anti-CDH 6 antibodies and anti-CDH 6 antibody-drug conjugates

文档序号：1590665 发布日期：2020-01-03 浏览：27次中文

阅读说明：本技术 抗cdh6抗体和抗cdh6抗体-药物缀合物 (anti-CDH 6 antibodies and anti-CDH 6 antibody-drug conjugates ) 是由齐藤敦子平田刚之中村健介于 2018-05-14 设计创作，主要内容包括：本发明解决提供以下的问题：结合CDH6的具有内化活性的抗体；由上述抗体和具有抗肿瘤活性的药物组成的抗体-药物缀合物；使用所述抗体-药物缀合物的药物,所述药物对肿瘤具有治疗作用；使用所述抗体、抗体-药物缀合物或药物治疗肿瘤的方法；等等。提供以下：具有内化活性的抗CDH6抗体；由上述抗体和具有抗肿瘤活性的药物组成的抗体-药物缀合物；和药物以及使用其治疗肿瘤的方法。(The present invention solves the problem of providing: an antibody that binds to CDH6 having internalization activity; an antibody-drug conjugate composed of the above antibody and a drug having an antitumor activity; a drug using the antibody-drug conjugate, which has a therapeutic effect on a tumor; a method of treating a tumor using the antibody, antibody-drug conjugate or drug; and so on. The following are provided: an anti-CDH 6 antibody having internalization activity; an antibody-drug conjugate composed of the above antibody and a drug having an antitumor activity; and medicaments and methods of using the same for treating tumors.)

1. 4 and having an internalizing ability that allows cellular uptake, or a functional fragment thereof.

2. The antibody or functional fragment of an antibody according to claim 1, which has competitive inhibitory activity against binding to the amino acid sequence shown in SEQ ID NO:4 against at least any one of the antibodies selected from the following antibodies (1) to (5):

(1) an antibody having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 53 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 56,

(2) an antibody having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69,

(3) an antibody having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73,

(4) an antibody having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73, and

(5) an antibody having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77.

3. The antibody or functional fragment of an antibody according to claim 1 or 2, comprising a CDRL1, a CDRL2 and a CDRL3 in any one of the following combinations (1) to (4):

CDRH1, CDRH2 and CDRH3 in any one of the following combinations (5) to (9):

(5) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:17, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:18, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:19,

(6) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:27, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:28, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:29,

(7) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:37, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:38, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:39,

(8) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:47, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:48, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:49, and

(9) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:17, 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.

4. An antibody or functional fragment of an antibody according to any one of claims 1 to 3, which comprises CDRL1, CDRL2 and CDRL3 and CDRH1, CDRH2 and CDRH3 in any one combination selected from the following combinations (1) to (5):

(1) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:13, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:14, as well as CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:17, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:18, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:19,

(2) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:22, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:23, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:24, as well as CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:27, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:28, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:29,

(3) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:32, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:33, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:34, as well as CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:37, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:38, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:39,

(4) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:42, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:43, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:44, and CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:47, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:48, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:49, and

(5) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:13, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:14, as well as CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:17, 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.

5. The antibody or functional fragment of an antibody according to any one of claims 1 to 4, which is humanized.

6. The antibody or functional fragment of an antibody according to any one of claims 1 to 5, which has any one light chain variable region selected from the following variable regions (1) to (4):

(1) the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:63,

(2) 67 from SEQ ID NO,

(3) an amino acid sequence having at least 95% or more sequence identity with the sequence of the framework region other than each CDR sequence in the amino acid sequences of (1) and (2), and

(4) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the sequence of the framework region other than each CDR sequence in the amino acid sequences of (1) to (3), and

any one of the heavy chain variable regions selected from the following variable regions (5) to (9):

(5) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:71,

(6) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75,

(7) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:79,

(8) an amino acid sequence having at least 95% or more sequence homology with a sequence of a framework region other than each CDR sequence among the amino acid sequences of (5) to (7), and

(9) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the sequence of the framework region except each CDR sequence in the amino acid sequences of (5) to (8).

7. The antibody or functional fragment of an antibody according to any one of claims 1 to 6, which comprises the light chain variable region and the heavy chain variable region of any one of the following combinations (1) to (4):

(1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO 71,

(2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75,

(3) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:67 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75, and

(4) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO 79.

8. The antibody or functional fragment of an antibody according to any one of claims 1 to 7, which has any one of the following combinations (1) to (4):

(1) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69,

(2) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73,

(3) a light chain consisting of the amino acid sequence from position 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence from position 20 to 471 in SEQ ID NO 73, and

(4) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77.

9. The antibody or functional fragment of an antibody according to claim 8, having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69.

10. The antibody or functional fragment of an antibody according to claim 8, having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73.

11. The antibody or functional fragment of an antibody according to claim 8, having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73.

12. The antibody or functional fragment of an antibody according to claim 8, having a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77.

13. A functional fragment of an antibody according to any one of claims 1 to 12, wherein said functional fragment is selected from Fab, F (ab ')2, Fab' and Fv.

14. A polynucleotide encoding the antibody or functional fragment of an antibody according to any one of claims 1 to 13.

15. The polynucleotide according to claim 14, which comprises a polynucleotide selected from any one of the following combinations (1) to (5):

(1) a polynucleotide encoding the light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO. 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 13 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 14, and a polynucleotide encoding the heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 17, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 18 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO. 19,

(2) a polynucleotide encoding the light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:22, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:23 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:24, and a polynucleotide encoding the heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:27, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:28 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:29,

(3) a polynucleotide encoding the light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:32, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:33 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:34, and a polynucleotide encoding the heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:37, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:38 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:39,

(4) a polynucleotide encoding the light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:42, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:43 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:44, and a polynucleotide encoding the heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:47, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:48 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO:49, and

(5) a polynucleotide encoding the light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO. 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 13 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 14, and a polynucleotide encoding the heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 17, 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.

16. The polynucleotide according to claim 14 or 15, comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 69.

17. The polynucleotide according to claim 14 or 15, comprising a polynucleotide encoding the light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 61 and a polynucleotide encoding the heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 73.

18. The polynucleotide according to claim 14 or 15, comprising a polynucleotide encoding the light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 65 and a polynucleotide encoding the heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 73.

19. The polynucleotide according to claim 14 or 15, comprising a polynucleotide encoding the light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 61 and a polynucleotide encoding the heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 77.

20. An expression vector comprising the polynucleotide of any one of claims 14 to 19.

21. A host cell transformed with the expression vector of claim 20.

22. The host cell of claim 21, wherein the host cell is a eukaryotic cell.

23. A method for producing an antibody or a functional fragment of an antibody of interest, comprising the steps of culturing the host cell according to claim 21 or 22, and collecting the antibody or the functional fragment of an antibody of interest from the culture obtained by the above steps.

24. The antibody or functional fragment of an antibody according to any one of claims 1 to 13, wherein the heavy or light chain has undergone one or two or more modifications selected from: n-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of methionine residues to the N-terminus, amidation of proline residues, conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid, and deletion of one or two amino acids from the carboxyl terminus.

25. The antibody of claim 24, wherein one or two amino acids are deleted from the carboxy-terminus of its heavy chain.

26. The antibody of claim 25, wherein one amino acid is deleted from each carboxy terminus of its two heavy chains.

27. The antibody of any one of claims 24 to 26, wherein the proline residue at the carboxy-terminal end of its heavy chain is further amidated.

28. The antibody or functional fragment of an antibody according to any one of claims 1 to 13 and 24 to 27, wherein sugar chain modifications are modulated to enhance antibody-dependent cellular cytotoxic activity.

29. An antibody-drug conjugate comprising an antibody or functional fragment of an antibody according to any one of claims 1 to 13 and 24 to 28 conjugated to a drug.

30. The antibody-drug conjugate of claim 29, wherein the drug is an anti-tumor compound.

31. The antibody-drug conjugate of claim 30, wherein the anti-neoplastic compound is an anti-neoplastic compound represented by the formula:

[ formula 1]

32. The antibody-drug conjugate of any one of claims 29 to 31, wherein the antibody is conjugated to the drug via a linker having any structure selected from the group consisting of formulae (a) to (f):

(a) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

(b) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

(d) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C(=O)-，

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C (= O) -, and

(f) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

Wherein the antibody is linked to the terminus of- (succinimid-3-yl-N) and the anti-neoplastic compound is linked to (a), (b), (e) or (f)-CH₂CH₂CH₂-C (= O) -moiety, (C) CH₂-O-CH₂-C (= O) -moiety or CH of (d)₂CH₂-O-CH₂-a carbonyl group of a C (= O) -moiety wherein the nitrogen atom of the amino group at position 1 is the linking position, GGFG represents an amino acid sequence consisting of glycine-phenylalanine-glycine linked through a peptide bond, and

- (succinimid-3-yl-N) -has a structure represented by the formula:

[ formula 2]

It is attached to the antibody at its position 3 and to the methylene group in the linker structure containing the structure at the nitrogen atom at position 1.

33. The antibody-drug conjugate of any one of claims 29 to 32, wherein the linker is represented by any one formula selected from the group consisting of the following formulae (c), (d), and (e):

(d) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C (= O) -, and

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

34. The antibody-drug conjugate of any one of claims 29 to 33, wherein the linker is represented by the following formula (c) or (e):

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

35. The antibody-drug conjugate of any one of claims 29 to 34, having a structure represented by the formula:

[ formula 3]

Wherein AB represents an antibody or a functional fragment of an antibody, n represents the average number of units of a drug-linker structure conjugated to the antibody per antibody, and the antibody is linked to the linker via a thiol group derived from the antibody.

36. The antibody-drug conjugate of any one of claims 29 to 34, having a structure represented by the formula:

[ formula 4]

37. The antibody-drug conjugate of any one of claims 29 to 36, wherein the antibody is an antibody comprising a light chain and a heavy chain selected from any one of the following combinations (1) to (4), or a functional fragment of the antibody:

(1) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69,

(2) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73,

(3) a light chain consisting of the amino acid sequence from position 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence from position 20 to 471 in SEQ ID NO 73, and

(4) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77.

38. The antibody-drug conjugate of claim 37, wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 69 or a functional fragment of said antibody.

39. The antibody-drug conjugate of claim 37, wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID No. 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID No. 77 or a functional fragment of said antibody.

40. The antibody-drug conjugate of any one of claims 29 to 39, wherein the heavy or light chain has undergone one or two or more modifications selected from: n-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of methionine residues to the N-terminus, amidation of proline residues, conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid, and deletion of one or two amino acids from the carboxyl terminus.

41. The antibody-drug conjugate of any one of claims 29 to 40, wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 1 to 10.

42. The antibody-drug conjugate of claim 41, wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 2 to 8.

43. The antibody-drug conjugate of claim 42, wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 5 to 8.

44. The antibody-drug conjugate of claim 43, wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 7 to 8.

45. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 29 to 44, a salt thereof, or a hydrate of the conjugate or the salt.

46. The pharmaceutical composition of claim 45, which is an anti-tumor drug.

47. The pharmaceutical composition of claim 46, wherein the tumor is a CDH 6-expressing tumor.

48. The pharmaceutical composition of claim 46 or 47, wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma.

49. A method for treating a tumor comprising administering to an individual any ingredient selected from the antibody-drug conjugate of any one of claims 29 to 44, a salt thereof, and a hydrate of the conjugate or the salt.

50. The method of treatment according to claim 49, wherein the tumor is a CDH 6-expressing tumor.

51. The therapeutic method of claim 49 or 50, wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma.

52. A method for treating a tumor comprising administering to an individual simultaneously, separately or sequentially a pharmaceutical composition comprising at least one component selected from the antibody-drug conjugate of any one of claims 29 to 44, a salt thereof, and a hydrate of the conjugate or the salt, and at least one antineoplastic drug.

53. A method for producing an antibody-drug conjugate, comprising the step of reacting the antibody or functional fragment of the antibody according to any one of claims 1 to 13 and 24 to 28, or the antibody or functional fragment of the antibody obtained by the production method according to claim 23, with a drug-linker intermediate compound.

Technical Field

The present invention relates to an anti-CDH 6 antibody that binds to CDH6 and has an internalization effect, a method of producing the anti-CDH 6 antibody, an antibody-drug conjugate comprising the antibody, an antitumor agent comprising the antibody-drug conjugate, and the like.

Background

Cadherins are glycoproteins that are present on the surface of cell membranes and function as cell-cell adhesion molecules through their calcium-dependent binding of the N-terminal extracellular domain, or as signaling molecules responsible for cell-cell interactions. Classical cadherins belong to the cadherin superfamily and are single-transmembrane proteins consisting of five extracellular domains (EC domains), one transmembrane domain and one intracellular domain. Based on their amino acid sequence homology, classical cadherins are classified into type I families (which are represented by E-cadherins and N-cadherins) and type II families.

Cadherin 6(CDH6) is a single transmembrane protein composed of 790 amino acids, which is classified as a type II cadherin family, and has N-terminal extracellular and C-terminal intracellular domains. The human CDH6 gene was first cloned in 1995 (non-patent document 1), and its sequence can be referred to under, for example, accession numbers NM _004932 and NP _004923 (NCBI).

CDH6 is specifically expressed in the brain or kidney at a developmental stage, and has been reported to play an important role in the loop formation of the central nervous system (non-patent documents 2 and 3) and the development of nephrons in the kidney (non-patent documents 4 and 5). The expression of CDH6 in normal tissues of adults is limited to renal tubules, bile duct epithelial cells, etc. of the kidney.

Meanwhile, it is known that CDH6 is specifically overexpressed at the tumor site in some types of human adult cancers. For human renal cell carcinoma, particularly renal clear cell carcinoma, the correlation of CDH6 expression with poor prognosis and its applicability as a tumor marker have been reported (non-patent documents 6 and 7). High expression of CDH6 has also been reported for human ovarian cancer (non-patent document 8). CDH6 has also been reported to be involved in epithelial-mesenchymal transition of human thyroid cancer (non-patent document 9). In addition, it has been reported that CDH6 is also expressed in human cholangiocarcinoma and human small cell lung cancer (non-patent documents 12 and 13).

Cancer ranks high among causes of death. Although the number of cancer patients is expected to increase as the population ages, the therapeutic needs have not been adequately met. The problems with conventional chemotherapeutic agents are: due to their low selectivity, these chemotherapeutic agents are toxic not only to tumor cells but also to normal cells and thus produce adverse reactions; and the chemotherapeutic agent cannot be administered in a sufficient amount and thus cannot sufficiently produce its effect. Therefore, in recent years, more selective molecular target drugs or antibody drugs have been developed which target molecules exhibiting a mutation or high expression characteristic in cancer cells, or target specific molecules involved in malignant transformation of cells.

Antibodies are highly stable in blood and specifically bind their target antigen. For these reasons, a decrease in adverse effects is expected, and a large number of antibody drugs have been developed for molecules highly expressed on the surface of cancer cells. One of the techniques that relies on the antigen-specific binding capacity of antibodies is the use of antibody-drug conjugates (ADCs). ADCs are conjugates in which an antibody that binds to an antigen expressed on the surface of a cancer cell and can internalize the antigen into the cell by this binding is conjugated to a drug having cytotoxic activity. The ADC can efficiently deliver a drug to cancer cells, and thus can be expected to kill cancer cells by accumulating the drug in the cancer cells (non-patent document 10 and patent documents 1 and 2). As for ADCs, for example, adcetris (tm) (bentuximab vedotin), which contains an anti-CD 30 monoclonal antibody conjugated with monomethyl auristatin E, has been approved as a therapeutic drug for hodgkin lymphoma and anaplastic large cell lymphoma. Furthermore, kadcula (tm) (trastuzumab maytansine) comprising an anti-HER 2 monoclonal antibody conjugated to maytansine is used to treat HER2 positive progressive or recurrent breast cancer.

The target antigens of ADCs suitable as antitumor drugs are characterized in that: the antigen is specifically and highly expressed on the surface of cancer cells, but is low or not expressed in normal cells; the antigen may be internalized into the cell; the antigen is not secreted from the cell surface, etc. The internalizing ability of an antibody depends on the target antigen and the nature of the antibody. It is difficult to predict an antigen binding site suitable for internalization from the molecular structure of a target, or to predict an antibody having high internalization ability from the binding strength, physical properties, etc. of the antibody. Therefore, an important challenge in developing ADCs with high potency is to obtain antibodies with high internalization ability against the target antigen (non-patent document 11).

An ADC comprising DM4 conjugated with an anti-CDH 6 antibody that specifically binds to EC domain 5(EC5) of CDH6 is referred to as an ADC targeting CDH6 (patent document 3).

CITATION LIST

Patent document

Patent document 1: WO2014/057687

Patent document 2: US2016/0297890

Patent document 3: WO2016/024195

Non-patent document

Non-patent document 1: shimoyama Y, et al, Cancer Research, 2206-

Non-patent document 2: inoue T, et al, development Biology, 183-

Non-patent document 3: osterhout J A, et al, Neuron, 632-639, 71, Aug 25, 2011

Non-patent document 4: cho E A, et al, Development, 803-

Non-patent document 5: mah S P, et al, development Biology, 38-53, 223, 2000

Non-patent document 6: paul R, et al, Cancer Research, 2741-

Non-patent document 7: shimazui T, et al, Cancer, 963-

Non-patent document 8: koebel M, et al, PLoS Medicine, 1749-

Non-patent document 9: gugnoni M, et al, Oncogene, 667-

Non-patent document 10: polakis P., pharmaceutical 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, epidemics, 780-

Non-patent document 13: yokoi S, et al, American Journal of Pathology, 207-.

Summary of The Invention

Technical problem

An object of the present invention is to provide an antibody specifically binding to CDH6 and having a high internalization activity, an antibody-drug conjugate comprising the antibody and having a high anti-tumor activity, a drug product comprising the antibody-drug conjugate and having a therapeutic effect on tumors, a method for treating tumors using the antibody, the antibody-drug conjugate or drug product, and the like.

Solution to the problem

The present inventors have intensively studied for achieving the above object, and found that, surprisingly, an antibody that specifically binds to extracellular domain 3 (also referred to as EC3 in the present specification) of CDH6 has an extremely high internalization activity against cells expressing CDH6 and can be used as an antibody for ADC. The present inventors have further found that an anti-CDH 6 antibody-drug conjugate comprising the above-described anti-CDH 6 antibody conjugated to a drug exerting toxicity in cells via a linker having a specific structure exhibits stronger anti-tumor activity than that of a conventional CDH 6-drug conjugate.

The present invention includes the following aspects of the invention:

[1] 4 and having an internalizing capacity allowing cellular uptake, or a functional fragment of said antibody;

[2] the antibody or a functional fragment of the antibody according to [1], which has a competitive inhibitory activity on binding to an amino acid sequence shown in SEQ ID NO:4 against at least any one of the antibodies selected from the following antibodies (1) to (5):

[3] the antibody or functional fragment of an antibody according to [1] or [2], which comprises CDRL1, CDRL2 and CDRL3 in any one combination selected from the following combinations (1) to (4):

CDRH1, CDRH2 and CDRH3 in any one of the following combinations (5) to (9):

[4] the antibody or functional fragment of an antibody according to any one of [1] to [3], which comprises CDRL1, CDRL2 and CDRL3 and CDRH1, CDRH2 and CDRH3 in any one of the following combinations (1) to (5):

[5] the antibody or functional fragment of an antibody according to any one of [1] to [4], which is humanized;

[6] the antibody or functional fragment of an antibody according to any one of [1] to [5], which has any one light chain variable region selected from the following variable regions (1) to (4):

(1) the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:63,

(2) 67 from SEQ ID NO,

(3) an amino acid sequence having at least 95% or more sequence identity with the sequence of the framework region other than each CDR sequence in the amino acid sequences of (1) and (2), and

any one of the heavy chain variable regions selected from the following variable regions (5) to (9):

(5) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:71,

(6) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75,

(7) the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:79,

(8) an amino acid sequence having at least 95% or more sequence homology with a sequence of a framework region other than each CDR sequence among the amino acid sequences of (5) to (7), and

(9) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the sequence of the framework region other than each CDR sequence in the amino acid sequences of (5) to (8);

[7] the antibody or functional fragment of an antibody according to any one of [1] to [6], which comprises the light chain variable region and the heavy chain variable region of any one of the following combinations (1) to (4):

(1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO 71,

(2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75,

(3) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:67 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:75, and

(4) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO 79;

[8] the antibody or functional fragment of an antibody according to any one of [1] to [7], which has any one of the following combinations (1) to (4):

(1) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69,

(2) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73,

(3) a light chain consisting of the amino acid sequence from position 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence from position 20 to 471 in SEQ ID NO 73, and

(4) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77;

[9] the antibody or functional fragment of an antibody according to [8], which has a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO: 69;

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

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

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

[13] a functional fragment of the antibody according to any one of [1] to [12], wherein the functional fragment is selected from the group consisting of Fab, F (ab ')2, Fab', and Fv;

[14] a polynucleotide encoding the antibody or the functional fragment of the antibody according to any one of [1] to [13 ];

[15] the polynucleotide according to [14], which comprises a polynucleotide selected from any one of the following combinations (1) to (5):

[16] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO:61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO: 69;

[17] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO. 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO. 73;

[18] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO. 65 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO. 73;

[19] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO:61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO: 77;

[20] an expression vector comprising the polynucleotide according to any one of [14] to [19 ];

[21] a host cell transformed with the expression vector according to [20 ];

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

[23] a method for producing an antibody or a functional fragment of an antibody of interest, which comprises the steps of culturing the host cell according to [21] or [22], and collecting the antibody or the functional fragment of an antibody of interest from the culture obtained by the above steps;

[24] the antibody or functional fragment of an antibody according to any one of [1] to [13], wherein the heavy or light chain has undergone one or two or more modifications selected from: n-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of methionine residue to N-terminus, amidation of proline residue, conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid, and deletion of one or two amino acids from the carboxyl terminus;

[25] the antibody according to [24], wherein one or two amino acids are deleted from the carboxy-terminal of the heavy chain thereof;

[26] the antibody of [25], wherein one amino acid is deleted from each carboxy terminus of both heavy chains thereof;

[27] the antibody according to any one of [24] to [26], wherein the proline residue at the carboxy-terminal end of its heavy chain is further amidated;

[28] the antibody or the functional fragment of the antibody according to any one of [1] to [13] and [24] to [27], wherein the sugar chain modification is modulated to enhance antibody-dependent cellular cytotoxicity activity;

[29] an antibody-drug conjugate comprising an antibody or functional fragment of an antibody according to any one of [1] to [13] and [24] to [28] conjugated to a drug;

[30] the antibody-drug conjugate according to [29], wherein the drug is an anti-tumor compound;

[31] the antibody-drug conjugate according to [30], wherein the anti-tumor compound is an anti-tumor compound represented by the following formula:

[ formula 1]

