MEDICAL USE OF ANTI-C MET ANTIBODY-CYTOTOXIC DRUG CONJUGATE

Abstract

The medical use of an anti-c Met antibody-cytotoxic drug conjugate is described. In particular, an anti-c-Met antibody, an antigen-binding fragment thereof, a chimeric antibody and a humanized antibody containing the anti-c-Met antibody CDRs, and an antibody-cytotoxic drug conjugate thereof or a pharmaceutically acceptable salt or solvate thereof are described. Also described are the use of a pharmaceutical composition containing the humanized anti-c-Met antibody, the antigen-binding fragment thereof, the antibody-cytotoxic drug conjugate thereof, or the pharmaceutically acceptable salt or solvate thereof as an anti-hepatoma drug.

Claims

1-36. (canceled)

37. A method of treating hepatic carcinoma in a subject in need thereof, the method comprising administering to the subject an antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-cytotoxic drug conjugate has a structure of formula (I):
Ab-[(L.sub.2)t-L.sub.1-D)]y(I) wherein: D is a cytotoxic drug; L.sub.1 and L.sub.2 are linker units; t is 0 or 1; y is 1-8; and Ab is an antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor, comprising: an antibody heavy chain variable region comprising an amino acid sequence having HCDR sequences of SEQ ID NO: 6, SEQ ID NO:7 and SEQ ID NO:8, or a mutant sequence thereof; and an antibody light chain variable region comprising an amino acid sequence having LCDR sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, or a mutant sequence thereof.

38. The method of claim 37, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor is a chimeric antibody or a humanized antibody, or antigen-binding fragment thereof.

39. The method of claim 38, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor is a humanized antibody, wherein the humanized antibody heavy chain variable region comprises heavy chain framework regions having FR1, FR2, FR3 and FR4 of the human germline heavy chain IGHV 3-33*01, or a mutant sequence thereof, and wherein the humanized antibody light chain variable region comprises heavy chain framework regions having FR1, FR2, FR3 and FR4 of the human germline light chain IGKV085 or IGKV4-1*01, or a mutant sequence thereof.

40. The method of claim 39, wherein the humanized antibody comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14 and 15, and comprises a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17 and 18.

41. The method of claim 37, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor comprises a combination of a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from any one of a) to c): a) Heavy chain variable region sequence of SEQ ID NO: 13, and light chain variable region sequence of SEQ ID NO: 16; b) Heavy chain variable region sequence of SEQ ID NO: 14, and light chain variable region sequence of SEQ ID NO: 17; and c) Heavy chain variable region sequence of SEQ ID NO: 15, and light chain variable region sequence of SEQ ID NO: 18.

42. The method of claim 38, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor is a humanized antibody, wherein the heavy chain constant region of the humanized antibody comprises a constant region derived from human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof, and wherein the light chain constant region of the humanized antibody comprises a constant region selected from the group consisting of human and human , or a variant thereof.

43. The method of claim 42, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor comprises a full-length heavy chain sequence selected from the group consisting of SEQ ID NOs: 23, 24 and 25 and sequences having at least 90% identity to SEQ ID NOs: 23, 24 or 25, and comprises a full-length light chain sequence selected from the group consisting of SEQ ID NOs: 26, 27 and 28 and sequences having at least 90% identity to SEQ ID NOs: 26, 27 and 28.

44. The method of claim 38, wherein the antibody or antigen-binding fragment thereof that specifically binds to c-Met receptor is a humanized antibody, wherein the humanized antibody comprises a combination of a full-length light chain amino acid sequence and a full-length heavy chain amino acid sequence selected from: Ab-9, comprising a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 26; Ab-10, comprising a heavy chain amino acid sequence of SEQ ID NO: 24 and a light chain amino acid sequence of SEQ ID NO: 27; and Ab-11, comprising a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 28.

45. The method of claim 37, wherein the antibody-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvate thereof is administered in a pharmaceutical composition, the pharmaceutical composition comprising the antibody-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable excipient, diluent or carrier.

46. The method of claim 37, wherein -L.sub.2- comprises formula (-L.sub.2-): ##STR00043## wherein: X.sub.1 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; X.sub.2 is selected from the group consisting of C.sub.1-6 alkyl, 3-8 membered cycloalkyl and 3-8 membered heterocyclyl; m is 0, 1, 2, 3, 4 or 5; and S is a sulfur atom.

47. The method of claim 37, wherein D is a cytotoxic agent selected from the group consisting of toxins, chemotherapeutic agents, antibiotics, radioisotopes and nucleolytic enzymes.

48. The method of claim 47, wherein D comprises formula (D): ##STR00044## or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof; wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.8, R.sup.9, R.sup.10, and R.sup.11 are each selected from the group consisting of hydrogen, halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; or any two of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are taken together with the carbon atoms to which they are attached to form a 3-8 membered cycloalkyl, and the rest are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and 3-8 membered cycloalkyl; R.sup.12 and R.sup.13 are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and halogen; R.sup.14 is selected from the group consisting of 6-8 membered aryl and 5-8 membered heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from the group consisting of hydrogen, halogen, hydroxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.15 is selected from the group consisting of halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, 3-8 membered cycloalkyl, carboxyl, C.sub.1-6 alkyl carbonyl and C.sub.1-6 alkoxy carbonyl; R.sup.16 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl.

49. The method of claim 48, wherein L.sub.2 comprises a linker selected from the group consisting of valine-citrulline (Val-Cit), 6-maleimido-caproyl (MC), P-aminobenzyloxycarbonyl (PAB) and 6-maleimido-caproyl-P-aminobenzyloxycarbonyl (MC-PAB).

50. The method of claim 37, wherein D is a maytansinoid.

51. The method of claim 50, wherein L.sub.2 is selected from the group consisting of N-succinimidyl 4-(2-pyridylthio) valerate, N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and N-succinimidyl (4-iodo-acetyl) aminobenzoate.

52. The method of claim 37, wherein D is a camptothecin alkaloid selected from the group consisting of camptothecin (CPT), 10-hydroxy-CPT, Irinotecan, SN-38 and topotecan.

53. The method of claim 52, wherein L.sub.2 is selected from the group consisting of valine-citrulline (Val-Cit), 6-maleimido-caproyl (MC), P-aminobenzyloxycarbonyl (PAB) and 6-maleimido-caproyl-P-aminobenzyloxycarbonyl (MC-PAB).

54. The method of claim 37, wherein the antibody-cytotoxic drug conjugate of formula (I) or the pharmaceutically acceptable salt or solvate thereof is an antibody-cytotoxic drug conjugate of formula (II) or a pharmaceutically acceptable salt or solvate thereof: ##STR00045## wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.8, R.sup.9, R.sup.10, and R.sup.11 are each selected from the group consisting of hydrogen, halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; or any two of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are taken together with the carbon atoms to which they are attached to form a 3-8 membered cycloalkyl, and the rest are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and 3-8 membered cycloalkyl; R.sup.12 and R.sup.13 are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and halogen; R.sup.14 is selected from the group consisting of 6-8 membered aryl and 5-8 membered heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from the group consisting of hydrogen, halogen, hydroxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.15 is selected from the group consisting of halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, 3-8 membered cycloalkyl, carboxyl, C.sub.1-6 alkyl carbonyl and C.sub.1-6 alkoxy carbonyl; R.sup.16 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; and Ab, t, y, L.sub.1, and L.sub.2 are as defined in claim 37.

55. The method of claim 37, wherein the antibody-cytotoxic drug conjugate of formula (I) or the pharmaceutically acceptable salt or solvate thereof is an antibody-cytotoxic drug conjugate of formula (III) or a pharmaceutically acceptable salt or solvate thereof: ##STR00046## wherein: R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.8, R.sup.9, R.sup.10, and R.sup.1 are each selected from the group consisting of hydrogen, halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; or any two of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are taken together with the carbon atoms to which they are attached to form a 3-8 membered cycloalkyl, and the rest are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and 3-8 membered cycloalkyl; R.sup.12 and R.sup.13 are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and halogen; R.sup.14 is selected from the group consisting of 6-8 membered aryl and 5-8 membered heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from the group consisting of hydrogen, halogen, hydroxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.15 is selected from the group consisting of halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, 3-8 membered cycloalkyl, carboxyl, C.sub.1-6 alkyl carbonyl and C.sub.1-6 alkoxy carbonyl; R.sup.16 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, alkyl, C.sub.i-6 alkoxy and 3-8 membered cycloalkyl; Ab and y are as defined in claim 37; and n is 3, 4, 5 or 6.

56. The method of claim 37, wherein the antibody-cytotoxic drug conjugate of formula (I) or the pharmaceutically acceptable salt or solvate thereof is an antibody-cytotoxic drug conjugate of formula (IV) or a pharmaceutically acceptable salt or solvate thereof: ##STR00047## wherein: R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.8, R.sup.9, R.sup.10, and R.sup.11 are each selected from the group consisting of hydrogen, halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; or any two of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are taken together with the carbon atoms to which they are attached to form a 3-8 membered cycloalkyl, and the rest are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and 3-8 membered cycloalkyl; R.sup.12 and R.sup.13 are each selected from the group consisting of hydrogen, C.sub.1-6 alkyl and halogen; R.sup.14 is selected from the group consisting of 6-8 membered aryl and 5-8 membered heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from the group consisting of hydrogen, halogen, hydroxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; R.sup.15 is selected from the group consisting of halogen, C.sub.2-6 alkenyl, C.sub.1-6 alkyl, 3-8 membered cycloalkyl, carboxyl, C.sub.1-6 alkyl carbonyl and C.sub.1-6 alkoxy carbonyl; R.sup.16 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; n is 3, 4, 5 or 6; X.sub.1 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; X.sub.2 is selected from the group consisting of C.sub.1-6 alkyl, 3-8 membered cycloalkyl and 3-8 membered heterocyclyl; and Ab and y are as defined in claim 37.

57. The method of claim 37, wherein the antibody-cytotoxic drug conjugate of formula (I) or the pharmaceutically acceptable salt or solvate thereof is an antibody-cytotoxic drug conjugate of formula (V) or a pharmaceutically acceptable salt or solvate thereof: ##STR00048## n is 3, 4, 5 or 6; X.sub.1 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, C.sub.1-6 alkyl, C.sub.1-6 alkoxy and 3-8 membered cycloalkyl; X.sub.2 is selected from the group consisting of C.sub.1-6 alkyl, 3-8 membered cycloalkyl and 3-8 membered heterocyclyl; and Ab, D, and y are as defined in claim 37.

58. The method of claim 37, wherein the antibody-cytotoxic drug conjugate of formula (I) is selected from the group consisting of: ##STR00049## ##STR00050## ##STR00051## or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab-9 is a humanized antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 26; Ab-10 is a humanized antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 24 and a light chain amino acid sequence of SEQ ID NO: 27; and Ab-11 is a humanized antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 25 and a light chain amino acid sequence of SEQ ID NO: 28; and y is 1, 2, 3, 4, 5, 6, 7 or 8.

