 antibody, the rG055 antibody, and the rG056 antibody Or, the monoclonal antibody competes with the binding of the rG061 antibody to CDH6 ( That is, the monoclonal antibody is an rG019 antibody, an rG055 antibody, an rG056 antibody, or By confirming that the specific epitope is Even if the sequence or structure of the target antibody has not been determined, the monoclonal antibody may be an anti-CDH6 antibody. It can be determined that the antibody binds to the same epitope as the antibody. If confirmed, the monoclonal antibody is rG019 antibody, rG055 antibody, rG056 antibody antibody or rG061 antibody, and has antigen-binding ability, biological activity, and / or internalization activity equivalent to that of the antibody. It is strongly expected that this will happen.

[0067] (3) Other antibodies The antibodies of the present invention include monoclonal antibodies against CDH6 as described above, as well as allogeneic antibodies against humans. Recombinant antibodies that have been artificially modified to reduce species antigenicity, for example , chimeric antibodies, humanized antibodies, human antibodies, etc. These antibodies can be produced using known methods.

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

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

[0070] Another example of a chimeric antibody derived from a rat anti-human CDH6 antibody is, for example, The rat anti-human CDH6 antibodies (e.g., rG019 antibody, rG055 antibody, rG056 1 to several residues, 1 to 3 residues, 1 to 2 residues in each light chain variable region of the rG061 antibody or the rG062 antibody), Preferably, a light chain comprising a light chain variable region in which one amino acid residue is substituted with another amino acid residue, and and one to several residues, one to three residues, one to two residues, preferably one residue, in each heavy chain variable region. and antibodies having heavy chains containing heavy chain variable regions in which one amino acid residue is replaced with another amino acid residue. The antibody may have a constant region of any human origin.

[0071] Another example of a chimeric antibody derived from a rat anti-human CDH6 antibody is, for example, The rat anti-human CDH6 antibodies (e.g., rG019 antibody, rG055 antibody, rG056 1 to 2 residues in any 1 to 3 CDRs in each light chain variable region of the a light chain comprising a light chain variable region in which one amino acid residue has been replaced with another amino acid residue, preferably and 1 to 2 residues, preferably 1 residue, in any 1 to 3 CDRs in each heavy chain variable region. Examples of antibodies include antibodies having heavy chains containing heavy chain variable regions in which one amino acid residue is replaced with another amino acid residue. The antibody may have a constant region of any human origin.

[0072] Examples of chimeric antibodies derived from the rG019 antibody include those having the amino acid sequence shown in SEQ ID NO: 10. a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15; and the antibody can be an antibody having a heavy chain comprising a heavy chain variable region comprising any human-derived constant domain. It may have a region.

[0073] Another example of a chimeric antibody derived from the rG019 antibody is, for example, the chimeric antibody shown in SEQ ID NO: 10. One to several residues, one to three residues, one to two residues, preferably one to several amino acid residues, in the light chain variable region consisting of the amino acid sequence a light chain comprising a light chain variable region in which one amino acid residue is replaced with another amino acid residue, and a light chain comprising a sequence 1 to several residues, 1 to 3 residues, 1 to 3 residues, or 1 in the heavy chain variable region consisting of the amino acid sequence shown in No. 15 and a heavy chain variable region in which at least two, preferably one, amino acid residue is substituted with another amino acid residue. The antibody may have a constant region of any human origin. .

[0074] Another example of a chimeric antibody derived from the rG019 antibody is, for example, the chimeric antibody shown in SEQ ID NO: 10. One or two residues (preferably) in any one to three CDRs in the light chain variable region consisting of the amino acid sequence a light chain comprising a light chain variable region in which one or more amino acids (preferably one or more residues) have been replaced with another amino acid residue; and any 1 to 3 amino acids in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15. One or two amino acid residues (preferably one residue) in the CDR are substituted with another amino acid residue. Examples of such antibodies include antibodies consisting of a heavy chain containing a heavy chain variable region, and the antibody can be any human-derived constant region. may have

[0075] Another example of a chimeric antibody derived from the rG019 antibody is, for example, the chimeric antibody shown in SEQ ID NO: 10. a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 58; The antibody may be an antibody of any human origin, which comprises a heavy chain having a heavy chain variable region consisting of the sequence The amino acid sequence shown in SEQ ID NO: 58 may have the same or similar constant region as SEQ ID NO: 15. A sequence in which the cysteine ​​residue in CDRH2 in the amino acid sequence shown in It is a column.

[0076] A specific example of a chimeric antibody derived from the rG019 antibody is the full-length light chain antibody shown in SEQ ID NO: 53. a light chain consisting of the amino acid sequence shown in SEQ ID NO: 56; and a heavy chain consisting of the full-length amino acid sequence shown in SEQ ID NO: 57. The chimeric anti-human CDH6 antibody is referred to herein as a "chimeric anti-human CDH6 antibody." The antibody is referred to as "G019 antibody," "chG019 antibody," or "chG019." The full-length amino acid sequence of the light chain of this antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 54, The full-length amino acid sequence of the heavy chain of the hG019 antibody is the nucleotide sequence shown in SEQ ID NO:57. will be loaded.

[0077] The amino acid sequence of the light chain variable region of the chG019 antibody is the same as that of the light chain variable region of the rG019 antibody. The chG019 antibody has the same amino acid sequence as that of the chG019 antibody shown in SEQ ID NO: 10. The light chain of the human body is CDRL1, which consists of the amino acid sequence shown in SEQ ID NO: 12; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 14 and the light chain CDRL1, CDRL2 and CDRL3 of rG019 are identical to those of the light chain of rG019. The light chain variable region amino acid sequence of the chG019 antibody is shown in SEQ ID NO: 55. It is encoded by a nucleotide sequence.

[0078] The heavy chain variable region amino acid sequence of the chG019 antibody is the amino acid sequence shown in SEQ ID NO:58. The heavy chain of the chG019 antibody consists of the amino acid sequence shown in SEQ ID NO: 17. CDRH1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 19 It has a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 58. The sequence is a sequence that protonates the cysteine ​​residue in CDRH2 in the amino acid sequence shown in SEQ ID NO: 15. The CDRH sequence is a sequence in which a phosphorus residue is substituted. 2 replaces the cysteine ​​residue in rG019 CDRH2 shown in SEQ ID NO: 18 with a proline residue. The amino acid sequence of the heavy chain variable region of the chG019 antibody is SEQ ID NO: 5 It is encoded by the nucleotide sequence shown in FIG.

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

[0080] Chimeric antibodies derived from the rat anti-human CDH6 antibody rG055 include, for example, those having SEQ ID NO: a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 20; and a chimeric antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown below, The antibody may have a constant region of any human origin.

[0081] Chimeric antibodies derived from the rat anti-human CDH6 antibody rG056 include, for example, those having SEQ ID NO: a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 30; and a chimeric antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown below, The antibody may have a constant region of any human origin.

[0082] Chimeric antibodies derived from the rat anti-human CDH6 antibody rG061 include, for example, those having SEQ ID NO: a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40; and a chimeric antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown below, The antibody may have a constant region of any human origin.

[0083] Humanized antibodies include those derived from complementarity determining regions (CDRs). Human-derived antibody with only the terminating region incorporated (Nat (See ure (1986) 321, pp. 522-525), and by CDR grafting method, In addition to the sequence of the antibody, some of the framework amino acid residues were also transplanted into a human antibody (International Publication No. No. 90 / 07861), and further, amino acids of some CDRs can be modified while maintaining antigen-binding ability. Examples include antibodies in which the amino acid sequence has been modified.

[0084] As used herein, the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody The humanized antibody derived from the antibody or chG019 antibody includes the rG019 antibody, the rG055 antibody, the rG 6 unique antibodies for either the 056 antibody, rG061 antibody, or chG019 antibody Any humanized antibody that retains all four CDR sequences and has internalization activity may be used. The humanized antibody is not limited to the above-mentioned humanized antibody. The humanized antibody may further contain a portion thereof as long as it has internalization activity. A portion of the amino acid sequence of the CDR may be modified.

[0085] Examples of humanized antibodies of the chG019 antibody include: (1) the antibody represented by SEQ ID NO: 63 or 67 (2) an amino acid sequence having at least 95% identity to the amino acid sequence of (1) above an amino acid sequence having the following structure (preferably, a sequence of a framework region other than each CDR sequence) (3) an amino acid sequence having at least 95% sequence identity with the above (1); An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (4) a light chain comprising a light chain variable region selected from the group consisting of: (5) the amino acid sequence of SEQ ID NO: 71, 75 or 79; an amino acid sequence having at least 95% identity to the CDR sequence (preferably each CDR sequence) A framework region sequence that has at least 95% sequence identity to the (6) the amino acid sequence of (4) above, in which one or several amino acids are missing a deletion, substitution or addition of an amino acid sequence according to any one of the following: Any combination of heavy chains containing chain variable regions may be included.

[0086] Alternatively, one of the heavy or light chains may be humanized and the other may be a light or heavy chain of a rat antibody or a chimeric antibody. Examples of such antibodies include: (1) SEQ ID NO: 63 or (2) is at least 95% of the amino acid sequence of (1) above. % or more (preferably, the framework sequences other than each CDR sequence) an amino acid sequence having at least 95% sequence identity to the sequence of the region), and 3) The amino acid sequence of (1) above in which one or several amino acids are deleted, substituted or added. a light chain comprising a light chain variable region selected from the group consisting of: (4) an amino acid sequence set forth in SEQ ID NO: 15, 25, 35, 45, or 58; (5) An amino acid sequence having at least 95% identity to the amino acid sequence of (4) above. (Preferably, at least 95% of the sequences of the framework regions other than each CDR sequence) (6) an amino acid sequence having the above sequence identity, and (7) an amino acid sequence of (4) above and an amino acid sequence in which one or several amino acids are deleted, substituted or added. Any combination of heavy chains comprising a heavy chain variable region described in any one of the above can be used. Other examples include: (1) the amino acid sequence set forth in SEQ ID NO: 10, 20, 30, or 40; 2) Amino acids having at least 95% identity to the amino acid sequence of (1) above sequences (preferably at least 9 sequences for the framework region sequences other than each CDR sequence) (3) an amino acid sequence having 5% or more sequence identity with the amino acid sequence of (1) above; and a sequence of amino acids in which one or several amino acids are deleted, substituted or added. (4) a light chain comprising a light chain variable region selected from any one of SEQ ID NOs: 71 and 72; (5) an amino acid sequence according to the above (4), Amino acid sequences with 95% or more identity (preferably, framework sequences other than each CDR sequence) an amino acid sequence having at least 95% sequence identity to the sequence of the target region, and and (6) the amino acid sequence of (4) above, in which one or several amino acids are deleted, substituted, or added. and a heavy chain variable region selected from the group consisting of the amino acid sequences Any combination of heavy chains may be included.

[0087] In addition, conservative amino acid substitutions are preferred for amino acid substitutions herein. Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; non-polar group = ara leucine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and uncharged polar family = glycine, asparagine, glutamine, cysteine Other suitable amino acid groups are: threonine, serine, threonine, tyrosine. Aliphatic hydroxy groups = serine and threonine; amide-containing groups = asparagus aliphatic group = alanine, valine, leucine and isoleucine; and aromatic groups = phenylalanine, tryptophan and tyrosine. The acid substitution is preferably carried out within a range that does not impair the properties of the substance having the original amino acid sequence.

[0088] An antibody having a suitable combination of the light chain and heavy chain includes the light chain variable region shown in SEQ ID NO: 63. The hL02 light chain variable region amino acid sequence (also referred to herein as the hL02 light chain variable region amino acid sequence) The light chain or the light chain variable region amino acid sequence shown in SEQ ID NO: 67 (hL03 light chain a light chain having a variable region amino acid sequence (also referred to as a variable region amino acid sequence) and a heavy chain having a variable region amino acid sequence (also referred to as a variable region amino acid sequence) set forth in SEQ ID NO: 71; The heavy chain variable region amino acid sequence (herein also referred to as the hH01 heavy chain variable region amino acid sequence) a heavy chain having a heavy chain variable region amino acid sequence shown in SEQ ID NO: 75 (hH02 heavy chain a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 79; The heavy chain variable region amino acid sequence (herein also referred to as the hH04 heavy chain variable region amino acid sequence) A preferred example is an antibody having a heavy chain comprising the light chain shown in SEQ ID NO: 63. A light chain having a variable region amino acid sequence and a heavy chain variable region amino acid sequence shown in SEQ ID NO: 71 an antibody comprising a heavy chain having the amino acid sequence of a light chain variable region shown in SEQ ID NO: 63; an antibody consisting of a light chain and a heavy chain having the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 75; A light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63 and a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 79 an antibody having a heavy chain having a heavy chain variable region amino acid sequence shown in SEQ ID NO: 67; A light chain having the variable region amino acid sequence shown in SEQ ID NO: 71 and a heavy chain having the variable region amino acid sequence shown in SEQ ID NO: 72 an antibody having a heavy chain having the amino acid sequence of SEQ ID NO: 67; an antibody comprising a light chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 75; or a light chain having the light chain variable region amino acid sequence set forth in SEQ ID NO: 67 and SEQ ID NO: 7 9. A preferred example is a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63. an antibody having a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 71; a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO: 63 and a heavy chain shown in SEQ ID NO: 75 An antibody comprising a heavy chain having a variable region amino acid sequence; a light chain variable region represented by SEQ ID NO: 63 a light chain having the amino acid sequence shown in SEQ ID NO: 79 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 79 or an antibody having a light chain variable region amino acid sequence shown in SEQ ID NO: 67. An antibody comprising a light chain and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 75. Examples include:

[0089] Another example of an antibody having a suitable combination of the light chain and heavy chain is the light chain shown in SEQ ID NO: 61. 21 to 24 of the full-length amino acid sequence of the hL02 light chain (also referred to herein as the full-length amino acid sequence of the hL02 light chain). A light chain consisting of the 233rd amino acid sequence or the full-length light chain amino acid sequence shown in SEQ ID NO: 65 The amino acids 21 to 233 of the full-length amino acid sequence of the hL03 light chain are also referred to herein as the full-length amino acid sequence of the hL03 light chain. a light chain consisting of the full-length amino acid sequence of SEQ ID NO: 69 ( The amino acid sequence from 20th to 471st of the hH01 heavy chain (also referred to as the full-length amino acid sequence of hH01) a heavy chain having the full-length amino acid sequence shown in SEQ ID NO: 73 (referred to herein as the full-length heavy chain of hH02); A heavy chain consisting of the amino acid sequence of positions 20 to 471 ... heavy chain consisting of the amino acid sequence of positions 20 to 471 of the amino acid sequence of positions 20 to 471 of the amino acid sequence of positions 20 to 471 of the amino acid sequence of positions 20 to 4 The full-length heavy chain amino acid sequence shown in No. 77 (referred to herein as the full-length heavy chain amino acid sequence of hH04) Examples of antibodies include antibodies having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of IgG1 (also referred to as IgG1). A preferred example is a light chain comprising the amino acid sequence from positions 21 to 233 of the full-length amino acid sequence of the light chain shown in SEQ ID NO: 61. and a light chain consisting of the amino acid sequence of SEQ ID NO: 69. An antibody having a heavy chain consisting of the amino acid sequence at position 471; and a light chain having the full length shown in SEQ ID NO: 61. a light chain consisting of the amino acid sequence of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 73; an antibody comprising a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the full-length heavy chain amino acid sequence; The light chain full-length amino acid sequence shown in SEQ ID NO: 61 consists of amino acids 21 to 233. and the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77. an antibody comprising a heavy chain consisting of the sequence: 21 to 23 of the full-length amino acid sequence of the light chain shown in SEQ ID NO: 65; A light chain consisting of the 233rd amino acid sequence and a heavy chain consisting of the full-length amino acid sequence shown in SEQ ID NO: 69 an antibody having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of SEQ ID NO: 65; a light chain consisting of the amino acid sequence of positions 21 to 233 of the full-length light chain amino acid sequence of SEQ ID NO: 7; From the heavy chain consisting of the amino acid sequence from 20th to 471st of the full-length heavy chain amino acid sequence shown in 3 an antibody comprising the amino acid sequence from 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 65; a light chain consisting of the amino acid sequence shown in SEQ ID NO: 77 and a heavy chain consisting of 20 to 47 amino acids of the full-length amino acid sequence shown in SEQ ID NO: 77 An example of a more preferred example is an antibody having a heavy chain consisting of the first amino acid sequence. , from 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 light chain consisting of amino acids 20 to 471 of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69. An antibody having a heavy chain consisting of the following sequence (referred to herein as "H01L02 antibody" or "H01L0") 2); the amino acid sequence from the 21st to the 233rd positions of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 a light chain consisting of the amino acid sequence of SEQ ID NO: 73 and a heavy chain consisting of 20 to 40 amino acids of the full-length amino acid sequence of SEQ ID NO: 73 An antibody having a heavy chain consisting of the 71st amino acid sequence (referred to herein as "H02L02 antibody"). or "H02L02"); 2 of the full-length amino acid sequence of the light chain shown in SEQ ID NO: 61 A light chain consisting of the amino acid sequence of positions 1 to 233 and a heavy chain consisting of the full-length amino acid sequence shown in SEQ ID NO: 77 An antibody (referred to herein as "H") having a heavy chain consisting of the amino acid sequence of positions 20 to 471 of the amino acid sequence or a light antibody set forth in SEQ ID NO: 65; a light chain consisting of the amino acid sequence from positions 21 to 233 of the full-length amino acid sequence of the light chain; The heavy chain consists of the amino acid sequence from 20 to 471 of the full-length heavy chain amino acid sequence shown. antibody (also referred to herein as "H02L03 antibody" or "H02L03"). The sequence of the H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody The columns are shown in Table 1.