[32] The antibody-drug conjugate according to any one of [29] to [31], wherein the antibody is conjugated to the drug via a linker having any structure selected from the group consisting of the following formulae (a) to (f):

(a) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

(b) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

(d) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C(=O)-，

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C (= O) -, and

(f) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

Wherein the antibody is linked to the terminus of- (succinimid-3-yl-N) and the anti-neoplastic compound is linked to the-CH of (a), (b), (e) or (f)₂CH₂CH₂-C (= O) -moiety, (C) CH₂-O-CH₂-C (= O) -moiety or CH of (d)₂CH₂-O-CH₂-a carbonyl group of a C (= O) -moiety wherein the nitrogen atom of the amino group at position 1 is the linking position, GGFG represents an amino acid sequence consisting of glycine-phenylalanine-glycine linked through a peptide bond, and

- (succinimid-3-yl-N) -has a structure represented by the formula:

[ formula 2]

It is attached to the antibody at its position 3 and to the methylene group in the linker structure containing the structure at the nitrogen atom at position 1;

[33] the antibody-drug conjugate according to any one of [29] to [32], wherein the linker is represented by any one formula selected from the group consisting of the following formulae (c), (d), and (e):

(d) - (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C (= O) -, and

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-；

[34] The antibody-drug conjugate according to any one of [29] to [33], wherein the linker is represented by the following formula (c) or (e):

(e) - (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-；

[35] The antibody-drug conjugate according to any one of [29] to [34], which has a structure represented by the following formula:

[ formula 3]

[36] the antibody-drug conjugate according to any one of [29] to [34], which has a structure represented by the following formula:

[ formula 4]

[37] the antibody-drug conjugate according to any one of [29] to [36], wherein the antibody is an antibody comprising a light chain and a heavy chain selected from any one of the following combinations (1) to (4) or a functional fragment of the antibody:

(1) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 69,

(2) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 73,

(3) a light chain consisting of the amino acid sequence from position 21 to 233 in SEQ ID NO 65 and a heavy chain consisting of the amino acid sequence from position 20 to 471 in SEQ ID NO 73, and

(4) a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO 61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in SEQ ID NO 77;

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

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

[40] the antibody-drug conjugate according to any one of [29] to [39], wherein the heavy or light chain has undergone one or two or more modifications selected from: n-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of methionine residue to N-terminus, amidation of proline residue, conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid, and deletion of one or two amino acids from the carboxyl terminus;

[41] the antibody-drug conjugate according to any one of [29] to [40], wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 1 to 10;

[42] the antibody-drug conjugate of [41], wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 2 to 8;

[43] the antibody-drug conjugate of [42], wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 5 to 8;

[44] the antibody-drug conjugate of [43], wherein the average number of units of the selected drug-linker structure per antibody conjugate ranges from 7 to 8;

[45] a pharmaceutical composition comprising the antibody-drug conjugate according to any one of [29] to [44], a salt thereof, or a hydrate of the conjugate or the salt;

[46] the pharmaceutical composition according to [45], which is an antitumor agent;

[47] the pharmaceutical composition of [46], wherein the tumor is a CDH 6-expressing tumor;

[48] the pharmaceutical composition according to [46] or [47], wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma;

[49] a method for treating a tumor comprising administering to an individual any ingredient selected from the antibody-drug conjugate according to any one of [29] to [44], a salt thereof, and a hydrate of the conjugate or the salt;

[50] the method of treating according to [49], wherein the tumor is a CDH 6-expressing tumor;

[51] the method of treatment according to [49] or [50], wherein the tumor is renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor, or neuroblastoma;

[52] a method for treating a tumor, comprising administering to an individual simultaneously, separately or sequentially a pharmaceutical composition comprising at least one component selected from the antibody-drug conjugate according to any one of [29] to [44], a salt thereof and a hydrate of the conjugate or the salt, and at least one antitumor drug;

[53] a method for producing an antibody-drug conjugate, comprising the step of reacting the antibody or the functional fragment of the antibody according to any one of [1] to [13] and [24] to [28] or the antibody or the functional fragment of the antibody obtained by the production method according to [23] with a drug-linker intermediate compound; and

[54] a method for producing an antibody-drug conjugate, which comprises a step of culturing the host cell according to [21] or [22], a step of collecting an antibody of interest or a functional fragment of the antibody from the culture obtained by the above steps, and a step of reacting the antibody or the functional fragment of the antibody obtained by the above steps with a drug-linker intermediate compound.

Advantageous effects of the invention

The anti-CDH 6 antibody of the present invention is characterized by specifically recognizing EC domain 3(EC3) of CDH6 and having high internalization activity. It can be expected that, by administration to a patient having cancer cells expressing CDH6, an anti-CDH 6 antibody-drug conjugate comprising the anti-CDH 6 antibody of the present invention conjugated to a drug exerting toxicity in cells via a linker having a specific structure achieves excellent anti-tumor effect and safety. In particular, the anti-CDH 6 antibody-drug conjugates of the present invention are useful as anti-tumor agents.

Brief Description of Drawings

FIG. 1 shows the results of flow cytometry examining the binding of four rat anti-CDH 6 monoclonal antibodies (clone numbers rG019, rG055, rG056 and rG061) or rat IgG controls to control cells or hCDH6 transfected 293T cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIG. 2-1 shows the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or the negative control antibody rat IgG2b against control cells or full-length hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIG. 2-2 shows the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or rat IgG controls against control cells or EC 1-deleted hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIGS. 2-3 show the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or rat IgG controls against control cells or EC 2-deleted hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIGS. 2-4 show the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or rat IgG controls against control cells or EC 3-deleted hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIGS. 2-5 show the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or rat IgG controls against control cells or EC 4-deleted hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIGS. 2-6 show the binding activity of four rat anti-CDH 6 monoclonal antibodies (rG019, rG055, rG056, and rG061) or rat IgG controls against control cells or EC 5-deleted hCDH 6-transfected 293 cells. The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIG. 3 shows the results of flow cytometry evaluating the expression of CDH6 on the cell membrane surface of 4 types of human tumor cell lines (human ovarian tumor cell lines NIH: OVCAR-3, PA-1 and ES-2 and human renal cell tumor cell line 786-O). The abscissa plots FITC fluorescence intensity, which indicates the amount of antibody bound, and the ordinate plots cell count.

FIG. 4 shows a graph in which four types of rat anti-CDH 6 antibodies (rG019, rG055, rG056 and rG061) or rat IgG control were evaluated for internalization activity in NIH: OVCAR-3 cells and 786-O cells using the anti-rat IgG reagent rat-ZAP conjugated to the toxin inhibiting protein synthesis (saporin) or the goat anti-rat IgG not conjugated to the toxin as a negative control, Fc (γ) fragment (specificity). The ordinate of the graph depicts ATP activity (RLU). Cell viability (%) is shown below each graph, calculated as relative viability when the number of viable cells in wells supplemented with negative control, but not rat-ZAP, is defined as 100%.

FIG. 5 shows the binding of the human chimeric anti-CDH 6 antibody chG019 to human CDH6 and monkey CDH 6. The abscissa plots the antibody concentration, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity.

[ FIG. 6-1] FIGS. 6-1 and 6-2 each show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02) or the negative control antibody human IgG1 against human CDH6, monkey CDH6, mouse CDH6 and rat CDH 6. The abscissa plots the antibody concentration, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity.

[ FIG. 6-2] FIGS. 6-1 and 6-2 each show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02) or the negative control antibody human IgG1 against human CDH6, monkey CDH6, mouse CDH6 and rat CDH 6. The abscissa plots the antibody concentration, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity.

FIG. 7-1 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control antibody hIgG1 against control cells or full-length hCDH 6-transfected 293 α cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIG. 7-2 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control antibody hIgG1 against control cells or EC 1-deleted hCDH 6-transfected 293 alpha cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIGS. 7-3 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control antibody hIgG1 against control cells or EC 2-deleted hCDH 6-transfected 293 alpha cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIGS. 7-4 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control antibody hIgG1 against control cells or EC 3-deleted hCDH 6-transfected 293 alpha cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIGS. 7-5 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control hIgG1 against control cells or EC 4-deleted hCDH 6-transfected 293 α cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIGS. 7-6 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control hIgG1 against control cells or EC 5-deleted hCDH 6-transfected 293 α cells. The abscissa plots APC fluorescence intensity, which indicates the amount of bound antibody. The ordinate plots cell count.

FIG. 8 shows the results of flow cytometry examining the expression of human CDH6 in a 786-O/hCDH6 stably expressing cell line and its parental cell line 786-O. The abscissa plots Alexa Fluor 647 fluorescence intensity, which indicates the amount of bound antibody, and the ordinate plots cell count.

FIG. 9 shows binding competition assays for four unlabeled humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 or negative control hIgG1 using (a) labeled NOV0712 or (b) labeled H01L 02. The abscissa plots the final concentration of unlabeled antibody added, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity.

FIG. 10-1 shows a graph in which four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 and a negative control antibody were evaluated for internalizing activity in NIH: OVCAR-3 cells using the anti-human IgG reagent Hum-ZAP conjugated to the toxin that inhibits protein synthesis (saporin) or the F (ab')2 fragment goat anti-human IgG, Fc (γ) fragment (specific) not conjugated to the toxin as a negative control. The ordinate of the graph depicts ATP activity (RLU). Cell viability (%) calculated as relative viability when the number of viable cells in wells supplemented with negative control other than Hum-ZAP was defined as 100% is shown below each graph.

Fig. 10-2 shows a graph in which internalization activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), an anti-CDH 6 antibody NOV0712 and a negative control antibody was evaluated in 786-O cells using an anti-human IgG reagent Hum-ZAP conjugated to a toxin that inhibits protein synthesis (saporin) or a F (ab')2 fragment goat anti-human IgG that is not conjugated to a toxin as a negative control, Fc (γ) fragment (specificity). The ordinate of the graph depicts ATP activity (RLU). Cell viability (%) calculated as relative viability when the number of viable cells in wells supplemented with negative control other than Hum-ZAP was defined as 100% is shown below each graph.

Fig. 10-3 shows graphs in which internalization activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH 6 antibody NOV0712 and a negative control antibody was evaluated in PA-1 cells using an anti-human IgG reagent Hum-ZAP conjugated to a toxin that inhibits protein synthesis (saporin) or a F (ab')2 fragment goat anti-human IgG that is not conjugated to a toxin as a negative control, Fc (γ) fragment (specificity). The ordinate of the graph depicts ATP activity (RLU). Cell viability (%) calculated as relative viability when the number of viable cells in wells supplemented with negative control other than Hum-ZAP was defined as 100% is shown below each graph.

FIG. 11 shows the results of evaluating the in vitro cell growth inhibitory activity of four humanized hG 019-drug conjugates (H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) or NOV0712-DM4 against PA-1 cells. The abscissa plots the concentration of the antibody-drug conjugate, and the ordinate plots the cell viability (%).

FIG. 12 shows the in vivo antitumor effect of four humanized hG 019-drug conjugates (H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) or NOV0712-DM 4. Evaluation was performed using an animal model in which a CDH 6-positive human kidney cell tumor cell line 786-O was inoculated in immunodeficient mice. The abscissa plots days and the ordinate plots tumor volume. The error range depicts Standard Error (SE) values.

FIG. 13 shows the in vivo anti-tumor effect of humanized hG 019-drug conjugate H01L02-DXd or NOV0712-DM4 or NOV 0712-DXd. Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the CDH 6-positive human ovarian tumor cell line PA-1. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

FIG. 14 shows the in vivo anti-tumor effect of humanized hG 019-drug conjugate H01L02-DXd or NOV0712-DM 4. Evaluation was carried out using an animal model in which immunodeficient mice were inoculated with the CDH 6-positive human ovarian tumor cell line NIH: OVCAR-3. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

FIG. 15 shows the in vivo anti-tumor effect of humanized hG 019-drug conjugate H01L02-DXd or NOV0712-DM 4. Evaluation was performed using an animal model in which a CDH 6-positive human kidney cell tumor cell line 786-O was inoculated in immunodeficient mice. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

FIG. 16 shows the in vivo anti-tumor effect of humanized hG 019-drug conjugate H01L02-DXd or NOV0712-DM 4. Evaluation was performed using an animal model in which a CDH 6-negative human ovarian tumor cell line ES-2 was inoculated in immunodeficient mice. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

[ description of the embodiments ]

Hereinafter, preferred embodiments for implementing the present invention will be described with reference to the accompanying drawings. It should be noted that the embodiments described below merely illustrate representative embodiments of the present invention, and the scope of the present invention should not be narrowly construed in view of these examples.

In the present specification, the term "cancer" is used with the same meaning as that of the term "tumor".

In the present specification, the term "gene" is used to include not only DNA but also mRNA and cDNA thereof and cRNA thereof.

In the present specification, the term "polynucleotide" or "nucleotide" is used to have the same meaning as that of nucleic acid, and also includes DNA, RNA, probes, oligonucleotides and primers. In this specification, the terms "polynucleotide" and "nucleotide" may be used interchangeably with each other, unless otherwise specified.

In the present specification, the terms "polypeptide" and "protein" may be used interchangeably with each other.

In the present specification, the term "cell" includes cells in individual animals and cells in culture.

In the present specification, the term "CDH 6" may be used to have the same meaning as that of CDH6 protein.

In this specification, human CDH6 is also referred to as "hCDH 6".

In the present specification, the term "cytotoxic activity" is used to mean causing pathological changes to cells in any given way. The term is intended not only to mean direct trauma but also to cause all types of structural or functional damage to cells, such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to cell mitotic apparatus and reduced activity of various types of enzymes.

In the present specification, the phrase "exerts toxicity in a cell" is used to indicate that toxicity is exhibited in a cell in any given manner. The term is not only intended to refer to direct trauma, but also to all types of structural, functional or metabolic influences which cause cells, such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to the mitotic apparatus of cells, reduction in the activity of various types of enzymes and inhibition of the effect of cell growth factors.

In the present specification, the term "functional fragment of an antibody", also referred to as "antigen-binding fragment of an antibody", is used to mean a partial fragment of an antibody having a binding activity to an antigen, including Fab, F (ab')2, scFv, diabody, linear antibody formed from an antibody fragment, multispecific antibody, and the like. Also included in the antigen-binding fragments of antibodies are Fab ', which are monovalent fragments of the variable regions of antibodies obtained by treating F (ab')2 under reducing conditions. However, the antigen-binding fragment of the antibody is not limited to these molecules as long as the antigen-binding fragment has an antigen-binding ability. These antigen-binding fragments include not only those obtained by treating the full-length molecules of the antibody protein with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

In the present specification, the term "epitope" is used to indicate a partial peptide or partial three-dimensional structure of CDH6 to which a particular anti-CDH 6 antibody binds. Such an epitope, i.e. the partial peptide of CDH6 described above, can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of the antigen are generated. For generating such partial structures, known oligopeptide synthesis techniques can be applied. For example, a series of polypeptides in which CDH6 has been truncated consecutively from its C-terminus or N-terminus by an appropriate length is produced by genetic recombination techniques well known to those skilled in the art. Thereafter, the reactivity of the antibody to these polypeptides was investigated and the recognition site was roughly determined. Thereafter, further shorter peptides are synthesized and their reactivity towards these peptides can be subsequently investigated in order to determine the epitope. When an antibody that binds to a membrane protein having a plurality of extracellular domains targets a three-dimensional structure composed of a plurality of domains as an epitope, the domain to which the antibody binds can be determined by changing the amino acid sequence of a specific extracellular domain, and thereby changing the three-dimensional structure. Epitopes that are part of the three-dimensional structure of an antigen bound to a particular antibody can also be determined by pointing out the amino acid residues of the antigen adjacent to the antibody via X-ray structural analysis.

In the present specification, the phrase "antibody binding to the same epitope" is used to mean an antibody binding to a common epitope. If the second antibody binds to a portion of the peptide or a portion of the three-dimensional structure bound by the first antibody, it can be determined that the first and second antibodies bind to the same epitope. Alternatively, the first and second antibodies may be determined to bind the same epitope by confirming that the second antibody competes with the first antibody for binding of the first antibody to the antigen (i.e., the second antibody interferes with the binding of the first antibody to the antigen), even though the specific sequence or structure of the epitope has not been determined. In the present specification, the phrase "binds to the same epitope" refers to a case where the binding of the first antibody and the second antibody to the common epitope is determined by any one or both of these determination methods. When the first antibody and the second antibody bind to the same epitope and further the first antibody has a special effect such as an antitumor activity or an internalizing activity, the second antibody can be expected to have the same activity as that of the first antibody.

In the present specification, the term "CDR" is used to mean the complementarity determining region. It is known that the heavy and light chains of an antibody molecule each have three CDRs. Such CDRs are also known as hypervariable regions and are located in the variable regions of the heavy and light chains of antibodies. These regions have a particularly highly variable primary structure and are separated into three sites on the primary structure of the polypeptide chain of each of the heavy and light chains. In the present specification, with respect to the CDRs of an antibody, the CDRs of a heavy chain are referred to as CDRH1, CDRH2, and CDRH3, respectively, from the amino-terminal side of the amino acid sequence of the heavy chain, and the CDRs of a light chain are referred to as CDRL1, CDRL2, and CDRL3, respectively, from the amino-terminal side of the amino acid sequence of the light chain. These sites are located close to each other in three-dimensional structure and determine the specificity of the antibody for the antigen to which it binds.

In the present invention, the phrase "hybridization under stringent conditions" is used to mean that hybridization is carried out at 68 ℃ in a commercially available hybridization solution, ExpressHyb hybridization solution (manufactured by Clontech Laboratories, Inc.), or under conditions (or equivalent thereto) in which hybridization is carried out at 68 ℃ in the presence of 0.7 to 1.0M NaCl using a DNA immobilization filter, and the resultant is then washed with a 0.1 to 2-fold concentration of SSC solution (in which 1-fold concentration of SSC consists of 150mM NaCl and 15mM sodium citrate) at 68 ℃ for identification.

In this specification, the term "one to several" is used to mean 1 to 10,1 to 9,1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 or 2.

1. CDH6

CDH6 (cadherin 6) is a single transmembrane protein composed of 790 amino acids, which is classified as a type II cadherin family, and which has N-terminal extracellular and C-terminal intracellular domains. The human CDH6 gene was first cloned in 1995 (non-patent document 1), and its sequence can be referred to under, for example, accession numbers NM _004932 and NP _004923 (NCBI).

The CDH6 protein used in the present invention may be directly purified from a cell expressing CDH6 of a human or non-human mammal (e.g., rat, mouse, or monkey), and may be used subsequently, or a cell membrane fraction of the above cell may be prepared and may be used as a CDH6 protein. Alternatively, CDH6 can also be obtained by in vitro synthesis or by genetic manipulation to allow host cells to produce CDH 6. From this genetic manipulation, the CDH6 protein can be obtained, in particular, by incorporating the CDH6 cDNA into a vector capable of expressing the CDH6 cDNA, and then synthesizing the CDH6 in a solution containing enzymes, substrates and energy materials required for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes, thereby allowing them to express CDH 6. Cells expressing CDH6 or cell lines expressing CDH6 based on the above genetic manipulations can also be used to present CDH6 protein. Alternatively, an expression vector that has incorporated CDH6 cDNA can be administered directly to the animal to be immunized, and CDH6 can be expressed in the animal so immunized.

In addition, the term "CDH 6" also includes a protein consisting of an amino acid sequence including substitution, deletion and/or addition of one or more amino acids in the amino acid sequence of CDH6 described above, and having biological activity equivalent to that of CDH6 protein.

The human CDH6 protein has the amino acid sequence shown in SEQ ID NO. 1. The extracellular region of the human CDH6 protein consists of: an extracellular domain 1 (also referred to as EC1 in the present specification) having an amino acid sequence of positions 54 to 159 in the amino acid sequence shown in SEQ ID No. 1, an extracellular domain 2 (also referred to as EC2 in the present specification) having an amino acid sequence of positions 160 to 268 in the amino acid sequence shown in SEQ ID No. 1, an extracellular domain 3 (also referred to as EC3 in the present specification) having an amino acid sequence of positions 269 to 383 in the amino acid sequence shown in SEQ ID No. 1, an extracellular domain 4 (also referred to as EC4 in the present specification) having an amino acid sequence of positions 384 to 486 in the amino acid sequence shown in SEQ ID No. 1, and an extracellular domain 5 (in the present specification, also known as EC 5). The amino acid sequences of EC1 to EC5 are shown in SEQ ID NOS: 2 to 6, respectively (Table 1).

2. Production of anti-CDH 6 antibodies

One example of an anti-CDH 6 antibody of the invention can include an anti-CDH 6 antibody that recognizes an amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 4 and has internalization activity. One example of an anti-CDH 6 antibody of the invention can include an anti-CDH 6 antibody that specifically recognizes an amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 4 and has internalization activity. One example of an anti-CDH 6 antibody of the invention can include an anti-CDH 6 antibody that recognizes an amino acid sequence consisting of the amino acid sequence shown in SEQ ID No. 4 and has internalization activity. An example of the anti-CDH 6 antibody of the present invention may include an anti-CDH 6 antibody that specifically recognizes an amino acid sequence consisting of the amino acid sequence shown in SEQ ID No. 4 and has an internalizing activity. The phrase "specifically recognizes an amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 4" or "specifically recognizes the EC3 domain" as applied to an antibody is used to mean that the antibody strongly recognizes or binds to the EC3 domain of CDH6 compared to the other extracellular domains of CDH 6.

The anti-CDH 6 antibodies of the invention can be derived from any species. Preferred examples of such species may include human, monkey, rat, mouse and rabbit. When the anti-CDH 6 antibody of the invention is derived from a species other than human, the anti-CDH 6 antibody is preferably chimeric or humanized by well-known techniques. The antibody of the invention may be a polyclonal antibody, or may be a monoclonal antibody, and preferably is a monoclonal antibody.

The anti-CDH 6 antibodies of the invention are antibodies that can target tumor cells. Specifically, the anti-CDH 6 antibody of the present invention has the property of recognizing tumor cells, binding to tumor cells, internalizing into tumor cells by cellular uptake, and/or the like. Accordingly, the anti-CDH 6 antibody of the present invention can be conjugated with a compound having anti-tumor activity via a linker to prepare an antibody-drug conjugate.

The binding activity of the antibody to tumor cells can be confirmed by flow cytometry. Uptake of the antibody into the tumor cells can be confirmed by: (1) an assay for observing cellular uptake of antibodies using secondary antibodies (fluorescently labeled) that bind to the antibodies (Cell Death and Differentiation 2008, 15, 751-761), (2) an assay for measuring the amount of fluorescence taken up by cells using secondary antibodies (fluorescently labeled) that bind to the antibodies (Molecular Biology of Cell Vol.15, 5268-5282, 12 months 2004), or (3) a Mab-ZAP assay using immunotoxins that bind to the antibodies, wherein the toxins are released upon cellular uptake, thereby inhibiting Cell growth (Bio technologies 28: 162-165, 1 month 2000). Recombinant conjugated proteins of the catalytic region of diphtheria toxin and protein G are useful as immunotoxins.