59. The method of claim 37, wherein the hepatic carcinoma is c-Met positive hepatic carcinoma or hepatic carcinoma which overexpresses c-Met.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0250] FIG. 1 is the curve tracing the tumor volume, showing the inhibition of hepatic carcinoma cells by ADC molecules of the present invention in a LI-03-0022 HCC PDX tumor model. The results show that the ADC molecule can achieve complete tumor inhibition by the introduced toxin, whereas the antibody alone cannot. The ADC drug of the invention has good tolerance in tumor-bearing animals; the data represent the mean value of a group, and the error bars represent the standard error of the mean value (SEM);

[0251] FIG. 2 is the IHC (immunohistochemical staining) score of LI-03-0010 (negative control) in a LI-03-0022 HCC PDX tumor model;

[0252] FIG. 3 is the IHC (immunohistochemical staining) score of LI-03-0022 tissue in a LI-03-0022 HCC PDX tumor model;

[0253] FIG. 4 is the curve tracing the tumor volume, showing the inhibition of hepatic carcinoma cells by ADC molecules of the present invention in a LI-03-0240 HCC PDX tumor model;

[0254] FIG. 5 is the IHC (immunohistochemical staining) score of LI-03-0010 (negative control) in a LI-03-0240 HCC PDX tumor model;

[0255] FIG. 6 is the IHC (immunohistochemical staining) score of LI-03-0240 tissue in a LI-03-0240 HCC PDX tumor model.

DETAILED DESCRIPTION OF THE INVENTION

[0256] Hereinafter, the present invention is illustrated in more details with reference to examples. The examples of the invention are merely used to exemplify the technical solution of the invention, and the merits and scope of the invention are not limited thereto.

[0257] In the examples or test examples of the present invention, where specific conditions are not described, the experiments are generally conducted under conventional conditions, or under conditions proposed by the manufacturers of the material or product. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Ausubel et al, Greene Publishing Associates, Wiley Interscience, NY. Where the source of the agents is not specifically given, the agents are commercially available.

EXAMPLES

Example 1. Clonal Expression of Antigen and Antibody

[0258] The antibodies (light and heavy chains) and antigens used in the present invention are constructed by overlapping extension PCR methods known in the art. The DNA fragment obtained by overlapping extension PCR was inserted into the expression vector pEE6.4 (Lonza Biologics) using HindIII/BstBI restriction sites, and expressed in 293F cells (Invitrogen, Cat # R790-07). The resulting recombinant protein was used for immunization or screening. The c-Met gene template was derived from origene Corporation (number RC217003). The DNA sequences cloned and expressed are as follows:

[0259] DNA sequence for fusion protein of human c-Met extracellular region (ECD) and murine Fc region (human c-Met ECD-mFc):

TABLE-US-00002 (SEQIDNO:1) atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttac cttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccg agatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaaca cccatccagaatgtcattctacatgagcatcacattttccttggtgccac taactacatttatgttttaaatgaggaagaccttcagaaggttgctgagt acaagactgggcctgtgctggaacacccagattgtttcccatgtcaggac tgcagcagcaaagccaatttatcaggaggtgtttggaaagataacatcaa catggctctagttgtcgacacctactatgatgatcaactcattagctgtg gcagcgtcaacagagggacctgccagcgacatgtctttccccacaatcat actgctgacatacagtcggaggttcactgcatattctccccacagataga agagcccagccagtgtcctgactgtgtggtgagcgccctgggagccaaag tcctttcatctgtaaaggaccggttcatcaacttctttgtaggcaatacc ataaattcttcttatttcccagatcatccattgcattcgatatcagtgag aaggctaaaggaaacgaaagatggttttatgtttttgacggaccagtcct acattgatgttttacctgagttcagagattcttaccccattaagtatgtc catgcctttgaaagcaacaattttatttacttcttgacggtccaaaggga aactctagatgctcagacttttcacacaagaataatcaggttctgttcca taaactctggattgcattcctacatggaaatgcctctggagtgtattctc acagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatact tcaggctgcgtatgtcagcaagcctggggcccagcttgctagacaaatag gagccagcctgaatgatgacattcttttcggggtgttcgcacaaagcaag ccagattctgccgaaccaatggatcgatctgccatgtgtgcattccctat caaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtga gatgtctccagcatttttacggacccaatcatgagcactgctttaatagg acacttctgagaaattcatcaggctgtgaagcgcgccgtgatgaatatcg aacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaat tcagcgaagtcctcttaacatctatatccaccttcattaaaggagacctc accatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggt ttctcgatcaggaccatcaacccctcatgtgaattttctcctggactccc atccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggc tacacactggttatcactgggaagaagatcacgaagatcccattgaatgg cttgggctgcagacatttccagtcctgcagtcaatgcctctctgccccac cctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaa tgcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaa ggttttcccaaatagtgcaccccttgaaggagggacaaggctgaccatat gtggctgggactttggatttcggaggaataataaatttgatttaaagaaa actagagttctccttggaaatgagagctgcaccttgactttaagtgagag cacgatgaatacattgaaatgcacagttggtcctgccatgaataagcatt tcaatatgtccataattatttcaaatggccacgggacaacacaatacagt acattctcctatgtggatcctgtaataacaagtatttcgccgaaatacgg tcctatggctggtggcactttacttactttaactggaaattacctaaaca gtgggaattctagacacatttcaattggtggaaaaacatgtactttaaaa agtgtgtcaaacagtattcttgaatgttataccccagcccaaaccatttc aactgagtttgctgttaaattgaaaattgacttagccaaccgagagacaa gcatcttcagttaccgtgaagatcccattgtctatgaaattcatccaacc aaatcttttattagtggtgggagcacaataacaggtgttgggaaaaacct gaattcagttagtgtcccgagaatggtcataaatgtgcatgaagcaggaa ggaactttacagtggcatgtcaacatcgctctaattcagagataatctgt tgtaccactccttccctgcaacagctgaatctgcaactccccctgaaaac caaagcctttttcatgttagatgggatcctttccaaatactttgatctca tttatgtacataatcctgtgtttaagccttttgaaaagccagtgatgatc tcaatgggcaatgaaaatgtactggaaattaagggaaatgatattgaccc tgaagcagttaaaggtgaagtgttaaaagttggaaataagagctgtgaga atatacacttacattctgaagccgttttatgcacggtccccaatgacctg ctgaaattgaacagcgagctaaatatagagtggaagcaagcaatttcttc aaccgtccttggaaaagtaatagttcaaccagatcagaatttcaca

[0260] DNA sequence for human c-Met extracellular Sema region and Flag-His tag (Human c-Met Sema-Flis):

TABLE-US-00003 (SEQIDNO:2) atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttac cttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccg agatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaaca cccatccagaatgtcattctacatgagcatcacattttccttggtgccac taactacatttatgttttaaatgaggaagaccttcagaaggttgctgagt acaagactgggcctgtgctggaacacccagattgtttcccatgtcaggac tgcagcagcaaagccaatttatcaggaggtgtttggaaagataacatcaa catggctctagttgtcgacacctactatgatgatcaactcattagctgtg gcagcgtcaacagagggacctgccagcgacatgtctttccccacaatcat actgctgacatacagtcggaggttcactgcatattctccccacagataga agagcccagccagtgtcctgactgtgtggtgagcgccctgggagccaaag tcctttcatctgtaaaggaccggttcatcaacttctttgtaggcaatacc ataaattcttcttatttcccagatcatccattgcattcgatatcagtgag aaggctaaaggaaacgaaagatggttttatgtttttgacggaccagtcct acattgatgttttacctgagttcagagattcttaccccattaagtatgtc catgcctttgaaagcaacaattttatttacttcttgacggtccaaaggga aactctagatgctcagacttttcacacaagaataatcaggttctgttcca taaactctggattgcattcctacatggaaatgcctctggagtgtattctc acagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatact tcaggctgcgtatgtcagcaagcctggggcccagcttgctagacaaatag gagccagcctgaatgatgacattcttttcggggtgttcgcacaaagcaag ccagattctgccgaaccaatggatcgatctgccatgtgtgcattccctat caaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtga gatgtctccagcatttttacggacccaatcatgagcactgctttaatagg acacttctgagaaattcatcaggctgtgaagcgcgccgtgatgaatatcg aacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaat tcagcgaagtcctcttaacatctatatccaccttcattaaaggagacctc accatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggt ttctcgatcaggaccatcaacccctcatgtgaattttctcctggactccc atccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggc tacacactggttatcactgggaagaagatcacgaagatcccattgaatgg cttgggctgcagacatttccagtcctgcagtcaatgcctctctgccccac cctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaa tgcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaa ggactacaaggacgacgacgacaagcatgtccaccatcatcaccatcact gattcgaa

[0261] DNA sequence for human c-Met ECD his tag (Human c-Met ECD-His) recombinant protein:

TABLE-US-00004 (SEQIDNO:3) atgaaggcccccgctgtgcttgcacctggcatcctcgtgctcctgtttac cttggtgcagaggagcaatggggagtgtaaagaggcactagcaaagtccg agatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaaca cccatccagaatgtcattctacatgagcatcacattttccttggtgccac taactacatttatgttttaaatgaggaagaccttcagaaggttgctgagt acaagactgggcctgtgctggaacacccagattgtttcccatgtcaggac tgcagcagcaaagccaatttatcaggaggtgtttggaaagataacatcaa catggctctagttgtcgacacctactatgatgatcaactcattagctgtg gcagcgtcaacagagggacctgccagcgacatgtctttccccacaatcat actgctgacatacagtcggaggttcactgcatattctccccacagataga agagcccagccagtgtcctgactgtgtggtgagcgccctgggagccaaag tcctttcatctgtaaaggaccggttcatcaacttctttgtaggcaatacc ataaattcttcttatttcccagatcatccattgcattcgatatcagtgag aaggctaaaggaaacgaaagatggttttatgtttttgacggaccagtcct acattgatgttttacctgagttcagagattcttaccccattaagtatgtc catgcctttgaaagcaacaattttatttacttcttgacggtccaaaggga aactctagatgctcagacttttcacacaagaataatcaggttctgttcca taaactctggattgcattcctacatggaaatgcctctggagtgtattctc acagaaaagagaaaaaagagatccacaaagaaggaagtgtttaatatact tcaggctgcgtatgtcagcaagcctggggcccagcttgctagacaaatag gagccagcctgaatgatgacattcttttcggggtgttcgcacaaagcaag ccagattctgccgaaccaatggatcgatctgccatgtgtgcattccctat caaatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtga gatgtctccagcatttttacggacccaatcatgagcactgctttaatagg acacttctgagaaattcatcaggctgtgaagcgcgccgtgatgaatatcg aacagagtttaccacagctttgcagcgcgttgacttattcatgggtcaat tcagcgaagtcctcttaacatctatatccaccttcattaaaggagacctc accatagctaatcttgggacatcagagggtcgcttcatgcaggttgtggt ttctcgatcaggaccatcaacccctcatgtgaattttctcctggactccc atccagtgtctccagaagtgattgtggagcatacattaaaccaaaatggc tacacactggttatcactgggaagaagatcacgaagatcccattgaatgg cttgggctgcagacatttccagtcctgcagtcaatgcctctctgccccac cctttgttcagtgtggctggtgccacgacaaatgtgtgcgatcggaggaa tgcctgagcgggacatggactcaacagatctgtctgcctgcaatctacaa ggttttcccaaatagtgcaccccttgaaggagggacaaggctgaccatat gtggctgggactttggatttcggaggaataataaatttgatttaaagaaa actagagttctccttggaaatgagagctgcaccttgactttaagtgagag cacgatgaatacattgaaatgcacagttggtcctgccatgaataagcatt tcaatatgtccataattatttcaaatggccacgggacaacacaatacagt acattctcctatgtggatcctgtaataacaagtatttcgccgaaatacgg tcctatggctggtggcactttacttactttaactggaaattacctaaaca gtgggaattctagacacatttcaattggtggaaaaacatgtactttaaaa agtgtgtcaaacagtattcttgaatgttataccccagcccaaaccatttc aactgagtttgctgttaaattgaaaattgacttagccaaccgagagacaa gcatcttcagttaccgtgaagatcccattgtctatgaaattcatccaacc aaatcttttattagtggtgggagcacaataacaggtgttgggaaaaacct gaattcagttagtgtcccgagaatggtcataaatgtgcatgaagcaggaa ggaactttacagtggcatgtcaacatcgctctaattcagagataatctgt tgtaccactccttccctgcaacagctgaatctgcaactccccctgaaaac caaagcctttttcatgttagatgggatcctttccaaatactttgatctca tttatgtacataatcctgtgtttaagccttttgaaaagccagtgatgatc tcaatgggcaatgaaaatgtactggaaattaagggaaatgatattgaccc tgaagcagttaaaggtgaagtgttaaaagttggaaataagagctgtgaga atatacacttacattctgaagccgttttatgcacggtccccaatgacctg ctgaaattgaacagcgagctaaatatagagtggaagcaagcaatttcttc aaccgtccttggaaaagtaatagttcaaccagatcagaatttcacacacc atcatcaccatcactgattcgaa