[0090] Combine sequences that show high identity with the above heavy chain amino acid sequences and light chain amino acid sequences By doing so, it is possible to select antibodies having biological activity equivalent to that of the above antibodies. Such identity is generally 80% or more, preferably 90% or more. identity, more preferably 95% or more identity, and most preferably 99% or more identity. The amino acid sequence of the heavy chain or light chain is identical to that of the non-heavy chain. Combining deleted or added amino acid sequences can also produce antibodies equivalent to the above antibodies. It is possible to select antibodies that have biological activity.

[0091] The identity between the two amino acid sequences was calculated using ClustalW version 2 (Lar kin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompso n JD, Gibson TJ and Higgins DG (2007), “Cl ustal W and Clustal X version 2.0”, Bioi nformatics.23(21):2947-2948) default parameters This can be determined by aligning the sequences using

[0092] In addition, in the full-length amino acid sequence of hL02 light chain shown in SEQ ID NO: 61, amino acids 1 to 20 are The amino acid sequence consisting of 21 to 128 amino acid residues is a signal sequence. The amino acid sequence consisting of amino acid residues 129 to 233 is a variable region. The amino acid sequence is the constant region. The full-length nucleotide sequence of the hL02 light chain shown in SEQ ID NO: 62 In this sequence, the nucleotide sequence consisting of nucleotides 1 to 60 encodes a signal sequence. The nucleotide sequence from nucleotides 61 to 384 encodes the variable region, The nucleotide sequence from nucleotides 85 to 699 encodes the constant region. .

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

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

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

[0096] The 1st to 19th amino acid residues in the full-length amino acid sequence of hH04 heavy chain shown in SEQ ID NO: 77 The amino acid sequence consisting of the 20th to 141st amino acid residues is a signal sequence. The amino acid sequence is a variable region consisting of amino acid residues 142 to 471. The sequence is the constant region. The full-length nucleotide sequence of hL04 heavy chain shown in SEQ ID NO: 78: The nucleotide sequence consisting of nucleotides 1 to 57 is a signal sequence, and nucleotides 58 to 4 The nucleotide sequence consisting of the 23rd nucleotide encodes the variable region, 424 The nucleotide sequence from nucleotide 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.) The antibodies of the present invention further include human antibodies that bind to CDH6. Anti-CDH6 human antibodies are human antibodies that contain only the gene sequence of antibodies derived from human chromosomes. The anti-CDH6 human antibody is a human chromosome fragment containing the genes for the heavy and light chains of a human antibody. A method using human antibody-producing mice (Tomizuka, K. et al., Na ture Genetics(1997)16,p.133-143,;Kuroiwa ,Y.et.al.,Nucl.Acids Res.(1998)26,p.3447 -3448;Yoshida,H.et.al.,Animal Cell Techn ology:Basic and Applied Aspects vol.10,p .69-73(Kitagawa, Y., Matsuda, T. and Iijima, S.eds.), Kluwer Academic Publishers, 1999. ;Tomizuka,K.et.al.,Proc.Natl.Acad.Sci.US A(2000)97, pp.722-727, etc.)

[0113] Such human antibody-producing mice specifically have endogenous immunoglobulin heavy and light chains. The locus was disrupted and replaced with a yeast artificial chromosome (Yeast artificial c Human immunoglobulin heavy and light chain genes are introduced via vectors such as chromosomes and YACs. Genetically modified animals into which gene loci have been introduced include knockout animals and transgenic animals. They can be produced by creating animals and crossbreeding these animals.

[0114] Furthermore, genes encoding the heavy and light chains of such human antibodies can be produced by genetic recombination techniques. A eukaryotic cell is transformed with a cDNA encoding the gene, preferably a vector containing the cDNA, and the gene is expressed as a gene encoding the gene. By culturing transformed cells that produce recombinant human monoclonal antibodies, The antibody can also be obtained from the culture supernatant.

[0115] Here, examples of the host include eukaryotic cells, preferably CHO cells, lymphocytes, and myeloma cells. Mammalian cells such as the above can be used.

[0116] In addition, we have obtained human antibodies derived from phage display selected from a human antibody library. How to (Wormstone, IMet.al, Investigative O phthalmology & Visual Science.(2002)43(7 ),p.2301-2308;Carmen,S.et.al.,Briefings in Functional Genomics and Proteomics(20 02),1(2),p.189-203;Siriwardena,D.et.al., See Ophthalmology (2002) 109(3), p.427-431, etc. ) is also known.

[0117] For example, the variable region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv). The phage display method (Nature Bio technology (2005), 23, (9), p. 1105-1116) It is possible.

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

[0119] Once the DNA sequence of the scFv that binds to the antigen is determined, an expression vector containing the sequence can be prepared. Human antibodies can be obtained by creating a human antibody vector and expressing it in a suitable host. (International Publication Nos. 92 / 01047, 92 / 20791, 93 / 06213, 93 / 11236, 93 / 19172, 95 / 01438, 95 / 153 No. 88, Annu. Rev. Immunol (1994) 12, p. 433-455, N ature Biotechnology(2005)23(9),p.1105-11 16).

[0120] The newly generated human antibodies include the rat anti-human CDH6 antibody, chimeric anti-CDH6 antibody, and Human CDH6 antibody or humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG055 Antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02 a partial peptide to which any one of the following antibodies binds: H02L02 antibody, H02L03 antibody, or H04L02 antibody If the human antibody binds to the peptide or partial conformation, the human antibody is a rat anti-human CDH6 antibody, It binds to the same epitope as the humanized anti-human CDH6 antibody or humanized anti-human CDH6 antibody. Alternatively, the rat anti-human CDH6 antibody, chimeric Anti-human CDH6 antibody or humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG05 5 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H0 2L02 antibody, H02L03 antibody, or H04L02 antibody) to CDH6 The human antibody competes with the rG019 antibody, rG055 antibody, rG065 antibody, G056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 anti antibody, H02L03 antibody or H04L02 antibody and CDH6, preferably EC3 of CDH6 By confirming that the sequence or structure of a specific epitope is Even if the human antibody has not been determined, the human antibody may be a rat anti-human CDH6 antibody, chimeric antibody, or the like, as described herein. It was found to bind to the same epitope as the humanized anti-human CDH6 antibody or humanized anti-human CDH6 antibody. In this specification, at least one of the determination methods If the newly created human antibody is determined to "bind to the same epitope" by , a rat anti-human CDH6 antibody, a chimeric anti-human CDH6 antibody, or a humanized anti-human CDH6 antibody described herein. It can be said that it "binds to the same epitope" as the human CDH6 antibody. When it is confirmed that the human antibody is a rat anti-human CDH6 antibody, a chimeric anti-human CDH6 antibody, or antibody or humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG055 antibody, rG0 56 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, It is expected to have biological activity equivalent to that of the H02L03 antibody or H04L02 antibody. do.

[0121] The chimeric, humanized, or human antibodies obtained by the above methods can be purified by known methods, etc. By this method, the binding ability to the antigen can be evaluated and a suitable antibody can be selected.

[0122] Another index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a thermal analysis that provides a good indicator of the relative structural stability of proteins. This is an instrument that can measure the denaturation midpoint (Tm) quickly and accurately. By measuring the Tm value and comparing the values, differences in thermal stability can be compared. It is known that the storage stability of antibodies correlates to some extent with their thermal stability. (Lori Burton,et.al.,Pharmaceutical Development opment and Technology(2007)12,p.265-273) Therefore, suitable antibodies can be selected using thermostability as an index. The targets are high yield in suitable host cells and low aggregation in aqueous solution. For example, the antibody with the highest yield does not necessarily exhibit the highest thermostability. Therefore, based on the above mentioned indicators, a comprehensive judgment is made to select the most suitable antibody for administration to humans. You need to select the body.

[0123] The antibody of the present invention also includes modified antibodies. Such modified antibodies are those that have been modified chemically or by other methods. means that the amino acid skeleton has been biologically modified. and chemical modifications of N-linked or O-linked carbohydrate chains. Examples of potential modifications include post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal glycosylation, etc.). End-processing, deamidation, aspartic acid isomerization, methionine oxidation, or N Pyroglutamic acid of terminal glutamine or N-terminal glutamic acid) These include those with an added methionine residue at the N-terminus due to expression in host cells. In addition, the antibody or antigen of the present invention may be labeled to enable detection or isolation, e.g. For example, enzyme-labeled products, fluorescent-labeled products, and affinity-labeled products are also included in the meaning of such modifications. Such modified antibodies of the present invention have improved antibody stability and blood retention, and reduced antigenicity. , and are useful for detecting or isolating antibodies or antigens.

[0124] In addition, it is possible to regulate sugar chain modification (glycosylation, defucosylation) of the antibody of the present invention. Antibody-dependent cellular cytotoxicity can be enhanced by glycosylation of antibodies. Techniques for adjusting decorations are described in International Publication Nos. 1999 / 54342 and 2000 / 61739. , 2002 / 31140, etc. are known, but are not limited to these. The antibodies of the invention also include antibodies in which the glycosylation has been regulated.

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

[0126] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, mammalian cells, such as monkey cells, are suitable for use as animal cells. luzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL -1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), Chinese hamster ovary cells (CHO cells, ATCC CCL-61) Folate reductase-deficient strain (Urlaub, G. and Chasin, LA Proc. N atl.Acad.Sci.USA(1980)77,p.4126-4220) and FreeStyle 293F cells (Invitrogen). .

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

[0128] The desired antibody gene is introduced into these cells by transformation, and the transformed cells The antibody is obtained by culturing the antibody in vitro. Yields may vary depending on the sequence, so use the yield as an indicator to select antibodies with equivalent binding activity. Therefore, it is possible to select antibodies that can be easily produced as pharmaceuticals. a step of culturing the transformed host cells, and extracting the target gene from the culture obtained in the step. 2. A method for producing an antibody comprising the step of collecting an antibody or a functional fragment of said antibody. Antibodies obtained by the method are also included.

[0129] In addition, the carboxyl-terminal lysine residue of the heavy chain of the antibody produced in mammalian cultured cells is deleted. It is known that (Journal of Chromatography A, 705:129-134(1995)), and also glycine at the carboxyl terminus of the heavy chain. Two lysine amino acid residues are deleted, and a new proline residue is added at the carboxyl terminal. It is known that amidation occurs (Analytical Biochemistry , 360:75-83(2007)). However, these deletions and modifications of the heavy chain sequence have not been shown to be effective against the antibody. The body's antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) Therefore, the antibody according to the present invention includes the modified antibody and the antibody. Also included are functional fragments of the heavy chain lacking one or two amino acids at the carboxyl terminus of the heavy chain. Deletions and amidated versions of the deletions (e.g., proline residues at the carboxyl terminal end) However, the heavy chains may be amidated, provided that the antigen-binding ability and effector function are maintained. As long as the deletion of the carboxyl terminal of the heavy chain of the antibody of the present invention is not limited to the above types. The two heavy chains constituting the antibody of the present invention are composed of a full-length and the above-mentioned deleted heavy chains. The heavy chain may be any one of the heavy chains selected from the group, or a combination of any two of the heavy chains. The quantitative ratio of each deletion may be determined depending on the type of cultured mammalian cell that produces the antibody of the present invention. Although the antibody of the present invention may be affected by the culture conditions and the amount of the heavy chain, the main components of the antibody of the present invention are both heavy chains. An example is a case where one amino acid residue at the carboxyl terminus is deleted.

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

[0131] The biological activity of an antibody generally includes antigen binding activity, which is the ability of the antibody to bind to an antigen. The ability to be internalized into cells expressing antigens, neutralize the activity of antigens, and enhance the activity of antigens. activity, antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and Examples of the phagocytosis that the antibody of the present invention possesses include phagocytosis-dependent cell-mediated phagocytosis (ADCP). The function is binding activity to CDH6, and preferably, binding to CDH6. Furthermore, the antibody of the present invention has the cell-internalizing activity. In addition, it may also have ADCC activity, CDC activity and / or ADCP activity.

[0132] The obtained antibodies can be purified to homogeneity. Separation and purification methods used for proteins can be used. For example, column chromatography -, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, By appropriately selecting and combining methods such as isoelectric focusing, antibodies can be separated and purified (S strategies for Protein Purification and C haracterization:A Laboratory Course Manu al,Daniel R.Marshak et al.eds.,Cold Spri ng Harbor Laboratory Press(1996);Antibod ies:A Laboratory Manual.Ed Harlow and Da vid Lane,Cold Spring Harbor Laboratory(1) 988)), but is not limited to these.

[0133] Chromatography includes affinity chromatography, ion exchange chromatography, chromatograph, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography Examples of suitable methods include chromatography, adsorption chromatography, and the like.

[0134] These chromatography methods use liquid chromatography such as HPLC and FPLC. This can be done.

[0135] Columns used for affinity chromatography include Protein A columns, Protein B columns, and Protein C columns. For example, a column using a protein A column can be used. Hyper D, POROS, Sepharose FF (Pharmacia), etc. Some examples include:

[0136] Antibodies can also be purified by using antigen-immobilized carriers and utilizing their binding to antigens. It is possible.

[0137] 3. Anti-CDH6 Antibody-Drug Conjugates (1) Drugs The anti-CDH6 antibody obtained in the above "2. Production of anti-CDH6 antibody" has a linker structure By binding the drug via the The drug may have a substituent or partial structure that can be bonded to the linker structure. There are no particular limitations as long as the conjugate is a drug. Examples of such drugs include substances with antitumor activity. , substances effective against blood diseases, substances effective against autoimmune diseases, anti-inflammatory Examples of such substances include antibacterial, antifungal, antiparasitic, antiviral, and antianesthetic substances. This can be done.

[0138] (1)-1 Antitumor compounds The compound conjugated to the anti-CDH6 antibody-drug conjugate of the present invention may be an antitumor compound. Examples of the antitumor compounds include compounds having antitumor effects. There are no particular restrictions on the substance, as long as it has a substituent or partial structure that can be bonded to the linker structure. The antitumor compound is capable of exerting its antitumor activity by cleaving part or all of the linker in tumor cells. The compound portion is released and the antitumor effect is exerted. The linker is cleaved at the site of binding to the drug. If this occurs, the antitumor compound is released in its original structure, and its original antitumor effect is exerted.