In the present specification, the term "high internalization capacity" is used to indicate that the survival rate of cells expressing CDH6 (as indicated by the ratio relative to the survival rate of cells without added antibody defined as 100%) to which the above-described antibody and saporin-labeled anti-rat IgG antibody have been administered is preferably 70% or less, and more preferably 60% or less.

The anti-tumor antibody-drug conjugate of the present invention comprises a conjugated compound exerting an anti-tumor effect. Thus, preferably, but not necessarily, the antibody itself should have an anti-tumor effect. For the purpose of specifically and/or selectively exerting the cytotoxicity of the antitumor compound in tumor cells, it is important and preferred that the antibody should have the property of internalizing and transferring into tumor cells.

anti-CDH 6 antibodies can be obtained by: an animal is immunized with a polypeptide serving as an antigen according to a method generally practiced in the art, and then an antibody produced in its living body is collected and purified. CDH6 that retains a three-dimensional structure is preferably used as the antigen. Examples of such methods may include DNA immunization methods.

The source of the antigen is not limited to a human, and therefore an animal can also be immunized with an antigen derived from a non-human animal such as a mouse or a rat. In this regard, antibodies suitable for human disease can be selected by testing the obtained antibodies that bind to the heterologous antigen for cross-reactivity with human antigens.

Furthermore, an antibody-producing cell that produces an antibody against an antigen can be fused with a myeloma cell according to a known method (for example, Kohler and Milstein, Nature (1975)256, 495-497; and Kennet, R. editor, Monoclonal Antibodies, 365-367, Plenum Press, N. Y. (1980)) to establish a hybridoma, thereby obtaining a Monoclonal antibody.

Hereinafter, a method of obtaining an antibody against CDH6 will be specifically described.

(1) Preparation of antigens

The antigen can be obtained by allowing the host cell to produce a gene encoding an antigenic protein according to genetic manipulation. Specifically, a vector capable of expressing an antigen gene is produced, and then the vector is introduced into a host cell to express the gene therein, and then, the expressed antigen can be purified. The antibody can also be obtained by a method of immunizing an animal using an antigen-expressing cell based on the above genetic manipulation or a cell line expressing the antigen.

Alternatively, instead of using the antigen protein, an antibody against the antigen protein may be produced therein by incorporating cDNA of the antigen protein into an expression vector, then administering the expression vector to an animal to be immunized, and expressing the antigen protein in the animal thus immunized.

(2) Production of anti-CDH 6 monoclonal antibodies

The anti-CDH 6 antibody used in the present invention is not particularly limited. For example, an antibody designated by an amino acid sequence shown in the sequence listing of the present application can be suitably used. anti-CDH 6 antibodies for use in the invention are desirably antibodies having the following properties:

(1) an antibody having the following properties:

(a) specifically binds CDH6, and

(b) has activity of internalizing into a cell expressing CDH6 by binding to CDH 6;

(2) the antibody according to the above (1) or the above antibody, wherein the CDH6 is human CDH 6; and

(3) specifically recognizes EC3 of human CDH6, and has internalization activity.

The method for obtaining the antibody against CDH6 of the present invention is not particularly limited as long as an anti-CDH 6 antibody can be obtained. CDH6 that retains its conformation is preferably used as the antigen.

One preferred example of the method for obtaining the antibody may include a DNA immunization method. The DNA immunization method is a method involving: an individual animal (e.g., mouse or rat) is transfected with an antigen expression plasmid and then expresses an antigen in the individual to induce immunity against the antigen. The transfection method includes a method of directly injecting plasmids into muscles, a method of injecting transfection reagents such as liposomes or polyethyleneimine into veins, a method using viral vectors, a method of injecting gold particles to which plasmids are attached using a gene gun, a hydrodynamic method of rapidly injecting a large amount of plasmid solution into veins, and the like. As a transfection method for injecting an expression plasmid into a muscle, as a method for increasing the expression level, a technique called in vivo electroporation is known, which involves applying electroporation to the intramuscular injection site of the plasmid (Aihara H, Miyazaki J. Nat Biotechnol. 1998, 9 months; 16 (9): 867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaree P, Branellec D, Schwartz B, Scherman D. Proc Natl Acad Sci U S.1999, 4 months 13 days; 96 (8): 4262-7). The method further increases expression levels by treating the muscle with hyaluronidase followed by intramuscular injection of plasmids (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ., Gene ther. 2001 Aug; 8 (16): 1264-70). In addition, the generation of hybridomas can be performed by a known method, and can also be performed using, for example, the hybridoma production system (cytopulse Sciences, Inc.).

Specific examples of obtaining monoclonal antibodies may also include the following procedures:

(a) an immune response may be induced by incorporating the CDH6 cDNA into an expression vector (e.g., pcdna3.1; Thermo Fisher Scientific Inc.) and administering the vector directly to the animal to be immunized (e.g., rat or mouse) by methods such as electroporation or using a gene gun, thereby expressing CDH6 in the animal. If it is desired to increase the antibody titer, vector administration by electroporation or the like may be carried out one or more times, preferably a plurality of times;

(b) collecting tissues (e.g., lymph nodes) containing antibody-producing cells from the above-mentioned animal in which an immune response has been induced;

(d) cell fusion between antibody-producing cells and myeloma;

(e) selecting a group of hybridomas producing the target antibody;

(f) cloning into single cells;

(g) optionally, culturing the hybridoma for mass production of the monoclonal antibody, or breeding an animal into which the hybridoma is inoculated; and/or

(h) The physiological activity (internalization activity) and binding specificity of the monoclonal antibody thus produced were investigated, or the characteristics of the antibody as a labeling agent were examined.

Examples of methods for measuring antibody titers used herein may include, but are not limited to, flow cytometry and cell ELISA.

Examples of the hybridoma strains thus established may include hybridomas rG019, rG055, rG056 and rG061 which produce anti-CDH 6 antibody. It should be noted that, in the present specification, the antibody produced by the hybridoma rG019 which produces the anti-CDH 6 antibody is referred to as "rG 019 antibody" or simply "rG 019", the antibody produced by the hybridoma rG055 is referred to as "rG 055 antibody" or simply "rG 055", the antibody produced by the hybridoma rG056 is referred to as "rG 056 antibody" or simply "rG 056", and the antibody produced by the hybridoma rG061 is referred to as "rG 061 antibody" or simply "rG 061".

The variable region of the light chain 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 encoded by the nucleotide sequence shown in SEQ ID NO. 11. The light chain variable region of the rG019 antibody had a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:12, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:13, and a CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14. The heavy chain variable region of the rG019 antibody consists of the amino acid sequence shown in SEQ ID NO. 15. The amino acid sequence of the heavy chain variable region of the rG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 16. The heavy chain variable region of the rG019 antibody had a CDRH1 consisting of the amino acid sequence shown in SEQ ID NO:17, a CDRH2 consisting of the amino acid sequence shown in SEQ ID NO:18, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 19. The sequences of the rG019 antibodies are shown in table 1.

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

The variable region of the light chain 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 encoded by the nucleotide sequence shown in SEQ ID NO: 31. The light chain variable region of the rG056 antibody has a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:32, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:33, and a CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 34. The heavy chain variable region of the rG056 antibody consists of the amino acid sequence shown in SEQ ID NO: 35. The amino acid sequence of the heavy chain variable region of the rG056 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 36. The heavy chain variable region of the rG056 antibody has a CDRH1 consisting of the amino acid sequence shown in SEQ ID No. 37, a CDRH2 consisting of the amino acid sequence shown in SEQ ID No. 38, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID No. 39. The sequences of rG056 antibodies are shown in table 1.

The variable region of the light chain of the rG061 antibody consisted of the amino acid sequence shown in SEQ ID NO: 40. The amino acid sequence of the variable region of the light chain of the rG061 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 41. The light chain variable region of the rG061 antibody had a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:42, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:43, and a CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44. The heavy chain variable region of the rG061 antibody consisted of the amino acid sequence shown in SEQ ID NO: 45. The amino acid sequence of the heavy chain variable region of the rG061 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 46. The heavy chain variable region of the rG061 antibody had a CDRH1 consisting of the amino acid sequence shown in SEQ ID No. 47, a CDRH2 consisting of the amino acid sequence shown in SEQ ID No. 48, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID No. 49. The sequences of rG061 antibodies are shown in table 1.

Further, in the case where the steps (a) to (h) in the above-mentioned "production of anti-CDH 6 antibody" are carried out again to obtain monoclonal antibodies independently, respectively, and in the case where monoclonal antibodies are obtained separately by other methods, an antibody having an internalization activity equivalent to that of rG019 antibody, rG055 antibody, rG056 antibody or rG061 antibody can be obtained. One example of such an antibody may include an antibody that binds to the same epitope to which the rG019, rG055, rG056, or rG061 antibodies bind. If the newly prepared monoclonal antibody binds to a partial peptide or a partial three-dimensional structure to which the rG019 antibody, the rG055 antibody, the rG056 antibody, or the rG061 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope to which the rG019 antibody, the rG055 antibody, the rG056 antibody, or the rG061 antibody binds. Furthermore, by confirming that the monoclonal antibody competes with the rG019, rG055, rG056, or rG061 antibody for binding of the antibody to CDH6 (i.e., the monoclonal antibody interferes with the binding of the rG019, rG055, rG056, or rG061 antibody to CDH6), it can be determined that the monoclonal antibody binds to the same epitope as the anti-CDH 6 antibody, even if the specific sequence or structure of the epitope has not been determined. When it is confirmed that the monoclonal antibody binds to the same epitope as the rG019, rG055, rG056, or rG061 antibody binds, it is strongly expected that the monoclonal antibody should have an antigen binding capacity, biological activity, and/or internalization activity equivalent to that of the rG019, rG055, rG056, or rG061 antibody.

(3) Other antibodies

The antibodies of the present invention also include genetically recombinant antibodies, such as chimeric, humanized and human antibodies, which are artificially modified for the purpose of reducing heterologous antigenicity to humans, as well as the monoclonal antibodies against CDH6 described above. These antibodies can be produced by known methods.

Examples of chimeric antibodies may include antibodies in which the variable and constant regions are heterologous to each other, such as those formed by conjugating the variable region of a mouse or rat derived antibody to a human derived constant region (see proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)).

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

Other examples of chimeric antibodies derived from the rat anti-human CDH6 antibody include antibodies consisting of: a light chain comprising a light chain variable region of 1 to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of an amino acid in the light chain variable region of each rat anti-human CDH6 antibody described in the specification (e.g., rG019 antibody, rG055 antibody, rG056 antibody, or rG061 antibody) substituted with other amino acid residues, and a heavy chain comprising a heavy chain variable region of 1 to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of an amino acid in its heavy chain variable region substituted with other amino acid residues. The antibody may have any given human-derived constant region.

Other examples of chimeric antibodies derived from the rat anti-human CDH6 antibody include antibodies consisting of: a light chain comprising a light chain variable region in which 1 or 2 residues, preferably 1 residue, of amino acids in any 1 to 3 CDRs in the light chain variable region of each rat anti-human CDH6 antibody (e.g., rG019 antibody, rG055 antibody, rG056 antibody, or rG061 antibody) described in this specification is substituted with other amino acid residues, and a heavy chain comprising a heavy chain variable region in which 1 or 2 residues, preferably 1 residue, of amino acids in any 1 to 3 CDRs in the heavy chain variable region thereof is substituted with other amino acid residues. The antibody may have any given human-derived constant region.

Examples of chimeric antibodies derived from the rG019 antibody include an antibody composed of a light chain comprising a light chain variable region composed of the amino acid sequence shown in SEQ ID NO:10 and a heavy chain comprising a heavy chain variable region composed of the amino acid sequence shown in SEQ ID NO: 15. The antibody may have any given human-derived constant region.

Other examples of chimeric antibodies derived from the rG019 antibody include antibodies consisting of: a light chain comprising the light chain variable region substituted with other amino acid residues for one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of the amino acids in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO. 10, and a heavy chain comprising the heavy chain variable region substituted with other amino acid residues for one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of the amino acids in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO. 15. The antibody may have any given human-derived constant region.

Other examples of chimeric antibodies derived from the rG019 antibody include antibodies consisting of: a light chain comprising a light chain variable region in which 1 or 2 residues (preferably 1 residue) of the amino acids in any 1 to 3 CDRs in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:10 are substituted with other amino acid residues, and a heavy chain comprising a heavy chain variable region in which 1 or 2 residues (preferably 1 residue) of the amino acids in any 1 to 3 CDRs in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO:15 are substituted with other amino acid residues. The antibody may have any given human-derived constant region.

Other examples of chimeric antibodies derived from the rG019 antibody include an antibody consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:10 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 58. The antibody may have any given human-derived constant region. The amino acid sequence shown in SEQ ID NO. 58 is a sequence in which the cysteine residue in CDRH2 is substituted with a proline residue in the amino acid sequence shown in SEQ ID NO. 15.

Specific examples of chimeric antibodies derived from the rG019 antibody include an antibody consisting of a light chain consisting of the full-length amino acid sequence of the light chain shown in SEQ ID NO:53 and a heavy chain consisting of the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 56. In the present specification, the chimeric anti-human CDH6 antibody is referred to as "chimeric G019 antibody", "chG 019 antibody" or "chG 019". The full-length amino acid sequence of the light chain of the chG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 54, and the full-length amino acid sequence of the heavy chain of the chG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 57.

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

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

The sequence of the chG019 antibody is shown in table 1.

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

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

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

Examples of humanized antibodies may include an antibody formed by incorporating only Complementarity Determining Regions (CDRs) into a human-derived antibody (see Nature (1986)321, p. 522. sup. 525), an antibody formed by incorporating amino acid residues from some framework regions and CDR sequences into a human antibody according to a CDR-grafting method (International publication No. WO90/07861), and an antibody formed by modifying the amino acid sequences of some CDRs while retaining the antigen-binding ability.

In the present specification, the humanized antibody derived from the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody or the chG019 antibody is not limited to a specific humanized antibody as long as the humanized antibody retains all of the 6 CDR sequences of the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody or the chG019 antibody and has an internalizing activity. The amino acid sequences of some CDRs of the humanized antibody may be further modified so long as they have internalizing activity.

Specific examples of humanized antibodies to the chG019 antibody may include any given combination of: a light chain comprising a light chain variable region consisting of any one of the amino acid sequences selected from the group consisting of: (1) 63 or 67, (2) an amino acid sequence having at least 95% or more identity to the above amino acid sequence (1) (preferably, an amino acid sequence having at least 95% or more sequence identity to a sequence of the framework region other than each CDR sequence), and (3) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above amino acid sequence (1); and a heavy chain comprising a heavy chain variable region consisting of any one amino acid sequence selected from the group consisting of: (4) 71, 75 or 79, (5) an amino acid sequence having at least 95% or more identity to the above-mentioned amino acid sequence (4) (preferably an amino acid sequence having at least 95% or more sequence identity to the sequence of the framework region except for each CDR sequence), and (6) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above-mentioned amino acid sequence (4).

Alternatively, antibodies having a humanized heavy or light chain and another chain derived from a murine antibody or a chimeric antibody may also be used. Examples of such antibodies may include any given combination of: a light chain comprising a light chain variable region consisting of any one of the amino acid sequences selected from the group consisting of: (1) 63 or 67, (2) an amino acid sequence having at least 95% or more identity to the above amino acid sequence (1) (preferably, an amino acid sequence having at least 95% or more sequence identity to a sequence of the framework region other than each CDR sequence), and (3) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above amino acid sequence (1); and a heavy chain comprising a heavy chain variable region consisting of any one amino acid sequence selected from the group consisting of: (4) 15, 25, 35, 45 or 58, (5) an amino acid sequence having at least 95% or more identity to the above-mentioned amino acid sequence (4) (preferably an amino acid sequence having at least 95% or more sequence identity to a sequence of the framework region other than each CDR sequence), and (6) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above-mentioned amino acid sequence (4). Other examples of such antibodies may include any given combination of: a light chain comprising a light chain variable region consisting of any one of the amino acid sequences selected from the group consisting of: (1) 10, 20, 30 or 40, (2) an amino acid sequence having at least 95% or more identity to the above amino acid sequence (1) (preferably an amino acid sequence having at least 95% or more sequence identity to the sequence of the framework region other than each CDR sequence), and (3) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above amino acid sequence (1); and a heavy chain comprising a heavy chain variable region consisting of any one amino acid sequence selected from the group consisting of: (4) 71, 75 or 79, (5) an amino acid sequence having at least 95% or more identity to the above-mentioned amino acid sequence (4) (preferably an amino acid sequence having at least 95% or more sequence identity to the sequence of the framework region except for each CDR sequence), and (6) an amino acid sequence comprising deletion, substitution or addition of one or several amino acids in the above-mentioned amino acid sequence (4).

The amino acid substitutions in the present specification are preferably conservative amino acid substitutions. Conservative amino acid substitutions are those that occur within a group of amino acids that are related to certain amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid and glutamic acid; basic group = lysine, arginine, and histidine; nonpolar groups = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and uncharged polar family = glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide-containing groups = asparagine and glutamine; aliphatic = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Such amino acid substitution is preferably carried out without impairing the properties of the substance having the original amino acid sequence.

Examples of antibodies having the above-described preferred combination of light and heavy chains include antibodies consisting of: a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO:63 (also referred to as hL02 light chain variable region amino acid sequence in the present specification) or a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO:67 (also referred to as hL03 light chain variable region amino acid sequence in the present specification), and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO:71 (also referred to as hH01 heavy chain variable region amino acid sequence in the present specification), a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO:75 (also referred to as hH02 heavy chain variable region amino acid sequence in the present specification) or a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO:79 (also referred to as hH04 heavy chain variable region amino acid sequence in the present specification). Preferred examples thereof include: an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO:63 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 71; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:63 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 75; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:63 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 79; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:67 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 71; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:67 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 75; and an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:67 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 79. More preferable examples thereof include: an antibody consisting of a light chain having the light chain variable region amino acid sequence shown in SEQ ID NO:63 and a heavy chain having the heavy chain variable region amino acid sequence shown in SEQ ID NO: 71; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:63 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 75; an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:63 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 79; and an antibody consisting of a light chain having the amino acid sequence of the light chain variable region shown in SEQ ID NO:67 and a heavy chain having the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 75.

Other examples of antibodies having the preferred combination of light and heavy chains described above include antibodies consisting of: a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 (in the present specification, also referred to as the hL02 light chain full-length amino acid sequence) or a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 (in the present specification, also referred to as the hL03 light chain full-length amino acid sequence) and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:69 (in the present specification, also referred to as the hH01 heavy chain full-length amino acid sequence), a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:73 (in the present specification, also referred to as the hH02 heavy chain amino acid sequence) or an amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO Also known as the full-length amino acid sequence of the hH04 heavy chain). Preferred examples thereof include: an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 69; an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 73; an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 77; an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 69; an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 73; and an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65, and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 77. More preferable examples thereof include: an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:69 (also referred to as "H01L02 antibody" or "H01L 02" in the present specification); an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:73 (also referred to as "H02L02 antibody" or "H02L 02" in the present specification); an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:77 (also referred to as "H04L02 antibody" or "H04L 02" in the present specification); and an antibody composed of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65, and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO:73 (in the present specification, also referred to as "H02L 03 antibody" or "H02L 03"). The sequences of the H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody are shown in table 1.

By combining sequences showing high identity to the above-mentioned heavy chain amino acid sequence and light chain amino acid sequence, an antibody having biological activity equivalent to each of the above-mentioned antibodies can be selected. This identity is typically 80% or more, preferably 90% or more, more preferably 95% or more and most preferably 99% or more identity. Furthermore, an antibody having biological activity equivalent to each of the above-mentioned antibodies can also be selected by combining the amino acid sequences of the heavy chain and the light chain comprising substitution, deletion or addition of one or several amino acid residues thereof with respect to the amino acid sequence of the heavy chain or the light chain.

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

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

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

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

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

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

In the present specification, tables 1-1 to 1-15 are also collectively referred to as Table 1.

Other examples of antibodies of the invention can include human antibodies that bind to CDH 6. An anti-CDH 6 human antibody refers to a human antibody having only the gene sequences of an antibody derived from a human chromosome. anti-CDH 6 human antibodies can be obtained by methods using human antibody-producing mice having human chromosome fragments that contain the heavy and light chain genes of a human antibody (see Tomizuka, K. et al, Nature Genetics (1997)16, p. 133-.

Such human antibody-producing mice can be specifically produced by using genetically modified animals, in which the loci of endogenous immunoglobulin heavy and light chains have been disrupted, and instead introducing the loci of human immunoglobulin heavy and light chains using Yeast Artificial Chromosome (YAC) vectors and the like, followed by generating knockout animals and transgenic animals from such genetically modified animals, and then breeding these animals to each other.

Alternatively, according to genetic recombination techniques, an anti-CDH 6 human antibody can also be obtained by transforming eukaryotic cells with cDNA encoding each of the heavy and light chains of such a human antibody, or preferably with a vector comprising the cDNA, and then culturing the transformed cells to produce a genetically modified human monoclonal antibody, whereby the antibody can be obtained from the culture supernatant.

In this context, eukaryotic cells, and preferably mammalian cells, such as CHO cells, lymphocytes or myelomas, for example, can be used as hosts.

Furthermore, methods for obtaining phage display-derived human antibodies that have been selected from human antibody libraries are also known (see Wormstone, IM et al, Investigative Ophthalmology & Visual Science (2002)43(7), p.2301-2308; Carmen, S. et al, Briefings in Functional Genomics and protocols (2002), 1 (2), p.189-.

For example, a phage display method can be applied, which comprises expressing the variable region of a human antibody as a single chain antibody (scFv) on the phage surface, and then selecting a phage that binds to an antigen (Nature Biotechnology (2005),23, (9), pp. 1105-.

By analyzing phage genes selected for their ability to bind to an antigen, the DNA sequence encoding the variable region of a human antibody that binds to the antigen can be determined.

Once the DNA sequence of scFv binding to an antigen has been determined, an expression vector having the above-mentioned sequence is produced, and then the produced expression vector is introduced into a suitable host and allowed to express therein, thereby obtaining a human antibody (International publication Nos. WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388, Annu. Rev. Immunol (1994)12, p. 433-455, Nature Biotechnology (2005)23 (9), p. 1105-1116).