Example 2. Binding Assay of Antibody and Antigen (ELISA)

[0262] This experiment uses enzyme linked immunosorbent assay to detect affinity of c-Met antibody (including supermatant of hybridonma or recombinant expressed monoclonal antibodies) to c-Met antigen in vitro.

[0263] Experimental procedures: Coating buffer (PBS; Hyclone, Cat No.: SH30256.01B) was used to dilute antigen (human c-Met-His, example 1) to 2 g/mL, which was added to a 96-well microplate at 100 L/well (Costar 9018, Cat No.:03113024) and incubated overnight at 4 C. The next day, the antibody-coated 96-well microplate was restored to room temperature and was washed three times with washing buffer (PBS+0.05% Tween 20 (Sigma, Cat No.:P1379). Blocking buffer was added at 200 L/well (PBS+1% BSA (Roche, Cat No.:738328) and the plate was incubated at 37 C. for 1 hour. The plate was then washed three times with washing buffer. The anti c-Met antibody to be tested was added to the 96-well microplate and was incubated for 1 hour at room temperature. The plate was then washed three times with washing buffer. Secondary antibody (Goat anti-Mouse IgG(H+L)(HRP) (Thermo, No.:31432) diluted with blocking buffer (10000 dilution) was added to the 96-well microplate at 100 L/well and the plate was incubated for 1 hour at room temperature. The plate was then washed three times and TMB chromogenic substrate (eBioscience REF:00-4201-56) was added to the 96-well microplate at 100 L/well. Stop solution 2N H.sub.2SO.sub.4 was added to the 96-well microplate at 100 L/well. The plate was read with plate reader at 450 nm.

Example 3. Production of Murine Monoclonal Antibody Cell Strain Against Human c-Met

[0264] Murine anti-human c-Met monoclonal cell lines were obtained by immunizing mice, fusion of spleen cells, and screening of hybridomas. This method is well-known in this field. Recombinant expressed antigen (human c-Met ECD-mFc, human c-Met Sema-flis, see example 1) was diluted to 1 mg/mL with PBS (Hyclone, Cat No.:SH30256.01B) and emulsified with Freund's adjuvant (the first immunization was performed with Freund's complete adjuvant, and the booster immunizations were performed with Freund's incomplete adjuvant); and injected into Balb/C mice subcutaneously (5 mice/group) with each mouse inoculated with 100 g antigen, and booster immunizations were given every two weeks. After the first booster immunization, mice serum was collected during 7 to 10 days after each booster immunization, and the titer was detected by ELISA (Methods were described in Example 2).

[0265] After immunization, mice with a serum titer higher than 1:10.sup.5 were selected for cell fusion. Mouse B-cells and myeloma cells (SP2/0, ATCC number:CRL-1581) were prepared respectively in aseptic conditions and counted. The two kinds of cells were mixed at a ratio of B-cells:SP2/0 of 1:4 and then were centrifuged (1500 r/min, 7 min). The supernatant was discarded and 1 mL of 50% polyethylene glycol (Supplier: SIGMA, Catalogue # RNBB306) was added. Next, 1 mL serum-free RPMI1640 (Supplier: GIBCO, Catalogue # C22400) was used for termination, and samples were centrifuged for 10 minutes. The supernatant was then discarded. The pellet was resuspended in RPMI1640 which comprised hybridoma cell growth factor (Supplier: Roche, Catalogue #1363735001), serum (Supplier: GIBCO, Catalogue # C20270) and HAT (Supplier: Invitrogen, Catalogue #21060-017). B-cells were plated on the plate at 10.sup.5 cells/well and each well was 100 L. The plate was placed in a cell incubator at 37 C. Three days later, 100 L of RPMI1640, which comprised hybridoma cell growth factor, serum and HT (Supplier: Invitrogen, Catalogue #11067-030) was added to each well. After 2 to 4 days, each well was replaced with 150 L RPMI1640 comprising hybridoma cell growth factor, serum and HT. The next day, positive clones were detected by ELISA (see Methods in Example 2).

[0266] Experimental Results:

TABLE-US-00005 TABLE 1 Detection of hybridoma fusion of mice immunized with human c-Met Clone No. Detection Results (OD450) Negative control 0.07 Ab-1 1.48 Ab-2 1.38 Ab-3 1.29 Ab-4 1.6 Ab-5 1.64 Ab-6 1.75 Ab-7 1.58 Ab-8 1.24

Example 4. Cloning of Anti c-Met Antibody Sequence

[0267] The cell line Ab-5 was selected for cDNA sequence cloning. The mAb was recombinantly expressed and subjected to various activity tests. The variable regions of the heavy chain and light chain of the antibody gene were amplified by reverse transcription PCR, and ligated to vector to obtain the heavy and light chain sequences of the monoclonal antibody by sequencing. First, an RNA purification kit (Qiagen company, Cat. No. 74134, see the instructions for this procedure) was used to extract total RNAs from the active single cell stain from example 3. Next, single stranded cDNA was prepared by the cDNA synthesis kit (Invitrogen company, Cat. No. 18080-051), which involves cDNA reverse transcription using Oligo-dT primers. The product served as a template, and the variable region sequence of the antibody heavy and light chain was synthesized by PCR. The products of PCR were cloned into the TA vector pMD-18T and then sequenced. The obtained heavy and light chain sequences of the antibody were separately cloned to expression vectors (see example 1), and the recombinant monoclonal antibody was expressed to prove its activity (see examples 2 and 3), followed by humanization.

TABLE-US-00006 Sequenceofmousehybridomacellmonoclonal antibodyAb-5: Heavychainvariableregion: (SEQIDNO:4) QVQLKQSGPGLVQPSQSLSITCTVSGFSLPNYGVHWVRQSPGKGLEWLGV IWSGGSTNYAAAFVSRLRISKDNSKSQVFFEMNSLQADDTAVYYCARNHD NPYNYAMDYWGQGTTVTVSS Lightchainvariableregion: (SEQIDNO:5) DIVLTQSPGSLAVYLGQRATISCRANKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPARFSGSGSGTDFTLNIHPLEEEDAATYYCQHSRDLPP TFGAGTKLELKR

[0268] The amino acid residues of VH/VL CDR of anti-human c-Met antibodies were determined and annotated by the Kabat numbering system.

[0269] CDR sequences of murine in the invention are shown in Table below:

TABLE-US-00007 TABLE2 CDRsequenceofMurineanti-sclerostinantibody Antibody Ab-5 HeavychainCDR1 NYGVH (SEQIDNO:6) HeavychainCDR2 VIWSGGSTNYAAAFVS (SEQIDNO:7) HeavychainCDR3 NHDNPYNYAMDY (SEQIDNO:8) LightchainCDR1 RANKSVSTSTYNYLH (SEQIDNO:9) LightchainCDR2 LASNLAS (SEQIDNO:10) LightchainCDR3 QHSRDLPPT (SEQIDNO:11)

Example 5. Humanization of Anti c-Met Antibody

[0270] The murine anti c-Met monoclonal antibody heavy and light chain sequences obtained from example 4 were aligned against an antibody database for homology, and a humanized antibody model was established then. The optimal humanized c-Met monoclonal antibody was selected as the preferred molecule of the invention according to the model for back-mutation. A crystal structure showing similar homology with the obtained murine candidate molecules was selected from the published database of mice Fab crystal structure models (e.g. PDB database), and a Fab crystal structure with high resolution (such as, less than 2.5 ) was selected; and the mouse Fab model was established. The murine antibody heavy and light chain sequences of the invention were aligned against the sequences in the model, and the constant sequence was maintained so that the structural model of the mouse antibody of the invention could be obtained. The variable amino acids might be potential sites for back-mutation. Swiss-pdb viewer software was used to run the mouse antibody structure model to optimize energy (minimization). Back-mutation was performed at different amino acid sites other than those in CDRs of the model, and the activities of the resultant humanized antibody and of the antibody without humanization were compared. A humanized antibody with good activity was maintained. The CDR region was further optimized, including mutations to prevent glycosylation, deamination, oxidation sites and so on. CDR regions of the optimized humanized anti c-Met antibody are shown in table below:

TABLE-US-00008 TABLE3 CDRsequenceoftheoptimizedantic-Metantibody Antibody Optimizedhumanizedantibody HeavyChainCDR1 NYGVH (SEQIDNO:6) HeavyChainCDR2 VIWSGGSTNYAAAFVS (SEQIDNO:7) HeavyChainCDR3 NHDNPYNYAMDY (SEQIDNO:8) LightChainCDR1 RADKSVSTSTYNYLH (SEQIDNO:12) LightChainCDR2 LASNLAS (SEQIDNO:10) LightChainCDR3 QHSRDLPPT (SEQIDNO:11)

[0271] Variable regions of the humanized heavy and light chain sequences are shown below:

TABLE-US-00009 1.Heavychainvariableregions Ab-9 (SEQIDNO:13) QVTLKESGPVLVKPTETLTLTCTVSGFSLPNYGVHWVRQPPGKALEWLAV IWSGGSTNYAAAFVSRLRISKDTSKSQVVFTMNNMDPVDTATYYCARNHD NPYNYAMDYWGQGTTVTVSS Ab-10 (SEQIDNO:14) QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAV IWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHD NPYNYAMDYWGQGTTVTVSS Ab-11 (SEQIDNO:15) QVQLVESGGGVVQPGRSLRLSCAASGFTLPNYGVHWVRQAPGKGLEWLAV IWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHD NPYNYAMDYWGQGTTVTVSS 2.Lightchainvariableregions Ab-9 (SEQIDNO:16) DIVLTQSPASLAVSPGQRATITCRANKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRDLPP TFGQGTKLEIKR Ab-10 (SEQIDNO:17) DIVLTQSPDSLAVSLGERATINCRADKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPP TFGQGTKLEIKR Ab-11 (SEQIDNO:18) DIVLTQSPDSLAVSLGERATINCRANKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPP TFGQGTKLEIKR