[0139] The anti-CDH6 antibody obtained in the above "2. Production of anti-CDH6 antibody" has a linker structure By binding the antitumor compound via It can be said that

[0140] One example of an antitumor compound that can be used in the present invention is camptothecin derivative E. Xatecan ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydrochloride 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] The compound can be suitably used. It can be easily obtained by the method described in Patent Publication No. 0297890 or other known methods, and the amino group at the 1-position can be easily obtained by the method described in Patent Publication No. 0297890 or other known methods. can be suitably used as a binding site to the linker structure. In some cases, a portion of the linker may be released in the tumor cell while still bound. It is a compound that also exhibits excellent antitumor effects.

[0143] Since exatecan has a camptothecin structure, it is easily absorbed in an acidic aqueous medium (e.g., pH 3 or so). ), the equilibrium shifts to the structure in which the lactone ring is formed (closed ring), while in a basic aqueous medium ( For example, at pH 10, the equilibrium is known to be biased toward the structure in which the lactone ring is opened (open ring isomer). Drugs containing exatecan residues corresponding to the closed and open ring structures have been developed. Conjugates are expected to have the same antitumor effect, and both are within the scope of the present invention. It goes without saying that it is included.

[0144] Other antitumor compounds include, for example, those listed in the Pharmacological Review ews, 68, p3-19, 2016), and Examples include doxorubicin, calchemycin, heamicine), dorastatin 10, monomethyl aurine Auristatins such as statin E (MMAE) and monomethylauristatin F (MMAF) Auristatins, maytansinoids such as DM1 and DM4 sinoids, pyrrolobenzodiazepines ne) dimeric SG2000 (SJG-136), a camptothecin derivative, SN- 38, Duocarmycins such as CC-1065, flaxseed Amanitin, Daunorubicin, Mitomycin C, Bleomycin, Cyclobacter pylori ciclosporin, vincristine, vinblastine, methotrexate, platinum-based antitumor agents ( Examples of suitable amines include sucralose, thiazolinone ... do.

[0145] In antibody-drug conjugates, the number of drugs bound to one antibody molecule determines its efficacy and safety. The production of antibody-drug conjugates is an important factor affecting the safety of the antibody-drug conjugates. The reaction conditions, such as the amounts of raw materials and reagents used, are specified to ensure a certain number of reactions. Unlike chemical reactions involving small molecules, a mixture of different numbers of drugs is obtained. The number of drugs bound to one antibody molecule is usually an average value, that is, the average number of drugs bound is In principle, unless otherwise specified, the present invention also Antibodies with specific binding numbers - Antibodies with specific drug binding numbers contained in a drug conjugate mixture Except when referring to drug conjugates, the number of drugs bound is an average value. The number of exatecan conjugated molecules can be controlled, and the average number of drugs bound to one antibody is Approximately 1 to 10 exatecans can be bound, preferably 2 to 8, 3 1 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, and more preferably 5 to 8. It is more preferable that the number of the nucleotides is 7 to 8, and even more preferable that the number of the nucleotides is 8. If this is the case, it will be possible to design a reaction for binding the required number of drugs to an antibody based on the description of the examples of this application. Therefore, it is possible to obtain antibody-drug conjugates with controlled binding of exatecan. can be done.

[0146] (2) Linker structure In the anti-CDH6 antibody-drug conjugate of the present invention, a drug is bound to an anti-CDH6 antibody. We will describe the linker structure that enables this.

[0147] In the antibody-drug conjugate of the present application, a linker that connects the anti-CDH6 antibody and the drug The structure is not particularly limited as long as it can be used as an antibody-drug conjugate. The linker structure can be selected appropriately depending on the purpose of use. , Publicly known literature (Pharmacol Rev 68:3-19, January 201 6, Protein Cell DOI 10.1007 / s13238-016-03 23-0, etc.), and more specific examples include VC (Vacuum Capsules) and valine-citrulline), MC (maleinamide caprylate Maleimidocaproyl), SMCC (Succinimidyl 4-(N- Maleimidomethyl)cyclohexane-1-carboxylate: succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-car boxylate), SPP (N-succinimidyl 4-(2-pyridyldithio)pentaerythritol N-succinimidyl 4-(2-pyridyldithio)pen Tanoate, SS (disulfide), SPDB (N-succinimide) N-succinimidyl 4-(2-pyridyldithio)butyrate 2-pyridyldithio)butyrate, SS / hydrazone, hydrazone, Mention may be made of carbonates.

[0148] Another example is described in US Patent Publication US2016 / 0297890. Linker structures (for example, those described in paragraphs

[0260] to

[0289] ) can be mentioned. The following structure can be suitably used. is the binding site for the antibody, and the right end is the binding site for the drug. GGF in the diet consists of glycine-glycine-phenylalanine-glycine (GGF). The amino acid sequence connected by peptide bonds is shown. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2O-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-. Examples include:

[0150] The antibody may be -(Succinimid-3-yl-N)-terminated (e.g., -(Succ inimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGF In the case of G-NH-CH2-O-CH2-C(=O)-, (-CH2CH2CH2C H2CH2-) binds at the opposite end (left end) of the molecule, and the antitumor compound -(Succinimid-3-yl-N) and the opposite end (the right end in the example above, C The bond is formed by the carbonyl group of H2-O-CH2-C(=O)-. d-3-yl-N)-' is a compound of the formula:

[0151] [ka]

[0152] The third position in this partial structure is the binding site for anti-CDH6 antibodies. The binding to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 of this structural part is a methylene group present in the linker containing this structure. It bonds to the carbon atom of

[0153] In the antibody-drug conjugate of the present invention in which the drug is exatecan, The drug-linker structure moiety is preferably linked to an antibody. The average number of structural moieties bound to one antibody may be 1 to 10, but it is preferred Preferably, it is 2 to 8, more preferably 5 to 8, and even more preferably 7 to 8. , and even more preferably 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2O-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-(NH-DX).

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

[0157] [ka]

[0158] The structure is represented by the formula: This indicates a group.

[0159] (3) Method for producing antibody-drug conjugates The antibodies that can be used in the antibody-drug conjugates of the present invention are those described above in "2. Anti-CDH The anti-CDH6 antibody having internalization activity and the antibody described in the "Production of Antibody No. 6" section and Examples There are no particular limitations as long as it is a functional fragment.

[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 are referred to as "compound of formula (1)", "compound (1)", etc. The compounds are similarly described.

[0161] (3)-1 Manufacturing method 1 Among the antibody-drug conjugates represented by the following formula (1), anti-C The CDH6 antibody and the linker structure are bonded to each other by reducing the anti-CDH6 antibody to form disulfides. The antibody in which the bond was converted to a sulfhydryl group was treated with a compound ( 2) (for example, it can be obtained by the method described in US2016 / 297890 (for example, the method described in paragraphs

[0336] to

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

[0162]

number

[0163] [wherein AB represents an antibody having a sulfhydryl group. where L 1 teeth, -(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-N H-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2O-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-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-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-.

[0168] In the above reaction scheme, in the antibody-drug conjugate (1), the drug is converted to a linker. Although it is described as a structure in which one structural portion up to the terminal binds to one antibody, This is a convenient description for the purpose of explanation, and in reality, the structural portion in question is present in one antibody molecule. This situation is the same in the following explanation of the manufacturing method. be.

[0169] That is, compound (2) which can be obtained by a known method (e.g., US2016 / 29 The method described in Patent Publication No. 7890 (for example, the method described in paragraphs

[0336] to

[0374] ) The antibody (3a) having a sulfhydryl group was reacted with the antibody (3b) The antibody-drug conjugate (1) can be produced by the above procedure.

[0170] The antibody (3a) having a sulfhydryl group can be obtained by a method well known to those skilled in the art (H ermanson, GT, Bioconjugate Techniques, pp. 56-136, pp.456-493, Academic Press (1996)). For example, Traut's reagent is used to react with the amino groups of antibodies; N-succinimidyl S- After reacting acetylthioalkanoates with the amino groups of the antibody, hydroxylamine N-succinimidyl 3-(pyridyldithio)propionate was reacted with Then, a reducing agent is applied; dithiothreitol, 2-mercaptoethanol, tris(2 2-carboxyethyl)phosphine hydrochloride (TCEP) reduction of interchain disulfide bonds to generate sulfhydryl groups; It can be, but is not limited to, these.

[0171] Specifically, TCEP was used as a reducing agent, and the ratio of TCEP to each intra-chain disulfide in the antibody was By using 0.3 to 3 molar equivalents of the chelating agent and reacting it with the antibody in a buffer containing the chelating agent, the antibody It is possible to obtain antibodies in which the intracellular interchain disulfides are partially or completely reduced. Examples of rate agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine These are used at a concentration of 1 mM to 20 mM. Buffer solutions include sodium phosphate, sodium borate, and sodium acetate solutions. In a specific example, the antibody is incubated at 4°C to 37°C for 1 to 4 hours. Has sulfhydryl groups that have been partially or completely reduced by reaction with TCEP Antibody (3a) can be obtained.

[0172] In addition, a reaction to add a sulfhydryl group to the drug-linker moiety is carried out here. The drug-linker moiety can be attached via a thioether bond.

[0173] Next, 2 to 20 molar equivalents of the compound (3a) are added to each antibody (3a) having a sulfhydryl group. Using the substance (2), an antibody-drug conjugate having 2 to 8 drugs bound to one antibody can be produced. Gate (1) can be produced. Specifically, an antibody (3) with a sulfhydryl group can be produced. A solution of compound (2) dissolved in a buffer solution containing a) may be added to react the compound. As the buffer solution, sodium acetate solution, sodium phosphate, sodium borate, etc. are used. The pH during the reaction is 5 to 9, and more preferably, the reaction is carried out at around pH 7. The solvent for dissolving compound (2) is dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridinyl An organic solvent such as NMP can be used. The solution was added at 1 to 20% v / v to a buffer containing an antibody (3a) having sulfhydryl groups. The reaction temperature is preferably 0 to 37°C, more preferably 10 to 25°C. The reaction time is 0.5 to 2 hours. The reaction is carried out by thiolating the unreacted compound (2). The thiol-containing reagent can be used to terminate the reaction by quenching with a thiol-containing reagent, for example: Cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, NA C was added in an amount of 1 to 2 molar equivalents relative to the compound (2) used, and the mixture was left at room temperature for 10 to 30 minutes. The reaction can be terminated by incubation.

[0174] (4) Identification of antibody-drug conjugates The produced antibody-drug conjugate (1) was concentrated and buffered by the following common procedure. antibody-drug interaction analysis. Identification of the product conjugate (1) can be performed.

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

[0176] (4)-2 Common Procedure B: Antibody Concentration Measurement UV measurement device (Nanodrop 1000, Thermo Fisher Scien Antibody concentrations were measured using a ELISA kit (Protein Immunosorbent Assay Kit, Inc.) according to the manufacturer's instructions. At that time, 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 NAP-25 column (Cat. No. 17) using Sephadex G-25 support -0852-02,GE Healthcare Japan Corporation ) was diluted with sodium chloride (50 mM) and EDTA (2 mM) according to the manufacturer's instructions. Phosphate buffer solution (50 mM, pH 6.0) containing EDTA (referred to herein as PBS6.0 / EDTA) Each NAP-25 column was equilibrated with 2.5 mL of antibody aqueous solution. After loading the column, the fraction (3.5 mL) eluted with 3.5 mL of PBS 6.0 / EDTA was separated. This fraction was concentrated using common procedure A, and the antibody concentration was measured using common procedure B. After that, the antibody concentration was adjusted to 20 mg / mL using PBS6.0 / EDTA.

[0178] (4)-4 Common Procedure D: Purification of Antibody-Drug Conjugates Commercially available acetate buffer (10 mM, pH 5.5; see below) containing Sorbitol (5%) The NAP-25 column was equilibrated with one of the following buffers: The antibody-drug conjugate reaction solution (approximately 2.5 mL) was placed on the AP-25 column. The antibody fraction was collected by eluting with a specified amount of buffer. The gel filtration purification procedure, in which the product is loaded onto a NAP-25 column and eluted with buffer, is repeated 2 to 3 times. This allows the removal of unbound drug linkers and small molecules (tris(2-carboxyethyl)phosphine). thiamin hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide The antibody-drug conjugate was obtained by removing the side chain.

[0179] (4)-5 Common Procedure E: Antibody Concentration and Single Antibody Molecule in Antibody-Drug Conjugates Measurement of the average number of drugs bound per The bound drug concentration in the antibody-drug conjugate is determined by the aqueous solution of the antibody-drug conjugate. After measuring the UV absorbance at two wavelengths of 280 nm and 370 nm, the following calculation was performed. By doing so, it can be calculated.

[0180] The total absorbance at a given wavelength is equal to the sum of the absorbances of all absorbing species present in the system [ The absorbance of the antibody and the drug differs depending on the conjugation. Assuming there is no change in the molar extinction coefficient of the compound, the antibody concentration in the antibody-drug conjugate The intensity and drug concentration 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,370C D +ε A,370 C A formula( 2) where A 280 indicates the absorbance of an aqueous solution of antibody-drug conjugate at 280 nm. death 、 A 370 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 is at 370 nm The absorbance of the antibody is shown, and A D,280 is the absorbance of the conjugate precursor at 280 nm Indicates the degree, 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 is 370 nm where ε is the molar extinction coefficient of the antibody at D,280 is the conjugate at 280 nm denotes the molar extinction coefficient of the precursor, ε D,370 is the conjugate precursor at 370 nm is the molar extinction coefficient of 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 Please prepare in advance. The values ​​(calculated estimates or actual values ​​obtained from UV measurements of the compound) are used. For example, , ε A,280 is calculated from the amino acid sequence of the antibody using a known calculation method (Protein Science ence, 1995, vol. 4, 2411-2423) ε A,370is usually zero. D,280 and ε D,370 The condiments used By measuring the absorbance of a solution in which a certain molar concentration of the dextrose precursor is dissolved, the Lambertian By Beer's law (absorbance = molar concentration x molar extinction coefficient x cell path length), A of the antibody-drug conjugate solution 280 and A 370 Measure these values By substituting into equations (1) and (2) and solving the simultaneous equations, C A and C D Ask for Furthermore, C D C A Divide by this to find the average number of drugs bound per antibody. It is possible.

[0182] (4)-6 Common Procedure F: Drug per antibody molecule in antibody-drug conjugates Measurement of average number of bonds (2) The average number of drugs bound per antibody molecule in an antibody-drug conjugate is calculated as described above. 4)-5 In addition to "Common Procedure E", high performance liquid chromatography (HP) using the following method The following is a summary of the results obtained by LC analysis. A method for measuring the average number of bound drugs by HPLC is described below. Depending on the binding mode between the antibody and the drug linker, the drug may be appropriately detected by HPLC. The average number of binding sites can be determined.

[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) was dissolved in dithiothreitol ( The mixture was mixed with an aqueous solution of DTT (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes. By bating the antibody, the disulfide bond between the light and heavy chains of the antibody-drug conjugate is cleaved. The digested 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. 7μm, 130Å; Waters, P / N 186002884) Column temperature: 80℃ Mobile phase A: 0.10% trifluoroacetic acid (TFA), 15% 2-propanol in water solution Mobile phase B: acetonitrile solution containing 0.075% TFA and 15% 2-propanol Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min) -17 minutes), 80%-14% (17 minutes - 17.01 minutes), 14% (17.01 minutes - 25 minutes) ) Sample injection volume: 10 μL F-3. Data Analysis F-3-1 Drug-binding to the light chain (L0) and heavy chain (H0) of an antibody that is not bound to a drug Light chain with drug-bound (light chain with drug-bound: L i ) and heavy chain (heavy chain with i drugs bound: H i ) increases in hydrophobicity in proportion to the number of bound drugs, and the retention time increases. For example, L0, L1, H0, H1, H2, and H3 are eluted in this order. Detected peaks are assigned to L0, L1, H0, H1, H2, or H3 by comparison. The number of drugs bound can be determined by those skilled in the art, but preferably, L0, L1 , H0, H1, H2, H3.