If a newly produced human antibody binds to a partial peptide or partial three-dimensional structure to which any of the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG055 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody) described in the specification binds, the human antibody can be determined to bind to the same epitope as the rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody, or humanized anti-human CDH6 antibody binds. Alternatively, by confirming that a human antibody competes for binding of the antibody to CDH6 with a rat anti-human CDH6 antibody, a chimeric anti-human CDH6 antibody, or a humanized anti-human CDH6 antibody (e.g., a rG019 antibody, a rG055 antibody, a rG056 antibody, a rG061 antibody, a chG019 antibody, a H01L02 antibody, a H02L02 antibody, a H02L03 antibody, or a H04L02 antibody) described in the present specification (e.g., the human antibody interferes with binding of the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody, the chG019 antibody, the H01L02 antibody, the H02L02 antibody, the H02L03 antibody, or the H04L02 antibody to CDH6 (preferably EC3 of CDH 6)), it can be determined that the human antibody binds to the rat anti-human CDH6, the chimeric anti-human CDH6 antibody, or the epitope of the same as the human antibody described in the present specification, even if the sequence is not determined to be the same as that the human antibody. In the present specification, when a human antibody "binds to the same epitope" is determined by at least any one of these determination methods, it is concluded that the newly prepared human antibody "binds to the same epitope" as the rat anti-human CDH6 antibody, the chimeric anti-human CDH6 antibody, or the humanized anti-human CDH6 antibody described in the present specification. When it is confirmed that a human antibody binds to the same epitope, it is expected that the human antibody should have biological activity equivalent to a rat anti-human CDH6 antibody, a chimeric anti-human CDH6 antibody, or a humanized anti-human CDH6 antibody (e.g., rG019 antibody, rG055 antibody, rG056 antibody, rG061 antibody, chG019 antibody, H01L02 antibody, H02L02 antibody, H02L03 antibody, or H04L02 antibody).

The chimeric antibody, humanized antibody or human antibody obtained by the above method is evaluated for its binding activity to an antigen according to a known method or the like, so that a preferred antibody can be selected.

One example of another indicator for comparing antibody properties may include the stability of the antibody. A Differential Scanning Calorimeter (DSC) is a device capable of rapidly and accurately measuring the thermal denaturation midpoint (Tm) which serves as a good indicator of the relative structural stability of a protein. By measuring the Tm value using DSC and comparing the obtained values, the difference in thermal stability can be compared. It is known that the storage stability of an antibody has a certain correlation with the thermostability of the antibody (Lori Burton et al, Pharmaceutical Development and Technology (2007)12, pages 265-273), and therefore, a preferred antibody can be selected using the thermostability as an index. Other examples of other indicators for selecting antibodies may include high yield in a suitable host cell and low aggregability in aqueous solution. For example, since the antibody having the highest productivity does not always exhibit the highest thermostability, it is necessary to select the antibody most suitable for administration to humans by comprehensively determining it based on the above-mentioned indexes.

The antibodies of the invention also include modifications of the antibodies. Such modifications are intended to mean antibodies of the invention, which are chemically or biologically modified. Examples of such chemical modifications include the attachment of chemical moieties to the amino acid backbone, and the chemical modification of N-linked or O-linked carbohydrate chains. Examples of such biological modifications include antibodies that have undergone post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, and conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid), and antibodies that have added a methionine residue to their N-terminus as a result of allowing expression using a prokaryotic host cell. In addition, such modifications are meant to also include labeled antibodies, such as enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies, capable of detecting or isolating the antibodies or antigens of the invention. Such modifications of the antibodies of the invention can be used to improve stability and retention in blood of the antibodies, reduce antigenicity, detect or isolate the antibodies or antigens, and the like.

Furthermore, by adjusting the sugar chain modification (glycosylation, defucosylation, etc.) bound to the antibody of the present invention, the antibody-dependent cellular cytotoxic activity can be enhanced. As techniques for modulating sugar chain modification of an antibody, those described in International publication Nos. WO1999/54342, WO2000/61739, WO2002/31140 and the like are known, but the techniques are not limited thereto. The antibody of the present invention also includes an antibody in which the above-described sugar chain modification has been modulated.

Once the antibody gene is isolated, the gene can be introduced into an appropriate host using an appropriate combination of host and expression vector to produce an antibody. A specific example of the antibody gene may be a combination of a gene encoding a heavy chain sequence of the antibody described in the specification and a gene encoding a light chain sequence of the antibody described therein. After transformation of the host cell, such heavy chain sequence gene and light chain sequence gene may be inserted into a single expression vector, or these genes may be each inserted into a different expression vector.

When a eukaryotic cell is used as the host, an animal cell, a plant cell, or a eukaryotic microorganism can be used. Specifically, examples of the animal cells may include mammalian cells such as COS cells as monkey cells (Gluzman, Y., Cell (1981)23, p.175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC number CRL-1658), dihydrofolate reductase-deficient Cell line of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci.U.S.A. (1980)77, p.4126-4220), and FreeStyle 293F cells (Invitrogen Corp.).

When prokaryotic cells are used as hosts, for example, Escherichia coli (E.coli) ((E.coli))Escherichia coli) Or Bacillus subtilis (A), (B)Bacillus subtilis)。

A gene of an antibody of interest is introduced into these cells for transformation, and the transformed cells are then cultured in vitro to obtain the antibody. In the above culture, there are cases where the yield differs depending on the sequence of the antibody, and therefore an antibody that is easily produced as a drug can be selected from antibodies having equivalent binding activity using the yield as an index. Accordingly, the antibody of the present invention also includes an antibody obtained by the above-described antibody production method, which includes a step of culturing the transformed host cell and a step of collecting the antibody of interest or a functional fragment of the antibody from the culture obtained in the above-described step.

It is known that the carboxy-terminal lysine residue of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)), and further, it is known that the two amino acid residues glycine and lysine at the carboxy-terminal of the heavy chain are deleted and the proline residue at the carboxy-terminal is amidated (Analytical Biochemistry, 360:75-83 (2007)). However, deletion and modification of these heavy chain sequences have no effect on the antigen-binding activity and effector function of the antibody (activation of complement, antibody-dependent cellular cytotoxicity, etc.). Therefore, the antibody according to the present invention also includes an antibody that has undergone the above-described modification, and a functional fragment of the antibody, and specific examples of such an antibody include a deletion mutant comprising a deletion of 1 or 2 amino acids at the carboxy terminus of the heavy chain, and a deletion mutant formed by amidating the above-described deletion mutant (e.g., a heavy chain in which a proline residue at the carboxy terminus is amidated). However, deletion mutants related to deletion of the carboxy terminus of the heavy chain of the antibody according to the present invention are not limited to the above deletion mutants as long as they retain antigen binding activity and effector function. The two heavy chains constituting the antibody according to the present invention may be any type of heavy chain selected from the group of a full-length antibody and the above deletion mutant, or may be a combination of any two types selected from the above group. The ratio of individual deletion mutants may be influenced by the type of mammalian cell cultured to produce the antibody of the present invention and the culture conditions. Examples of the main component of the antibody according to the present invention may include an antibody in which one amino acid residue is deleted at each carboxyl terminal of two heavy chains.

Examples of the antibody isotype of the present invention may include IgG (IgG1, IgG2, IgG3, and IgG 4). Among them, IgG1 and IgG4 are preferable.

Examples of the biological activity of an antibody may generally include antigen-binding activity, activity of internalization into a cell expressing an antigen by binding to the antigen, activity of neutralizing the antigen activity, activity of enhancing the antigen activity, antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent phagocytosis (ADCP). The function of the antibodies according to the invention is the binding activity to CDH6, and preferably the activity of internalization into cells expressing CDH6 by binding to CDH 6. Furthermore, the antibody of the present invention may have ADCC activity, CDC activity and/or ADCP activity, as well as cell internalization activity.

The obtained antibody can be purified to a homogeneous state. For the isolation and purification of the antibody, an isolation and purification method for a general protein may be used. For example, column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis and isoelectric focusing are appropriately selected and combined with each other so that Antibodies can be isolated and purified (stratgies for protein Purification and chromatography: A Laboratory Course Manual, edited by DanielR. Marshak et al, Cold Spring Harbor Laboratory Press (1996) and Antibodies: organism Manual, Ed Harbor and David Lane, Cold Spring Harbor Laboratory (1988)), but examples of the isolation and Purification method are not limited thereto.

Examples of the chromatography may include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.

These chromatographic techniques may be performed using liquid chromatography such as HPLC or FPLC.

Examples of the column for affinity chromatography may include a protein a column and a protein G column. Examples of columns involving the use of protein a may include Hyper D, POROS and Sepharose f.f. (Pharmacia).

In addition, using an antigen-immobilized carrier, an antibody can be purified by utilizing the binding activity of the antibody to an antigen.

3. anti-CDH 6 antibody-drug conjugates

(1) Medicine

The anti-CDH 6 antibody obtained in the above "production of anti-CDH 6 antibody" may be conjugated to a drug through a linker moiety to prepare an anti-CDH 6 antibody-drug conjugate. The drug is not particularly limited as long as it has a substituent or partial structure that can be attached to the linker structure. Depending on the conjugated drug, the anti-CDH 6 antibody-drug conjugate can be used for various purposes. Examples of such a drug may include a substance having an antitumor activity, a substance effective for hematological diseases, a substance effective for autoimmune diseases, an anti-inflammatory substance, an antimicrobial substance, an antifungal substance, an antiparasitic substance, an antiviral substance and an anti-anesthetic substance.

(1) -1 antitumor Compounds

Examples of using an anti-tumor compound as a compound conjugated in the anti-CDH 6 antibody-drug conjugate of the present invention will be described below. The antitumor compound is not particularly limited as long as the compound has an antitumor effect and has a substituent or partial structure that can be attached to the linker structure. After cleavage of part or all of the linker in the tumor cell, part of the antitumor compound is released, so that the antitumor compound exhibits an antitumor effect. When the linker is cleaved at the site of attachment to the drug, the anti-tumor compound is released in its original structure to exert its original anti-tumor effect.

The anti-CDH 6 antibody obtained in the above "production of anti-CDH 6 antibody" may be conjugated with an anti-tumor compound through a linker moiety to prepare an anti-CDH 6 antibody-drug conjugate.

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

[ formula 5]

The compound can be easily obtained by, for example, a method described in U.S. patent publication No. US2016/0297890 or other known methods, and the amino group at position 1 can be preferably used as a linking site to a linker structure. Further, the irinotecan can be released in the tumor cells while the portion of the linker remains attached thereto. However, even in this state, the compound exerts an excellent antitumor effect.

Since irinotecan has a camptothecin structure, it is known that the equilibrium shifts to a structure having a lactone ring formed (closed ring) in an acidic aqueous medium (e.g., pH of about 3), and to a structure having an lactone ring opened (open ring) in a basic aqueous medium (e.g., pH of about 10). Drug conjugates having introduced the residues of irinotecan corresponding to such closed-loop and open-loop structures are also expected to have equivalent antitumor effects, and needless to say, any such drug conjugates are included within the scope of the present invention.

Other examples of anti-tumor compounds may include anti-tumor compounds described in the literature (pharmaceutical Reviews, 68, pages 3-19, 2016). Specific examples thereof may include doxorubicin, calicheamicin, dolastatin 10, auristatins, such as monomethyl auristatin e (mmae) and monomethyl auristatin f (mmaf); maytansinoids, such as DM1 and DM 4; pyrrolobenzodiazepine dimer SG2000 (SJG-136), camptothecin derivative SN-38, duocarmycins such as CC-1065, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antineoplastic agents (cisplatin and its derivatives) and paclitaxel and its derivatives.

In antibody-drug conjugates, the number of conjugated drug molecules per antibody molecule is a key factor that has an impact on its efficacy and safety. The production of antibody-drug conjugates is performed by specifying reaction conditions, such as the amounts of starting materials and reagents used for the reaction, so as to obtain a constant number of conjugated drug molecules. Unlike the chemical reaction of low molecular weight compounds, mixtures containing different numbers of conjugated drug molecules are often obtained. The number of conjugated drug molecules per antibody molecule is defined and expressed as the average, i.e. the average number of conjugated drug molecules. Unless otherwise indicated, i.e., in addition to representing antibody-drug conjugates having a specific number of conjugated drug molecules contained in a mixture of antibody-drug conjugates having different numbers of conjugated drug molecules, the number of conjugated drug molecules according to the present invention also generally represents an average value. The number of irinotecan molecules conjugated to the antibody molecule is controllable and, as the average number of conjugated drug molecules per antibody, can conjugate about 1 to 10 irinotecan molecules. The number of molecules of irinotecan is preferably from 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8, more preferably from 5 to 8, further preferably from 7 to 8, and still more preferably 8. It is noted that one skilled in the art can design reactions for conjugating a desired number of drug molecules to antibody molecules based on the description of the examples herein, and can obtain antibody-drug conjugates with a controlled number of conjugated irinotecan molecules.

(2) Joint structure

The linker structure conjugating the drug to the anti-CDH 6 antibody in the anti-CDH 6 antibody-drug conjugate of the present invention will now be described.

In the antibody-drug conjugate of the present application, the linker structure to conjugate the anti-CDH 6 antibody to the drug is not particularly limited as long as the resulting antibody-drug conjugate can be used. The joint structure may be appropriately selected and used according to the purpose of use. An example of the linker structure may include linkers described in known documents (Pharmacol Rev 68: 3-19, 2016, 1 month, Protein Cell DOI 10.1007/s13238-016 0323-0, etc.). Further specific examples thereof may include VC (valine-citrulline), MC (maleimidocaproyl), SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate), SPP (N-succinimidyl 4- (2-pyridyldithio) valerate, SS (disulfide), SPDB (N-succinimidyl 4- (2-pyridyldithio) butyrate, SS/hydrazone, and carbonate.

Another example may include the linker structure described in U.S. patent publication No. US2016/0297890 (as one example, those described in paragraphs [0260] to [0289] thereof). Any joint structure given below may be preferably used. It should be noted that the left end of the structure is the attachment site to the antibody, and the right end thereof is the attachment site to the drug. Further, GGFG in the linker structure given below represents an amino acid sequence consisting of glycine-phenylalanine-glycine (GGFG) linked by peptide bonds.

- (succinylidene)amine-3-yl-N) -CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

More preferably the following:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

Still more preferably the following:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

Antibodies are attached to the terminus of- (succinimidyl-3-yl-N) (e.g., with "-CH)₂CH₂CH₂CH₂CH₂- (succinimid-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂The end opposite to the end of linkage in-C (= O) - "(left end)), and an antitumor compound is linked to the end opposite to the end of antibody linkage- (succinimidyl-3-yl-N) (in the above examples, CH at the right end)₂-O-CH₂-carbonyl of C (= O) -). "- (succinimid-3-yl-N) -" has a structure represented by the formula:

[ formula 6]

Position 3 of the partial structure is the attachment position of the anti-CDH 6 antibody. This linkage to the antibody at position 3 is characterized by the formation of a thioether bond. The nitrogen atom at position 1 of the moiety is attached to a carbon atom comprising a methylene group present within the linker of the structure.

In the antibody-drug conjugate of the present invention having irinotecan as a drug, a drug-linker moiety having any of the structures given below is preferably used for conjugation to the antibody. For these drug-linker moieties, the average number of conjugates per antibody may be 1 to 10, and preferably 2 to 8, more preferably 5 to 8, further preferably 7 to 8, and still more preferably 8.

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-(NH-DX)，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-(NH-DX)，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-(NH-DX)，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C(=O)-(NH-DX)，

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C (= O) - (NH-DX), and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-(NH-DX)。

More preferably the following:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-(NH-DX)，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C (= O) - (NH-DX), and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-(NH-DX)。

Still more preferably the following:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C (= O) - (NH-DX), and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-(NH-DX)。

- (NH-DX) has a structure represented by the following formula:

[ formula 7]

And it represents a group derived by removing one hydrogen atom of the amino group at position 1 of irinotecan.

(3) Methods of producing antibody-drug conjugates

The antibody that can be used in the antibody-drug conjugate of the present invention is not particularly limited as long as it is an anti-CDH 6 antibody or a functional fragment of the antibody having internalization activity, as described above in the section "production of anti-CDH 6 antibody" and examples.

Next, a typical method of producing the antibody-drug conjugate of the present invention will be described. It should be noted that in the following description, "compound number" shown in each reaction scheme is used to represent a compound. Specifically, each compound is referred to as "a compound of formula (1)", "a compound (1)", and the like. The same is true for the other compound numbers.

(3) -1 production method 1

An antibody-drug conjugate represented by formula (1) given below (in which an anti-CDH 6 antibody is linked to a linker structure via a thioether) can be produced by: an antibody having a thiol group converted from a disulfide bond by reducing an anti-CDH 6 antibody is reacted with a compound (2), which compound (2) can be obtained by a known method (for example, can be obtained by a method described in patent publication US2016/297890 (for example, a method described in paragraphs [0336] to [0374 ]). For example, the antibody-drug conjugate can be produced by:

[ expression 1]

Wherein AB represents an antibody having a thiol group, wherein

L¹Has a structure represented by- (succinimidin-3-yl-N) -, and

L¹' represents a maleimide group represented by the following formula:

[ formula 8]

-L¹-L^XHas a structure represented by any one of the following formulae:

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

Among them, the following are more preferable:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C(=O)-，

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂-O-CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

Further preferred is the following:

- (succinimidin-3-yl-N) -CH₂CH₂CH₂CH₂CH₂-C(=O)-GGFG-NH-CH₂-O-CH₂-C (= O) -, and

- (succinimidin-3-yl-N) -CH₂CH₂-C(=O)-NH-CH₂CH₂O-CH₂CH₂O-CH₂CH₂-C(=O)-GGFG-NH-CH₂CH₂CH₂-C(=O)-。

In the above reaction scheme, the antibody-drug conjugate (1) can be understood as having a structure in which one structural portion from the drug to the end of the linker is linked to one antibody. However, this description is given for convenience, and there are actually many cases in which a plurality of the above-described structural portions are linked to one antibody molecule. The same is true for the explanation of the generation method described below.

Specifically, the antibody-drug conjugate (1) can be produced by reacting the compound (2) obtainable by a known method (e.g., obtainable by a method described in patent publication US2016/297890 (e.g., obtainable by a method described in paragraphs [0336] to [0374 ])) with the antibody (3a) having a thiol group.

The antibody having a thiol group (3a) can be obtained by a method well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). Examples of such methods may include, but are not limited to: reacting the Traut reagent with the amino group of the antibody; reacting N-succinimidyl S-acetylthioalkanoate with the amino group of the antibody, followed by reaction with hydroxylamine; reacting N-succinimidyl 3- (pyridyldithio) propionate with an antibody, followed by reaction with a reducing agent; the antibody is reacted with a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to reduce interchain disulfide bonds in the antibody, thereby forming thiol groups.

Specifically, an antibody having a partially or fully reduced interchain disulfide bond can be obtained by using 0.3 to 3 molar equivalents of TCEP as a reducing agent for each interchain disulfide bond in the antibody and reacting the reducing agent with the antibody in a buffer containing a chelating agent. Examples of the chelating agent may include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The chelating agent may be used at a concentration of 1mM to 20 mM. As the buffer solution, a solution of sodium phosphate, sodium borate, sodium acetate, or the like can be used. As a specific example, the antibody (3a) having a partially or completely reduced thiol group can be obtained by reacting the antibody with TCEP at 4 ℃ to 37 ℃ for 1 to 4 hours.

It should be noted that by performing an addition reaction of a thiol group with a drug-linker moiety, the drug-linker moiety may be conjugated through a thioether bond.

Then, using 2 to 20 molar equivalents of compound (2) for each antibody (3a) having a thiol group, an antibody-drug conjugate (1) in which 2 to 8 drug molecules are conjugated per antibody can be produced. Specifically, a solution containing the compound (2) dissolved therein may be added to a buffer solution containing the antibody (3a) having a thiol group for reaction. In this regard, sodium acetate solution, sodium phosphate, sodium borate solution, etc. may be used as the buffer solution. The pH of the reaction is 5 to 9, and more preferably, the reaction may be carried out at around pH7. An organic solvent such as dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), Dimethylacetamide (DMA), or N-methyl-2-pyrrolidone (NMP) can be used as a solvent for dissolving the compound (2). The reaction can be carried out by adding a solution containing the compound (2) dissolved in an organic solvent to a buffer solution containing the antibody (3a) having a thiol group at 1 to 20% v/v. The reaction temperature is 0 to 37 ℃, more preferably 10 to 25 ℃, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by inactivating the reactivity of the unreacted compound (2) using a thiol-group-containing reagent. Thiol-containing agents are, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC to the compound (2) used and incubating the resulting mixture at room temperature for 10 to 30 minutes.

(4) Identification of antibody-drug conjugates

The resulting antibody-drug conjugate (1) can be concentrated, buffer exchanged, purified and the antibody concentration and the average number of conjugated drug molecules per antibody molecule can be measured according to the following conventional procedures to identify the antibody-drug conjugate (1).

(4) -1 general procedure a: concentration of aqueous solutions of antibodies or antibody-drug conjugates

To Amicon Ultra (50,000 MWCO, Millipore Corporation) containers, solutions of antibody or antibody-drug conjugate were added and the solutions of antibody or antibody-drug conjugate were concentrated by centrifugation (centrifugation at 2000G to 3800G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

(4) -2 conventional procedure B: measurement of antibody concentration

The measurement of antibody concentration was performed according to the method defined by the manufacturer using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.). In this connection, the absorption coefficient at 280nm (1.3 mLmg) which differs between antibodies was used^-1cm^-1To 1.8 mLmg^-1cm^-1)。

(4) -3 general procedure C: buffer exchange of antibodies

NAP-25 column using Sephadex G-25 vector (catalog No. 17-0852-02, GE Healthcare Japan Corporation) was equilibrated with phosphate buffer (50mM, pH6.0) containing sodium chloride (50mM) and EDTA (2mM) (referred to as PBS6.0/EDTA in the present specification) according to the manufacturer's defined method. An aqueous antibody solution was applied in an amount of 2.5mL per NAP-25 column, and then a fraction eluted with 3.5mL of PBS6.0/EDTA (3.5mL) was collected. The fraction was concentrated by conventional procedure a. After measuring the antibody concentration using conventional procedure B, the antibody concentration was adjusted to 20mg/mL using PBS 6.0/EDTA.

(4) 4 general procedure D: purification of antibody-drug conjugates

The NAP-25 column is equilibrated with any commercially available buffer solution, e.g., acetate buffer (10mM, pH 5.5; referred to herein as ABS) containing sorbitol (5%). An aqueous reaction solution (about 2.5mL) of the antibody-drug conjugate was applied to a NAP-25 column, and then eluted with a manufacturer-defined amount of buffer solution to collect an antibody fraction. The gel filtration purification process, in which the collected fraction was applied to the NAP-25 column again and eluted with a buffer solution, was repeated 2 or 3 times in total to obtain an antibody-drug conjugate, excluding unconjugated drug linker and low molecular weight compounds (tris (2-carboxyethyl) phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide).