[0272] The humanized heavy and light chain sequences were recombined with IgG Fc regions to obtain the humanized anti c-Met monoclonal antibody of the invention. The Fc sequence used was selected optionally from the following sequences:

TABLE-US-00010 Heavychainconstantregion: (SEQIDNO:19) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQIDNO:20) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQIDNO:21) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK Lightchainconstantregion: (SEQIDNO:22) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0273] The above antibodies were cloned, expressed and purified by gene cloning and recombinant expression, respectively. The humanized antibodies Ab-9, Ab-10 and Ab-11 with best activity were finally selected by ELISA (Example 2) and in vitro binding activity assay (Example 6). The sequences are shown below:

TABLE-US-00011 Ab-9humanizedantibody: Heavychain: (SEQIDNO:23) QVTLKESGPVLVKPTETLTLTCTVSGFSLPNYGVHWVRQPPGKALEWLAV IWSGGSTNYAAAFVSRLRISKDTSKSQVVFTMNNMDPVDTATYYCARNHD NPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Lightchain: (SEQIDNO:26) DIVLTQSPASLAVSPGQRATITCRANKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSRDLPP TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Ab-10humanizedantibody: Heavychain: (SEQIDNO:24) QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAV IWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHD NPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Lightchain: (SEQIDNO:27) DIVLTQSPDSLAVSLGERATINCRADKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPP TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Ab-11humanizedantibody: Heavychain: (SEQIDNO:25) QVQLVESGGGVVQPGRSLRLSCAASGFTLPNYGVHWVRQAPGKGLEWLAV IWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHD NPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Lightchain: (SEQIDNO:28) DIVLTQSPDSLAVSLGERATINCRANKSVSTSTYNYLHWYQQKPGQPPKL LIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPP TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Example 6. Detection of In Vitro Binding Activity of Anti c-Met Humanized Antibody

[0274] The humanized antibodies of the invention were analyzed for their in vitro activity by ELISA (Example 2), and also analyzed for their binding with the cell line MKN45, which highly expresses c-Met, and for their affinity to c-Met antigen (BIACore assay).

[0275] A FACS method was used to detect the binding activity of c-Met humanized antibodies with the cell line MKN45, which highly expresses c-Met.

[0276] MKN45 cells (JCRB, Cat No.: JCRB0254) were resuspended in RPMI1640 medium (GIBCO, Cat No.: 11835-030) which contains 10% (v/v) fetal calf serum (FBS GIBCO, Cat No.: 10099-141) and Penicillin/Streptomycin (GIBCO, Cat No.: 15070-063) to reach 10,000,000 cells/mL. 2 mL of resuspended MKN45 cells was added to 96-well microtiter plate (Corning, Cat No.: 3799) at 150,000 cells/well, and 8 concentrations of c-Met antibody (5-fold gradient diluted, starting from 20 g/mL) were added to the corresponding wells, and the final volume was 100 L. The plate was incubated for 1 hour at 4 C. FACS buffer (PBS comprising 2.5% (v/v) FBS (Hyclone, Cat: SH30256.01B)) was added. The plate was centrifuged under 4 C. at 1300 rmp for 4 minutes, and the supernatant was discarded. This procedure was repeated three times. 100 L of secondary antibodies (Fluorescence labeled goat-anti-mouse secondary antibodies with 1:200 dilution, Biolegend, Cat No. 405307; Fluorescence labeled anti-human secondary antibody with 1:30 dilution, Biolegend, Cat No. 409304) were added to each well, and the plate was incubated for 1 hour at 4 C. FACS buffer was added, the plate was centrifuged under 4 C. at 1300 rpm for 4 minutes, the supernatants were discarded; and this procedure was repeated three times. 200 L FACS buffer was added to resuspend the cells, and the prepared samples were detected by flow cytometry (BD FACS Array).

[0277] The affinity of c-Met antibody to c-Met antigen Sema-His was detected by surface plasmon resonance (SPR) in the invention.

[0278] Anti-mouse IgG (GE Life Sciences catalog # BR-1008-38) or anti-human IgG (GE Life Sciences catalog # BR-1008-39) antibodies were respectively diluted to 30 g/mL and 50 g/mL by sodium acetate solution pH 5.0 (GE Healthcare, Cat # BR-1003-51). An amino coupling kit (GE Life Sciences, Cat # BR100050) was immobilized onto the test channels and control channels on a CM5 chip (GE Life Sciences catalog # BR-1000-12), and the coupling level was set at 15000 RU. The c-Met antibody was diluted with Running buffer PBS (Hyclone, Cat # SH30256.01B)+0.05% P20 (GE Life Sciences, Cat # BR-1000-54) to 1.5 g/mL. Antigen Sema-His was diluted to 200 nM with running buffer, and then diluted at a 1:2 dilution with the same buffer until 0.78 nM was reached. The diluted antibody passed through the test channel for 1 minute at a speed of 30 L/min, and the antigen passed through the test channels and control channels for 3 minutes at the same speed. 10 minutes after dissociation, the flow speed was adjusted to 10 L/min, and regeneration buffer was passed through test channels and control channels for 3 minutes. Data was fitted by BiaEvaluation 4.1 after double deduction, and the fitting model was a 1:1 Langmuir model.

[0279] Experimental Results:

TABLE-US-00012 TABLE 4 Binding activity of humanized anti c-Met antibodies Humanized antibody Ab-9 Ab-10 Ab-11 ELISA assay (EC.sub.50, nM) 0.13 0.39 0.2

TABLE-US-00013 TABLE 5 Binding activity and affinity of humanized anti c-Met antibodies to MKN45 cells and to antigen MKN45/FCAS Binding affinity to antigen Humanized antibody activity (nM) Biacore(nM) Ab-9 1.6 4 Ab-10 1.23 8

[0280] Conclusion: The above experimental results show that the binding activity of humanized antibodies with antigen was within 0.13-8 nM, and the results may vary depending on the detection methods used. The results show that humanized anti c-Met antibodies maintain the binding activity of the parent antibodies prior to humanization.

Example 7. Endocytosis of Anti c-Met Antibody

[0281] Antibodies of the invention bind to human c-Met, and have very good in vitro activity and good activity in inhibiting tumor activity in vivo. In addition, the antibodies do not have agonist activity, or have very weak agonist activity. In order to detect whether the antibodies would be internalized into the cell along with human c-Met once bound to human c-Met, human gastric cancer cell line MKN45 (JCRB, Cat No.: JCRB0254) expressing c-Met was used for evaluation.

[0282] MKN45 cells were resuspended to 10,000,000 cells/mL in RPMI 1640 medium (GIBCO, Cat No.: 11835-030), which contains 10% (v/v) FBS (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063). 2 mL resuspended MKN45 cells were added to a 96-well microtiter plate with 250,000 cells/well, and 10 g/mL of c-Met antibody was added to the corresponding wells and the final volume was 100 L. The plate was incubated at 4 C. for 1 hour. FACS buffer (phosphate buffer solution including 2.5% fetal bovine serum; Hyclone, Cat: SH30256.01B) was added and the plate was centrifuged at 4 C., 1300 rpm for 4 minutes. The supernatant was discarded and this procedure was repeated three times. 100 L secondary antibody solution (Fluorescence labeled goat anti mouse secondary antibodies at 1:200 dilution, Biolegend, Cat #405307; Fluorescence labeled anti-human secondary antibody at 1:30 dilution, Biolegend, Cat #409304) was added into each well. The plate was incubated at 4 C. for 1 hour. FACS buffer was added and the plate was centrifuged at 4 C., 1300 rpm for 4 minutes. The supernatant was discarded and this procedure was repeated three times. Complete cell culture medium (RPMI 1640 medium with 10% FBS) was added and was incubated at 37 C. in 5% CO.sub.2 for 0, 0.5, 1, 2, 4 hours. 5 L 7-AAD (Biolegend, Cat:420403) was added to 100 L FACS buffer which was added to each well, and the plate was incubated at 4 C. for 30 minutes. FACS buffer was added and the plate was centrifuged at 4 C., 1300 rpm for 4 minutes. The supernatant was discarded and this procedure was repeated three times. 200 L Stripping buffer (0.05 M glycine, pH 3.0; 0.1 M NaCl, mixed at 1:1 (v/v)) was added to each well. The cells were resuspended and were incubated for 7 minutes at room temperature. The cells were centrifuged at room temperature at 1300 rpm for 4 minutes, and the supernatant was discarded. 200 L neutralizing wash buffer (0.15M trihydroxymethyl aminomethane, pH 7.4) was added to each well, the cells were resuspended and centrifuged at room temperature at 1300 rpm for 4 minutes, and the supernatant was discarded. 200 L FACS buffer was added and the cells were resuspended. The prepared samples were detected by flow cytometry (BD FACS Calibur). The results are shown in the table below.

Endocytosis of c-Met antibody %=(intensity of fluorescence at each time pointmean intensity of fluorescence at time 0)/mean intensity of fluorescence at time 0.

[0283] Experimental Results:

TABLE-US-00014 TABLE 6 Evaluation of endocytosis of humanized anti c-Met antibodies of the present invention (endocytosis %) Humanized antibody 0 h 0.5 h 1 h 2 h 4 h hIgG (control)* 0 0.9 4.4 4.9 3.6 Ab-9 0 26 32 32 31 Ab-10 0 24 38 53 59 *4.9% and 3.6% in control group were due to experimental error (background value), and was classified as no endocytosis.

[0284] Experimental Conclusion:

[0285] The experimental results in the table above show that antibodies of the invention have good endocytosis while they do not have agonist activity. Once bound with target cells, both antibodies and receptors were rapidly internalized into target cells, and the maximum value was reached within 2-4 hours.

Example 8. Analysis of the Biophysical Stability of Anti c-Met Antibodies

[0286] To evaluate the biophysical stability of the anti c-Met antibodies of the invention, such as the presence of glycosylation and deamination sites and stability, LC-MS analysis was used.

[0287] The molecular weight of the heavy and light chains was directly detected by LC-MS to analyze glycosylation. Deamination was analyzed by LC-MS at 4 C. for long time (at least 3 months), or at 40 C. for 21 days under an accelerated condition. Samples treated with different conditions were diluted to 2 mg/mL with pH 7.2 Tris-HCl; final concentrations of 10 mM of TCEP and 6M of urea (AMRESCO, Cat #0378).sub.3 were added, then the samples were incubated for 20 minutes at 37 C. IAA (Sigma-Aldrich, Cat # I1149) with a final concentration of 20 mM was added and was incubated for 15 minutes in darkness to protect the sulfhydryl group. The pH of the sample was adjusted by dilution with Tris-HCl, pH 7.2, and protease (Sigma-Aldrich, Cat # T6567) was added at a ratio by weight of 10:1 (protein: enzyme). The samples were incubated at 37 C. for 25 minutes, and then formic acid with a final concentration of 0.1% (Fluca, Cat #94318) was added to terminate the reactions. Samples were centrifuged and analyzed by LC-MS.