[0186] F-3-2 Because the drug linker has UV absorption, the light Correct the peak area values ​​using the molar extinction coefficients of the lower chain, the heavy chain, and the drug linker according to the following formula: cormorant.

[0187]

number

[0188]

number

[0189] Here, the molar extinction coefficients (280 nm) of the light and heavy chains of each antibody were calculated using a known method. (Protein Science, 1995, vol.4, 2411-242 3) allows the use of values ​​estimated from the amino acid sequences of the light and heavy chains of each antibody. In the case of H01L02, the molar extinction coefficient of the light chain is 31, according to the amino acid sequence. The molar extinction coefficient of the heavy chain was estimated to be 710 and 79990. The molar extinction coefficient of the CAR (280 nm) was calculated by dividing each drug linker by mercaptoethanol or N- The maleimide group was converted to a succinimide thioether by reaction with acetylcysteine. The molar extinction coefficient (280 nm) of the compound was used. The wavelength at which the absorbance was measured was determined by a method known to those skilled in the art. However, it is preferable that the wavelength is a wavelength at which the peak of the antibody can be measured. More preferably, it is 280 nm.

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

[0191]

number

[0192] F-3-4 Average number of drugs bound per antibody molecule in antibody-drug conjugates , is calculated according to the following formula:

[0193] Average number of drug bonds = (L0 peak area ratio x 0 + L1 peak area ratio x 1 + H0 peak area ratio x0 + H1 peak area ratio x1 + H2 peak area ratio x2 + H3 peak area ratio x3) / 100 x2 In order to ensure the amount of antibody-drug conjugate, the same antibody-drug conjugate was prepared under similar conditions. Mix multiple antibody-drug conjugates with similar average drug numbers (e.g., ±1). In this case, the average number of drugs must fall within the average number of drugs before mixing. circle.

[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] where AB represents the anti-CDH6 antibody disclosed herein, and the sulfhydryl group derived from the antibody. The linker is connected to the compound via a hydroxyl group, where n is the so-called DAR (Drug-to-Arrow). Synonymous with o-Antibody Ratio, which indicates the drug-antibody ratio per antibody. That is, it shows the number of drugs bound to one antibody molecule, but this is the average value, i.e., the average drug binding The compound represented by [Chemical Formula 9] and [Chemical Formula 10] of the present invention is a compound represented by [Chemical Formula 11] and [Chemical Formula 12]. For organo-drug conjugates, n may be 2 to 8, as determined by common procedure F. Preferably, it is 5 to 8, more preferably 7 to 8, and even more preferably 8. do.

[0200] An example of the antibody-drug conjugate of the present invention is the antibody-drug conjugate shown in the above formula [Chemical Formula 9] or [Chemical Formula 10]. In the structure shown, the antibody represented by AB is selected from the group consisting of the following (a) to (g): an antibody-drug comprising the heavy and light chain antibody or a functional fragment thereof selected from any one of claims 1 to 4; Examples of suitable compounds include: (a) the amino acid sequence from positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69. an antibody having a heavy chain consisting of the amino acid sequence; (b) the amino acid sequence from positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73. an antibody having a heavy chain consisting of the amino acid sequence; (c) the amino acid sequence from positions 21 to 233 of the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77. an antibody having a heavy chain consisting of the amino acid sequence; (d) 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 light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69. an antibody having a heavy chain consisting of the amino acid sequence; (e) 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 light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73. an antibody having a heavy chain consisting of the amino acid sequence; (f) 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 light chain consisting of the 20th to 471st amino acids of the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77. an antibody comprising a heavy chain consisting of the amino acid sequence; or (g) The heavy or light chain undergoes N-linked glycosylation, O-linked glycosylation, or N-terminal processing. ligation, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation , addition of methionine residues to the N-terminus, amidation of proline residues, N-terminal glutamine and N-terminal glutamine Post-translational modifications, such as pyroglutamylation of amino acids, and carboxyl-terminal modifications and one or more modifications selected from the group consisting of one or two amino acid deletions, An antibody according to any one selected from the group consisting of a) to (f).

[0201] 4. Pharmaceuticals The anti-CDH6 antibody of the present invention described in the above section "2. Production of anti-CDH6 antibody" and the Examples The antibody and functional fragment of the antibody bind to CDH6 on the surface of tumor cells and have internalization activity. Therefore, it is being used as a medicine, either alone or in combination with other drugs, to treat renal cell tumors and ovarian cancers. Cancers such as tumors, for example, renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous carcinoma Adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, It can be used as a therapeutic agent for mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma. do.

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

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

[0204] The anti-CDH6 antibody-drug conjugates of the present invention described in the above section "3. Anti-CDH6 antibody-drug conjugates" and Examples Among CDH6 antibody-drug conjugates, those with antitumor activity such as cytotoxicity as drugs For those using drugs that have internalization activity, anti-CDH6 antibodies and / or the anti-CDH6 antibodies It is a conjugate of a functional fragment of a cytotoxic drug with antitumor activity such as cytotoxic activity. Since it exhibits antitumor activity against cancer cells expressing DH6, it is expected to be useful as a medicine, especially in It can be used as a therapeutic and / or preventive agent for cancer.

[0205] The anti-CDH6 antibody-drug conjugate of the present invention may be left in the air or may be recrystallized or decomposed. When the product is purified, it absorbs moisture or absorbs water, The compounds or pharmacologically acceptable salts containing such water may be hydrated. It is included in the invention.

[0206] When the anti-CDH6 antibody-drug conjugate of the present invention has a basic group such as an amino group, In this case, a pharmacologically acceptable acid addition salt can be formed, if desired. Examples of addition salts include hydrofluoride, hydrochloride, hydrobromide, and hydroiodide. Hydrohalides; inorganic acid salts such as nitrates, perchlorates, sulfates, and phosphates; methanesulfonate lower alkanes such as fluorosulfonates, trifluoromethanesulfonates, and ethanesulfonates; sulfonates; aryl sulfonates such as benzenesulfonates and p-toluenesulfonates Acid salts; formates, acetates, trifluoroacetates, malates, fumarates, succinates, Organic acid salts such as enoates, tartrates, oxalates, maleates, etc.; or ornithine salts, Examples include amino acid salts such as glutamate and aspartate.

[0207] The anti-CDH6 antibody-drug conjugate of the present invention has an acidic group such as a carboxy group. In such cases, pharmacologically acceptable base addition salts can be formed as desired. Suitable base addition salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts. alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts Inorganic salts: dibenzylamine salts, morpholine salts, phenylglycine alkyl ester salts, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, Cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine Amine salts, diethanolamine salts, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethyl Examples include organic amine salts such as ammonium salt.

[0208] The present invention also relates to an antibody-drug conjugate in which one or more of the atoms constituting the antibody-drug conjugate are isotopically linked to the isotope of that atom. The isotope may include an anti-CDH6 antibody-drug conjugate substituted with a radioisotope. There are two types of isotopes: isotopes of hydrogen (2 H and 3H), carbon isotopes (11C, 13C and 14C), nitrogen isotopes (13N and 15N), oxygen isotopes (15O, 17O, and 18O), fluorine isotopes (18F), etc. The composition comprising an isotope-labeled antibody-drug conjugate can be For example, therapeutic agents, preventive agents, research reagents, assay reagents, diagnostic agents, in vivo imaging diagnostic agents, etc. Isotopically labeled antibody-drug conjugates and isotopically labeled antibody-drug conjugates are useful as therapeutic agents. Mixtures of the antibody-drug conjugates in any proportion are also encompassed by the present invention. The antibody-drug conjugates labeled with the carboxyl group can be prepared by methods known in the art, e.g., by By using an isotope-labeled raw material instead of the raw material in the manufacturing method of the present invention described below, It can be manufactured more easily.

[0209] In vitro cytocidal activity can be measured, for example, by cell proliferation inhibition. For example, cancer cell lines overexpressing CDH6 were cultured, and various concentrations of anti-CDH6 antibodies were added to the culture system. DH6 antibody-drug conjugate was added, and focus activity, colony formation, and spheroid formation were measured. The inhibitory activity against idioblast proliferation can be measured, for example, in renal cell tumors and oocytes. By using cancer cell lines derived from renal cell tumors, cell proliferation inhibition in renal cell tumors and ovarian tumors The activity can be examined.

[0210] The therapeutic effect against cancer in in vivo experimental animals has been demonstrated by, for example, Anti-CDH6 antibody-drug conjugates were administered to nude mice implanted with tumor cell lines expressing CDH6. For example, renal cell carcinoma, renal clear coat Cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, or thyroid cancer cells were cultured in immunodeficient mice. By using animal models transplanted into mice, we have been able to investigate the effects of renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, The effect of treatment on ovarian cancer, ovarian serous adenocarcinoma, or thyroid cancer can be measured.

[0211] The types of cancer to which the anti-CDH6 antibody-drug conjugate of the present invention can be applied include: There are no particular limitations on the cancer as long as the target cancer cells express CDH6. For example, renal cell carcinoma (e.g. renal clear cell carcinoma or papillary renal cell carcinoma), ovarian cancer, ovarian serous gland Cancer, thyroid cancer, bile duct cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, Examples of cancers that may be mentioned include uterine cancer, pancreatic cancer, Wilms' tumor, and neuroblastoma, but CDH6 The present invention is not limited to these cancers as long as they express the above-mentioned gene. More preferred examples of cancers include renal cell carcinoma (RCC) and For example, renal clear cell carcinoma, papillary renal cell carcinoma, or ovarian cancer can be mentioned.

[0212] The anti-CDH6 antibody-drug conjugate of the present invention can be suitably administered to mammals. The animal may be any animal, but is more preferably a human.

[0213] The present invention is used in a pharmaceutical composition comprising an anti-CDH6 antibody-drug conjugate. The substances to be administered are those that are comparable in dosage and concentration to those commonly used in this field. It is possible to appropriately select and apply from among others.

[0214] The anti-CDH6 antibody-drug conjugates of the present invention contain one or more pharmaceutically compatible components. For example, the pharmaceutical composition may be administered as a pharmaceutical composition containing one or more of the following: The above pharmaceutical carriers (e.g., sterile liquids (e.g., water and oils (petroleum, animal, plant, or oils of synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.) Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, are also suitable liquid carriers. Suitable pharmaceutical excipients can be used as pharmaceuticals, particularly for injectable solutions. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH adjusting agents. Buffering agents may be included. Examples of suitable pharmaceutical carriers are found in "Re: Published in "Mington's Pharmaceutical Sciences" The formulation corresponds 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. Methods of administration include intradermal, intramuscular, intraperitoneal, intravenous, and transdermal. Routes of administration include, but are not limited to, the following: administration by infusion or bolus injection In certain preferred embodiments, the antibody-drug conjugate Administration of the drug is by injection. Parenteral administration is the preferred route of administration.

[0216] In an exemplary embodiment, the pharmaceutical composition is a pharmaceutical composition adapted for intravenous administration to humans. The composition is formulated according to routine procedures, typically a composition for intravenous administration. is a solution in sterile isotonic aqueous buffer. If necessary, the drug may also be dissolved in water. Contains a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the ingredients are packaged in a container (e.g., an ampoule or sachet indicating the quantity of active agent). as a dry lyophilized powder or anhydrous concentrate in a sealed container, either individually or in units or mixed together in a dosage form. If so, it may be, for example, an infusion bottle containing sterile pharmaceutical-grade water or saline. When the above medicine is administered by injection, it can be administered with an amount of sterile water for injection or saline. A sample may be provided, for example, so that the ingredients can be mixed prior to administration.

[0217] The pharmaceutical composition of the present invention includes a pharmaceutical composition containing only the anti-CDH6 antibody-drug conjugate of the present application. or an anti-CDH6 antibody-drug conjugate and at least one of the The anti-CDH6 antibody-drug combination of the present invention may also be a pharmaceutical composition containing a cancer therapeutic agent other than the anti-CDH6 antibody-drug combination of the present invention. Jugate can also be administered with other cancer treatments, which may increase the anti-cancer effect. Other anti-cancer drugs used for this purpose are antibody-drug conjugates. The drug may be administered to an individual simultaneously, separately, or sequentially, or each administration may be performed in a single dose. The administration interval may be varied. Tyrosine kinase inhibitors, including nitinib and regorafenib, and Palb CDK4 / 6 inhibitors, including ociclib, and HSP90 inhibitors, including TAS-116 Antibody, MEK inhibitors including MEK162, nivolumab, pembrolizumab, ipilimumab Examples of these include immune checkpoint inhibitors, including mabs, which have antitumor activity. There is no limitation as long as the drug has the function.

[0218] Such pharmaceutical compositions are available in lyophilized form as formulations with the selected composition and required purity. When formulating as a lyophilized preparation, The formulation may contain suitable formulation additives used in the field of medicine. Similarly, in the case of liquid preparations containing various formulation additives used in this field, It can be formulated as follows.

[0219] The composition and concentration of the pharmaceutical composition vary depending on the administration method. The anti-CDH6 antibody-drug conjugates contained in the antibody-drug conjugates have the ability to bind to the antigen. The affinity of the antibody to the antigen, i.e., the dissociation constant (Kd value), is high (K The lower the d value, the more effective the drug can be even at a smaller dose. When determining the dosage of the antibody-drug conjugate, the antibody-drug conjugate and the antibody The dosage can also be determined based on the affinity of the antibody to the antigen. When the adjugate is administered to humans, for example, about 0.001 to 100 mg / kg is used. It may be administered once or multiple times at intervals of once every 1 to 180 days. 50 mg / kg, more preferably 1 to 50 mg / kg, 1 to 30 mg / kg, 1 to 2 0mg / kg, 1~15mg / kg, 2~50mg / kg, 2~30mg / kg, 2~2 0 mg / kg or 2 to 15 mg / kg once every 1 to 4 weeks, preferably once every 2 to 3 weeks It may be administered multiple times at intervals of 10 min. [Example]

[0220] The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto. Furthermore, these should not be interpreted in any restrictive manner. In the following examples, unless otherwise specified, each operation related to genetic manipulation is performed in accordance with the "Moreki" Molecular Cloning" (Sambrook, J ., Fritsch, E.F. and Maniatis, T., Cold Spring (Harbor Laboratory Press, 1989) and other methods used by those skilled in the art, or using commercially available reagents and kits. When using a commercially available product, the procedure was carried out according to the instructions of the product. Free reagents, solvents and starting materials 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 encoding human CDH6 protein (NP_004923) Mammalian genes were prepared using a recombinant human genomic DNA (OriGene, RC217889) according to methods known to those skilled in the art. By incorporating it into the current vector, the human CDH6 expression vector pcDNA3.1-hC DH6 was constructed. Human CDH6 ORF (Open Reading Frame) The amino acid sequence is shown in SEQ ID NO:1.

[0222] cDNA expression vector encoding mouse CDH6 protein (NP_031692) (OriGene, MC221619) according to methods known to those skilled in the art. By incorporating it into the expression vector, mouse CDH6 expression vector pcDNA3.1- The mouse CDH6 and p3xFLAG-CMV-9-mCDH6 were constructed. The amino acid sequence of the 6 ORF is shown in SEQ ID NO:7.

[0223] cDNA expression vector encoding rat CDH6 protein (NP_037059) (OriGene, RN211850) were used to generate the cDNA fragments, which were then cloned using methods known to those skilled in the art. The human CDH6 expression vector pc was synthesized by incorporating it into a mammalian expression vector according to the method. DNA3.1-rCDH6 and p3xFLAG-CMV-9-rCDH6 were generated. The amino acid sequence of rat CDH6 ORF is shown in SEQ ID NO:8.