(4) -5 general procedure E: measurement of antibody concentration and average number of conjugated drug molecules per antibody molecule in antibody-drug conjugates

The conjugate drug concentration in the antibody-drug conjugate can be calculated as follows: UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths of 280nm and 370nm was measured, and then the calculation as shown below was performed.

The total absorbance at any given wavelength is equal to the sum of the absorbances of all light absorbing chemicals present in the system [ the additivity of the absorbances ]. Therefore, the antibody concentration and the drug concentration in the antibody-drug conjugate are represented by the following equations, based on the assumption that the molar absorption coefficients of the antibody and the drug are unchanged before and after the conjugation of the antibody and the drug.

A₂₈₀ = A_D,280+ A_A,280 = ε_D,280C_D+ ε_A,280C_AEquation (1)

A₃₇₀ = A_D,370 + A_A,370 = ε_D,370C_D + ε_A,370C_AEquation (2).

In this context, A₂₈₀Represents the absorbance at 280nm of an aqueous solution of the antibody-drug conjugate, A₃₇₀Represents the absorbance at 370nm of an aqueous solution of the antibody-drug conjugate, A_A，280Denotes the absorbance of the antibody at 280nm, A_A，370Denotes the absorbance of the antibody at 370nm, A_D，280Denotes the absorbance at 280nm of the conjugate precursor, A_D，370Denotes the absorbance, ε, of the conjugate precursor at 370nm_A，280Denotes the molar absorptivity, ε, of the antibody at 280nm_A，370Represents the molar absorptivity, ε, of an antibody at 370nm_D，280Denotes the molar absorptivity, ε, of the conjugate precursor at 280nm_D，370Denotes the molar absorption coefficient at 370nm of the conjugate precursor, C_ADenotes the concentration of antibody in the antibody-drug conjugate, and C_DIndicates the drug concentration in the antibody-drug conjugate.

In this context, with respect to ε_A,280，ε_A,370，ε_D,280And ε_D,370A previously prepared value (based on a calculated estimate or a measured value obtained by UV measurement of the compound) is used. For example, ε can be estimated from the amino acid sequence of an antibody by a known calculation method (protein science, 1995, vol.4, 2411-2423)_A,280。ε_A,370Typically zero. Epsilon_D,280And ε_D,370Can be obtained by measuring the absorbance of a solution in which the conjugate precursor used is dissolved at a molar concentration according to Lambert-Beer's law (absorbance = molar concentration ✕ molar absorption coefficient ✕ cell channel length). C_AAnd C_DCan be measured by measuring A of an aqueous solution of the antibody-drug conjugate₂₈₀And A₃₇₀And then determined by solving simultaneous equations (1) and (2) by replacing these values. Furthermore, by C_DIs divided by C_AThe conjugated drug molecules per antibody can be determinedAverage of (d).

(4) -6 conventional procedure F: measurement of average number of conjugated drug molecules per antibody molecule in antibody-drug conjugate- (2)

In addition to the above-described "(4) -5 routine procedure E", the average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate can also be determined by High Performance Liquid Chromatography (HPLC) analysis using the following method. Hereinafter, a method of measuring the average number of conjugated drug molecules by HPLC when an antibody is conjugated to a drug linker by a disulfide bond will be described. Referring to this method, one skilled in the art can suitably measure the average number of conjugated drug molecules by HPLC, depending on the manner of linkage between the antibody and the drug linker.

F-1 preparation of samples for HPLC analysis (reduction of antibody-drug conjugates)

The antibody-drug conjugate solution (about 1mg/mL, 60. mu.L) was mixed with an aqueous solution of Dithiothreitol (DTT) (100mM, 15. mu.L). The disulfide bond between the light and heavy chains of the antibody-drug conjugate was cleaved by incubating the mixture at 37 ℃ for 30 minutes. The resulting sample was used for HPLC analysis.

HPLC analysis of F-2

HPLC analysis was performed under the following measurement conditions.

HPLC system: agilent 1290 HPLC System (Agilent Technologies, Inc.)

A detector: ultraviolet absorption spectrometer (measuring wavelength: 280nm)

Column: ACQUITY UPLC BEH phenyl (2.1 ✕ 50mm, 1.7 μm, 130 angstroms; Waters Corp., P/N186002884)

Column temperature: 80 deg.C

Mobile phase A: aqueous solution containing 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol

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 min), 80% -14% (17 min-17.01 min) and 14% (17.01 min-25 min)

Sample injection: 10 μ L.

F-3. data analysis

F-3-1. light chain bound to drug molecules (light chain linked to i drug molecules: L) in comparison to unconjugated antibody light chain (L0) and heavy chain (H0)_i) And a heavy chain binding to a drug molecule (heavy chain linked to i drug molecules: h_i) Exhibit higher hydrophobicity in proportion to the number of conjugated drug molecules and thus have a greater retention time. Thus, these chains elute in the order of, for example, L0 and L1 or H0, H1, H2 and H3. By comparison with retention times of L0 and H0, a detection peak can be assigned to any one of L0, L1, H0, H1, H2, and H3. The number of conjugated drug molecules can be defined by the person skilled in the art, but is preferably L0, L1, H0, H1, H2 and H3.

F-3-2. since the drug linker has UV absorption, the peak area values are corrected in response to the number of conjugated drug linker molecules using the molar absorption coefficients of the light or heavy chain and the drug linker according to the following expression.

In this connection, the value estimated from the amino acid sequence of the light chain or heavy chain of each antibody can be used as the molar absorption coefficient (280nm) of the light chain or heavy chain of the antibody by a known calculation method (Protein Science, 1995, vol.4, 2411-2423). In the case of H01L02, molar absorption coefficients 31710 and 79990 were used as estimates of the light and heavy chains, respectively, depending on the amino acid sequence of the antibody. The actually measured molar absorptivity (280nm) of a compound in which a maleimide group has been converted to a succinimide thioether by the reaction of each drug linker with mercaptoethanol or N-acetylcysteine was used as the molar absorptivity (280nm) of the drug linker. The wavelength for the absorbance measurement may be appropriately set by those skilled in the art, but is preferably a wavelength capable of measuring an antibody peak, more preferably 280 nm.

F-3-3. calculating the peak area ratio (%) of each chain according to the following expression to obtain the sum of corrected values of peak areas.

F-3-4. the average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate was calculated according to the following expression.

Average number of conjugated drug molecules = (L)₀Peak area ratio X0 + L₁Peak area ratio X1 + H₀Peak area ratio X0 + H₁Peak area ratio X1 + H₂Peak area ratio X2 + H₃Peak area ratio x 3)/100 x 2.

It should be noted that, in order to ensure the amount of antibody-drug conjugate, a plurality of antibody-drug conjugates having almost the same average number of conjugated drug molecules (e.g., about ± 1) that have been produced under similar conditions may be mixed to prepare one new lot. In this case, the average number of drug molecules of the new batch falls between the average number of drug molecules before mixing.

One specific example of the antibody-drug conjugate of the present invention may include an antibody-drug conjugate having a structure represented by the following formula:

[ formula 9]

Or an antibody-drug conjugate having a structure represented by the following formula:

[ formula 10]

In this aspect, AB represents an anti-CDH 6 antibody disclosed in the specification, and the antibody is conjugated to the drug linker through a thiol group derived from the antibody. In this regard, n has the same meaning as the so-called DAR (drug-to-antibody ratio) and represents the drug-to-antibody ratio per antibody. In particular, n represents the number of conjugated drug molecules per antibody molecule, which is a defined number and expressed as an average, i.e. the average number of conjugated drug molecules. In the case of the antibody-drug conjugate represented by [ formula 9] or [ formula 10] of the present invention, n may be 2 to 8, and preferably 5 to 8, more preferably 7 to 8, and still more preferably 8 in the measurement by the conventional procedure F.

An example of the antibody-drug conjugate of the present invention may include an antibody-drug conjugate having a structure represented by the above formula [ formula 9] or [ formula 10] (wherein the antibody represented by AB includes any one antibody selected from the following antibodies (a) to (g), or a functional fragment of the antibody) or a pharmacologically acceptable salt of the antibody-drug conjugate:

(a) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 69;

(b) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 73;

(c) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:61 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 77;

(d) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 69;

(e) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 73;

(f) an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 233 in the full-length amino acid sequence of the light chain shown in SEQ ID NO:65 and a heavy chain consisting of the amino acid sequence of positions 20 to 471 in the full-length amino acid sequence of the heavy chain shown in SEQ ID NO: 77; and

(g) any one antibody selected from the group consisting of antibodies (a) to (f), wherein the heavy or light chain comprises one or two or more modifications selected from post-translational modifications represented by: n-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of methionine residues to the N-terminus, amidation of proline residues and conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid and deletion of one or two amino acids at the carboxyl terminus.

4. Medicine

Since the anti-CDH 6 antibody or functional fragment of the antibody of the present invention described in the above section or example of "production of anti-CDH 6 antibody" binds to CDH6 on the surface of tumor cells and has an internalizing activity, it can be used as a drug, and particularly as a therapeutic agent for cancers, e.g., renal cell carcinoma or ovarian tumor, e.g., renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, wilms' tumor or neuroblastoma, alone or in combination with another drug.

In addition, the anti-CDH 6 antibodies or functional fragments of antibodies of the invention can be used to detect cells expressing CDH 6.

In addition, since the anti-CDH 6 antibody or functional fragment of the antibody of the present invention has an internalizing activity, it can be applied as an antibody in an antibody-drug conjugate.

When a drug having an anti-tumor activity (such as a cytotoxic activity) is used as a drug, the anti-CDH 6 antibody-drug conjugate of the present invention described in the above section "3. anti-CDH 6 antibody-drug conjugate" and examples is a conjugate of an anti-CDH 6 antibody and/or a functional fragment of an antibody having an internalizing activity and a drug having an anti-tumor activity (such as a cytotoxic activity). Since the anti-CDH 6 antibody-drug conjugate shows anti-tumor activity on cancer cells expressing CDH6, it can be used as a drug, and particularly as a therapeutic and/or prophylactic agent for cancer.

The anti-CDH 6 antibody-drug conjugates of the present invention can absorb moisture or have adsorbed water, e.g., become a hydrate when left in air or subjected to recrystallization or purification procedures. Such aqueous compounds or pharmacologically acceptable salts are also included in the present invention.

When the anti-CDH 6 antibody-drug conjugate of the present invention has a basic group such as an amino group, it can form a pharmacologically acceptable acid addition salt, if necessary. Examples of such acid addition salts may include: hydrohalides such as hydrofluorides, hydrochlorides, hydrobromides and hydroiodides; inorganic acid salts such as nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as ornithine, glutamate and aspartate.

When the anti-CDH 6 antibody-drug conjugate of the present invention has an acidic group such as a carboxyl group, it can form a pharmacologically acceptable base addition salt, if necessary. Examples of such base addition salts may include: alkali metal salts such as sodium, potassium and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; inorganic salts such as ammonium salts; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N' -dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N- (2-phenylethoxy) amine salt, piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt.

The invention may also include anti-CDH 6 antibody-drug conjugates in which one or more of the atoms comprising the antibody-drug conjugate is replaced with an isotope of an atom. There are two types of isotopes: radioactive isotopes and stable isotopes. Examples of isotopes may include isotopes of hydrogen(s) ((²H and³H) isotopes of carbon (C:)¹¹C，¹³C and¹⁴C) isotopes of nitrogen (¹³N and¹⁵n), isotopes of oxygen (¹⁵O，¹⁷O and¹⁸isotopes of O and fluorine: (¹⁸F) In that respect Compositions comprising antibody-drug conjugates labeled with such isotopes are useful, for example, as therapeutic agents, prophylactic agents, research agents, assay agents, diagnostic agents, and in vivo diagnostic imaging agents. All of each isotopically labeled antibody-drug conjugate, as well as mixtures of isotopically labeled antibody-drug conjugates in any given ratio, are encompassed by the present invention. The antibody-drug conjugate labeled with an isotope may be prepared, for example, according to a method known in the art by using a starting material labeled with an isotope (instead of the raw material used for the production method of the present invention mentioned below).

For example, in vitro cytotoxicity can be measured based on activity in inhibiting cell proliferation responses. For example, cancer cell lines overexpressing CDH6 were cultured and anti-CDH 6 antibody-drug conjugates were added to the culture system at different concentrations. Thereafter, its inhibitory activity on lesion formation, colony formation and spheroid growth can be measured. In this context, for example, by using a cancer cell line derived from a renal cell tumor or an ovarian tumor, the cell growth inhibitory activity against the renal cell tumor or the ovarian tumor can be examined.

The in vivo therapeutic effect on cancer can be measured in experimental animals, for example, by administering an anti-CDH 6 antibody-drug conjugate to nude mice that have been inoculated with a tumor cell line highly expressing CDH6, and then measuring changes in cancer cells. In this context, for example, the therapeutic effect on renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, or thyroid carcinoma can be measured by using an animal model derived from immunodeficient mice by inoculating cells derived from renal cell carcinoma-, renal clear cell carcinoma-, papillary renal cell carcinoma-, ovarian carcinoma-or thyroid carcinoma-origin.

The type of cancer to which the anti-CDH 6 antibody-drug conjugate of the present invention is applied is not particularly limited, as long as the cancer expresses CDH6 in the cancer cells to be treated. Examples thereof may include renal cell carcinoma (e.g., renal clear cell carcinoma or papillary renal cell carcinoma), ovarian cancer, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, wilms' tumor, and neuroblastoma, although the cancer is not limited thereto, as long as the cancer expresses CDH 6. More preferred examples of cancer may include renal cell carcinoma (e.g., renal clear cell carcinoma and papillary renal cell carcinoma) and ovarian cancer.

The anti-CDH 6 antibody-drug conjugate of the present invention can be preferably administered to a mammal, and more preferably to a human.

The substance used in the pharmaceutical composition comprising the anti-CDH 6 antibody-drug conjugate of the present invention may be suitably selected from pharmaceutical additives and other substances generally used in the art (in terms of application dose or application concentration) and then used.

The anti-CDH 6 antibody-drug conjugates of the invention can be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible components. For example, Pharmaceutical compositions typically comprise one or more Pharmaceutical carriers (e.g., sterile liquids (e.g., water and oils including those of petroleum and animal, vegetable, or synthetic origin (e.g., peanut, soybean, mineral, and sesame oils)). when a Pharmaceutical composition is administered intravenously, water is the more typical carrier.

Various delivery systems are known and they may be used to administer the anti-CDH 6 antibody-drug conjugates of the invention. Examples of routes of administration may include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. For example, administration can be by injection or bolus injection. According to a particularly preferred embodiment, the administration of the above antibody-drug conjugate is performed by injection. Parenteral administration is the preferred route of administration.

According to representative embodiments, the pharmaceutical composition is prescribed as a pharmaceutical composition suitable for intravenous administration to a human according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile and isotonic aqueous buffer solutions. If necessary, the drug may also contain a solubilizing agent and a local anesthetic to relieve pain in the area of injection (e.g., lidocaine). Typically, the above ingredients are provided separately or together in admixture in a unit dosage form as a freeze-dried powder or anhydrous concentrate contained in a container obtained by sealing, for example, an ampoule or sachet representing the active dose. When the drug is administered by injection, it can be administered using, for example, an injection vial containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the above ingredients are mixed with each other before administration.

The pharmaceutical composition of the present invention may be a pharmaceutical composition comprising only the anti-CDH 6 antibody-drug conjugate of the present application, or may be a pharmaceutical composition comprising an anti-CDH 6 antibody-drug conjugate and at least one other cancer therapeutic agent. The anti-CDH 6 antibody-drug conjugates of the invention can also be administered with other cancer therapeutics, and can thereby enhance anti-cancer effects. Other anti-cancer agents for this purpose may be administered to the individual simultaneously, separately or sequentially along with the antibody-drug conjugate. In addition, the other anti-cancer agent and the anti-CDH 6 antibody-drug conjugate can each be administered to the subject at different administration intervals. Examples of such cancer therapeutic agents may include tyrosine kinase inhibitors including imatinib, sunitinib, and regorafenib, CDK4/6 inhibitors including palbociclib, HSP90 inhibitors including TAS-116, MEK inhibitors including MEK162, and immune checkpoint inhibitors including nivolumab, pembrolizumab, and priomab, although the cancer therapeutic agent is not limited thereto as long as the drug has an antitumor activity.

Such pharmaceutical compositions may be prepared as lyophilized formulations or as liquid formulations having the selected composition and requisite purity. The pharmaceutical composition prepared as a freeze-dried preparation may be a preparation containing appropriate pharmaceutical additives used in the art. Likewise, a liquid formulation may be prepared such that the liquid formulation contains various pharmaceutical additives used in the art.

The composition and concentration of the pharmaceutical composition also varies depending on the method of administration. With respect to the affinity of the anti-CDH 6 antibody-drug conjugate for antigen, that is, the dissociation constant (Kd value) of the anti-CDH 6 antibody-drug conjugate from antigen, contained in the pharmaceutical composition of the present invention, the pharmaceutical composition can exert drug efficacy even if its applied dose is reduced as the affinity increases (that is, the Kd value is low). Therefore, the applied dose of the antibody-drug conjugate can also be determined by setting the applied dose based on the state of affinity of the antibody-drug conjugate for the antigen. When the antibody-drug conjugate of the present invention is administered to a human, it may be administered once at a dose of, for example, about 0.001 to 100mg/kg or multiple times at intervals of 1 to 180 days. It may be administered preferably at a dose of 0.1 to 50mg/kg, and more preferably at a dose of 1 to 50mg/kg, 1 to 30 mg/kg, 1 to 20 mg/kg, 1 to 15 mg/kg, 2 to 50mg/kg, 2 to 30 mg/kg, 2 to 20 mg/kg or 2 to 15 mg/kg, administered in multiple administrations at intervals of 1 to 4 weeks, preferably 2 to 3 weeks.

Examples

Hereinafter, the present invention will be specifically described in the following examples. However, these examples are not intended to limit the scope of the present invention. Furthermore, these examples should not be construed in any way as limiting. It should be noted that in the following examples, unless otherwise specified, various operations concerning genetic manipulation were performed according to the method described in "Molecular Cloning" (Sambrook, j., Fritsch, e.f. and manitis, t., Cold Spring Harbor Laboratory Press, published in 1989) or other methods described in an experimental manual used by those skilled in the art, or when a commercially available reagent or kit was used, the examples were carried out according to the instructions included in the commercially available products. In this specification, reagents, solvents and starting materials are readily available from commercial sources, unless otherwise specified.

Example 1: obtaining a rat anti-human CDH6 antibody with internalization Activity

1) -1 construction of human, mouse, rat and cynomolgus CDH6 expression vectors

cDNA was incorporated into a vector for mammalian expression using a cDNA expression vector (OriGene Technologies inc., RC217889) encoding human CDH6 protein (NP _004923) according to a method known to those skilled in the art to produce human CDH6 expression vector pcdna3.1-hCDH 6. The amino acid sequence of the human CDH6 ORF (open reading frame) is shown in SEQ ID NO: 1.

The cDNA was incorporated into a vector for mammalian expression using a cDNA expression vector (OriGene Technologies inc., MC221619) encoding the mouse CDH6 protein (NP _031692) according to methods known to those skilled in the art to generate the mouse CDH6 expression vectors pcdna3.1-mCDH6 and p3xFLAG-CMV-9-mCDH 6. The amino acid sequence of the mouse CDH6 ORF is shown in SEQ ID NO 7.

The cDNA was incorporated into a vector for mammalian expression using a cDNA expression vector (OriGene Technologies inc., RN211850) encoding each cDNA portion of rat CDH6 protein (NP _037059) according to a method known to those skilled in the art to generate human CDH6 expression vectors pcdna3.1-rCDH6 and p3xFLAG-CMV-9-rCDH 6. The amino acid sequence of the rat CDH6 ORF is shown in SEQ ID NO 8.

A cDNA encoding the cynomolgus CDH6 protein was cloned using primer 1 (5'-CACCATGAGAACTTACCGCTACTTCTTGCTGCTC-3') (SEQ ID NO:85) and primer 2 (5'-TTAGGAGTCTTTGTCACTGTCCACTCCTCC-3') (SEQ ID NO:86) using cDNA synthesized from total cynomolgus kidney RNA as a template. It was confirmed that the obtained sequence corresponded to the extracellular region of cynomolgus monkey CDH6 (NCBI, XP — 005556691.1). It was also confirmed that the sequence corresponded to the full-length sequence of cynomolgus monkey CDH6 (EHH54180.1) registered in EMBL. The cDNA was incorporated into a vector for mammalian expression according to methods known to those skilled in the art to produce the cynomolgus CDH6 expression vector pcDNA3.1-cynoCDH 6. The amino acid sequence of the cynomolgus CDH6 ORF is shown in SEQ ID NO 9.

EndoFree Plasmid Giga Kit (Qiagen NV) was used to generate large quantities of the resulting Plasmid DNA.

1) -2 immunization

For immunization, WKY/Izm female rats (Japan SLC, Inc.) were used. First, the lower limb of each rat was pretreated with hyaluronidase (Sigma-Aldrich co. LLC), and then the human CDH6 expression vector pcdna3.1-hCDH6 produced in example 1) -1 was injected intramuscularly to the same site. Subsequently, in vivo electroporation was performed using ECM830 (BTX) at the same site using a two-needle electrode. The same in vivo electroporation was repeated approximately once every two weeks, and thereafter, lymph nodes or spleens were collected from rats and then used to produce hybridomas.

1) -3 Generation of hybridomas

According to the electrofusion, lymph node cells or spleen cells were fused with a mouse myeloma SP2/0-ag14 Cell (ATCC, number CRL-1581) using LF301 Cell Fusion Unit (BEX Co., Ltd.), and then the cells were suspended and diluted with ClonaCell-HY selection Medium D (StemCell Technologies Inc.), and then at 37 ℃ and 5% CO₂Culturing under the conditions of (1). Single hybridoma colonies appearing in the medium were collected as monoclonal hybridomas, then suspended in ClonaCell-HY selection Medium E (StemCell Technologies Inc.), and then incubated at 37 ℃ and 5% CO₂Culturing under the conditions of (1). After moderate proliferation of the cells, frozen stocks of individual hybridoma cells were generated, while using the resulting hybridoma culture supernatants to screen for hybridomas producing anti-human CDH6 antibody.