[0288] BiopharmaLynx was used to analyze the presence of deamination. Extracted Ion Chromatogram (EIC) was obtained from MS data by searching native peptide comprising deamination site and modified product, and then extracting parent ion. Peak area was obtained by integration, and the percentages of deamination and oxidation product were calculated.

[0289] Experimental Results:

TABLE-US-00015 TABLE 7 Evaluation of physical stability of the humanized anti c-Met antibody of the invention Analysis The antibody of deamination# of the Molecular weight of light chain* 4 C., 40 C., invention Detected value Estimated value 3.5 months 21 days Ab-9 25940 23907 0.66 Ab-10 23828 23832 0.3 *Heavy chains all involve glycosylation, and molecular weight was consistent with the expected value. #Percentage of deaminated molecules (%). 0.66-1.0% is within the background of detection. : not tested.

[0290] Experimental conclusion: The above results show that the antibodies of the invention are stable and have good physical properties.

Example 9. Anti-c-Met Antibody Ab-10 Conjugated to Toxin MC-MMAF (No. 1)

[0291] The anti-c-Met antibodies of the present invention have inhibitory activity against receptor binding, without having agonist activity, show endocytosis activity into targeted cells, as well as physical stability. These properties make the antibodies of the invention particularly suitable for the preparation of ADC drugs when conjugated to toxins for the treatment of c-Met expressing cancers. The coupling process is shown below:

##STR00029##

[0292] Step 1. Thioacetic acid S-(3-carbonyl propyl) ester (0.7 mg, 5.3 mol) was dissolved in acetonitrile solution (0.9 mL), for use. The thioacetic acid S-(3-carbonyl propyl) ester in acetonitrile prepared above was added into acetic acid/sodium acetate buffer pH=4.3 (10.35 mg/mL, 9.0 mL, 0.97 mmol) containing Ab-10 monoclonal antibody, and sodium borohydride aqueous solution (14.1 mg, 224 mol, 1.0 mL) was added dropwise with shaking for 2 hours at 25 C. At the end of the reaction, desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution pH 6.5), and product 1b solution was collected and was concentrated to 10 mg/mL directly for the next reaction.

[0293] Step 2. hydroxylamine hydrochloride solution (2.0M, 0.35 mL) was added into 11.0 mL of 1b solution with shaking for 30 minutes at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution pH 6.5), and the captioned product Ab-10 monoclonal antibody-propyl mercaptan 1c solution was collected (6.17 mg/mL, 14.7 mL).

[0294] Step 3. The compound MC-MMAF (1.1 mg, 1.2 mol; prepared by method published in PCT patent WO2005081711) was dissolved in acetonitrile (0.3 mL) and was added in Ab-10 monoclonal antibody-propyl mercaptan 1c solution (6.17 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution pH 6.5). The captioned product ADC-1 (3.7 mg/mL, 4.7 mL) in PBS buffer was obtained by filtration through a 0.2 m filter under aseptic conditions, and then frozen stored at 4 C.

[0295] Q-TOF LC/MS: characteristic peak: 148119.2 (M.sub.Ab+0D), 149278.1 (M.sub.Ab+1D), 150308.1 (M.sub.Ab+2D), 151314.1 (M.sub.Ab+3D). The amount of the conjugated toxin per antibody (DAR) was calculated by analysis and the mean value was y=1.7.

Example 10. Anti-c-Met Antibody Ab-10 Conjugated with Toxin MC-VC-PAB-MMAE (No. 2)

[0296] ##STR00030##

[0297] The compound MC-VC-PAB-MMAE (1.6 mg, 1.2 mol; prepared by the method disclosed in PCT patent application WO2004010957) was dissolved in acetonitrile (0.3 mL) and was added into Ab-10 monoclonal antibody-propyl mercaptan 1c solution (6.17 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution, pH 6.5). The captioned product ADC-2 in PBS buffer (3.6 mg/mL, 4.8 mL) was obtained by filtration through a 0.2 m filter under aseptic conditions, and then frozen stored at 4 C.

[0298] Q-TOF LC/MS: characteristic peak: 148118.4 (M.sub.Ab+0D), 149509.2 (M.sub.Ab+1D), 150903.1 (M.sub.Ab+2D), 152290.4 (M.sub.Ab+3D), 153680.7 (M.sub.Ab+4D). The amount of the conjugated toxin per antibody (DAR) was calculated by analysis and the mean value is y=1.8.

Example 11. Anti-c-Met Antibody Ab-10 Conjugated with Toxin MC-VC-PAB-MMAF (No. 3)

[0299] ##STR00031##

[0300] The compound MC-VC-PAB-MMAF (1.6 mg, 1.2 mol; prepared as method disclosed in PCT patent application WO2005081711) was dissolved in acetonitrile (0.3 mL) and was added to Ab-10 monoclonal antibody-propyl mercaptan 1c solution (6.17 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-3 (3.5 mg/mL, 4.9 mL) in PBS buffer was obtained by filtration through a 0.2 m filter under aseptic conditions, and then frozen stored at 4 C.

[0301] Q-TOF LC/MS: characteristic peak: 148119.1 (M.sub.Ab+0D), 149525.3 (M.sub.Ab+1D), 150930.7 (M.sub.Ab+2D), 152335.2 (M.sub.Ab+3D), 153739.8 (M.sub.Ab+4D). The amount of the conjugated toxin per antibody (DAR) was calculated by analysis and the mean value was y=1.6.

Example 12. Anti-c-Met Antibody Ab-10 Conjugated Toxin with MC-MMAE (No. 4)

[0302] ##STR00032##

[0303] The compound MC-MMAE (1.2 mg, 1.2 mol; prepared as method disclosed in patent application US7/750/116B1) was dissolved in acetonitrile (0.3 mL) and was added to Ab-10 monoclonal antibody-propyl mercaptan 1c solution (6.17 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-4 in PBS buffer (3.4 mg/mL, 5.0 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then frozen stored at 4 C.

[0304] Q-TOF LC/MS: characteristic peak: 148118.6 (M.sub.Ab+0D), 149104.3 (M.sub.Ab+1D), 150090.1 (M.sub.Ab+2D), 151075.8 (M.sub.Ab+3D). The amount of the conjugated toxin per antibody (DAR) was calculated by analysis and the mean value was y=1.6.

Example 13. Anti-c-Met Antibody Ab-9 Conjugated with Toxin MC-MMAE (No. 5)

[0305] ##STR00033##

[0306] Step 1. Thioacetic acid S-(3-carbonyl propyl) ester (0.7 mg, 5.3 mol) was dissolved in 0.9 mL acetonitrile solution, for use. The thioacetic acid S-(3-carbonyl propyl) ester in acetonitrile prepared above was added into acetic acid/sodium acetate buffer containing Ab-9 monoclonal antibody (10.85 mg/mL, 9.0 mL, 0.976 mmol), and sodium borohydride aqueous solution (14.1 mg, 224 mol, 1.0 mL) was added dropwise with shaking for 2 hours at 25 C. At the end of the reaction, desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5), and the captioned product 5b solution was collected and concentrated to 10 mg/mL directly for the next reaction.

[0307] Step 2. hydroxylamine hydrochloride solution (2.0M, 0.35 mL) was added into 5b solution (11.0 mL) with shaking for 30 minutes at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5), and the captioned product Ab-9 monoclonal antibody-propyl mercaptan 5c solution was collected (6.2 mg/mL, 15.0 mL).

[0308] Step 3. the compound MC-MMAE (1.1 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL) and was added to Ab-9 monoclonal antibody-propyl mercaptan 5c solution (6.2 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-5 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then frozen stored at 4 C.

[0309] Q-TOF LC/MS: characteristic peak: 150530.9 (M.sub.Ab+OD), 151915.7 (M.sub.Ab+1D), 153333.6 (M.sub.Ab+2D), 154763.4 (M.sub.Ab+3D), 156271.9 (M.sub.Ab+4D). The amount of conjugated toxin per antibody (DAR) was calculated by analysis and the mean value was y=1.5.

Example 14. Anti-c-Met Antibody Ab-9 Conjugated with Toxin MC-MMAF (No. 6)

[0310] ##STR00034##

[0311] The compound MC-MMAF (1.1 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL) and was added to Ab-9 monoclonal antibody-propyl mercaptan 5c solution (6.17 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-6 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then frozen stored at 4 C.

[0312] Q-TOF LC/MS: characteristic peak: 150537.8 (M.sub.Ab+OD), 152087.9 (M.sub.Ab+1D), 153486.5 (M.sub.Ab+2D), 154911.7 (M.sub.Ab+3D), 156499.9 (M.sub.Ab+4D). The amount of conjugated toxin per antibody (DAR) was calculated by analysis and the mean value was y=1.7.

Example 15. Anti-c-Met Antibody Ab-9 Conjugated with Toxin MC-VC-PAB-MMAF (No. 7)

[0313] ##STR00035##

[0314] The compound MC-VC-PAB-MMAF (1.6 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL) and was added to Ab-9 monoclonal antibody-propyl mercaptan 5c solution (6.2 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-7 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then stored at 4 C.

[0315] Q-TOF LC/MS: characteristic peak: 150537.8 (M.sub.Ab+0D), 152087.9 (M.sub.Ab+1D), 153486.5 (M.sub.Ab+2D), 154911.7 (M.sub.Ab+3D), 156499.9 (M.sub.Ab+4D). The amount of conjugated toxin per antibody (DAR) was gained by analysis and the mean value was y=1.8.

Example 16. Anti-c-Met Antibody Ab-9 Conjugated with Toxin MC-VC-PAB-MMAE (No. 8)

[0316] ##STR00036##

[0317] The compound MC-VC-PAB-MMAE (1.6 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL) and was added to Ab-9 monoclonal antibody-propyl mercaptan 5c solution (6.2 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-8 in PBS buffer (3.8 mg/mL, 4.6 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then frozen stored at 4 C.

[0318] Q-TOF LC/MS: characteristic peak: 150508.6 (M.sub.Ab+OD), 151903.6 (M.sub.Ab+1D), 153314.5 (M.sub.Ab+2D), 154747.8 (M.sub.Ab+3D), 156039.5 (M.sub.Ab+4D). The amount of conjugated toxin per antibody (DAR) was gained by analysis and the mean value was y=1.6.

Example 17. Anti-c-Met Antibody Ab-9 Conjugated with Toxin SN-38 (No. 9)

[0319] ##STR00037##

[0320] The compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL) and was added to Ab-9 monoclonal antibody-propyl mercaptan 5c solution (6.2 mg/mL, 3.0 mL) with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product ADC-11 in PBS buffer (3.7 mg/mL, 4.5 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then stored at 4 C.

[0321] Q-TOF LC/MS: characteristic peak: 150537.1 (M.sub.Ab+0D), 151786.6 (M.sub.Ab+1D), 152948.6 (M.sub.Ab+2D), 154161.7 (M.sub.Ab+3D), 155365.9 (M.sub.Ab+4D), 156477.8 (M.sub.Ab+5D).