[0224] The cDNA encoding the cynomolgus monkey CDH6 protein was cloned from the cynomolgus monkey kidney tot cDNA synthesized from the RNA was used as a template for primer 1 (5'-CACCATG AGAACTTACCGCTACTTCTTGCTGCTC-3' (SEQ ID NO: 85) and and primer 2 (5'-TTAGGAGTCTTTGTCACTGTCCACTC CTCC-3' (SEQ ID NO: 86). The extracellular domain of CDH6 from Macaca chinensis (NCBI, XP_005556691.1) is identical to that of the CDH6 from Macaca chinensis (NCBI, XP_005556691.1). In addition, we confirmed that the cynomolgus monkey CDH6 (EHH5418) registered at EMBL 0.1) and confirmed that the full-length sequence was identical. Cynomolgus CDH6 expression vector pcDNA3.1 The amino acid sequence of the cynomolgus monkey CDH6 ORF was determined. Shown in number 9.

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

[0226] 1)-2 Immunity Female WKY / Izm rats (Japan SLC) were used for immunization. After pre-treating the lower leg with Hyaluronidase (SIGMA-ALDRICH) The human CDH6 expression vector pcDNA3.1-h prepared in Example 1)-1 was inserted into the same site. CDH6 was injected intramuscularly. Subsequently, a two-needle electroporation was performed using ECM830 (BTX). In vivo electroporation was performed at the same site using a 1000-kJ / cm2 electrode. After repeated in vivo electroporation, lymph nodes or spleens were harvested from the rats. The isolated cells were used to prepare hybridomas.

[0227] 1)-3 Hybridoma production Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells (ATC C, No.CRL-1 581) and LF301 Cell Fusion Unit After electrofusion using ClonaCell-HY Selective Immunoprecipitation (BEX), on Medium D (StemCell Technologies), The diluted cells were cultured at 37°C in 5% CO2. The clones were recovered as monoclones and subjected to ClonaCell-HY Selection. Suspend in Medium E (StemCell Technologies) for 37 min. After the cells had grown sufficiently, each hybridoma was isolated. Frozen cell stocks were prepared, and the resulting hybridoma culture supernatant was used for anti-human CDH antibody testing. 6 were used to screen 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 (HEK293-derived cells expressing integrin αv and integrin β3) Stable cell line) was cultured at 5x10 in DMEM medium containing 10% FBS. 5 cells / mL Lipofectamine 2000 (Thermo Fisher Scientific) was prepared. The 293α cells were transfected with p cDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 or negative As a control, pcDNA3.1 DNA was introduced into a 96-well plate ( After dispensing 100 μL into each tube (Corning), the tube was incubated for 37 min in DMEM medium containing 10% FBS. The cells were cultured at ℃ and 5% CO2 for 24 to 27 hours. The resulting transfected cells were in an adherent state. The cells were used in Cell-ELISA as they were.

[0229] 1)-4-2 Cell-ELISA After removing the culture supernatant of the 293α cells transfected with the expression vector prepared in Example 1)-4-1, p cDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 or pcD Hybridoma culture supernatant was added to each of the NA3.1-transfected 293α cells, and the cells were incubated at 4°C for 1 hour. The cells in the wells were washed once with 5% FBS-containing PBS(+), and then added to 5% FBS-containing PBS(+). Anti-Rat IgG-Peroxide diluted 500-fold with PBS(+) containing BS The enzyme antibody produced in rabbit (SIGMA) The cells in the wells were then incubated for 3 hours at 4°C with 5% FBS-containing PBS(+). After washing twice, OPD coloring solution (OPD dissolution solution (0.05 M trisodium citrate, 0 1M disodium hydrogen phosphate 12H2O (pH 4.5) in o-phenylenediamine disodium salt Acid salt (Wako Pure Chemical Industries, Ltd.), and H2O2 were added at 0.4 mg / mL and 0.6% (v / v), respectively. The color reaction was carried out with occasional stirring, and 1 The color reaction was stopped by adding 100 μL / well of 10 M HCl, and then the plate was read using a plate reader. The absorbance at 490 nm was measured using an ENVISION (PerkinElmer). Compared with control pcDNA3.1-transfected 293α cells, pcDNA3.1-hCD H6 and pcDNA3.1-cynoCDH6 expression vector-transfected 293α cells Hybridomas producing culture supernatants with higher absorbance were selected from human and cynomolgus monkey C A hybridoma was selected as one that produced an antibody that binds to DH6.

[0230] 1)-5 Flow cytometry of selectively binding antibodies to cynomolgus monkey CDH6 cleaning 1)-5-1 Preparation of antigen gene-expressing cells for flow cytometry analysis 5 × 10 293T cells 4 cells / cm 2 225cm 2 Flask (Sumitomo Bakelite) The cells were seeded on a 10% FBS-containing DMEM medium at 37°C and 5% CO2. The 293T cells were cultured overnight under the same conditions. As a negative control, pcDNA3.1 was transfected with Lipofectamine 2000. The vectors were then introduced into the cells, and the cells were further cultured overnight at 37°C and 5% CO2. 293T cells were incubated with TrypLE Express (Thermo Fisher Scientific) After treatment with 5% FBS (Enftific), the cells were washed with DMEM containing 10% FBS. The cells were suspended in PBS containing FBS. The resulting cell suspension was used for flow cytometry analysis. did.

[0231] 1)-5-2 Flow cytometry analysis Human and cynomolgus monkey CDH selected by Cell-ELISA in Example 1)-4 Binding of antibodies produced by hybridomas producing antibodies that bind to CDH6 in cynomolgus monkeys The binding specificity was further confirmed by flow cytometry. The suspension of transiently expressing 293T cells was centrifuged, the supernatant was removed, and then the hybrid The cells were suspended in the supernatant of the IgG culture medium and left to stand at 4°C for 1 hour. The cells were then washed twice with PBS containing 5% FBS. After washing, the cells were diluted 500-fold with PBS containing 5% FBS. ITC conjugate (SIGMA) was added and suspended, and the mixture was allowed to stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, 2 μg / mL 7-aminoactin PBS containing 5% FBS containing mycin D (Molecular Probes) and analyzed by flow cytometry (FC500: Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). After excluding actinomycin D-positive dead cells by gating, FITC fluorescence of live cells was measured. A histogram of the intensity was created. Fluorescence intensity histogram of pcDNA3.1-cynoCDH6-transfected 293T cells Hybridomas that produce antibodies with a histogram shifted to the strong fluorescence intensity side were selected from the cell membrane surface. As a result, a hybridoma producing an antibody that specifically binds to cynomolgus monkey CDH6 expressed on the surface of the Selected.

[0232] 1)-6 Determination of the isotype of rat monoclonal antibodies Among the rat anti-CDH6 antibody-producing hybridomas selected in Example 1)-5, human and clones rG019 and rG020, which were shown to bind strongly and specifically to monkey CDH6. G055, rG056, and rG061 were selected, and the isotype of each antibody was identified. Heavy chain subclass and light chain type are RAT MONOCLONAL ANTIBODY Determined by ISOTYPING TEST KIT (DS Pharma Biomedical Co., Ltd.) As a result, four clones, rG019, rG055, rG056, and rG061, were identified. The subclass was confirmed to be IgG2b and the type was kappa chain.

[0233] 1)-7 Preparation of rat anti-CDH6 antibody 1)-7-1 Preparation of culture supernatant The rat anti-human CDH6 monoclonal antibody was purified from the hybridoma culture supernatant. First, rat anti-CDH6 monoclonal antibody-producing hybridomas were isolated from ClonaCell- HY Selection Medium E(StemCell Technolog After growing the cells in sufficient amounts in Ultra Low IgG FBS (Ther Hybridoma (Fisher Scientific) was added at 20%. The medium was replaced with SFM (Thermo Fisher Scientific), and The culture supernatant was collected and passed through a 0.8 μm filter. Insoluble matter was removed by passing through a 0.2 μm filter.

[0234] 1)-7-2 Purification of rat anti-CDH6 antibody The antibody (rat anti-CDH6) was isolated from the culture supernatant of the hybridoma prepared in Example 1)-7-1. Antibodies (rG019, rG055, rG056, rG061) were incubated with Protein G The product was purified by affinity chromatography on a Protein G column (GE Heal) The antibody was adsorbed onto a column (Thcare Biosciences), and the column was washed with PBS. Elution was performed with 0.1M glycine / hydrochloric acid aqueous solution (pH 2.7). After adding HCl (pH 9.0) to adjust the pH to 7.0-7.5, al UF Filter Device VIVASPIN20 (molecular weight cutoff UF30 K, Sartorius) in HBS (25 mM histidine / 5% sorbitol) The antibody was concentrated at a concentration of 1 mM. Finally, the concentration was adjusted to 100 mg / mL. The mixture was filtered using a filter (IUS) to obtain a purified sample.

[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 binding of the rat anti-CDH6 antibody prepared in Example 1)-7 to human CDH6 was confirmed by flow cytometry. The pcDNA3.1-hCDH6 prepared in Example 1)-1 was evaluated by the ELISA method. 293T cells (ATCC) were transfected with Lipofectamine 2000 (Thermo F Transiently transfected cells were incubated at 37°C, 5% CO2 using a PBS containing 1000 ng / ml of ethanol. After overnight culture under the conditions, a cell suspension was prepared. After centrifugation and removal of the supernatant, the rat anti-CDH6 monoclonal antibody prepared in Example 1)-7 was added. Four antibodies (clone numbers: rG019, rG055, rG056, and rG061) or Rat IgG control (R&D Systems) was added to a final concentration of 10 ng / mL. The mixture was resuspended in PBS and left to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, Anti-Rat IgG (whole molecule) diluted 50-fold with PBS containing e)-FITC antibody produced in rabbit(SIGM A) was added to the suspension and allowed to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, Detection was performed using a flow cytometer (FC500: Beckman Coulter). Data analysis was performed using FlowJo (TreeStar). The results are shown in Figure 1. In the histogram of Figure 1, the horizontal axis is the FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis is the number of cells. The shaded histogram shows the negative control 2 cells that were not transfected with hCDH6. The histogram shown with a solid white line indicates the results of hCDH6-transfected 29T cells. This shows the case of using 3T cells. Fluorescence was generated by antibody binding to hCDH6 on the cell surface. The rat IgG control does not bind to any of the cells. As a result, the four rat anti-CDH6 monoclonal antibodies produced were identified as pcDNA3.1-hC It was confirmed that it binds to DH6-transfected 293T cells.

[0236] 2)-2 Analysis of the CDH6 binding site of rat anti-CDH6 antibodies by flow cytometry analysis 2)-2-1 Construction of human CDH6 domain deletion expression vectors The full-length extracellular domain 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), EC5 (SEQ ID NO: 6) There are genes in which one of the five EC domains is deleted from the full-length human CDH6. The gene was synthesized by GeneArt and expressed in the mammalian expression vector p The vector was inserted into 3xFLAG-CMV-9 (Sigma-Aldrich) and then subjected to EC Expression vectors for each domain deletion were constructed by deleting EC1 to EC5, respectively.

[0237] 2)-2-2 Flow cytometry of rat anti-CDH6 antibody using domain-deleted antibodies Antibody epitope analysis Flow cytometry using 293α cell lines transfected with each EC domain deletion vector Analysis identified the binding epitope of rat anti-human CDH6 antibody. and cell line 2, in which an integrin β3 expression vector was stably transfected into HEK293 cells. The 93α cell line was treated with the expression vectors for each domain deletion vector prepared in Example 2)-2-1, and pcDNA3.1-hCDH6 expressing full-length human CDH6 were transfected with Lipofectamin. Transient analysis was performed using ine 2000 (Thermo Fisher Scientific) The cells were transfected into the cells and cultured overnight at 37°C in 5% CO2, after which a cell suspension was prepared. The transfected 293α cell suspension was centrifuged, the supernatant was removed, and the rat Four anti-CDH6 monoclonal antibodies (clone numbers: rG019, rG055, and rG0 56 and rG061) or rat IgG control (R&D Systems) at a final concentration The mixture was added at a concentration of 20 nM, suspended, and left to stand at 4°C for 1 hour. Washed twice with PBS containing 5% FBS. Afterwards, the cells were incubated with anti-rat IgG (who le molecule)-FITC antibody produced in r The mixture was suspended in 5% FBS-containing P After washing twice with BS, the cells were analyzed by a flow cytometer (Canto II: BD Biosciences) Detection was performed using FlowJo (TreeStar). The results are shown in Figures 2-1 to 2-6. In the histograms of Figures 2-1 to 2-6, the horizontal axis The vertical axis indicates the FITC fluorescence intensity, which represents the amount of antibody binding, and the vertical axis indicates the number of cells. The white circle indicates the results when negative control 293α cells without gene transfection were used. The histograms shown with solid lines were obtained using full-length hCDH6 or 293 cells lacking each EC domain. The figure shows the cases where the antibody bound to full-length hCDH6 or each EC domain deletion on the cell surface. The rat IgG control does not bind to any of the transfected cells. The four rat anti-CDH6 monoclonal antibodies we generated were: full-length hCDH6, EC1-deleted hCDH6, and It binds to the EC2 deletion mutant, EC4 deletion mutant, and EC5 deletion mutant, but not to the EC3 deletion mutant. These results suggest that the four rat anti-CDH6 monoclonal antibodies inhibit the EC of hCDH6. It was shown to specifically bind to epitope 3.

[0238] 2)-3 Internalization activity of rat anti-CDH6 antibody 2)-3-1 Confirmation of CDH6 expression in human tumor cell lines In order to select CDH6-positive human tumor cell lines to be used for evaluating the obtained antibodies, a public database was used. CDH6 expression information was searched for from the data, and CDH6 expression on the cell membrane surface was detected by flow cytometry. The expression of 6 was evaluated in human ovarian tumor cell lines NIH: OVCAR-3, PA-1, and ES-2. and human renal cell carcinoma cell line 786-O (all obtained from ATCC) at 37°C, 5% CO After culturing under the conditions, a cell suspension was prepared. The cells were centrifuged, the supernatant was removed, and the resulting solution was collected using a commercially available Anti-human CDH6 antibody (MABU2715, R&D Systems) or negative control Mouse IgG1 (BD Pharmingen) was added as a control to a final concentration of 50 μg / mL. The cells were suspended in PBS and left to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, F(ab')2 Fragment of FITC- conjugated Goat Anti-mouse immunoglobuli The cells were suspended in PBS containing 5% FBS and allowed to stand at 4°C for 1 hour. After washing twice, the cells were analyzed by a flow cytometer (Canto II: BD Biosciences). The detection was performed using FlowJo (TreeStar). Data analysis was performed using FlowJo (TreeStar). This is shown in Figure 3. In the histogram of Figure 3, the horizontal axis is the FITC fluorescence intensity, which represents the amount of antibody binding. The vertical axis indicates the number of cells. The shaded histograms are stained with the negative control mIgG1. The histogram shown with a solid white line represents staining with anti-human CDH6 antibody. The figure shows that the fluorescence intensity increased when the antibody bound to hCDH6 on the cell surface. The mIgG1 control did not bind to either cell. AR-3, PA-1, and 786-O cell lines endogenously express CDH6 on the cell surface. On the other hand, the ES-2 cell line was shown to 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 antibody binds to a toxin (saporin) that inhibits protein synthesis. Combined anti-rat IgG reagent Rat-ZAP (ADVANCED TARGETING The results were evaluated using the human CDH6-positive ovarian tumor cell line NIH :OVCAR-3 (ATCC) 4x10 3 cells / well in a 96-well plate The cells were seeded onto a plate and cultured overnight at 37°C in 5% CO2. Tumor cell line 786-O (ATCC) was 1x10 3 96 wells with cells / well The cells were seeded onto a plate and cultured overnight. The next day, rat anti-CDH6 antibody (final concentration: 1 nM) or , and rat IgG2b antibody (R&D Systems) was added as a negative control antibody. In addition, Rat-ZAP (final concentration: 0.5 nM) or, as a negative control, Toxin-free Goat Anti-Rat IgG, Fc(gamma) F ragment Specific(JACKSON IMMUNORESEARCH) (final concentration: 0.5 nM) was added, and the cells were cultured for 3 days at 37°C under 5% CO2 conditions. Cell numbers were determined using CellTiter-Glo TM Luminescent Cell Via ATP activity (RLU) was measured using the Ability Assay (Promega). In this evaluation, Rat-ZAP was found to be dependent on the internalization activity of rat anti-CDH6 antibody. It is taken up into the vesicles and releases saporin, which inhibits protein synthesis, into the cells. The cell proliferation suppression effect of the addition of anti-CDH6 antibody was observed in place of Rat-ZAP. The number of viable cells in the wells containing the negative control was set at 100%. Figure 4 shows a graph and a table of cell viability. It was shown to bind to CDH6 and cause its internalization.