1) -4 screening of antibody-producing hybridomas according to the cell-ELISA method

1) -4-1 preparation of antigen Gene-expressing cells for cell-ELISA

5 × 10 in DMEM medium supplemented with 10% FBS⁵293 α cells (stably expressing cell lines derived from HEK293 cells expressing integrin α v and integrin β 3) were prepared per 10 mL. DNA of pcDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 or pcDNA3.1 as a negative control was introduced into 293 α cells according to the transduction procedure using Lipofectamine 2000(Thermo Fisher Scientific Inc.), and the cells were dispensed to 96-well plates (Corning Inc.) at an amount of 100 μ L/well. Thereafter, the cells were incubated at 37 ℃ and 5% CO₂Is cultured in DMEM medium supplemented with 10% FBS for 24 to 27 hours. The obtained transfected cells were used in a cell-ELISA in an adherent state.

1) -4-2 cell-ELISA

The culture supernatant of the 293 α cell transfected with the expression vector prepared in example 1) -4-1 was removed, and then the culture supernatant from each hybridoma was added to the 293 α cell transfected with pcDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 or pcDNA3.1. The cells were allowed to stand at 4 ℃ for 1 hour. Cells in wells were washed once with PBS (+) supplemented with 5% FBS, and then anti-rat IgG-peroxidase antibody (Sigma-Aldrich co. LLC) produced in rabbits, which had been diluted 500-fold with PBS (+) supplemented with 5% FBS, was added to the wells. The cells were allowed to stand at 4 ℃ for 1 hour. Cells in wells were washed three times with PBS (+) supplemented with 5% FBS, and then OPD developing solution (obtained by mixing o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H was added to wells at 100 μ L/well₂O₂Was dissolved in OPD solution (0.05M trisodium citrate, 0.1M disodium phosphate 12-hydrate; pH 4.5) to bring the material to 0.4 mg/ml and 0.6% (v/v), respectively). The color reaction was carried out with occasional stirring. Thereafter, 1M HCl was added to the plate to terminate the color reaction (100. mu.L/well), and then the absorbance at 490nm was measured using a plate reader (ENVISION: Perkinelmer, Inc.). As hybridomas producing antibodies that bind to human CDH6 and cynomolgus CDH6, hybridomas producing culture supernatants that showed higher absorbance in 293 α cells transfected with pcdna3.1-hCDH6 or pcdna3.1-cynoCDH6 expression vectors than in 293 α cells transfected with control pcdna3.1 were selected.

1) -5 Selective screening of antibodies binding to cynomolgus CDH6 according to flow cytometry

1) -5-1 preparation of cells expressing antigenic genes for flow cytometry analysis

293T cells at 5X 10⁴Individual cell/cm²Inoculating at 225cm²Flasks (Sumitomo Bakelite CO., Ltd.), and then cells were incubated at 37 ℃ and 5% CO₂Was cultured overnight in DMEM medium supplemented with 10% FBS. Using Lipofectamine 2000, pcDNA3.1-cynoCDH6 or pcDNA3.1 as a negative control was introduced into 293T cells and incubated at 37 ℃ and 5% CO₂Further culturing the cells overnight under the conditions of (1). 293T cells transfected with each vector were treated with TrypLE Express (Thermo Fisher scientific Corp.) and the cells were washed with DMEM supplemented with 10% FBS and then suspended in PBS supplemented with 5% FBS. The cell suspension obtained was used for flow cytometry analysis.

1) -5-2 flow cytometry analysis

The binding specificity of the antibodies produced by the human CDH 6-and cynomolgus monkey CDH 6-binding antibody-producing hybridomas that have been selected by the cell-ELISA in examples 1) -4 for cynomolgus monkey CDH6 was further confirmed by flow cytometry. The suspension of transiently expressed 293T cells prepared in example 1) -5-1 was centrifuged, and then the supernatant was removed. Thereafter, the cells were suspended by adding culture supernatant from each hybridoma. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding anti-mouse IgG FITC conjugate (Sigma-Aldrich co. LLC) diluted 500-fold with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed 2 times with PBS supplemented with 5% FBS and then resuspended in PBS supplemented with 5% FBS and 2 μ g/mL 7-amino actinomycin D (molecular probes, Inc.), followed by detection using a flow cytometer (FC500; Beckman Coulter, Inc.). Data were analyzed using FlowJo (Tree Star, Inc.). Output of 7-amino actinomycin D positive cells by gating, histogram of FITC fluorescence intensity of live cells was generated after dead cells were removed from the assay. Hybridomas producing antibodies specifically binding to cynomolgus monkey CDH6 expressed on the surface of the cell membrane were selected based on the results in which the histogram of the antibodies in 293T cells transfected with pcdna3.1-cynoCDH6 shifted to the side of strong fluorescence intensity compared to 293T cells transfected with control pcdna3.1.

1) Determination of the isotype of the 6 rat monoclonal antibody

Clones rG019, rG055, rG056 and rG061, which showed specific and strong binding to human CDH6 and monkey CDH6, were selected from the hybridomas producing rat anti-CDH 6 antibodies selected in examples 1) -5, and the isotype of each antibody was identified. The heavy chain subclasses and light chain types of the antibodies were determined using ratonoclone ANTIBODY ISOTYPING TEST KIT (DS Pharma biological co., Ltd.). As a result, all of the 4 clones rG019, rG055, rG056 and rG061 were confirmed to have heavy chains of IgG2b subclass and light chains of κ chain type.

1) -7 preparation of rat anti-human CDH6 antibody

1) Production of supernatant of-7-1 culture

Rat anti-human CDH6 monoclonal antibody was purified from hybridoma culture supernatants. First, the volume of each Hybridoma producing the rat anti-CDH 6 monoclonal antibody was sufficiently increased with clonocell-HY selection medium E (StemCell Technologies Inc.), and then the medium was replaced with Hybridoma SFM (Thermo Fisher scientific Corp.) to which 20% Ultra Low IgG FBS (Thermo Fisher scientific Corp.) had been added. Thereafter, the hybridomas were cultured for 4 to 5 days. The resulting culture supernatant was harvested, and insoluble matter was removed therefrom by passing through a 0.8- μm filter and a 0.2- μm filter.

1) Purification of-7-2 rat anti-CDH 6 antibody

The antibody (rat anti-CDH 6 antibody (rG019, rG055, rG056, or rG061)) was purified from the culture supernatant of the hybridoma prepared in example 1) -7-1 according to protein G affinity chromatography. The antibody was adsorbed on a protein G column (GE healthcare biosciences Corp.), then the column was washed with PBS, and then the antibody was eluted with 0.1M glycine/HCl aqueous solution (pH 2.7). 1M Tris-HCl (pH 9.0) was added to the eluent, so that the pH was adjusted to 7.0 to 7.5. Thereafter, the buffer was replaced with HBSor (25mM histidine/5% sorbitol, pH6.0) using a centrifugal UF Filter Device VIVASPIN20 (molecular weight cut-off: UF30K, Sartorius Inc.) while concentrating the antibody so that the concentration of the antibody was adjusted to 1 mg/mL. Finally, the antibody was filtered through a Minisart-Plus filter (Sartorius Inc.) to obtain a purified sample.

Example 2: in vitro evaluation of rat anti-CDH 6 antibody

2) -1 assessment of binding Capacity of rat anti-CDH 6 antibody by flow cytometry

The rat anti-CDH 6 antibodies generated in examples 1) -7 were evaluated for human CDH 6-binding activity by flow cytometry. pcDNA3.1-hCDH6 produced in example 1) -1 was transiently introduced into 293T cells (ATCC) using Lipofectamine 2000(Thermo Fisher Scientific Inc.). Cells were incubated at 37 ℃ and 5% CO₂Overnight, and thereafter a cell suspension was prepared. The suspension of transfected 293T cells was centrifuged and then the supernatant was removed. Thereafter, the total amount of the 4 rat anti-CDH 6 monoclonal antibodies (clone numbers: each of rG019, rG055, rG056, and rG061) or rat IgG control (R)&D Systems, Inc.) (final concentration: 10 ng/mL) of the suspension cells. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding 50-fold dilution of anti-rabbit IgG (whole molecule) -FITC antibody (Sigma-aldrich co. LLC) produced in rabbits with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and subsequently detected using a flow cytometer (FC500; Beckman Coulter, Inc.). Data were analyzed using FlowJo (Tree Star, Inc.). The results are shown in FIG. 1. In the histogram of fig. 1, the abscissa plots FITC fluorescence intensity, which indicates the amount of bound antibody, and the ordinate plots cell count. The shaded histogram shows the use of negative control 293T cells that were not transfected with hCDH6, and the open solid histogram shows the use of 293T cells transfected with hCDH 6. As seen, fluorescence intensity was enhanced by binding of the antibody to hCDH6 on the cell surface. The rat IgG control did not bind to any of the cells. As a result, it was confirmed that the 4 kinds of generated rat anti-CDH 6 monoclonal antibodies bind to 293T cells transfected with pcDNA3.1-hCDH 6.

2) -2 analysis of CDH 6-binding site of rat anti-CDH 6 antibody by flow cytometry

2) Construction of expression vector for each domain deletion mutant of-2-1 human CDH6

The full-length extracellular region of human CDH6 has five extracellular domains, EC1 (SEQ ID NO:2), EC2 (SEQ ID NO:3), EC3 (SEQ ID NO:4), EC4 (SEQ ID NO:5) and EC5 (SEQ ID NO: 6). The gene to be expressed so that each of the five EC domains can be deleted from full-length human CDH6 was synthesized by GeneArt and incorporated into the p3 xfag-CMV-9 vector for mammalian expression (Sigma-Aldrich co. LLC) according to methods known to those skilled in the art to produce expression vectors lacking each domain deletion mutant of any of EC1 to EC 5.

2) -2-2 epitope analysis of rat anti-CDH 6 antibody by flow cytometry using domain deletion mutants

The epitope to which the rat anti-human CDH6 antibody binds was identified by flow cytometry analysis using 293 α cell lines transfected with each EC domain deleted vector. Each of the domain deletion mutant expression vectors generated in examples 2) -2-1 or pcdna3.1-hCDH6 used to express full-length human CDH6 was transiently introduced into 293 α cell line, which is a cell line derived from HEK293 cells by stable transfection with integrin α v and integrin β 3 expression vectors, using Lipofectamine 2000(Thermo Fisher Scientific Inc.). Cells were incubated at 37 ℃ and 5% CO₂Overnight, and thereafter a cell suspension was prepared. The suspension of transfected 293 α cells was centrifuged and then the supernatant was removed. Thereafter, the total amount of the 4 rat anti-CDH 6 monoclonal antibodies (clone numbers: each of rG019, rG055, rG056, and rG061) or rat IgG control (R)&D Systems, Inc.) (final concentration: 20 nM) of cells in suspension. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding 50-fold dilution of anti-rabbit IgG (whole molecule) -FITC antibody (Sigma-Aldrich co.llc) produced in rabbits with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS, followed by flow cytometry (Canto II; BD Biosciences)And (6) detecting. Data were analyzed using FlowJo (Tree Star, Inc.). The results are shown in FIGS. 2-1 to 2-6. In the histograms of fig. 2-1 to 2-6, FITC fluorescence intensity is plotted on the abscissa, which indicates the amount of bound antibody, and cell count is plotted on the ordinate. The shaded histogram shows untransfected 293 α cells using the negative control, and the open solid histogram shows the use of 293 cells expressing either full-length hCDH6 or deletion mutants of each EC domain. Fluorescence intensity was enhanced when the antibody bound to full-length hCDH6 or each EC domain deletion mutant on the cell surface. The rat IgG control did not bind to any transfected cells. The 4 generated rat anti-CDH 6 monoclonal antibodies bound to full-length hCDH6, the EC1 deletion mutant, the EC2 deletion mutant, the EC4 deletion mutant, and the EC5 deletion mutant, but not to the EC3 deletion mutant. From the results, it was confirmed that 4 rat anti-CDH 6 monoclonal antibodies specifically bind to hCDH6 with EC3 as an epitope.

2) -3 internalization Activity of rat anti-CDH 6 antibody

2) Demonstration of CDH6 expression in 3-1 human tumor cell lines

To select CDH 6-positive human tumor cell lines for evaluation of the obtained antibodies, CDH6 expression information was retrieved in a known database, and the expression of CDH6 on the cell membrane surface was evaluated by flow cytometry. Human ovarian tumor cell lines NIH OVCAR-3, PA-1 and ES-2 and human renal cell tumor cell line 786-O (all obtained from ATCC) were each treated at 37 ℃ and 5% CO₂And thereafter preparing a cell suspension. The cells were centrifuged and then the supernatant was removed. Thereafter, a commercially available anti-human CDH6 antibody (MABU2715, R) was added&D Systems, Inc.) or mouse IgG1 (BD Pharmingen) as a negative control (final concentration: 50 μ g/mL) of the suspension cells. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding F (ab')2 fragment of FITC-conjugated goat anti-mouse immunoglobulin (Dako) which had been diluted 50-fold with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS, and then detected using a flow cytometer (Canto II; BD Biosciences). Data were analyzed using FlowJo (Tree Star, Inc.). Results displayIn fig. 3. In the histogram of fig. 3, the abscissa plots FITC fluorescence intensity, which indicates the amount of bound antibody, and the ordinate plots cell count. The shaded histogram shows the negative control mIgG1 for staining and the open solid histogram shows anti-human CDH6 antibody for staining. As seen, fluorescence intensity was enhanced by binding of the antibody to hCDH6 on the cell surface. The mIgG control did not bind to any cells. As a result, it was confirmed that NIH-OVCAR-3, PA-1 and 786-O cell lines endogenously express CDH6 on the cell surface. On the other hand, ES-2 cell line was shown not to express CDH 6.

2) Evaluation of internalization Activity of 3-2 rat anti-CDH 6 antibody

The internalization activity of the rat anti-CDH 6 antibody was assessed using the anti-rat IgG reagent rat-zap (advanced Targeting systems) conjugated to a toxin that inhibits protein synthesis (saporin). Specifically, human CDH 6-positive ovarian tumor cell line NIH: OVCAR-3 (ATCC) at 4 x 10³Individual cells/well were seeded in 96-well plates and then at 37 ℃ and 5% CO₂Cultured overnight under the conditions of (1). Human CDH 6-Positive Kidney cell tumor cell line 786-O (ATCC) at 1X 10³Individual cells/well were seeded on 96-well plates and then cultured overnight. The next day, each rat anti-CDH 6 antibody (final concentration: 1 nM) or rat IgG2b antibody (R) as a negative control antibody&D Systems, Inc.) were added to the plates. rat-ZAP (final concentration: 0.5 nM) or goat anti-rat IgG unconjugated with toxin as a negative control, Fc (gamma) fragment specific (Jackson ImmunoResearch Laboratories, Inc.) (final concentration: 0.5 nM) was further added to the plate, and the cells were incubated at 37 ℃ and 5% CO₂Cultured for 3 days. The number of viable cells was measured by quantifying ATP activity (RLU) using CellTiter-glo (TM) luminescent cell viability assay (Promega Corp.). In this evaluation, rat-ZAP was taken up into cells in a manner dependent on the internalizing activity of the rat anti-CDH 6 antibody, so that saporin, which inhibits protein synthesis, was released into the cells, thereby inhibiting cell growth. The cell growth inhibition by addition of anti-CDH 6 antibody was indicated by relative survival when the number of viable cells in wells supplemented with negative control instead of rat-ZAP was defined as 100%. FIG. 4 showsGraphs and tables of cell viability. As a result, it was demonstrated that the rat anti-CDH 6 antibody binds to CDH6 and causes internalization.

Example 3: determination of nucleotide sequence of cDNA encoding the variable region of rat anti-CDH 6 antibody ]

3) Amplification and sequencing of-1 rG019 heavy chain variable region and light chain variable region gene fragments

3) -1-1 preparation of Total RNA from G019

To amplify the cDNA encoding each variable region of rG019, total RNA was prepared from G019 using TRIzol reagent (Ambion, Inc.).

3) -1-2 amplification of cDNA encoding rG019 heavy chain variable region by 5' -RACE PCR and determination of nucleotide sequence

The cDNA encoding the heavy chain variable region was amplified using approximately 1. mu.g of the total RNA prepared in example 3) -1-1 and the SMARTER RACE cDNA amplification kit (Clontech Laboratories, Inc.). As a primer for amplifying cDNA of the variable region of the rG019 heavy chain gene according to PCR, UPM (Universal primer A mix: included together with the SMARTer RACEcDNA amplification kit) and a primer designed from the sequence of the known rat heavy chain constant region were used.

The cDNA encoding the heavy chain variable region amplified by 5' -RACE PCR was cloned into a plasmid, and thereafter, the nucleotide sequence of the cDNA for the heavy chain variable region was subjected to sequence analysis.

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

3) -1-3 amplification of cDNA encoding the rG019 light chain variable region by 5' -RACE PCR and determination of the nucleotide sequence

Amplification and sequencing were carried out by the same method as applied in examples 3) -1-2. However, as a primer for amplifying cDNA of the variable region of the rG019 light chain gene according to PCR, UPM (Universal primer A mix: included together with the SMARTERRACE cDNA amplification kit) and a primer designed from the sequence of the known rat light chain constant region were used.

The nucleotide sequence of the cDNA determined to encode the light chain variable region of rG019 is shown in SEQ ID NO. 11, and the amino acid sequence thereof is shown in SEQ ID NO. 10.

3) Amplification and sequencing of-2 rG055 heavy chain variable region and light chain variable region gene fragments

The sequence was determined by the same method as that applied in example 3) -1.

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

3) Amplification and sequencing of-3 rG056 heavy chain variable region and light chain variable region gene fragments

The sequence was determined by the same method as that applied in example 3) -1.

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

3) Amplification and sequencing of-4 rG061 heavy chain variable region and light chain variable region gene fragments

The sequence was determined by the same method as that applied in example 3) -1.

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

Example 4: production of human chimeric anti-CDH 6 antibody chG019

4) Construction of-1 human chimeric anti-CDH 6 antibody chG019 expression vector

4) Construction of-1-1 chimeric and humanized light chain expression vector pCMA-LK

An about 5.4-kb fragment obtained by digesting plasmid pcDNA3.3-TOPO/LacZ (Invitrogen Corp.) with restriction enzymes XbaI and PmeI was combined with a DNA fragment comprising a DNA sequence (SEQ ID NO:50) encoding a human light chain signal sequence and a human kappa chain constant region using an In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.) to produce pcDNA3.3/LK.

The neomycin expression unit was removed from pcDNA3.3/LK to construct pCMA-LK.

4) -1-2 construction of chimeric and humanized IgG1 type heavy chain expression vector pCMA-G1

A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove therefrom a DNA sequence encoding a light chain signal sequence and a human kappa chain constant region was combined with a DNA fragment comprising a DNA sequence (SEQ ID NO:51) encoding a human heavy chain signal sequence and a human IgG1 constant region using an In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.) to construct pCMA-G1.

4) Construction of-1-3 chG019 heavy chain expression vector

A DNA fragment (GENEART) from nucleotide positions 36 to 440 in the nucleotide sequence of the chG019 heavy chain shown in SEQ ID NO:57 was synthesized. The synthesized DNA fragment was inserted into the site of pCMA-G1 that had been cut with the restriction enzyme BlpI using the In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.) to construct the chG019 heavy chain expression vector. It should be noted that for the chG019 heavy chain, CDR sequences with proline substituted for cysteine were used to prevent accidental disulfide bonds.

4) Construction of-1-4 chG019 light chain expression vector

A DNA fragment (GENEART) comprising the DNA sequence (SEQ ID NO:52) encoding the light chain of chG019 was synthesized. The synthesized DNA fragment was combined with a DNA fragment which had been obtained by digesting pCMA-LK with XbaI and PmeI to remove therefrom a DNA sequence encoding a light chain signal and a human kappa chain constant region, using an In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.) to construct a chG019 light chain expression vector.

4) Production and purification of-2 human chimeric anti-CDH 6 antibody chG019

4) Production of-2-1 chG019

FreeStyle 293F cells (Invitrogen Corp.) were cultured and passaged according to the manual. Mixing 1.2X 10⁹FreeStyle 293F cells (Invitrogen Corp.) from several logarithmic growth phases were seeded in 3-L Fernbach ErOn a lenmeyerFlask (Corning Inc.), followed by expression of the medium with FreeStyle 293 (Invitrogen Corp.) at 2.0X 10⁶Individual cells/mL dilution. To 40 ml of Opti-Pro SFM medium (Invitrogen Corp.), 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector and 1.8 mg of polyethyleneimine (Polyscience #24765) were added, and the resulting mixture was gently stirred. After 5 min incubation, the mixture was added to FreeStyle 293F cells. Cells were incubated at 90rpm in 8% CO₂The culture was cultured in an incubator at 37 ℃ for 4 hours with shaking, and then 600mL of EX-CELL VPRO medium (SAFC Biosciences Inc.), 18mL of GlutaMAX I (GIBCO) and 30mL of YeastolateUltrafiltte (GIBCO) were added to the culture. Cells were incubated at 8% CO₂The culture was further cultured in an incubator at 37 ℃ for 7 days with shaking at 90 rpm. The culture supernatant obtained was filtered through a disposable capsule filter (Advantec # CCS-045-E1H).

4) Purification of (E) -2-2 chG019

The antibody was purified from the culture supernatant obtained in example 4) -2-1 by the one-step method according to rProtein A affinity chromatography. The culture supernatant was applied to a column filled with MabSelectSuRe (GE healthcare biosciences Corp.) which had been equilibrated with PBS, and thereafter, the column was washed with PBS in an amount of two or more times the column volume. Subsequently, the antibody was eluted with 2M arginine hydrochloride solution (pH 4.0), so that antibody-containing fractions were collected. The fractions were dialyzed (Thermo Fisher Scientific Inc., Slide-A-Lyzer analysis Cassette) so as to replace the buffer with HBSor (25mM histidine/5% sorbitol, pH 6.0). The antibody was concentrated using a Centrifugal UF Filter DeviceVIVASPIN20 (molecular weight cut-off: UF10K, Sartorius Inc.) so that the concentration of IgG was adjusted to 5 mg/ml or higher. Finally, the antibody was filtered through a Minisart-Plus filter (Sartorius Inc.) to obtain a purified sample.

4) Evaluation of binding Activity of human chimeric anti-CDH 6 antibody chG019

The CDH 6-binding activity of the human chimeric anti-CDH 6 antibody chG019 purified in 4) -2 was confirmed by flow cytometry. pcDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6, or pcDNA3.1 produced in example 1) -1 was transiently introduced into 293 α cells using Lipofectamine 2000. Will be thinCells were incubated at 37 ℃ and 5% CO₂Overnight, and thereafter a cell suspension was prepared. chG019 was added to each suspension of these cells. The cells were allowed to stand at 4 ℃ for 1 hour. Thereafter, the cells were washed twice with PBS supplemented with 5% FBS, and then suspended by adding PE-labeled F (ab')2 fragment anti-human IgG, Fc γ antibody (Jackson ImmunoResearch Laboratories, Inc.) that had been diluted 500-fold with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. The cells were washed twice with 5% FBS-supplemented PBS and then resuspended in 5% FBS-supplemented PBS prior to detection using a flow cytometer (Canto II; BD Biosciences). Data were analyzed using FlowJo (Tree Star, Inc.). As shown in FIG. 5, the chG019 did not bind to the 293T cell transfected with pcDNA3.1 used as a negative control, but did bind to the 293T cell transfected with pcDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 in an antibody concentration-dependent manner. In fig. 5, the abscissa plots the antibody concentration, and the ordinate plots the amount of bound antibody based on the average fluorescence intensity. It is apparent from this result that chG019 specifically binds to human CDH6 and cynomolgus monkey CDH6 with nearly equivalent binding activity.