[0322] The mean value was y=2.6.

Example 18. Anti-c-Met Antibody Ab-10 Conjugated with Toxin (No. 10)

1. Preparation of Toxin

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)N,3-dimethyl-2-((S)-3-methyl-2-(methyl amino)butyramide)butyramide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl) propionic acid

[0323] ##STR00038## ##STR00039##

Step 1

Preparation of (S)-tert-butyl-2-amino-3-(2-fluorophenyl) propanoic acid

[0324] Starting material (S)-2-amino-3-(2-fluorophenyl)propanoic acid 12a (400 mg, 2.18 mmol, prepared according to the known method in Advanced Synthesis & Catalysis, 2012, 354(17), 3327-3332) was dissolved in 10 mL of tert-butyl acetate. Perchloric acid (300 mg (70%), 3.3 mmol) was added and stirred at room temperature for 16 hours. Water (6 mL) was added after the reaction, and the solution was separated. The organic phase was washed with saturated sodium bicarbonate solution (5 mL). The aqueous phase was adjusted to pH=8 with saturated sodium bicarbonate solution, and was then extracted with dichloromethane (5 mL3), and the organic phase was combined. The reaction mixture was then washed successively with water (3 mL) and saturated sodium chloride solution (5 mL), dried with anhydrous sodium sulfate, filtered; and the filtrate was concentrated under reduced pressure. The crude product compound 12b was obtained (390 mg, yellow, oily) and was subjected to the next reaction directly without purification.

Step 2

Preparation of (1S,3S,5S)-tert-butyl 3-((1R,2R)-3(((S)-1-(t-butoxy)-3-(2-fluorophenyl)-1-carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonyl propyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid

[0325] The starting material (2R,3R)-3-((1S,3S,5S)-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methyl propionate 12e (100 mg, 0.334 mmol) was dissolved in the mixture of dichloromethane (6 mL) and dimethylformamide (V/V=5:1), and then crude product (S)-tert-butyl 2-amino-3-(2-fluorophenyl) propionate 12b (80 mg, 0.334 mmol) was added. N,N-diisopropylethylamine (0.29 mL, 1.67 mmol) and 2-(7-azabenzotriazol)-N,N,N,N-tetramethyluronium hexafluorophosphate (152.3 mg, 0.40 mmol) were added to the mixture. The mixture was stirred for 1 hour under argon atmosphere at room temperature. After the reaction, water (10 mL) was added and stirred, and layers were separated. The layer of dichloromethane was washed by saturated sodium chloride solution (10 mL), and dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography using eluent system B to obtain the captioned product compound 12c (173 mg, clear liquid, the yield was 99.5%).

[0326] MS m/z (ESI): 521.2 [M+1]

Step 3

Preparation of (S)-tert-butyl-2-((2R,3R)-3-((1S,3S,5S),-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl) propionic acid

[0327] The starting material (1S,3S,5S)-tert-butyl-3-((1R,2R)-3-(((S)-1-(t-butoxy)-3(2-fluorophenyl)-1-carbonylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-carbonyl propyl)-2-azabicyclo[3.1.0]hexane-2-carboxylic acid 12c (173 mg, 0.33 mmol) was dissolved in dioxane (2 mL), and hydrogen chloride dioxane solution (5.6M, 0.21 mL, 1.16 mmol) was added. The mixture was stirred for 1 hour under argon atmosphere at room temperature, and was placed in a 0 C. refrigerator for 12 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, and dichloromethane (5 mL) was added to dilute the reaction mixture. Saturated sodium bicarbonate solution (10 mL) was added and the mixture was stirred for 10 minutes. The product was layered and the aqueous phase was extracted by dichloromethane (5 mL3). Dichloromethane layers were combined and were washed by saturated sodium chloride solution (10 mL), dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The crude product of the captioned compound 12d (77 mg, yellow liquid) was obtained and directly subjected to the next reaction without purification.

[0328] MS m/z (ESI):421.2 [M+1]

Step 4

Preparation of (S)-tert-butyl-2-((2R,3R)-3-((1S,3S,5S)-2-(5S,8S,11S,12R)-11-((S)-sec-butly)-1-(9H-fluorene-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxygen-4,7,10-triazatetradecyl-14-acyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl) propionic acid

[0329] Crude product (S)-tert-butyl-2-((2R,3R)-3-((1S,2S,5S)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropionamide)-3-(2-fluorophenyl) propionic acid 12d (77 mg, 0.183 mmol) and (5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluorene-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxo-4,7,10-triazatetradecyl-14-carboxylic acid 12i (116.8 mg, 0.183 mmol, prepared by methods published in patent application WO 2013072813) were dissolved in a mixture of dichloromethane (6 mL) and dimethylformamide (V/V=5:1). N,N-diisopropylethylamine (0.16 mL, 0.915 mmol) and 2-(7-azabenzotriazol)-N,N,N,N-tetramethyluronium hexafluorophosphate (84 mg, 0.22 mmol) were added to the mixture. The reaction mixture was stirred for 1 hour under argon atmosphere at room temperature. After the reaction, water (10 mL) was added and stirred, and layers were separated. The layer of dichloromethane was washed by saturated sodium chloride solution (10 mL), dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure. The residues were purified by silica gel column chromatography using eluent system B to obtain the captioned product compound 12e (190.5 mg, yellow viscous) with a yield of 100%.

[0330] MS m/z (ESI): 1040.6 [M+1]

Step 5

Preparation of (S)-tert-butyl-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)N,3-dimethyl-2-((5)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl) propionic acid

[0331] The starting material (S)-tert-butyl-2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-sec-butly)-1-(9H-fluorene-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-tricarbonyl-2-oxo-4,7,10-triazatetradecyl-14-acyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methyl propionamide)-3-(2-fluorophenyl) propionic acid 12e (190.5 mg, 0.183 mmol) was dissolved in dichloromethane (1.5 mL) and diethylamine (2 mL) was added. The mixture was stirred for 3 hours under argon atmosphere at room temperature. After the reaction, the reaction mixture was concentrated under reduced pressure and the crude captioned product compound 12f (150 mg, yellow viscous) was obtained. Products were directly subjected to the next reaction without purification.

[0332] MS m/z (ESI): 818.5 [M+1]

Step 6

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propionic acid

[0333] The crude product compound (S)-tert-butyl-2-((2R,3R)-3-((1 S,3 S,5S)-2-((3R,4S,5S)-4-((S)N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluoro phenyl)propionic acid 12f (150 mg, 0.183 mmol) was dissolved in dioxane (1 mL), and hydrogen chloride in dioxane (5.6M, 3 mL) was added. The mixture was stirred for 12 hours under argon atmosphere at room temperature. After the reaction, the reaction solution was concentrated under reduced pressure with ether solvent. The residues were purified by high performance liquid chromatography to obtain the captioned product compound 12g (28 mg, white powder with yield of 20%).

[0334] MS m/z (ESI): 762.7[M+1]

[0335] .sup.1H NMR (4.0 MHz, CD.sub.3OD): 7.38-7.18 (m, 2H), 7.13-7.01 (m, 2H), 4.80-4.67 (m, 2H) 4.30-4.15 (m, 1H), 4.13-4.01 (m, 1H), 3.96-3.83 (m, 2H), 3.75-3.60 (m, 2H), 3.42-3.11 (m, 9H), 3.06-2.95 (m, 1H), 2.70-2.58 (m, 4H), 2.28-2.01 (m, 4H), 1.88-1.70 (m, 3H), 1.57-1.25 (m, 4H), 1.22-0.95 (m, 18H), 0.92-0.80 (m, 4H), 0.78-0.65 (nm, 1H).

2. Preparation of Toxin Intermediates

(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl hexanamide)-3-methyl butanamide)-N,3-dimethyl butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl)propionic acid

[0336] ##STR00040##

[0337] The starting material (S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)-2-azabicyclo[3.1.0]hexane-3-yl)-3-methoxy-2-methylpropanamide)-3-(2-fluorophenyl) propionic acid 12g (25 mg, 0.033 mmol) was dissolved in dichloromethane (3 mL) and N,N-diisopropylethylamine (0.029 mL, 0.164 mmol) was added. The reaction system was dropwise added with 6-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride 4b in dichloromethane (11.3 mg, 0.049 mmol) prepared previously under argon atmosphere, in an ice-bath, and the reaction was performed for 3 hours at room temperature. After the reaction, water (5 mL) was added and the mixture was stirred for 20 minutes, until layered, and the organic layer was dried with anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residues were purified by high performance liquid chromatography to obtain the captioned product compound 12h (7 mg, yellow viscous, with the yield of 22.4%).

[0338] MS m/z (ESI): 955.4 [M+1]

[0339] .sup.1H NMR (400 MHz, CD.sub.3OD): 7.36-7.30 (m, 1H), 7.29-7.21 (m, 1H), 7.17-7.02 (m, 2H), 6.83-6.79 (m, 2H), 4.81-4.71 (m, 2H), 4.69-4.55 (m, 2H), 4.25-4.15 (m, 1H), 4.13-4.04 (m, 1H), 3.96-3.85 (m, 2H), 3.70-3.61 (m, 1H), 3.55-3.46 (m, 3H), 3.40-3.21 (m, 4H), 3.18-3.10 (m, 2H), 3.07-2.96 (m, 4H), 2.67-2.56 (m, 2H), 2.54-2.34 (m, 3H). 2.29-2.17 (m, 2H), 2.10-1.99 (m, 1H), 1.89-1.57 (m, 7H) 1.52-1.28 (m, 6H), 1.21-1.11 (m, 4H), 1.07-0.96 (m, 6H), 0.95-0.81 (m, 12H), 0.80-0.69 (m, 1H).

3. Preparation of Antibody-Toxin Conjugate

[0340] ##STR00041##

[0341] Compound 12h (1.2 mg, 1.2 mol) was dissolved in acetonitrile (0.3 mL). Ab-10 monoclonal antibody-propylmercaptan 1c solution (6.17 mg/mL, 3.0 mL) was added with shaking for 4 hours at 25 C., and then desalination and purification were done on a Sephadex G25 gel column (Elution phase: 0.05M of PBS solution which pH is 6.5). The captioned product compound ADC-12 in PBS buffer (3.3 mg/mL, 5.0 mL) was obtained by filtration through a 0.2 m filter under aseptic condition, and then frozen stored at 4 C.

[0342] Q-TOF LC/MS: characteristic peak: 148119.6 (M.sub.Ab+OD), 149150.5 (M.sub.Ab+1D), 150221.1 (M.sub.Ab+2D), 151265.1 (M.sub.Ab+3D), 152314.3 (M.sub.Ab+4D).

[0343] Mean value: y=1.6.

[0344] With reference to examples 9-18, No. 11-12 ADC compounds were prepared.

##STR00042##

Example 19. The Inhibitory Effect of Anti-c-Met Antibody Toxin Conjugate (ADC) Molecules on the Proliferation of Hepatic Carcinoma Cells

[0345] Test samples: certain antibody compounds of the present invention; the chemical names and preparation methods can be found in the preparation example of each compound.

[0346] The inhibitory effect of molecules of the present invention on cell proliferation was tested by the CCK method, and the in vitro activity of ADC molecules of the present invention was evaluated according to IC50.