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

[0241] 3)-1-2 cDNA containing the heavy chain variable region of rG019 by 5'-RACE PCR Amplification of A and determination of nucleotide sequence The cDNA containing the heavy chain variable region was amplified using the total R Approximately 1 μg of NA and SMARTer RACE cDNA Amplification The PCR was performed using a PCR kit (Clontech). UPM (Universal Primer) was used as a primer for amplifying cDNA by PCR. Primer A Mix:SMARTer RACE cDNA Amplifica (included in the PCR Kit) and a primer designed from the known sequence of the constant region of the rat heavy chain. We used mer.

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

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

[0244] 3)-1-3 cDNA containing the light chain variable region of rG019 by 5'-RACE PCR Amplification of A and determination of nucleotide sequence The procedure was carried out in the same manner as in Example 3)-1-2, except that the variable region of the light chain gene of rG019 was The primers used to amplify the cDNA of the region by PCR were UPM (Univers al Primer A Mix:SMARTer RACE cDNA Amplif The IgG1A1 gene was designed based on the known rat light chain constant region sequence. Primers were used.

[0245] The nucleotide sequence of the cDNA encoding the variable region of the light chain of rG019 was determined. The sequence is shown in column number 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 fixed The sequence was determined in the same manner as in Example 3)-1.

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

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

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

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

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

[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 Restriction of the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) A fragment of approximately 5.4 kb obtained by digestion with the enzymes XbaI and PmeI and the sequence of SEQ ID NO: D containing a DNA sequence encoding the human light chain signal sequence and the human κ chain constant region shown in 50 The NA fragment was cloned using the In-Fusion Advantage PCR Cloning Kit (Cl The DNA was ligated using a DNA polymerase (Ontech) to prepare pcDNA3.3 / LK.

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

[0254] 4)-1-2 Construction of chimeric and humanized IgG1-type heavy chain expression vector pCMA-G1 Construction pCMA-LK was digested with XbaI and PmeI to extract the light chain signal sequence and the human kappa chain constant sequence. The DNA fragment from which the region was removed, the human heavy chain signal sequence shown in SEQ ID NO: 51, and the human A DNA fragment containing the DNA sequence encoding the IgG1 constant region was added to the In-Fusion Ad The fragments were ligated using the vantage PCR cloning kit (Clontech). pCMA-G1 was constructed.

[0255] 4)-1-3 Construction of chG019 heavy chain expression vector Nucleotide numbers 36 to 57 of the nucleotide sequence of the chG019 heavy chain are shown in SEQ ID NO:57. The DNA fragment shown in 440 was synthesized (GENEART). D PCR cloning kit (Clontech) was used to restrict pCMA-G1. The chG019 duplication was created by inserting a synthesized DNA fragment into the site cleaved with the enzyme BlpI. The chG019 heavy chain expression vector was constructed. Note that the chG019 heavy chain contains a disulfide bond to prevent unexpected disulfide bonds. Therefore, a sequence in which cysteine ​​in the CDR was substituted with proline was used.

[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) In-Fusion HD PCR Cloning Kit (C The synthesized DNA fragment and pCMA-LK were ligated with XbaI and Pm The DNA fragments were digested with eI to remove the light chain signal sequence and the human κ chain constant region, and then ligated. The chG019 light chain expression vector was constructed by the above procedure.

[0257] 4)-2 Production and purification of human chimeric anti-CDH6 antibody chG019 4)-2-1 Production of chG019 FreeStyle 293F cells (Invitrogen) were cultured according to the manufacturer's instructions. Subculture and culture were performed. 1.2 × 10 9 FreeStyle 293 F cells (Invitrogen) were cultured in 3L Fernbach Erlenmeyer Flask (CORNING) was seeded with FreeStyle293 express Dilute with ion medium (Invitrogen) to 2.0 × 10 6 cells / ml 40 ml of Opti-Pro SFM medium (Invitrogen) was added to 0 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector, and 1.8 mg of Pol Add ethyleneimine (Polyscience #24765) and gently The mixture was stirred thoroughly and left for a further 5 minutes before being added to the FreeStyle 293F cells. After 4 hours of shaking culture in an incubator at 37℃ and 8% CO2 at 90 rpm, 600 ml EX-CELL VPRO medium (SAFC Biosciences), 18 ml GlutaMAX I (GIBCO) and 30 ml of Yeastolate Ult Add rafiltrate (GIBCO) and incubate at 37°C in an 8% CO2 incubator. The culture supernatant was then transferred to Disposable Ca Filter with a psule filter (Advantec #CCS-045-E1H). Ta.

[0258] 4)-2-2 Purification of chG019 The culture supernatant obtained in Example 4)-2-1 was subjected to rProtein A affinity chromatography. The culture supernatant was purified by a single step of MabSelect chromatography. tSuRe packed column (GE Healthcare Bioscience) After applying the solution to a column (product of the manufacturer), the column was washed with PBS in an amount equal to or greater than twice the column volume. The antibody was eluted with arginine hydrochloride solution (pH 4.0) and the fractions containing the antibody were collected. The sample was dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis) lysis cassette) in HBS (25 mM histidine / 5% sodium chloride solution). The buffer was replaced with Centrifugal UF (Centrifugal UF, pH 6.0). Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sar The antibody was concentrated using a centrifuge (Morius) to adjust the IgG concentration to 5 mg / ml or more. The purified san was filtered through an inisart-Plus filter (Sartorius). It was a pull.

[0259] 4)-3 Binding evaluation of the human chimeric anti-CDH6 antibody chG019 4)-2 The CDH6 binding activity of the purified human chimeric anti-CDH6 antibody chG019 was investigated using flow cytometry. The pcDNA3.1-hC prepared in Example 1)-1 was confirmed by the cytometry method. DH6, or pcDNA3.1-cynoCDH6, or pcDNA3.1, respectively. 93α cells were transiently transfected with Lipofectamine 2000 and incubated at 37°C. After overnight culture under 5% CO2 conditions, cell suspensions were prepared. After adding chG019 and leaving it at 4°C for 1 hour, the plate was washed twice with PBS containing 5% FBS. PE-labeled F(ab')2 Fragment diluted 500-fold in 5% FBS-containing PBS Anti-human IgG, Fcγ antibody (JACKSON IMMUNORESEARCH) was added. The cells were washed twice with 5% FBS-containing PBS and then resuspended in 5% FBS-containing PBS. The cells were resuspended in PBS containing BS and analyzed by a flow cytometer (Canto II, BD Biosciences). Detection was performed using FlowJo (TreeStar). As shown in Figure 5, chG019 was transfected with pcDNA3.1, a negative control. It did not bind to 3T cells, and pcDNA3.1-hCDH6 and pcDNA3.1-cyno The antibody bound to CDH6-transfected 293T cells in a concentration-dependent manner. The vertical axis represents the amount of binding as mean fluorescent intensity (Mean Fluorescent Intensity). These results show that chG019 has a potent inhibitory effect on human and cynomolgus monkey CDH6. It can be seen that the two antibodies bind specifically to each other and the binding activity is almost the same.

[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 )) was used. It has high sequence identity to the heavy and light chain variable regions of chG019. Protein Data Bank(Nuc.Acid Res.35,D301-D 303(2007)) (PDB ID: 2I9L) was used as a template. BioLuminate (Schrodinger) protein structure analysis program The test was performed using a fluororesin (manufactured by the company).

[0261] 5)-1-2 Design of the amino acid sequence for humanized hG019 chG019 was synthesized using CDR grafting (Proc. Natl. Acad. Sci. U SA 86, 10029-10033 (1989)). t al.(Sequences of Proteins of Immunolog ical Interest,5th Ed.Public Health Servi ce National Institutes of Health,Bethesd a, MD. (1991)) and human gamma chain subgroup 1 The consensus sequence for kappa chain subgroup 1 is in the framework region of chG019. These were selected as acceptors for the heavy and light chains, respectively, because they have high identity to The donor residues to be transferred onto the acceptor were selected according to the method described by Queen et al. (Pro c.Natl.Acad.Sci.USA 86,10029-10033(1989) ) and were selected by analyzing the three-dimensional models with reference to the criteria given by

[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 is set forth in SEQ ID NO: 69. The nucleotide sequence of hH02 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: 71. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 is set forth in SEQ ID NO: 73. 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. do.

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

[0264] 5)-4 Design of humanized hG019 by combining heavy and light chains The antibody consisting of hH01 and hL02 is referred to as "H01L02 antibody" or "H01L02." The antibody consisting of hH02 and hL02 is referred to as "H02L02 antibody" or "H02L02." An antibody consisting of hH02 and hL03 is referred to as "H02L03 antibody" or "H02L0 The antibody consisting of hH04 and hL02 is referred to as "H04L02 antibody" or "H04 It is called "L02".

[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 The nucleotide sequence of the humanized hG019-H01 type heavy chain shown in SEQ ID NO: 70 The DNA fragments shown in nucleotide numbers 36 to 440 were synthesized (GENEART). Example 4) Construct a humanized hG019-H01 type heavy chain expression vector in the same manner as in -1-3 did.

[0266] 5)-5-1-2 Construction of humanized hG019-H02 type heavy chain expression vector The nucleotide sequence of the humanized hG019-H02 type heavy chain shown in SEQ ID NO: 74 The DNA fragments shown in nucleotide numbers 36 to 440 were synthesized (GENEART). Example 4) Construct a humanized hG019-H02 type heavy chain expression vector in the same manner as in -1-3 did.

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

[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 The nucleotide sequence of the humanized hG019-L02 type light chain shown in SEQ ID NO: 62 The variable region of the humanized hG019-L02 type light chain shown in octets 37 to 399 A DNA fragment containing the coding sequence was synthesized (GENEART). pCMA- LK was digested with the restriction enzyme BsiWI and a synthetic DNA fragment was inserted into it. A modified hG019-L02 type light chain expression vector was constructed.

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

[0270] 5)-5-3 Preparation of humanized hG019 5)-5-3-1 Production of H01L02, H02L02, H02L03, and H04L02 It was produced in the same manner as in Example 4)-2-1. By combining them, H01L02, H02L02, H02L03, and H04L02 were produced. .

[0271] 5)-5-3-2 H01L02, H02L02, H02L03, H04L02 2s tep purification The culture supernatant obtained in Example 5)-5-3-1 was subjected to rProtein A affinity chromatography. The cells were purified in two steps using chromatography and ceramic hydroxyapatite. The supernatant was transferred to a column (GE Healthcare) packed with MabSelectSuRe equilibrated with PBS. After applying the solution to a column (manufactured by Litecare Bioscience), the volume of the column was increased by more than two times. The column was washed with PBS. Then, the antibody was dissolved in 2 M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was dialyzed (Thermo Scientific, Sli Buffer solution to PBS using a de-A-Lyzer Dialysis Cassette After the pH change, the buffer was changed to 5 mM sodium phosphate / 50 mM MES / pH 7.0. After diluting 5 times with 5mM NaPi / 50mM MES / 30mM NaCl / pH A ceramic hydroxyapatite column (Japan Bio) equilibrated with buffer solution 7.0 was used. Rad, Bio-Scale CHT Type-1 Hydroxyapatite The solution was applied to a column. A linear gradient elution with sodium chloride was performed to obtain the antibody. The fractions containing the lysosomes were collected and dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) The buffer was replaced with 25mM histidine / 5% sorbitol, pH 6.0. Ta. Centrifugal UF Filter Device VIVASPIN2 The antibody was concentrated with 0 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 2 Finally, the solution was filtered through a Minisart-Plus filter (Sart The mixture was filtered through a filter (Orius) to obtain a purified sample.

[0272] [Reference Example 1: Production of anti-CDH6 antibody NOV0712] The anti-CDH6 antibody NOV0712 used in the examples was The full-length amino acid sequences of the light chain and heavy chain of NOV0712 are listed in the International Publication No. It was prepared with reference to SEQ ID NO: 235 and SEQ ID NO: 234 of Patent Publication No. 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 Nucleotide number 36 of the nucleotide sequence of the heavy chain of NOV0712 set forth in SEQ ID NO:84 A DNA fragment containing the heavy chain variable region of NOV0712 shown in nucleotides 1 to 428 was synthesized (G (ENEART Co., Ltd.) The heavy chain expression vector of NOV0712 was prepared in the same manner as in Example 4)-1-3. The heavy chain of NOV0712 expressed by the NOV0712 heavy chain expression vector was constructed. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 83. In the amino acid sequence shown in 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 Nucleotide number 37 of the nucleotide sequence of the light chain of NOV0712 set forth in SEQ ID NO:82 D containing the DNA sequence encoding the light chain variable region of NOV0712 shown in The NA fragment was synthesized (GENEART). A light chain expression vector for NOV0712 was constructed. The amino acid sequence of the light chain of NOV0712 expressed by the IgG1 vector is shown in SEQ ID NO: 81. In the amino acid sequence shown in Figure 1, the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal It is a rule array.

[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 Anti-CD40 antibody was isolated from the culture supernatant obtained in Reference Example 1)-2-1 in the same manner as in Example 4)-2-2. The H6 antibody NOV0712 was purified (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 Antibody and antigen (Recombinant Human CDH6 Fc His chim era, R&D Systems) and dissociation constant measurements were performed using Biacore T200 ( GE Healthcare Biosciences) to ligate the antigen to the immobilized anti-His antibody. The capture method is performed by capturing the antibody as an analyte and measuring it with the antibody. Anti-histidine antibody (His capture kit, GE Healthcare Biosciences) was used. ence) to the sensor chip CM5 (GE Healthcare Biosciences) and Approximately 1000RU was covalently bound using the pulling method. The reference cell was also immobilized in the same way. The running buffer was HBS-P+ (10 mM) supplemented with 1 mM CaCl. HEPES pH7.4, 0.15M NaCl, 0.05% Surfactant The antigen was added to the chip on which the anti-histidine antibody was immobilized for 60 seconds. Then, a dilution series of antibody solutions (0.391-100nM) was added at a flow rate of 30μl / min for 300 seconds. The dissociation phase was monitored for 600 seconds. Glycine solution pH 1.5 containing 10 μl of glycine was added twice for 30 seconds at a flow rate of 10 μl / min. Data analysis was performed using analysis software (BIAevaluation software , version 4.1) Steady State Affinity model The dissociation constant (KD) was calculated using the above formula. The results are shown in Table 2.