Example 5: generation of humanized anti-CDH 6 antibody

5) -1 design of humanized versions of anti-CDH 6 antibodies

5) Molecular modeling of the-1-1 chG019 variable region

Molecular modeling of the variable region of chG019 utilizes a method called homology modeling (Methods in Enzymology, 203, 121-. A commercially available protein three-dimensional structure analysis program BioLuminate (manufactured by Schrodinger, LLC) was performed using, as a template, a structure (PDB ID:2I9L) having high sequence identity with the heavy and light chain variable regions of chG019 registered in a protein database (nuc. acid res.35, D301-D303 (2007)).

5) Design of amino acid sequence of-1-2 humanized hG019

chG019 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA86, 10029-. The consensus Sequences of human gamma chain subgroup 1 and kappa chain subgroup 1, determined by KABAT et al (Sequences of Proteins of Immunological Interest, published Health Service National Institutes of Health, Bethesda, MD. (1991)), share a high degree of identity with the framework regions of chG019 and, based thereon, are selected as acceptors for the heavy and light chains, respectively. The donor residues to be grafted onto the recipient are selected by reference to a standard analytical three-dimensional model given, for example, by Queen et al (Proc. Natl. Acad. Sci. USA86, 10029-.

5) Humanization of the-2 chG019 heavy chain

The three heavy chains thus designed were designated hH01, hH02, and hH 04. The full-length amino acid sequence of the hH01 heavy chain is shown in SEQ ID NO: 69. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO 69 is shown in SEQ ID NO 70. The full-length amino acid sequence of heavy chain hH02 is shown in SEQ ID NO. 73. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 73 is shown in SEQ ID NO. 74. The full-length amino acid sequence of heavy chain hH04 is shown in SEQ ID NO 77. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO 77 is shown in SEQ ID NO 78.

5) Humanization of the-3 chG019 light chain

The two light chains thus designed were designated hL02 and hL 03. The full-length amino acid sequence of the hL02 light chain is shown in SEQ ID NO 61. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO 61 is shown in SEQ ID NO 62. The full-length amino acid sequence of light chain hL03 is shown in SEQ ID NO 65. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO 65 is shown in SEQ ID NO 66.

5) -4 design of humanized hG019 by combining heavy and light chains

The antibody composed of hH01 and hL02 was designated as "H01L02 antibody" or "H01L 02". The antibody composed of hH02 and hL02 was designated as "H02L02 antibody" or "H02L 02". The antibody composed of hH02 and hL03 was designated as "H02L 03 antibody" or "H02L 03". The antibody consisting of hH04 and hL02 was designated as "H04L02 antibody" or "H04L 02".

5) Expression of-5 humanized anti-CDH 6 antibody

5) Construction of-5-1 humanized hG019 heavy chain expression vector

5) Construction of-5-1-1 humanized hG019-H01 type heavy chain expression vector

A DNA fragment (GENEART) from nucleotide position 36 to 440 in the nucleotide sequence of the humanized hG019-H01 type heavy chain shown in SEQ ID NO:70 was synthesized. The humanized hG019-H01 type heavy chain expression vector was constructed by the same method as that applied in examples 4) -1-3.

5) Construction of-5-1-2 humanized hG019-H02 type heavy chain expression vector

A DNA fragment (GENEART) from nucleotide position 36 to 440 in the nucleotide sequence of the humanized hG019-H02 type heavy chain shown in SEQ ID NO:74 was synthesized. The humanized hG019-H02 type heavy chain expression vector was constructed by the same method as that applied in examples 4) -1-3.

5) Construction of-5-1-3 humanized hG019-H04 type heavy chain expression vector

A DNA fragment (GENEART) from nucleotide position 36 to 440 in the nucleotide sequence of the humanized hG019-H04 type heavy chain shown in SEQ ID NO:78 was synthesized. The humanized hG019-H04 type heavy chain expression vector was constructed by the same method as that applied in examples 4) -1-3.

5) Construction of-5-2 humanized hG019 light chain expression vector

5) Construction of-5-2-1 humanized hG019-L02 type light chain expression vector

A DNA fragment (GENEART) comprising a DNA sequence encoding the variable region of the humanized hG019-L02 type light chain from nucleotide positions 37 to 399 in the nucleotide sequence of the humanized hG019-L02 type light chain shown in SEQ ID NO:62 was synthesized. The synthesized DNA fragment was inserted into the site of pCMA-LK which had been cut with the restriction enzyme BsiWI using In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.) to construct a humanized hG019-L02 type light chain expression vector.

5) Construction of-5-2-2 humanized hG019-L03 type light chain expression vector

A DNA fragment (GENEART) comprising a DNA sequence encoding the variable region of the humanized hG019-L03 type light chain from nucleotide positions 37 to 399 in the nucleotide sequence of the humanized hG019-L03 type light chain shown in SEQ ID NO:66 was synthesized. A humanized hG019-L03 type light chain expression vector was constructed by the same method as that applied in example 5) -5-2-1.

5) Preparation of (E) -5-3 humanized hG019

5) Production of-5-3-1H 01L02, H02L02, H02L03 and H04L02

The antibody was produced by the same method as that applied in example 4) -2-1. H01L02, H02L02, H02L03 and H04L02 were produced by combining the heavy and light chains shown in examples 5) -4.

5) Two-step purification of-5-3-2H 01L02, H02L02, H02L03 and H04L02

The antibody was purified from the culture supernatant obtained in example 5) -5-3-1 by a two-step process, i.e., by rProtein a affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a column packed with MabSelectSuRe (manufactured by GE Healthcare Biosciences corp.) which had been equilibrated with PBS, and thereafter, the column was washed with PBS in an amount of two or more times the column volume. Subsequently, the antibody was eluted using 2M arginine hydrochloride solution (pH 4.0). Antibody-containing fractions were dialyzed (Thermo Fisher Scientific inc., Slide-a-Lyzer analysis Cassette) so as to replace the buffer with PBS. The antibody solution was diluted 5-fold with 5mM sodium phosphate/50 mM MES/pH7.0 buffer and then applied to a ceramic hydroxyapatite column (Bio-Rad Laboratories, inc., Bio-Scale CHT 1-type hydroxyapatite column) that had been equilibrated with 5mM NaPi/50 mM MES/30 mM NaCl/pH 7.0 buffer. Elution was performed with a linear concentration gradient of sodium chloride so that antibody-containing fractions were collected. This fraction was dialyzed (Thermo Fisher scientific Inc., Slide-A-Lyzer analysis Cassette) so as to replace the buffer with HBsor (25mM histidine/5% sorbitol, pH 6.0). The antibody was concentrated with Centrifugal UF Filter Device VIVASPIN20 (molecular weight cut-off: UF10K, Sartorius Inc.), thereby adjusting the IgG concentration to 20 mg/ml. Finally, the antibody was filtered through a Minisart-Plus filter (Sartorius Inc.) to obtain a purified sample.

Reference example 1: production of anti-CDH 6 antibody NOV0712

The anti-CDH 6 antibody NOV0712 used in the examples was produced by reference to the full-length light chain and full-length heavy chain amino acid sequences (SEQ ID NO:235 and SEQ ID NO:234, respectively, in International publication No. WO 2016/024195) of NOV0712 described in International publication No. WO 2016/024195.

Reference example 1) -1 anti-CDH 6 antibody NOV0712

Reference example 1) -1-1 construction of anti-CDH 6 antibody NOV0712 heavy chain expression vector

A DNA fragment (GENEART) encoding the variable region of the NOV0712 heavy chain from nucleotide position 36 to 428 in the nucleotide sequence of the NOV0712 heavy chain shown in SEQ ID NO:84 was synthesized. The NOV0712 heavy chain expression vector was constructed by the same method as that applied in examples 4) -1-3. The amino acid sequence of the NOV0712 heavy chain expressed from the NOV0712 heavy chain expression vector is shown in SEQ ID NO 83. 83, the amino acid sequence consisting of the amino acid residues in positions 1 to 19 is a signal sequence.

Reference example 1) -1-2 construction of anti-CDH 6 antibody NOV0712 light chain expression vector

A DNA fragment (GENEART) comprising the DNA sequence encoding the variable region of the NOV0712 light chain from nucleotide positions 37 to 405 in the nucleotide sequence of the NOV0712 light chain shown in SEQ ID NO:82 was synthesized. The NOV0712 light chain expression vector was constructed by the same method as that applied in example 5) -5-2-1. The amino acid sequence of the NOV0712 light chain expressed from the NOV0712 light chain expression vector is shown in SEQ ID NO: 81. In the amino acid sequence shown in SEQ ID NO:81, the amino acid sequence consisting of the amino acid residues at positions 1 to 20 is a signal sequence.

Reference example 1) -2 preparation of anti-CDH 6 antibody NOV0712

Reference example 1) -2-1 production of anti-CDH 6 antibody NOV0712

NOV0712 was produced by the same method as applied in example 4) -2-1.

Reference example 1) -2-2 one-step purification of anti-CDH 6 antibody NOV0712

anti-CDH 6 antibody NOV0712 was purified from the culture supernatant obtained in reference example 1) -2-1 by the same method as that applied in example 4) -2-2 (reference concentration: 5 mg/l HBSor).

Example 6: in vitro evaluation of humanized hG019 and NOV0712

6) -1 evaluation of binding Activity of humanized hG019

6) -1-1 human CDH6 antigen binding Capacity of humanized hG019

Antibodies and antigens (recombinant human CDH6 Fc His chimera, R&D Systems, Inc.) the dissociation constant between the D Systems, Inc.) was measured by using Biacore T200 (GE Healthcare Biosciences Corp.) according to a capture method comprising capturing an antigen as a ligand with an immobilized anti-His antibody, and then measuring the dissociation constant using the antibody as an analyte. Approximately 1000 RU of anti-histidine antibody (His capture kit, GE Healthcare Biosciences Corp.) was covalently bound to the sensor chip CM5 (GE Healthcare Biosciences Corp.) by an amine coupling method. The antibody was also immobilized to the reference chamber in the same manner as described above. Supplemented with 1mM CaCl₂HBS-P + (10mM HEPES pH 7.4, 0.15M NaCl, 0.05% surfactant P20) was used as running buffer. Antigen was added to the anti-histidine antibody-immobilized chip for 60 seconds, and then a dilution series of the antibody (0.391 to 100 nM) was added at a flow rate of 30. mu.l/min for 300 seconds. Subsequently, the dissociation phase was monitored for 600 seconds. As regeneration solution, glycine solution (pH 1.5) supplemented with 5M MgCl2 was added twice at a flow rate of 10. mu.l/min for 30 seconds. The steady-state affinity model in the analysis software (BIAevaluation software, version 4.1) was used in the data analysis and the dissociation constant (KD) was calculated. The results are shown in table 2.

[ Table 2]

	Antibodies	KD(M)
			1	H02L02	1.5E-09
2	H02L02	1.1E-09
			3	H02L03	1.4E-09
4	H04L02	1.1E-09

6) -1-2 binding Activity against human, monkey, mouse or rat CDH6

pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, p3xFLAG-CMV-9-mCDH6 or p3xFLAG-CMV-9-rCDH6 produced in example 1) -1 were transiently introduced into 293 α cells using Lipofectamine 2000(Thermo Fisher Scientific Inc.). Cells were incubated at 37 ℃ and 5% CO₂Overnight, and thereafter a cell suspension was prepared. Untransfected 293 α cells were used as negative controls. The suspension of 293 α cells produced as described above was centrifuged, and then the supernatant was removed. Thereafter, the humanized hG019 antibodies prepared in examples 5) -5-3 were added by adding 4 humanized hG019 antibodies (clone numbers: H01L02, H02L02, H02L03 and H04L02) or human IgG1 control (Calbiochem). The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding 500-fold of anti-human IgG, Fc (γ) PE goat F (ab') (Jackson immunoresearch laboratories, Inc.) that had been diluted with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS, and then detected using a flow cytometer (Canto II; BD Biosciences). Data were analyzed using FlowJo (Tree Star, Inc.). In FIGS. 6-1 and 6-2, the abscissa plots the antibody concentration, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity. Human Ig as a negative control, as shown in FIGS. 6-1 and 6-2The G1 control did not bind to any CDH6 transfected cells. The 4 humanized hG019 antibodies (clone nos. H01L02, H02L02, H02L03 and H04L02) bound to human CDH6 and cynomolgus monkey CDH6, but neither mouse CDH6 nor rat CDH 6. None of the antibodies bound to the cells transfected with the empty vector pcDNA3.1 used as a negative control. On the other hand, international publication No. WO2016/024195 discloses that the NOV0712 antibody exhibits all of the binding activities against human CDH6, cynomolgus monkey CDH6, mouse CDH6 and rat CDH 6. As a result, it was confirmed that the 4 humanized hG019 antibodies obtained in the present specification were anti-CDH 6 antibodies, which exhibited binding characteristics different from those of the NOV0712 antibody.

6) Analysis of CDH 6-binding site of-2 humanized hG019 and NOV0712

6) -2-1 epitope analysis Using Domain deletion mutants

Each of the domain deletion mutant expression vectors generated in examples 2) -2-1 or pcDNA3.1-hCDH6 for expression of full-length human CDH6 was transiently introduced into cells using Lipofectamine 2000(Thermo Fisher Scientific Inc.). Cells were incubated at 37 ℃ and 5% CO₂Overnight, and thereafter a cell suspension was prepared. The suspension of transfected 293 α cells was centrifuged and then the supernatant was removed. Thereafter, the humanized hG019 antibodies prepared in examples 5) -5-3 were added by adding 4 humanized hG019 antibodies (clone numbers: H01L02, H02L02, H02L03 and H04L02), the anti-CDH 6 antibody NOV0712 which had been prepared in reference example 1, or human IgG1 (Calbiochem) as a negative control. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS and then suspended by adding APC-anti-human IgG goat F (ab')2 (Jackson ImmunoResearch Laboratories, Inc.) that had been diluted 500-fold with PBS supplemented with 5% FBS. The cells were allowed to stand at 4 ℃ for 1 hour. Cells were washed twice with PBS supplemented with 5% FBS, and then detected using a flow cytometer (Canto II; BD Biosciences). Data were analyzed using FlowJo (Tree Star, Inc.). The results are shown in FIGS. 7-1 to 7-6. In the histograms of fig. 7-1 to 7-6, APC fluorescence intensity is plotted on the abscissa, which indicates the amount of bound antibody, and cell count is plotted on the ordinate. Shaded histogram display useNegative controls were untransfected 293 α cells, and the open solid line histogram shows the use of 293 α cells expressing either full-length hCDH6 or deletion mutants of each EC domain. Fluorescence intensity was enhanced when the antibody bound to full-length hCDH6 or each EC domain deletion mutant on the cell surface. The human IgG1 control did not bind to any transfected cells. The 4 humanized hG019 antibodies (clone nos. H01L02, H02L02, H02L03 and H04L02) bound full-length hCDH6, the EC1 deletion mutant, the EC2 deletion mutant, the EC4 deletion mutant and the EC5 deletion mutant, but not the EC3 deletion mutant. Specifically, the 4 humanized hG019 antibodies were shown to specifically bind to hCDH6 with EC3 as the epitope. On the other hand, anti-CDH 6 antibody NOV0712 bound full-length hCDH6, EC1 deletion mutant, EC2 deletion mutant, EC3 deletion mutant and EC4 deletion mutant, but not EC5 deletion mutant. Specifically, it was demonstrated that the anti-CDH 6 antibody NOV0712 specifically binds hCDH6 with EC5 as an epitope. This is consistent with the epitope information described in international publication No. WO2016/024195 for NOV 0712. From this result, it was confirmed that the 4 humanized hG019 antibodies obtained in the present specification are anti-CDH 6 antibodies, which exhibit characteristics different from those of NOV 0712.

6) Binding competition assay for the-2-2 antibody

6) Production of a Stable expression cell line of-2-1786-O/hCDH 6

A786-O/hCDH 6 stable expression cell line was generated by infection of 786-O cells (ATCC) with recombinant retrovirus for full-length human CDH6 expression. A human CDH 6-expressing retroviral vector (pQCXIN-hCDH6) was generated by using a cDNA expression vector (origene technologies Inc., RC217889) encoding human CDH6 protein (NP _004923) and integrating the cDNA into the retroviral vector pQCXIN (Clontech Laboratories, Inc.) according to methods known to those skilled in the art. pQCXIN-hCDH6 was transiently introduced into retroviral packaging cells, RetroPack PT67 (Clontech Laboratories, Inc.) using FuGene HD (Promega Corp.). After 48 hours, the culture supernatant containing the recombinant retrovirus was recovered and then added to a 786-O cell culture system so that the cells were infected. From 3 days after infection, at 37 ℃ and 5% CO₂Under conditions of (2) in a culture supplemented with G418 (Gibco) (final concentration: 50 mg/mL)Infected cells were cultured in medium and screened with drug to establish the cell line 786-O/hCDH6 stably expressing human CDH 6. High expression of human CDH6 in the stable expression line was confirmed by flow cytometry in the same manner as applied in example 2) -3-1 (fig. 8). A 500-fold dilution of goat anti-mouse IgG1 secondary antibody Alexa Fluor 647 (Thermo Fisher Scientific Inc.) with PBS supplemented with 5% FBS was used as the antibody for detection. The results are shown in fig. 8. In the histogram of fig. 8, the abscissa plots Alexa Fluor 647 fluorescence intensity, which indicates the amount of bound antibody, and the ordinate plots cell count. The shaded histogram shows the negative control mIgG1 for staining and the open solid histogram shows anti-human CDH6 antibody for staining. As seen, fluorescence intensity was enhanced by binding of the antibody to hCDH6 on the cell surface. The mIgG control did not bind to any cells. As a result, it was demonstrated that the 786-O/hCDH6 stably expressing cell line expressed human CDH6 at a higher degree than the parental line 786-O cells.

6) 2-2-2 binding competition assay Using labeled H01L02 and labeled NOV0712

Labeled H01L02 and labeled NOV0712 were generated using an Alexa Fluor 488 monoclonal antibody labeling kit (Thermo Fisher Scientific Inc.). The cell suspension of the 786-O/hCDH6 stable expression cell line produced in 7) -2-2-1 was centrifuged, and then the supernatant was removed. Thereafter, the antibody was purified by adding labeled NOV0712 or labeled H01L02 (final concentration: 5 nM) and further adding 4 humanized hG019 antibodies (clone No.: H01L02, H02L02, H02L03 and H04L02) or the anti-CDH 6 antibody NOV0712 which had been prepared in reference example 1 or human IgG1 (Calbiochem) as a negative control (final concentration: as shown in the abscissa of fig. 9) to suspend the cells. The cells were allowed to stand at 4 ℃ for 1 hour. Thereafter, the cells were washed twice with PBS supplemented with 5% FBS, followed by detection using a flow cytometer (Canto II; BD Biosciences). Data were analyzed using FlowJo (Tree Star, Inc.). The results are shown in fig. 9. The abscissa plots the final concentration of unlabeled antibody added, and the ordinate plots the amount of bound antibody based on the mean fluorescence intensity. When unlabeled NOV0712 was added to cells supplemented with labeled NOV0712, the amount of bound labeled antibody was reduced by replacing it with unlabeled antibody in a concentration-dependent manner of addition, as they compete with each other for binding to the same epitope. On the other hand, even if each of the 4 humanized hG019 antibodies or human IgG1 as a negative control was added to the cells supplemented with labeled NOV0712, the amount of bound labeled antibody did not change, indicating that the epitopes of these antibodies were different and therefore did not compete for binding with each other. Likewise, when each of the 4 unlabeled humanized hG019 antibodies was added to cells supplemented with labeled H01L02, the amount of bound labeled antibody was reduced by replacing it with unlabeled antibody in an additive concentration-dependent manner, as they competed with each other for binding to the same epitope. On the other hand, even if NOV0712 or human IgG1 as a negative control were added to cells supplemented with labeled H01L02, there was no change in the amount of bound labeled antibody, indicating that the epitopes of these antibodies were different and therefore did not compete for binding with each other.

6) Evaluation of internalization Activity of-3 humanized hG019 and NOV0712

The internalizing activity of humanized hG019 and NOV0712 was assessed using the anti-human IgG reagent Hum-zap (advanced Targeting systems) conjugated to a toxin that inhibits protein synthesis (saporin). Specifically, human CDH 6-positive ovarian tumor cell line NIH: OVCAR-3 (ATCC) at 4 x 10³Individual cells/well were seeded in 96-well plates and then at 37 ℃ and 5% CO₂Cultured overnight under the conditions of (1). Human CDH 6-Positive Kidney cell tumor cell line 786-O (ATCC) at 1X 10³Individual cells/well were seeded on 96-well plates and then cultured overnight. Human CDH 6-Positive ovarian tumor cell line PA-1 (ATCC) at 1X 10³Individual cells/well were seeded in 96-well plates and then at 37 ℃ and 5% CO₂Cultured overnight under the conditions of (1). The next day, each anti-CDH 6 antibody (final concentration: 1 nM) or human IgG1 antibody (Calbiochem) as a negative control antibody was added to the plate. Hum-ZAP (final concentration: 0.5 nM) or F (ab')2 fragment goat anti-human IgG unconjugated with toxin as a negative control, Fc (gamma) fragment specific (Jackson ImmunoResearch Laboratories, Inc.) (final concentration: 0.5 nM) was further added to the plate andcells were incubated at 37 ℃ and 5% CO₂Cultured for 3 days. The number of viable cells was measured by quantifying ATP activity (RLU) using the CellTiter-glo (TM) luminescent cell viability assay. In this evaluation, Hum-ZAP is absorbed into cells in a manner dependent on the internalizing activity of the humanized anti-CDH 6 antibody, so that saporin, which inhibits protein synthesis, is released into the cells, thereby inhibiting cell growth. The cell growth inhibitory effect brought about by the addition of anti-CDH 6 antibody was indicated by relative survival when the number of viable cells in wells supplemented with negative control instead of Hum-ZAP was defined as 100%. FIGS. 10-1 to 10-3 each show graphs and tables of cell viability. In this experiment, antibodies with strong internalization activity are believed to provide low cell survival. As a result, the 4 humanized hG019 antibodies had internalization rates of approximately 50 to 75% predicted from cell viability of all 3 cell lines. Thus, the 4 humanized hG019 antibodies exhibited very high internalization activity and much higher internalization activity than that of NOV 0712. From the viewpoint of the mechanism of drug action of ADC, an antibody having higher internalization activity is considered to be more suitable as an ADC antibody.