[0347] Cell proliferation was measured using Cell Counting Kit (Dojindo Chemical Technologies, LTD Cat # CK04) (operated according to the instructions). The cells and the corresponding media used are shown in the table below:

TABLE-US-00016 cell line culture media Cat. No. HepG2 EMEM + 10% FBS Cell bank, the Chinese academy of science, Cat# TCHu 72 Hep3B EMEM + 10% FBS Cell bank, the Chinese academy of science, Cat# TCHu106 SK-HEP-1 EMEM + 10% FBS Cell bank, the Chinese academy of science, Cat# TCHu109 HCCLM3 DMEM + 10% FBS Jiangsu Howson pharmaceutical co., LTD QGY-7701 DMEM + 10% FBS Shanghai Bioleaf biotechnology co., LTD SMMC-7721 DMEM + 10% FBS Shanghai Bioleaf biotechnology co., LTD Bel-7402 DMEM + 10% FBS Shanghai Bioleaf biotechnology co., LTD

[0348] Experimental Procedures:

[0349] During the experiment, 2-3 mL trypsin was introduced to perform digestion for 2-3 min. After the cells were completely digested, the digested cells were eluted by adding 10-15 mL of complete medium, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Then, the cells were resuspended by adding 10-20 mL medium to prepare single cell suspensions, and the cell density was adjusted to 410.sup.4 cells/mL. 0.1 mL of the above cell suspension was added to each well of a 96-well cell culture plate, which was incubated at 37 C. in a 5% CO.sub.2 incubator, the media was removed 24 hours later, and 90 L media containing 2% FBS was added to each well. The samples to be tested were diluted with PBS to different concentration gradients, added with 10 L per well, and incubated at 37 C. in a 5% CO.sub.2 incubator for 72 hours. 10 L of CCK8 was added to each well, incubation was continued for another 2 hours in the incubator, the OD450 was detected by a microplate reader (VICTOR 3, PerkinElmer), and data analysis was performed using GraphPad Prism (version 5.0) software.

[0350] Experimental Results:

TABLE-US-00017 TABLE 8 The inhibitory effects of the molecules of the invention on proliferation of hepatic carcinoma cells ADC-1 Ab-10 Maximum Maximum Inhibition Inhibition cell line tumor type IC.sub.50 (nM) (%) IC.sub.50 (nM) (%) HepG2 hepatic 81.1 25 80.90 23 carcinoma Hep3B hepatic 109.4 27 >1000 0 carcinoma Sk-hep-1 hepatic 40.8 65 >1000 0 carcinoma QGY-7701 hepatic 9.1 100 >1000 0 carcinoma SMMC-7721 hepatic 21.7 100 >1000 0 carcinoma Bel-7402 hepatic 0.35 83 0.50 34 carcinoma HCCLM3 hepatic 1.1 85 3.20 24 carcinoma

[0351] Experimental Conclusion:

[0352] Based on the above table, the experimental results show that the ADC-1 drug molecule of the present invention has an inhibitory effect on the hepatic carcinoma cell line superior to that of an anti-c-Met antibody of the present invention, indicating that the ADC drug of the present invention has a better inhibitory effect on the proliferation of hepatic carcinoma cells.

Example 20: The Efficacy of ADC Drugs of the Present Invention on Subcutaneous Xenografted Tumor of Human Hepatic Carcinoma HCCLM3 in Nude Mice

[0353] Test sample: certain antibodies and ADC compounds of the present invention; the chemical names and preparation methods can be found in the preparation example of each compound.

[0354] Experimental animals: BALB/cA-nude mice, 21-28 days, male, purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Production license number: SCXK (Shanghai) 2013-0017, No311613700000089. Feeding environment: SPF level.

[0355] Preparation of Test Solutions:

[0356] ADC-12 was dissolved into a 20 mg/mL solution with water for injection, and each aliquot was stored in a 80 C. refrigerator, then diluted to the corresponding concentrations with 0.1% BSA in saline before use.

[0357] An Ab-10 antibody stock solution (antibody concentration of 16.3 mg/mL) was diluted with 0.1% BSA in saline, and each aliquot was stored in a 80 C. refrigerator.

[0358] Experimental Procedures:

[0359] Nude mice were subcutaneously inoculated with human hepatic carcinoma HCCLM3 cells. Once the tumors were grown to 100-150 mm.sup.3, the animals were randomly divided into groups (D0), 10 in each group. The tumor volume was measured 2-3 times per week, the mice were weighed, and the data were recorded.

[0360] The tumor volume (V) was calculated according to the formula:

[0361] V=1/2ab.sup.2, wherein a and b represent length and width, respectively;

[0362] T/C(%)=(TT.sub.0)/(CC.sub.0)100, wherein T and C are tumor volume at the end of the test; T.sub.0 and C.sub.0 are the tumor volume at the start of the test.

[0363] Experimental Results:

TABLE-US-00018 TABLE 9 Inhibitory test of molecules of the present invention on subcutaneous xenografted tumors of human hepatic carcinoma HCCLM3 in nude mice P Mean Mean tumor value volume of volume of % inhibition (d21) Compound admini- tumor (mm.sup.3) tumor (mm.sup.3) T/C rate (vs partial complete Groups stration D0 SEM D21 SEM D21 D21% blank) regression regression Solvent D0 125.3 4.0 2367.4 193.3 0 0 ADC-12 D0 125.5 3.1 1556.6 99.0 64 36 0.002 0 0 (1 mg/kg) ADC-12 D0 123.3 3.8 425.8 78.6 13 87 **0.000 1 0 (3 mg/kg) ADC-12 D0 121.5 5.7 27.2 12.8 78 178 **0.000 3 6 (10 mg/kg) Ab-10 D0,3,7,1 123.9 3.5 1565.1 150.5 64 36 0.004 0 0 (10 mg/kg) 0,14,17 D0: first administration time; P value, compared with solvent; **P < 0.01; all use Student's t test; number of mice at the start of the test: n = 10; short line indicates that the data is empty.

[0364] Experimental Conclusion:

[0365] Based on the data in the above table, ADC-12 (1, 3, 10 mg/kg, iv, D0) inhibited the growth of subcutaneous xenografted tumors (c-Met-expressing human hepatic carcinoma HCCLM3) in nude mice in a dose-dependent manner; tumor inhibition rates were 36%, 87%, and 178% (D21) respectively, 1 out of 10 tumors exhibited partial regression in 3 mg/kg dose group, 3 out of 10 tumors exhibited partial regression and 6 out of 10 tumors exhibited complete regression in 10 mg/kg dose group (D21). By 43 days after the first administration (D42), there were still 7 out of 10 tumors showing complete regression in the 10 mg/kg dose group. As for the Ab-10 antibody stock solution (10 mg/kg, IV, twice per week for 6 times), the tumor inhibition rate against HCCLM3 was 36%; tumor-bearing mice tolerated the above drugs well, and symptoms such as loss of weight were not observed. In comparison, ADC-12 was significantly more effective against HCCLM3 than Ab-10 antibody stock solution.

[0366] Therefore, ADC-12 had a significant inhibitory effect on subcutaneous xenograft tumors of c-Met human hepatic carcinoma HCCLM3 in nude mice, effectively inhibited tumor growth, caused partial or complete regression of tumors, and the tumor inhibition rate was increased along with the increase of dosage, inhibitory effect was more significant; Ab-10 antibody stock solution significantly inhibited HCCLM3 and effectively inhibited tumor growth. With the same dosage, ADC-12 was significantly more effective against HCCLM3 than Ab-10 antibody stock solution.

Example 21. Anti-Tumor Effect of ADC Drugs of the Present Invention on LI-03-0022 HCC Patient-Derived Tumor Transplantation (PDX) Model in BALB/c Nude Mice

[0367] Test sample: certain antibodies, ADC compounds of the present invention, the chemical names and preparation methods can be found in the preparation example of each compound.

[0368] Experimental animals: BALB/cA-nude mice, 6-8 weeks, 18 females, purchased from Shanghai Sippr-BK Lab Animal Co. Ltd., production license number: SCXK (Shanghai) 2013-0016, animal certificate No.: 2008001658891; Feeding environment: SPF level.

[0369] Preparation of Test Solutions:

[0370] The drug ADC-12 was dissolved into a 20 mg/mL solution with water for injection, and each aliquot was stored in a 80 C. refrigerator, then diluted to the corresponding concentrations with 5% glucose solution before use;

[0371] An Ab-10 antibody stock solution (antibody concentration of 16.3 mg/mL) was diluted with 5% glucose solution, each aliquot was stored in a 80 C. refrigerator; then diluted to the corresponding concentration with 5% glucose solution before use.

[0372] The particular preparation method is shown in the table below:

TABLE-US-00019 Packing concentration compound size preparation method (mg/mL) storage Solvent 5% glucose solution 4 C. ADC-12 40 mg/vial 2.0 mL sterile water was injected into a vial, 20.0 80 C. and ADC-12 powder was slowly added; the mixture was gently stirred until a 20 mg/mL clear stock solution was obtained; the stock solution was divided into 20 aliquots which were stored at 80 C. (0.1 mL/vial). ADC-12 20 mg/mL the stored stock solution (0.1 mL, 20 mg/mL) 1.0 RT injection was diluted with 1.9 mL of 5% solution glucose solution; the solution was freshly prepared at the time of each injection before use. Ab-10 16.3 mg/mL, Ab-10 antibody solution was evenly divided 5.0 RT antibody 5 mL/vial into several aliquots and stored at 80 C.; solution 0.613 mL stored stock solution was diluted with 1.386 mL of 5% glucose solution; the solution was freshly prepared at the time of each injection before use. Ab-10 16.3 mg/mL, Ab-103 antibody solution was evenly 1.0 RT antibody 5 mL/vial divided into several aliquots and stored at 80 C.; solution 0.12 mL stock solution was diluted with 1.836 mL of 5% glucose solution the solution was freshly prepared at the time of each injection before use.

[0373] Establishment of LI-03-0022 HCC PDX Tumor Model:

[0374] An LI-03-0022 HCC (hepatic cellular cancer, hepatic carcinoma) PDX tumor model (patient-derived tumor xenograft model, PDX) was originally established on a clinical tissue sample surgically resected from patients with hepatic carcinoma (from Shanghai Oriental Hepatobiliary Hospital) and was implanted in nude mice and defined as generation 0 (P0). Implantation of tumor generation 0 (P0) was defined as generation 1 (P1); the generation was thus defined according to the order continuously implanted in nude mice. FP3 tumors were recovered from P2T patients, the next generation from FP5 was defined as FP6, and so on; FP5 tumor tissue was used for this study.