[0278] [Table 2]

[0279] 6)-1-2 Binding to human, monkey, mouse, and rat CDH6 pcDNA3.1-hCDH6 and pcDNA3.1-cyn prepared in Example 1)-1 oCDH6, p3xFLAG-CMV-9-mCDH6, p3xFLAG-CMV-9- rCDH6 was transiently transfected into 293α cells using Lipofectamine 2000. After overnight culture at 37°C and 5% CO2, a cell suspension was prepared. As a control, non-transfected 293α cells were used. The 93α cell suspension was centrifuged, the supernatant was removed, and the humanized h93α cell suspension prepared in Example 5)-5-3 was added. Four G019 antibodies (clone numbers: H01L02, H02L02, H02L03, and H04L02), or human IgG1 control (Calbiochem) was added. The mixture was resuspended in PBS and left to stand at 4°C for 1 hour. After washing twice with PBS containing 5% FBS, Anti-human Fcg PE goat F( ab') (Jackson Laboratory) was added and suspended, and the mixture was left to stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were analyzed by a flow cytometer (Canto II). Detection was performed using FlowJo (BD Biosciences). Data analysis was performed using FlowJo (Tre In Figures 6-1 and 6-2, the horizontal axis represents the antibody concentration, and the vertical axis represents the result. The total amount is expressed as mean fluorescent intensity. As shown in Figures 6-1 and 6-2, the negative control, human IgG1 control The four humanized hG019 antibodies did not bind to any of the CDH6 gene-transfected cells. (Clone numbers are H01L02, H02L02, H02L03 and H04L02) Binds to human and cynomolgus monkey CDH6, but not to mouse or rat CDH6 Neither antibody bound to the cells transfected with the empty vector pcDNA3.1, which was used as a negative control. On the other hand, the NOV0712 antibody binds to human, cynomolgus monkey, mouse, and rat CDH WO 2016 / 024195 has shown that it exhibits binding activity to all six of these proteins. As a result, the four humanized hG019 antibodies obtained herein are different from the NOV0712 antibody. It was shown that the anti-CDH6 antibody exhibited binding properties.

[0280] 6)-2 Analysis of the CDH6 binding sites of humanized hG019 and NOV0712 6)-2-1 Epitope analysis using domain-deleted antibodies The expression vectors for each domain deletion vector prepared in Example 2)-2-1 and the full-length hCDH6 were The expressing pcDNA3.1-hCDH6 was transfected with Lipofectamine 2000 (Th Transient transfection was performed using a chemiluminescent microscope (Fisher Scientific) and incubated at 37°C for 5 min. After overnight culture under 5% CO2, a cell suspension was prepared. The cell suspension was centrifuged, the supernatant was removed, and the humanized hG01 prepared in Example 5)-5-3 was added. 9 Four antibodies (clone numbers: H01L02, H02L02, H02L03, and H04 L02), or the anti-CDH6 antibody NOV0712 prepared in Reference Example 1, or a negative control Add human IgG1 (Calbiochem) as a suspension and let stand at 4°C for 1 hour. After washing twice with 5% FBS-containing PBS, the cells were diluted 500 times with 5% FBS-containing PBS. Diluted APC-anti-human IgG goat F(ab')2 (Jack The mixture was suspended in 5% FBS (son laboratory) and left to stand at 4°C for 1 hour. After washing twice with PBS containing 100 μL of PBS, the cells were analyzed by a flow cytometer (Canto II: BD Biosciences). Detection was performed using FlowJo (TreeStar). The results are shown in Figures 7-1 to 7-6. The horizontal axis shows the fluorescence intensity of APC, which indicates the amount of antibody binding, and the vertical axis shows the number of cells. The graph shows the results when non-transfected negative control 293α cells were used, and the white The histograms shown with solid lines are for full-length hCDH6 or EC domain-deleted 293α cells. The antibody binds to the full-length hCDH6 or each EC domain deletion on the cell surface. The human IgG1 control binds to both transfected cells. No. Four humanized hG019 antibodies (clone numbers: H01L02, H02L02, H H02L03 and H04L02) are full-length hCDH6, EC1 deletion, EC2 deletion, E It binds to the C4 deletion and EC5 deletion, but not to the EC3 deletion. Four humanized hG019 antibodies specifically bind to the EC3 epitope of hCDH6 On the other hand, the anti-CDH6 antibody NOV0712 inhibited full-length hCDH6 and EC1-deleted hCDH6. It binds to the EC2 deletion mutant, EC3 deletion mutant, and EC4 deletion mutant, but not to the EC5 deletion mutant. In other words, the anti-CDH6 antibody NOV0712 antibody does not bind to the epitope. It has been shown to specifically bind to the nucleotide sequence of the nucleotide sequence described in WO 2016 / 024195. This result is consistent with the epitope information of NOV0712 published in the journal. 2 and the four humanized hG019 antibodies obtained herein are anti-CDH6 antibodies exhibiting different properties. It was shown that...

[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 developed by inducing human CDH6 expression in 786-O cells (ATCC). 6 was produced by infecting the recombinant retrovirus for full-length expression of human CDH6. The retroviral vector (pQCXIN-hCDH6) expresses the human CDH6 protein (N P_004923) was encoded by a cDNA expression vector (OriGene RC2178 89) to generate the retroviral vector pQCXIN(CL) according to methods known to those skilled in the art. The gene was generated by incorporating it into FuGene HD (Promega ONTECH). ) using retroviral packaging cells RetroPack PT67 (CLON pQCXIN-hCDH6 was transiently introduced into the TECH cells, and the recombinant retrovirus was detected 48 hours later. The culture supernatant containing the virus was collected and added to a 786-O cell culture system, which infected the cells. From 3 days after infection, the medium was supplemented with G418 (Gibco) at a final concentration of 50 mg / mL. The infected cells were cultured at 37°C under 5% CO2 conditions and drug selection was performed to identify human C A cell line 786-O / hCDH6 stably expressing DH6 was established. Example 2)-3- As in 1, high expression of human CDH6 in stable expression cells was confirmed by flow cytometry ( (Figure 8) The detection antibody was Goat ant, diluted 500-fold in 5% FBS-containing PBS. i-Mouse IgG1 Secondary Antibody Alexa Fl A Thermo Fisher Scientific 647 was used. The results are shown in Figure 8. In the histogram of Figure 8, the horizontal axis is Alexa, which represents the amount of antibody binding. The vertical axis indicates the fluorescence intensity of Fluor 647, and the vertical axis indicates the number of cells. The histogram shown is stained with the anti-IgG1 control, mIgG1. The results are shown for staining with hCDH6 antibody. The mIgG1 control did not bind to any of the cells. As a result, the 786-O / hCDH6 stable expressing cell line showed no significant changes compared to the parent 786-O cells. It was shown that the cells express high levels of human CDH6.

[0282] 6)-2-2-2 Binding competition using labeled H01L02 and labeled NOV0712 Combined Assay Alexa Fluor 488 Monoclonal Antibody Lab Using a labeling kit (Thermo Fisher), labeled H01L02 and 7)-2-2-1 786-O / hCD was prepared. The cell suspension of the H6 stable expressing cell line was centrifuged, the supernatant was removed, and then the labeled NOV0712 Alternatively, labeled H01L02 was added to a final concentration of 5 nM, and the solution prepared in Example 5)-5-3 was further added. Four humanized hG019 antibodies (clone numbers: H01L02, H02L02, H02L03, H02L04, H02L05, H02L06, H02L07, H02L08, H02L09, H02L10, H02L11, H02L12, H02L13, H02L14, H02L15, H02L16, H 03 and H04L02), or the anti-CDH6 antibody NOV0712 prepared in Reference Example 1, Alternatively, human IgG1 (Calbiochem) was used as a negative control, and the horizontal axis of Figure 9 shows the results. The cells were added to the final concentration of 1000 mg / ml, suspended in water, and left to stand at 4°C for 1 hour. The cells were then washed twice with PBS containing 5% FBS. Afterwards, the cells were analyzed 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 shows the final concentration of unlabeled antibody when added, and the vertical axis shows the amount of binding measured as Mea nFluorescent Intensity (mean fluorescence intensity) When unlabeled NOV0712 was added to cells treated with V0712, the same Since the antibody has an epitope and competes with the unlabeled antibody, the antibody is replaced by the unlabeled antibody in a concentration-dependent manner. Instead, the amount of labeled antibody binding decreases. When four types of humanized hG019 antibodies or human IgG1 as a negative control were added to the Since there was no change in the amount of binding of the labeled antibody, these antibodies have different epitopes and bind differently. Similarly, cells to which labeled H01L02 was added were treated with labeled When four unmodified humanized hG019 antibodies were added, they had the same epitope and competed for binding. Therefore, the amount of labeled antibody bound to the unlabeled antibody increases depending on the added concentration. On the other hand, when cells were treated with labeled H01L02, NOV0712 or negative control Even if human IgG1 was added as a control, the amount of bound labeled antibody did not change. This indicates 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 determined by the toxin that inhibits protein synthesis. Hum-ZAP (ADVANCED TA) is an anti-human IgG reagent conjugated with saporin. The results were evaluated using a GETING SYSTEM. 4x10 tumor cell line NIH:OVCAR-3 (ATCC) 3 cells / well The cells were seeded onto a 96-well plate and cultured overnight at 37°C in 5% CO2. DH6-positive renal cell carcinoma cell line 786-O (ATCC) at 1x10 3 cells / wel The human CDH6-positive ovarian tumor cell line P A-1 (ATCC) 1x10 3 Seed cells / well into a 96-well plate The cells were then cultured overnight at 37°C in 5% CO2. The next day, anti-CDH6 antibody (final concentration: 1 nM), or human IgG1 antibody (Calbiochem) as a negative control antibody. Furthermore, Hum-ZAP (final concentration: 0.5 nM) or a negative control was added. Toxin-free F(ab')2 Fragment Goat Anti-h uman IgG,Fc(gamma) Fragment Specific(JAC KSON IMMUNORESEARCH (final concentration: 0.5 nM) was added and the mixture was incubated for 3 days. The cells were cultured at 7°C and 5% CO2. The number of viable cells was determined using CellTiter-Glo. TM ATP activity by Luminescent Cell Viability Assay This evaluation was performed by quantifying the RLU (reduced unit of unit time). Hum-ZAP is then taken up into the cell, and saporin, which inhibits protein synthesis, is released into the cell. The release of CDH6 inhibits cell proliferation. Cell proliferation suppression effect of adding anti-CDH6 antibody The number of viable cells in the wells to which the negative control was added instead of Hum-ZAP was calculated by dividing the number by 100. The relative survival rates were expressed as %. Figures 10-1 to 10-3 show graphs and tables of cell survival rates. In this experiment, it is believed that antibodies with strong internalization activity show low cell viability. As a result, NOV0712 showed no cell proliferation in any of the four humanized hG019 antibodies or in any of the three cell lines. The internalization rate predicted from the cell viability was approximately 50-75%, indicating very high internalization activity. , and shows even higher intrinsic activity compared to NOV0712. Therefore, antibodies with high internalization activity are considered more suitable for ADCization.

[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] Reduction of antibody: H01L02 prepared in Example 5 was subjected to the common procedure B described in Production Method 1. (280nm absorption coefficient: 1.53mLmg -1 cm -1 Using C, P The solution was adjusted to 9.85 mg / mL with BS6.0 / EDTA. 0.231 mL of TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (per antibody molecule) 6.0 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, I nc.; 0.0855 mL) was added. The pH of this solution was confirmed to be within 7.0 ± 0.1. After confirming the antibody's activity, the antibody was incubated at 37°C for 2 hours to separate the disulfides between the antibody's intrachain regions. The amide bond was reduced.

[0287] Conjugation of antibody and drug linker: Incubate the above solution at 15°C for 10 minutes. Then, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]amine 10 mM dimethyl sulfoxide of (2-oxoethoxymethyl)glycinamide Add the solution (0.386 mL; 10 equivalents per antibody molecule) and incubate at 15°C for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) ) was added and stirred at room temperature for a further 20 minutes to terminate the reaction of the drug linker.

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

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

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

[0291] [ka]

[0292] Reduction of antibody: H02L02 prepared in Example 5 was subjected to the common procedure B described in Production Method 1. (280nm absorption coefficient: 1.51mLmg -1 cm -1 Using C, P The solution was adjusted to 9.95 mg / mL with BS6.0 / EDTA. 0.234 mL of TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (per antibody molecule) 6.0 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, I nc.; 0.0855 mL) was added. The pH of this solution was confirmed to be within 7.0 ± 0.1. After confirming the antibody's activity, the antibody was incubated at 37°C for 2 hours to separate the disulfides between the antibody's intrachain regions. The amide bond was reduced.

[0293] Conjugation of antibody and drug linker: Incubate the above solution at 15°C for 10 minutes. Then, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]amine 10 mM dimethyl sulfoxide of (2-oxoethoxymethyl)glycinamide Add the solution (0.389 mL; 10 equivalents per antibody molecule) and incubate at 15°C for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0350 mL; 9 equivalents per antibody molecule) ) was added and stirred at room temperature for a further 20 minutes to terminate the reaction of the drug linker.

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

[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%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 5.9; measured by standard procedure F Average number of drugs bound per antibody molecule (n): 7.6.

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

[0297] [ka]

[0298] Reduction of antibody: H02L03 prepared in Example 5 was subjected to the common procedure B described in Production Method 1. (280nm absorption coefficient: 1.53mLmg -1 cm -1 Using C, P The solution was adjusted to 9.86 mg / mL with BS6.0 / EDTA. 0.270 mL of TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (per antibody molecule) 7.0 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, I nc.; 0.0855 mL) was added. The pH of this solution was confirmed to be within 7.0 ± 0.1. After confirming the antibody's activity, the antibody was incubated at 37°C for 2 hours to separate the disulfides between the antibody's intrachain regions. The amide bond was reduced.

[0299] Conjugation of antibody and drug linker: Incubate the above solution at 15°C for 10 minutes. Then, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]amine 10 mM dimethyl sulfoxide of (2-oxoethoxymethyl)glycinamide Add the solution (0.386 mL; 10 equivalents per antibody molecule) and incubate at 15°C for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) ) was added and stirred at room temperature for a further 20 minutes to terminate the reaction of the drug linker.

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

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

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

[0303] [ka]

[0304] Reduction of antibody: H04L02 prepared in Example 5 was subjected to the common procedure B described in Production Method 1. (280nm absorption coefficient: 1.53mLmg -1 cm -1 Using C, P The solution was adjusted to 9.86 mg / mL with BS6.0 / EDTA. 0.232 mL of TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (per antibody molecule) 6.0 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, I nc.; 0.0855 mL) was added. The pH of this solution was confirmed to be within 7.0 ± 0.1. After confirming the antibody's activity, the antibody was incubated at 37°C for 2 hours to separate the disulfides between the antibody's intrachain regions. The amide bond was reduced.

[0305] Conjugation of antibody and drug linker: Incubate the above solution at 15°C for 10 minutes. Then, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]amine 10 mM dimethyl sulfoxide of (2-oxoethoxymethyl)glycinamide Add the solution (0.386 mL; 10 equivalents per antibody molecule) and incubate at 15°C for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) ) was added and stirred at room temperature for a further 20 minutes to terminate the reaction of the drug linker.

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

[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%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 5.8; measured by standard procedure F Average number of drugs bound per antibody molecule (n): 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 Common procedure B described in Manufacturing Method 1 (280 nm extinction coefficient: 1.51 mL mg -1 cm -1 ) and C, 20 mM HEPES 8.1 (LIFE TECHNOL HEPES, 1M Buffer Solution (20 mL) manufactured by OGIES After adjusting the pH to 8.1 with 1M sodium hydroxide and adding distilled water to make 1 L, the The solution was adjusted to 1 mL and incubated at 20°C for 10 minutes. 10 mM 1-(2,5-dioxopyrrolidin-1-yloxy)-2,5-dioxopyrrolidin-1-yloxybenzoate described in Patent Publication No. 5 1-Oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfone Acid DMA solution (0.366 mL; 5.2 equivalents per antibody molecule), 10 mM N2- Deacetyl-deacetyl-N2-(4-methyl-4-mercapto-1-oxopentyl) -Maytansine (DM4) in DMA solution (0.366 mL; 6.8 equivalents per antibody molecule) The resulting mixture was incubated at 20°C for 16 hours, and the drug was then added to the 100 ml of ... Next, 1M acetic acid solution was added to adjust the pH to 5.0, and the mixture was further incubated at room temperature. The mixture was stirred at RT for 20 minutes to terminate the drug linker reaction.