Example 7: generation of humanized hG 019-drug conjugates

7) Production of antibody-drug conjugate H01L 02-DXd-1

Step 1: antibody-drug conjugates (1)

[ formula 11]

Reduction of the antibody: by using the general procedure B described in production method 1 (using 1.53 mLmg)^-1cm^-1As 280nm absorptivity) and C, H01L02 produced in example 5 was adjusted to 9.85 mg/mL with PBS 6.0/EDTA. To this solution (5.7 mL) was added an aqueous solution (0.231 mL; 6.0 equivalents per antibody molecule) of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) and a 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0855 mL). After confirming that the solution has a pH within 7.0. + -. 0.1, the chains in the antibody are reduced by incubating the solution at 37 ℃ for 2 hoursAn inter-disulfide bond.

Conjugation between antibody and drug linker: the above solution was incubated at 15 ℃ for 10 minutes. Subsequently, adding theretoN- [6- (2, 5-dioxo-2, 5-dihydro-1)H-pyrrol-1-yl) hexanoyl]glycylglycyl-L-phenylalanyl-N-(2-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1-ylH,12H-benzo [2]de]Pyrano [3',4':6,7]Indoxazino [1,2-b]Quinolin-1-yl]Amino } -2-oxoethoxy) methyl]Glycinamide in dimethylsulfoxide at 10mM (0.386 mL; 10 equivalents per antibody molecule) and the resulting mixture was incubated at 15 ℃ for 1 hour to conjugate the drug linker to the antibody. Subsequently, an aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) of 100mM NAC (Sigma-Aldrich Co. LLC) was added thereto, and the resulting mixture was further stirred at room temperature for 20 minutes to terminate the reaction of the drug linker.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H01L 02-ADC".

And (3) characterization: using the general procedure E described in Generation method 1 (using ε)_D,280= 5440 and ε_D,370= 21240), the following characteristic values were obtained.

Antibody concentration: 2.26 mg/mL, antibody yield: 42.9 mg (76%), average number of conjugated drug molecules per antibody molecule (n) measured by general procedure E: 5.9, and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure F: 7.7.

7) production of-2 antibody-drug conjugate H02L02-DXd

Step 1: antibody-drug conjugates (2)

[ formula 12]

Reduction of the antibody: by using the general procedure B described in Generation method 1 (using 1.51 mLmg)^-1cm^-1As absorption coefficient of 280nm) And C, H02L02 generated in example 5 was adjusted to 9.95 mg/mL with PBS 6.0/EDTA. To this solution (5.7 mL) was added an aqueous solution (0.234 mL; 6.0 equivalents per antibody molecule) of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) and 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0855 mL). After confirming that the solution has a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody is reduced by incubating the solution at 37 ℃ for 2 hours.

Conjugation between antibody and drug linker: the above solution was incubated at 15 ℃ for 10 minutes. Subsequently, adding theretoN- [6- (2, 5-dioxo-2, 5-dihydro-1)H-pyrrol-1-yl) hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1-ylH,12H-benzo [2]de]Pyrano [3',4':6,7]Indoxazino [1,2-b]Quinolin-1-yl]Amino } -2-oxoethoxy) methyl]Glycinamide in dimethylsulfoxide at 10mM (0.389 mL; 10 equivalents per antibody molecule) and the resulting mixture was incubated at 15 ℃ for 1 hour to conjugate the drug linker to the antibody. Subsequently, an aqueous solution (0.0350 mL; 9 equivalents per antibody molecule) of 100mM NAC (Sigma-Aldrich Co. LLC) was added thereto, and the resulting mixture was further stirred at room temperature for 20 minutes to terminate the reaction of the drug linker.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H02L 02-ADC".

And (3) characterization: using the general procedure E described in Generation method 1 (using ε)_D,280= 5440 and ε_D,370= 21240), the following characteristic values were obtained.

Antibody concentration: 2.61 mg/mL, antibody yield: 49.6 mg (87%), average number of conjugated drug molecules per antibody molecule (n) measured by general procedure E: 5.9, and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure F: 7.6.

7) production of 3 antibody-drug conjugate H02L03-DXd

Step 1: antibody-drug conjugates (3)

[ formula 13]

Reduction of the antibody: by using the general procedure B described in production method 1 (using 1.53 mLmg)^-1cm^-1As 280nm absorptivity) and C, H02L03 produced in example 5 was adjusted to 9.86 mg/mL with PBS 6.0/EDTA. To this solution (5.7 mL) was added an aqueous solution (0.270 mL; 7.0 equivalents per antibody molecule) of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) and 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0855 mL). After confirming that the solution has a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody is reduced by incubating the solution at 37 ℃ for 2 hours.

Conjugation between antibody and drug linker: the above solution was incubated at 15 ℃ for 10 minutes. Subsequently, adding theretoN- [6- (2, 5-dioxo-2, 5-dihydro-1)H-pyrrol-1-yl) hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1-ylH,12H-benzo [2]de]Pyrano [3',4':6,7]Indoxazino [1,2-b]Quinolin-1-yl]Amino } -2-oxoethoxy) methyl]Glycinamide in dimethylsulfoxide at 10mM (0.386 mL; 10 equivalents per antibody molecule) and the resulting mixture was incubated at 15 ℃ for 1 hour to conjugate the drug linker to the antibody. Subsequently, an aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) of 100mM NAC (Sigma-Aldrich Co. LLC) was added thereto, and the resulting mixture was further stirred at room temperature for 20 minutes to terminate the reaction of the drug linker.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H01L 02-ADC".

And (3) characterization: using general procedure E described in Generation method 1 ((using ε)_D,280= 5440 and ε_D,370= 21240), the following characteristic values were obtained.

Antibody concentration: 2.71 mg/mL, antibody yield: 51.4 mg (91%), average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure E: 5.7, and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure F: 7.6.

7) production of-4 antibody-drug conjugate H04L02-DXd

Step 1: antibody-drug conjugates (4)

[ formula 14]

Reduction of the antibody: by using the general procedure B described in production method 1 (using 1.53 mLmg)^-1cm^-1As 280nm absorptivity) and C, H04L02 generated in example 5 was adjusted to 9.86 mg/mL with PBS 6.0/EDTA. To this solution (5.7 mL) was added an aqueous solution (0.232 mL; 6.0 equivalents per antibody molecule) of 10mM TCEP (Tokyo Chemical Industry Co., Ltd.) and 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0855 mL). After confirming that the solution has a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody is reduced by incubating the solution at 37 ℃ for 2 hours.

Conjugation between antibody and drug linker: the above solution was incubated at 15 ℃ for 10 minutes. Subsequently, adding theretoN- [6- (2, 5-dioxo-2, 5-dihydro-1)H-pyrrol-1-yl) hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S) -9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1H, 12H-benzo [ de]Pyrano [3',4':6,7]Indoxazino [1,2-b]Quinolin-1-yl]Amino } -2-oxoethoxy) methyl]Glycinamide in dimethylsulfoxide at 10mM (0.386 mL; 10 equivalents per antibody molecule) and the resulting mixture was incubated at 15 ℃ for 1 hour to conjugate the drug linker to the antibody. Subsequently, an aqueous solution (0.0347 mL; 9 equivalents per antibody molecule) of 100mM NAC (Sigma-Aldrich Co. LLC) was added thereto, and the resulting mixture was further stirred at room temperature for 20 minutes to terminate the reaction of the drug linker.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 19 mL of a solution containing the title antibody-drug conjugate "H04L 02-ADC".

And (3) characterization: using the general procedure E described in Generation method 1 (using ε)_D,280= 5440 and ε_D,370= 21240), the following characteristic values were obtained.

Antibody concentration: 2.56 mg/mL, antibody yield: 48.7 mg (87%), average number of conjugated drug molecules per antibody molecule (n) measured by general procedure E: 5.8, and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure F: 7.6.

reference example 2: production of NOV 0712-drug conjugates

Reference example 2) -1 production of antibody-drug conjugate NOV0712-DM4

Antibody-drug conjugates (5)

Conjugation between antibody and drug linker: by using the general procedure B described in Generation method 1 (using 1.51 mLmg)^-1cm^-1As 280nm absorption coefficient) and C, NOV0712 generated in reference example 1 was adjusted to 9.7 mg/mL with 20mM HEPES8.1 (HEPES, 1M buffer solution (20 mL) manufactured by Life Technologies corp. was pH-adjusted to 8.1 with 1M sodium hydroxide, and then brought to 1L with distilled water). The solution was incubated at 20 ℃ for 10 minutes. Subsequently, a 10mM solution of 1- (2, 5-dioxopyrrolidin-1-yloxy) -1-oxo-4- (pyridin-2-yldisulfanyl) butane-2-sulfonic acid described in WO2016/024195 in DMA (0.366 mL; 5.2 equivalents per antibody molecule), a 10mM solution of N2-deacetyl-N2- (4-methyl-4-mercapto-1-oxopentyl) -maytansine (DM4) in DMA (0.366 mL; 6.8 equivalents per antibody molecule) and 0.243 mL of DMA were added thereto, and the resulting mixture was incubated at 20 ℃ for 16 hours to conjugate the drug linker to the antibody. Subsequently, an aqueous solution of 1M acetic acid was added thereto to adjust the pH to 5.0, and the obtained mixture was further stirred at room temperature for 20 minutes to terminate the reaction of the drug linker.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 28 mL of a solution containing the title antibody-drug conjugate "NOV0712-DM 4".

And (3) characterization: using the general procedure E described in Generation method 1 (using ε)_A,280= 200500，ε_A,252 = 76295，ε_D,280= 43170, and ∈_D,252= 23224), the following characteristic values were obtained.

Antibody concentration: 2.58 mg/mL, antibody yield: 72.2 mg (93%), and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure E: 3.0.

reference example 2) -2 production of antibody-drug conjugate NOV0712-DXd

Step 1: antibody-drug conjugates (6)

[ formula 15]

Reduction of the antibody: by using the general procedure B described in Generation method 1 (using 1.5 mLmg)^-1cm^-1As 280 nm absorptivity) and C, NOV0712 produced in reference example 1 was adjusted to 9.26 mg/mL with PBS 6.0/EDTA. To this solution (6.6 mL) was added an aqueous solution (0.254 mL; 6.0 equivalents per antibody molecule) of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) and 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0990 mL). After confirming that the solution has a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody is reduced by incubating the solution at 37 ℃ for 2 hours.

Conjugation between antibody and drug linker: the above solution was incubated at 15 ℃ for 10 minutes. Subsequently, adding theretoN- [6- (2, 5-dioxo-2, 5-dihydro-1)H-pyrrol-1-yl) hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1-ylH,12H-benzo [ 2]de]Pyrano [3',4':6,7]Indoxazino [1,2-b]Quinolin-1-yl]Amino } -2-oxoethoxy) methyl]Glycinamide in 10 mM solution in dimethyl sulfoxide (0.381 mL; 9 equivalents per antibody molecule) and the resulting mixture was incubated at 15 ℃ for 1 hour to conjugate the drug linker to the antibody. Followed byTo this, 100 mM NAC (Sigma-Aldrich Co. LLC) in water (0.0381 mL; 9 equivalents per antibody molecule) was added, and the resulting mixture was further stirred at room temperature for 20 minutes to terminate the drug linker reaction.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 23.5 mL of a solution containing the title antibody-drug conjugate "NOV 0712-ADC".

And (3) characterization: using the general procedure E described in Generation method 1 (using ε)_D,280= 5440 and ε_D,370= 21240), the following characteristic values were obtained.

Antibody concentration: 2.26 mg/mL, antibody yield: 56.4 mg (92%), average number of conjugated drug molecules per antibody molecule (n) measured by general procedure E: 6.4, and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure F: 7.8.

reference example 3: production of H01L02-DM4

Reference example 3) -1 production of antibody-drug conjugate H01L02-DM4

Antibody-drug conjugates (7)

Conjugation between antibody and drug linker: by using the general procedure B described in production method 1 (using 1.53 mLmg)^-1cm^-1As 280 nm absorption coefficient) and C, H01L02 generated in example 5 was adjusted to 9.8 mg/mL with 20 mM HEPES8.1 (HEPES, 1M buffer solution (20 mL) manufactured by Life Technologies corp. was pH-adjusted to 8.1 with 1M sodium hydroxide, and then brought to 1L with distilled water). The solution was incubated at 20 ℃ for 10 minutes. Subsequently, a 10 mM solution of 1- (2, 5-dioxopyrrolidin-1-yloxy) -1-oxo-4- (pyridin-2-yldisulfanyl) butane-2-sulfonic acid described in WO2016/024195 in DMA (0.062 mL; 11.5 equivalents per antibody molecule) and a 10 mM solution of N2-deacetyl-N2- (4-methyl-4-mercapto-1-oxopentyl) -maytansine (DM4) in DMA (0.082 mL; 15.1 equivalents per antibody molecule) were added thereto, and the resulting mixture was incubated at 20 ℃ for 18 hours to conjugate the drug linker to the antibody. Subsequently, an aqueous solution of 1M acetic acid was added thereto to adjust the pH to 5.0, and the obtained mixture was further stirred at room temperatureThe reaction of the drug linker was terminated in 20 minutes.

And (3) purification: the above solution was purified by general procedure D described in production method 1 to obtain 3.5 mL of a solution containing the title antibody-drug conjugate "H01L02-DM 4".

And (3) characterization: using the general procedure E described in production method 1 (using ε a,280 = 223400, ε a,252 = 85646, ε D,280 = 4317, and ε D,252 = 23224), the following eigenvalues were obtained.

Antibody concentration: 1.97 mg/mL, antibody yield: 6.90 mg (88%), and the average number of conjugated drug molecules per antibody molecule (n) as measured by general procedure E: 3.6.

example 8: evaluation of in vitro Activity of antibody-drug conjugates ]

8) -1 evaluation of the in vitro cell growth inhibitory Activity of antibody-drug conjugates against CDH 6-Positive human tumor cell line

CDH 6-positive human ovarian tumor cell line PA-1 at 2X 10³Individual cells/100. mu.L/well were seeded in 10% FBS-supplemented MEM media on 96-well plates, and then cells were plated at 37 ℃ and 5% CO₂Cultured overnight under the conditions of (1). The next day, each of the 4 humanized hG 019-drug conjugates produced in example 7 (clone names: H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) or the NOV 0712-drug conjugate (NOV0712-DM4) produced in reference example 2 was added to the cells so that the final concentration was 0.0001 (nM) to 100 (nM). After 4 days of culture, the number of viable cells was measured by quantifying ATP using CellTiter-glo (TM) luminescent cell viability assay (Promega Corp.). Fig. 11 shows the concentration-dependent cell growth inhibitory activity when each antibody-drug conjugate was added to cells. From this result, it was confirmed that the 4 humanized hG 019-drug conjugates exhibited growth inhibitory activity against tumor cells at a lower addition concentration than that of NOV 0712-drug conjugates, and had high antitumor activity.

Example 9: in vivo anti-tumor Effect of antibody-drug conjugates

The anti-tumor effect of the antibody-drug conjugates was evaluated by inoculation with CDH 6-positive human tumor cell line cells using an animal model derived from immunodeficient mice. BALB/c nude mice (CAnN. Cg-Foxnl [ nu ]/CrlCrlj [ Foxnlnnu/Foxnlnnu ], Charles River Laboratories Japan Inc.) and SCID mice (CB17/Icr-Prkdc [ SCID ]/CrlCrlj, Charles River Laboratories Japan Inc.) at 4 to 5 weeks were adapted under SPF conditions for 3 days or more and then used in the experiment. Mice were fed a sterilized solid diet (FR-2, Funabashi farm co., Ltd) and sterilized tap water (which had been prepared by adding 5 to 15ppm sodium hypochlorite solution to tap water). The major and minor diameters of the inoculated tumors were measured twice weekly using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and then the volume of the tumor was calculated according to the following expression.

Tumor volume (mm)³) = 1/2X Long diameter (mm) X short diameter (mm)]²。

Each antibody-drug conjugate was diluted with ABS buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5) (Nacalai Tesque, Inc.) and the dilution was administered intravenously to the tail of each mouse at the dose shown in each example. ABS buffer was administered to the control group (vehicle group) in the same manner as described above. Six mice per group were used in the experiment.

9) -1 antitumor action- (1)

CDH 6-positive human renal cell tumor cell line 786-O (ATCC) (the CDH6 expression of which was confirmed in examples 2) -3-1) was suspended in Matrigel (Corning Inc.), and the cell suspension was suspended at 5X 10⁶Individual cell doses were inoculated subcutaneously into the right flank region of each male SCID mouse (day 0). On day 18, mice were randomly grouped. On the day of grouping, each of the 4 antibody-drug conjugates produced in example 7 (clone names: H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) or NOV0712-DM4 produced in reference example 2 was intravenously administered to the tail of each mouse at a dose of 3 mg/kg. The results are shown in fig. 12. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

In this tumor model, NOV0712-DM4 did not show significant anti-tumor effects. All 4 antibody-drug conjugates produced in example 7 reduced tumor volume after administration, exerted significant tumor regression, and maintained tumor regression for 24 days after administration (figure 12).

9) -2 antitumor action- (2)

CDH 6-positive human ovarian tumor cell line PA-1(ATCC), the CDH6 expression of which has been confirmed in examples 2) -3-1, was suspended in Matrigel (Corning Inc.), and the cell suspension was washed at 8.5X 10⁶A dose of individual cells was inoculated subcutaneously into the right flank region of each female nude mouse (day 0). On day 11, mice were randomly grouped. On the day of grouping, the antibody-drug conjugate produced in example 7, H01L02-DXd, or NOV0712-DM4 or NOV0712-DXd produced in reference example 2, was administered intravenously to the tail of each mouse at doses of 1 and 3 mg/kg. The results are shown in fig. 13. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

In this tumor model, NOV0712-DM4 showed no anti-tumor effect at any dose of 1 and 3 mg/kg. On the other hand, H01L02-DXd significantly reduced tumor volume after administration at doses of 1 and 3mg/kg, and exerted tumor regression effects (fig. 13). The H01L02 antibody and NOV0712 antibody obtained in the present specification were conjugated to the same drug DXd, and the drug effects of the resulting samples were compared. As a result, H01L02-DXd exerted a stronger antitumor effect than that of NOV0712-DXd at both doses of 1 and 3 mg/kg. Specifically, the H01L02 antibody of the present invention was demonstrated to be an antibody of an antibody-drug conjugate as an antitumor agent superior to the NOV0712 antibody (fig. 13).

9) -3 anti-tumor action- (3)

CDH 6-positive human ovarian tumor cell line NIH: OVCAR-3 (ATCC), the CDH6 expression of which has been confirmed in examples 2) -3-1, was suspended in Matrigel (Corning Inc.), and the cell suspension was suspended at 1X 10⁷A dose of individual cells was inoculated subcutaneously into the right flank region of each female nude mouse (day 0). On day 22, mice were randomly grouped. On the day of grouping, the antibody-drug conjugate produced in example 7, H01L02-DXd, or NOV0712-DM4 produced in reference example 2, was administered intravenously to the tail of each mouse at a dose of 1 or 3 mg/kg. The results are shown in fig. 14.The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

NOV0712-DM4 showed no anti-tumor effect at the dose of 1 mg/kg and at the dose of 3mg/kg, although tumor regrowth was observed from 2 weeks after administration. On the other hand, H01L02-DXd significantly inhibited the increase in tumor volume after administration both at doses of 1 and 3mg/kg, and tumor growth inhibition was sustained over a long period of 31 days after administration, especially at a dose of 3mg/kg (fig. 14).

The tumor growth inhibitory effect of NOV0712-DM4 produced in reference example 2 or H01L02-DM4 produced in reference example 3 was evaluated in the same manner as described above using PA-1 cells. H01L02-DM4 further reduced tumor volume than NOV0712-DM 4. Therefore, the H01L02 antibody of the present invention is an antibody of an antibody-drug conjugate as an antitumor agent superior to the NOV0712 antibody.

9) -4 anti-tumor action- (4)

CDH 6-positive human renal cell tumor cell line 786-O (ATCC) (the CDH6 expression of which was confirmed in examples 2) -3-1) was suspended in Matrigel (Corning Inc.), and the cell suspension was suspended at 5X 10⁶Individual cell doses were inoculated subcutaneously into the right flank region of each male SCID mouse (day 0). On day 20, mice were randomly grouped. On the day of grouping, the antibody-drug conjugate produced in example 7, H01L02-DXd, or NOV0712-DM4 produced in reference example 2, was administered intravenously to the tail of each mouse at a dose of 1 or 3 mg/kg. The results are shown in fig. 15. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

In this tumor model, NOV0712-DM4 did not exhibit significant anti-tumor effects at any dose of 1 and 3 mg/kg. On the other hand, H01L02-DXd reduced tumor volume after administration at both doses of 1 and 3mg/kg, and showed significant tumor regression, especially at the dose of 3mg/kg, and sustained tumor regression for 20 days after administration (fig. 15).

9) -5 antitumor action- (5)

CDH 6-negative human ovarian tumor cell line ES-2 (ATCC) (the absence of CDH6 expression of which was confirmed in examples 2) -3-1) was suspended in physiological saline, and the cell suspension was diluted at 1X 10⁶A dose of individual cells was inoculated subcutaneously into the right flank region of each female nude mouse (day 0). On day 7, mice were randomly grouped. On the day of grouping, the antibody-drug conjugate produced in example 7, H01L02-DXd, or NOV0712-DM4 produced in reference example 2, was administered intravenously to the tail of each mouse at a dose of 1 or 3 mg/kg. The results are shown in fig. 16. The abscissa plots days and the ordinate plots tumor volume. The error range depicts the SE value.

In this tumor model that does not express CDH6, H01L02-DXd and NOV0712-DM4 did not exhibit anti-tumor effects at any dose. From this result, the anti-tumor effect of the antibody-drug conjugate in the CDH 6-positive tumor model demonstrated in examples 9) -1, 9) -2, 9) -3 and 9) -4 was an effect dependent on the expression of CDH6 in tumor cells. Therefore, the antibody-drug conjugate of the present invention is considered to be a selective and safe antitumor drug, which specifically exhibits antitumor effect against CDH 6-positive tumors, while causing no cytotoxicity against normal tissues that are CDH 6-negative (fig. 16).

Industrial applicability

The present invention provides anti-CDH 6 antibodies having internalization activity and antibody-drug conjugates comprising the same. The antibody-drug conjugate can be used as a therapeutic drug for cancer and the like.