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

[0310] Characterization: Common procedure E(ε A,280 =200500 、 ε A,2 52 =76295, ε D,280 =43170 、 and ε D,252 =23224) The following characteristic values ​​were obtained using the Antibody concentration: 2.58 mg / mL, antibody yield: 72.2 mg (93%), measured using common procedure E The average number of drugs bound per antibody molecule (n) was determined to be 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 subjected to the same procedure as described in Production Method 1. B (280 nm extinction coefficient: 1.51 mL mg -1 cm -1 ) and C, The solution was adjusted to 9.26 mg / mL with PBS 6.0 / EDTA. 0 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.254 mL; per antibody molecule) 6.0 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc. (0.0990 mL) was added. The pH of this solution was confirmed to be within 7.0 ± 0.1. After confirming this, the antibody was incubated at 37°C for 2 hours to separate the disulfide bonds in the interchain region. The hydroxyl bonds were reduced.

[0314] Conjugation of antibody and drug linker: Incubate the above solution at 15°C for 10 minutes. Then, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]amine 10 mM dimethyl sulfoxide of (2-oxoethoxymethyl)glycinamide Add the solution (0.381 mL; 9 equivalents per antibody molecule) and incubate at 15°C for 1 hour. The drug linker was then attached to the antibody. Idrich Co. LLC) aqueous solution (0.0381 mL; 9 equivalents per antibody molecule) was added and stirred at room temperature for a further 20 minutes to terminate the reaction of the drug linker.

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

[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%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 6.4; measured by standard procedure F Average number of drugs bound per antibody molecule (n): 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 Common procedure B described in Manufacturing Method 1 (280 nm extinction coefficient: 1.53 mL mg -1 cm - 1 ) and C, 20 mM HEPES 8.1 (LIFE TECHNOLOGY Add 100 mL of GIES HEPES 1M Buffer Solution (20 mL) 9.8 mg / mM (pH adjusted to 8.1 with 1 M sodium hydroxide, then distilled water to 1 L) The mixture was then adjusted to 1 L and incubated at 20°C for 10 minutes. 10 mM 1-(2,5-dioxopyrrolidin-1-yloxy)-2-hydroxybenzoate described in Patent Publication No. )-1-Oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid DMA solution (0.062 mL; 11.5 equivalents per antibody molecule), 10 mM N2- Deacetyl-N2-(4-methyl-4-mercapto-1-oxopentyl)-maytansine Add 0.082 mL of DM4 (15.1 equivalents per antibody molecule) to the mixture. The mixture was incubated at 20°C for 18 hours to allow the drug linker to bind to the antibody. An aqueous solution of acetic acid was added to adjust the pH to 5.0, and the mixture was stirred at room temperature for 20 minutes to allow the drug linker to react. was stopped.

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

[0319] Characterization: Common procedure E (ε = 223400, ε = 223400) described in Preparation Method 1 52=85646, εD,280=4317, and εD,252=23224) The following characteristic values ​​were obtained using the material: Antibody concentration: 1.97 mg / mL, antibody yield: 6.90 mg (88%), measured using common procedure E The average number of drugs bound per antibody molecule (n) was determined to be 3.6.

[0320] [Example 8: In vitro activity evaluation of antibody-drug conjugates] 8)-1 In vivo evaluation of antibody-drug conjugates against CDH6-positive human tumor cell lines Antiproliferative activity evaluation CDH6-positive human ovarian tumor cell line PA-1 was cultured at 2 × 10 in MEM medium containing 10% FBS. 3 Cells were seeded into a 96-well plate at 100 μL per well and incubated at 37°C, 5% The cells were cultured overnight under CO2 conditions. The next day, the humanized hG019-drug conjugate prepared in Example 7 was added. Four types of jugate (clone names: H01L02-DXd, H02L02-DXd, H02 L03-DXd and H04L02-DXd), or NOV0712- prepared in Reference Example 2 The drug conjugate (NOV0712-DM4) was added at final concentrations ranging from 0.0001 (nM) to 10 After 4 days of culture, the number of viable cells was counted using CellTiter-G lo TM Luminescent Cell Viability Assay(Pr ATP was measured by omega-3 quantification. The humanized hG019-drug exhibited a concentration-dependent cell growth inhibitory activity. The four conjugates showed lower loadings compared to the NOV0712-drug conjugate. The concentration indicates tumor cell proliferation inhibitory activity, indicating high antitumor activity.

[0321] Example 9: In vivo antitumor effect of antibody-drug conjugates The antitumor effect of the antibody-drug conjugate was confirmed by immunohistochemistry of CDH6-positive human tumor cell lines. The results were evaluated using an animal model in which the cells were transplanted into 4-5 week old BALB / c nude mice. Mouse (CAnN.Cg-Foxnl[nu] / CrlCrlj[Foxnlnu / Fo xnlnu], Charles River, Japan) and SCID mice (CB17 / Icr-P rkdc[scid] / CrlCrlj (Charles River, Japan) was used for the experiment. The mice were acclimated to the F condition for more than 3 days. They were fed sterilized tap water (5-15 ppm hypochlorite) and The major and minor diameters of the transplanted tumor were measured electronically. Measured twice a week using a digital caliper (CD-15CX, Mitutoyo Corp.) The 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 stored in ABS buffer (10 mM Acetate Buffer er, 5% Sorbitol, pH 5.5 (NACALAI) and The doses shown in the examples were administered intravenously into the tail vein. ABS buffer was administered in the same manner. Six mice per group were used in the experiment.

[0322] 9)-1 Antitumor effect (1) CDH6-positive human renal cell tumor cell line in which CDH6 expression was confirmed in Example 2)-3-1 786-O (ATCC) was suspended in Matrigel (Corning) and 5 × 10 6 cells were subcutaneously implanted into the right flank of male SCID mice (Day 0), and randomly assigned to groups on Day 18. On the day of grouping, four types of antibody-drug conjugates (clot-type) prepared in Example 7 were administered. The line names are H01L02-DXd, H02L02-DXd, H02L03-DXd and H 04L02-DXd), or NOV0712-DM4 prepared in Reference Example 2 at 3 mg / kg The results are shown in Figure 12. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the error bar The circles indicate the SE values.

[0323] NOV0712-DM4 did not show significant antitumor effects in this tumor model. All four types of antibody-drug conjugates prepared in Example 7 showed a decrease in tumor volume after administration. Significant tumor regression was observed, and the tumor regression effect continued for 24 days after administration (FIG. 12).

[0324] 9)-2 Antitumor effect (2) CDH6-positive human ovarian tumor cell line P, in which CDH6 expression was confirmed in Example 2)-3-1 A-1 (ATCC) was suspended in Matrigel (Corning) and 8.5 × 10 6 cells The mice were subcutaneously implanted into the right flank of female nude mice (Day 0), and randomly assigned to groups on Day 11. On the day of grouping, the antibody-drug conjugate H01L02 prepared in Example 7 was administered. -DXd or NOV0712-DM4 or NOV0712-DX prepared in Reference Example 2 The results are shown in Figure 13. The horizontal axis represents the number of days. The vertical axis indicates tumor volume, and the error bar indicates SE value.

[0325] NOV0712-DM4 was effective in this tumor model at both 1 and 3 mg / kg doses. On the other hand, H01L02-DXd showed no antitumor effect at both 1 and 3 mg / kg. Even at this dose, the tumor volume was significantly reduced after administration, demonstrating the tumor shrinkage effect (Figure 13). In addition, the H01L02 antibody or NOV0712 antibody obtained herein may be administered with the same drug DX. When comparing the efficacy of the samples conjugated with 1 and d, H01L02-DXd was Both doses of 3 mg / kg showed stronger antitumor effects than NOV0712-DXd. That is, the H01L02 antibody of the present invention has a stronger antitumor activity than the NOV0712 antibody. It was shown that this antibody is superior to the antibody in the antibody-drug conjugate (Figure 1). 13).

[0326] 9)-3 Antitumor effect (3) CDH6-positive human ovarian tumor cell line N, in which CDH6 expression was confirmed in Example 2)-3-1 IH: OVCAR-3 (ATCC) cells were suspended in Matrigel (Corning) at 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0) and randomly assigned on Day 22. On the day of grouping, the antibody-drug conjugate H prepared in Example 7 was administered. 01L02-DXd or NOV0712-DM4 prepared in Reference Example 2 was added at 1 and 3 mg / The results are shown in Figure 14. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the The difference range indicates the SE value.

[0327] NOV0712-DM4 showed no antitumor effect at 1 mg / kg, and Although H0 showed antitumor effects, tumor regrowth was observed two weeks after administration. 1L02-DXd significantly increased tumor volume after administration at both 1 and 3 mg / kg doses. In particular, at 3 mg / kg, the tumor growth suppression effect was maintained for a long period of 31 days after administration. This continued (Figure 14).

[0328] In addition, the same procedure was carried out using PA-1 cells as in Reference Example 2. The tumor growth inhibitory effect of H01L02-DM4 prepared in Reference Example 3 was evaluated. H01L0 of the present invention reduced tumor volume more than NOV0712-DM4. 2 antibody has a higher efficacy as an antibody-drug conjugate as an antitumor agent than the NOV0712 antibody. It was a superior antibody.

[0329] 9)-4 Antitumor effect (4) CDH6-positive human renal cell tumor cell line in which CDH6 expression was confirmed in Example 2)-3-1 786-O (ATCC) was suspended in Matrigel (Corning) and 5 × 10 6 cells were subcutaneously implanted into the right flank of male SCID mice (Day 0), and randomly assigned to groups on Day 20. On the day of grouping, the antibody-drug conjugate H01L0 prepared in Example 7 was 2-DXd or NOV0712-DM4 prepared in Reference Example 2 was administered at 1 and 3 mg / kg. The results are shown in Figure 15. The horizontal axis represents the number of days, the vertical axis represents the tumor volume, and the error bars are The SE value is shown.

[0330] NOV0712-DM4 was effective in this tumor model at both 1 and 3 mg / kg doses. On the other hand, H01L02-DXd showed no significant antitumor effect at 1 and 3 mg / kg. The tumor volume decreased after administration at all doses, and particularly at 3 mg / kg, significant tumor regression was observed. The tumor regression effect was sustained for 20 days after administration (Figure 15).

[0331] 9)-5 Antitumor effect (5) CDH6-negative human ovary confirmed not to express CDH6 in Example 2)-3-1 Tumor cell line ES-2 (ATCC) was suspended in saline and 1 × 10 6 cells of a female wildebeest The mice were subcutaneously implanted into the right flank (Day 0), and randomly assigned to groups on Day 7. On the day of grouping, the antibody-drug conjugate H01L02-DXd or NOV0712-DM4 prepared in Reference Example 2 was administered intravenously 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 bar indicates the SE value. .

[0332] In this tumor model, which does not express CDH6, H01L02-DXd and NOV0712- DM4 showed no antitumor effect at any dose. Antibody-drug interactions in CDH6-positive tumor models shown in 1, 9)-2, 9)-3, and 9)-4 The antitumor effect of conjugates is dependent on CDH6 expression in tumor cells. It exhibits antitumor effects specifically against CDH6-positive tumors and does not cause cytotoxicity in CDH6-negative normal tissues. It is considered to be a selective and safe antitumor agent that does not cause tumor growth (Figure 16). [Industrial Applicability]

[0333] According to the present invention, an anti-CDH6 antibody having internalization activity and an antibody-drug conjugate containing said antibody are provided. The antibody-drug conjugate is used as a therapeutic agent for cancer, etc. It is possible.

Claims

1. An antibody-drug conjugate in which a drug is bound to 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; The present invention comprises a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and 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, 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, an antibody or an antigen-binding fragment thereof, The antibody-drug conjugate.

2. An antibody-drug conjugate in which a drug is bound to 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; The present invention comprises a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and 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, 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, an antibody or an antigen-binding fragment thereof, The antibody-drug conjugate.

3. 3. The antibody-drug conjugate according to 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.

4. The antibody or antigen-binding fragment thereof specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and has the ability to be internalized into cells; The present invention comprises a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and 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, 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, It is an antibody, The antibody-drug conjugate of claim 1.

5. The antibody or antigen-binding fragment thereof specifically binds to the amino acid sequence set forth in SEQ ID NO: 4 and has the ability to be internalized into cells; The present invention comprises a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and 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, 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, It is an antibody, The antibody-drug conjugate of claim 2.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the heavy chain or light chain of the antibody or antigen-binding fragment thereof has undergone one or more modifications selected from the group consisting of glycosylation to an N-linkage, glycosylation to an O-linkage, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, 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.

7. The antibody-drug conjugate of claim 6, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

8. The antibody-drug conjugate of claim 7, wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains.

9. The antibody-drug conjugate according to any one of claims 6 to 8, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment of the antibody is an antibody or antigen-binding fragment of the antibody in which glycosylation has been regulated to enhance antibody-dependent cellular cytotoxicity.

11. The antibody-drug conjugate according to any one of claims 1 to 10, wherein the drug is an antitumor compound.

12. The antitumor compound has the formula: 【Chemistry 1】 The antibody-drug conjugate according to claim 11, which is an antitumor compound represented by the formula:

13. The antibody and the drug are represented by the following formulas (a) to (f): (a)-(Succinimid-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-、 (b)-(Succinimid-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-、 (c)-(Succinimid-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-、 (d)-(Succinimid-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -O-CH ２ -C(=O)-、 (e)-(Succinimid-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-, and (f)-(Succinimid-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-、 The antibody-drug conjugate according to any one of claims 1 to 12, wherein the antibody-drug conjugate is bound via a linker having a structure selected from the group consisting of: (Here, the antibody is attached at the -(Succinimid-3-yl-N) end. The antitumor compound is a compound having a -CH ２ CH ２ CH ２ -C(=O)- moiety, CH ２ -O-CH ２ -C(=O)- moiety or CH of (d) ２ CH ２ -O-CH ２ It binds to the carbonyl group of the —C(═O)— moiety. In the above formula, GGFG represents an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by peptide bonds. -(Succinimid-3-yl-N)- has the following formula: 【Chemistry 2】 The structure is represented by the formula: Figure imgf000012_0001, which binds to the antibody at position 3 and binds to the methylene group in the linker structure containing the nitrogen atom at position 1.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein the linker is represented by the following formula (c) or (e): (c)-(Succinimid-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-、 (e)-(Succinimid-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-。

15. The following formula: 【Transformation 3】 The antibody-drug conjugate of any one of claims 1 to 14, having the structure shown below: Here, AB represents an antibody or a functional fragment of the antibody. n represents the average number of drug-linker structures bound to the antibody per antibody. The antibody and linker are bound via a sulfhydryl group derived from the antibody.

16. The following formula: 【Chemistry 4】 The antibody-drug conjugate of any one of claims 1 to 14, having the structure shown below: Here, AB represents an antibody or a functional fragment of the antibody. n represents the average number of drug-linker structures bound to the antibody per antibody. The antibody and linker are bound via a sulfhydryl group derived from the antibody.

17. The antibody-drug conjugate according to any one of claims 1 to 16, wherein the average number of selected drug-linker structures bound per antibody is in the range of 1 to 10.

18. The antibody-drug conjugate of claim 17, wherein the average number of selected drug-linker structures bound per antibody is in the range of 2 to 8.

19. The antibody-drug conjugate of claim 18, wherein the average number of selected drug-linker structures bound per antibody is in the range of 5 to 8.

20. The antibody-drug conjugate of claim 19, wherein the average number of the selected drug-linker structures bound per antibody is 7 to 8.

21. The following formula: 【Transformation 5】 wherein AB represents an antibody comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, 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, and comprising 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. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.

22. The following formula: 【Transformation 6】 wherein AB represents an antibody comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14, 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, and comprising a light chain consisting of an amino acid sequence having 95% or more identity to the amino acid sequence of amino acids 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 amino acids 20 to 471 of SEQ ID NO:

73. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.

23. The antibody-drug conjugate of claim 21 or 22, wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains of the antibody.

24. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 23, a salt thereof, or a hydrate thereof.

25. 25. The pharmaceutical composition according to claim 24, which is an antitumor drug.

26. The pharmaceutical composition according to claim 25, wherein the tumor is a tumor that expresses CDH6.

27. The pharmaceutical composition according to claim 25 or 26, characterized in that 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 carcinoma, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma.

Images