[0375] Experimental Procedure:

[0376] (1) Tumor implantation: Each mouse was s.c. implanted with LI-03-0022 FP5 tumor sections (about 30 mm.sup.3) on the right side to develop tumors, and 32 days after tumor implantation, the mean tumor size was close to 183.20 mm.sup.3. Since then, treatment was started with 6 tumor-bearing mice in each group, and the experimental procedure for mice was carried out according to the predetermined protocol in the experimental design of the following table:

TABLE-US-00020 administration dosage volume, concentration administration administration Group N.sup.a treatment mg/kg mL/kg.sup.b mg/mL mode time .sup.c 1 6 solvent 10 iv BIW 2 weeks 2 6 ADC-12 10 10 1.0 iv BIW 2 injection weeks solution 3 6 Ab-10 10 or 10 1.0 iv BIW 2 50 5.0 weeks 1.sup.st-2.sup.nd dosing, 10 mg/kg; 3.sup.rd-4.sup.th dosing, 50 mg/kg Note: .sup.aN is the number of animals in each group; .sup.bthe administration volume was adjusted based on 10 L/g body weight; .sup.c BIW is twice a week; iv is intravenously;

[0377] After 2 hours, the last administration was performed, blood samples were collected from all the mice without anticoagulation treatment, and about 50 L of serum was collected for PK analysis. At the end of the study, tumor samples were collected from 2 animals from the solvent group and 2 animals from the sample group. Animals were divided into two groups: one for FFPE (Formalin-Fixed and Parrffin-Embedded tissue was referred as FFPE sample) and IHC (Immunohistochemistry); the other for Frozen in liquid nitrogen.

[0378] (2) Observation and recording: The tumor volume was measured 2-3 times per week, the mice were weighed, and the data were recorded.

[0379] (3) Tumor measurement and endpoint

[0380] The endpoint was mainly dependent on whether the tumor growth was delayed or whether the mouse could be cured. The tumor volume was measured twice a week with a caliper in two dimensions (in mm.sup.3);

[0381] The tumor volume (V) was calculated as:

[0382] V=0.5ab.sup.2, wherein a and b represent the long and short diameter of the tumor, respectively;

[0383] The tumor volume was used to calculate T-C value, T/C value, T-C value by T (the mean time required for the tumor in treatment group to reach 1000 mm.sup.3, in days) and C (the mean time required for the tumor in control group to reach the same size, in days); the T/C value (percentage) was used as an indicator of antitumor efficacy, in particular T=T.sub.i/T.sub.0, C=C.sub.i/C.sub.0, T.sub.i is the mean tumor volume of the treatment group on a certain day, T.sub.0 is the mean tumor volume of the treatment group at the beginning of treatment, C.sub.i is the mean tumor volume of the solvent control group at the same time as T.sub.i, and V.sub.0 is the mean tumor volume of the solvent group at the beginning of treatment.

[0384] (4) Data analysis: Summary statistics, including mean and standard error (SEM), volumetric analysis of differences in tumor volume between different groups, and volumetric analysis of drug interactions after the last administration (day 14 after grouping) performed by the data obtained at optimal treatment time point, one-way variance analysis was performed to compare tumor volume and tumor weight between groups; when non-significant F-statistic was obtained (p=0.061, treatment variance vs. error variance), Dunnett's T (double-sided) inter-group comparison was performed; all data were analyzed using SPSS 17.0, P<0.05 was considered statistically significant.

[0385] Experimental Results:

TABLE-US-00021 TABLE 10-1 The change of tumor volume over time tumor volume (mm.sup.3).sup.a ADC-12 injection Ab-10 solution 1st-2nd 10 mg/kg Day.sup.b solvent 10 mg/kg 3rd-4th 50 mg/kg 0 184 32 184 28 .sup.164 31.sup.c 3 351 62 242 37 293 52 7 606 98 155 29 569 85 10 879 193 107 18 719 108 14 1,605 367 52 12 1,067 152 17 41 11 1,725 195 21 29 10 1,896 283 24 16 8 28 7 3 31 6 3 34 4 2 37 4 2 41 14 9 42 15 9 .sup.ais mean standard error; .sup.bis the number of days after beginning the treatment; .sup.cis n = 5; short line, , means that the animals in the corresponding group were sacrificed at this time, no data were obtained.

TABLE-US-00022 TABLE 10-2 Analysis of the inhibitory effect of molecules of the present invention on tumor growth of human hepatic carcinoma subcutaneous xenograft tumor in BALB/c nude mice Day 14 T-C Day 21 tumor (day) tumor size T/C.sup.b at 1000 p size p Sample (mm.sup.3).sup.a (%) mm.sup.3 value (mm.sup.3).sup.a value.sup.c solvent 1,605 367 ADC-12 52 12 3.25 >32 0.001 29 10 injection solution (10 mg/kg) Ab-10 1,067 152 74.47 3 0.252 1,896 283 <0.001 (1.sup.st-2.sup.nd, 10 mg/kg; 3.sup.rd-4.sup.th, 50 mg/kg).sup.d

[0386] a is meanstandard error; b is tumor growth inhibition, calculated by dividing the mean tumor volume of the treatment group by the mean tumor volume of the control group, T/C must be less than or equal to 50%; c is the p value calculated on the basis of tumor size; d means 3-5 tumor-bearing animals in each group.

TABLE-US-00023 TABLE 10-3 Immunohistochemistry (IHC) of c-Met staining No. PDX model IHC score 1 LI-03-0010 (negative control) 0 2 LI-03-0022 2+

[0387] The particular staining area is shown in FIG. 2 and FIG. 3 of the specification.

[0388] Note: 0 means unstained, + means light staining, ++ means medium staining, +++ means dark staining; the entire section was read under microscope, the percentage of different staining intensity in cells was determined by visual evaluation, and the H score was calculated as 1(% of +cells)+2(% of ++cells)+3(% of +++cells); then the scoring standard was used to evaluate the figure: the score of 0-0.3 is weakly positive, 0.3-1.5 is moderate positive and 1.5-3 is strongly positive.

[0389] Experimental Conclusion:

[0390] The results of the above table demonstrate that the ADC drug of the present invention is significantly stronger than the antibody for inhibiting the proliferation of hepatic carcinoma cells; treatment with ADC-12 injection solution resulted in significant antitumor activity with a mean tumor volume of 52.3 mm.sup.3 (T/C value=3.25%, p=0.001), when compared with the solvent group; the tumor growth volume was controlled within 1000 mm.sup.3 and delayed by 32 days; however, treatment with Ab-10 antibody solution resulted in only minimal antitumor activity, the mean tumor size was controlled within 1,067 mm.sup.3 (T/C value=74.47%) and delayed by 3 days; there was no statistically significant difference when compared with the solvent group (p=0.252). The LI-03-0022 HCC PDX model had an impression score of 2+ for c-Met protein expression, indicating that the expression level of c-Met protein in the model is strongly positive and can be used for further research in vivo. Therefore, the ADC drug of the present invention had significant antitumor activity in a LI-03-0022 HCC patient-derived tumor transplantation (PDX) model study, and was well tolerated in tumor-bearing animals.

Example 22. Anti-Tumor Effect of ADC Drug of the Present Invention on a LI-03-0240 HCC Patient-Derived Tumor Transplantation (PDX) Model in BALB/c Nude Mice

[0391] Test sample: certain antibodies and ADC compounds of the present invention; the chemical names and preparation methods can be found in the preparation example of each compound.

[0392] Experimental animals: BALB/cA-nude mice, 6-8 weeks, 30 females, purchased from Shanghai Sippr-BK Lab Animal Co. Ltd., production license number: SCXK (Shanghai) 2013-0016, animal certificate No.: 2008001658004; Feeding environment: SPF level.

[0393] Preparation of Test Solutions:

[0394] The drug ADC-12 lyophilized powder was dissolved as 20 mg/mL solution with water for injection, and each aliquot was stored in a 80 C. refrigerator, then diluted to corresponding concentrations with 5% glucose solution before use;

[0395] An Ab-10 antibody stock solution (concentration of 16.3 mg/mL) was diluted with 5% glucose solution, each aliquot was stored in a 80 C. refrigerator; then diluted to corresponding concentrations with 5% glucose solution before use.

[0396] The particular preparation method is shown in the table below:

TABLE-US-00024 packing concentration compound size preparation method mg/mL storage solvent 5% glucose solution RT ADC-12 40 mg/vial 2.0 mL sterile water was injected into vial, and 20 80 C. ADC-12 lyophilized powder was slowly added; the mixture was gently stirred until 20 mg/mL clear stock solution was obtained; the stock solution was divided into 20 aliquots which were stored at 80 C. ADC-12 20 mg/mL 0.14 mL ADC-12 was added into a vial, and diluted 1 RT injection with 2.66 mL 0.5% glucose solution to obtain 1 mg/mL solution working solution. ADC-12 1 mg/mL 0.2 mL ADC-12 injection solution was added into a 0.1 RT injection vial, and diluted with 1.8 mL 0.5% glucose solution solution to obtain 0.1 mg/mL working solution. The solution was prepared before each administration. ADC-12 1 mg/mL 0.6 mL ADC-12 injection solution was added into a 0.3 RT injection vial, and diluted with 1.4 mL 0.5% glucose solution solution to obtain 0.3 mg/mL working solution. The solution was prepared before each administration. Ab-10 16.3 mg/mL, Ab-10 antibody solution was evenly divided into 1 RT antibody 5 mL/vial several aliquots and stored at 80 C. solution 0.12 mL Ab-10 antibody solution was diluted with 1.836 mL of 5% glucose solution to obtain 1 mg/mL working solution. The solution was prepared before each administration.

[0397] Establishment of LI-03-0240 HCC PDX Tumor Model:

[0398] An LI-03-0240 HCC (hepatic cellular cancer, hepatic carcinoma) PDX tumor model (patient-derived tumor xenograft model, PDX) was originally established on clinical tissue sample surgically resected from patients with hepatic carcinoma (from Shanghai Oriental Hepatobiliary Hospital) which was implanted in nude mice and defined as generation 0 (P0). Implantation of tumor generation 0 (P0) was defined as generation 1 (P1); the generation was thus defined according to the order continuously implanted in nude mice. FP3 tumors were recovered from P2T patients, the next generation from FP5 was defined as FP6, and so on; FP5 tumor tissue was used for this study.

[0399] Experimental Procedure:

[0400] (1) Tumor implantation: Each mouse was s.c. implanted with LI-03-0240 FP5 tumor sections (about 30 mm.sup.3) on the right side to develop tumors, and 15 days after tumor implantation, the mean tumor size was close to 151.79 mm.sup.3. Since then, treatment was started with 6 tumor-bearing mice in each group, and the experimental procedure for mice was carried out according to the predetermined protocol in the experimental design of the following table

TABLE-US-00025 administration dosage volume, concentration administration administration group N.sup.a treatment mg/kg mL/kg.sup.b mg/mL mode time.sup.c 1 6 solvent 10 iv BIW 2 weeks 2 6 ADC-12 1 10 0.1 iv BIW 2 injection weeks solution 3 6 ADC-12 3 10 0.3 iv BIW 2 injection weeks solution 4 6 ADC-12 10 10 1.0 iv BIW 2 injection weeks solution 5 6 Ab-10 10 10 1.0 iv BIW 2 weeks Note: .sup.aN is the number of animals in each group; .sup.bthe dosage volume was adjusted based on 10 L/g body weight; .sup.cBIW is twice a week; iv is intravenously;

[0401] After 2 hours, the last administration was performed, blood samples were collected from all the mice without anticoagulation treatment, and about 50 L of serum was collected for PK analysis; at the end of the study, tumor samples were collected from 2 animals from the solvent group and 2 animals from the sample group. Animals were divided into two groups: one for FFPE (Formalin-Fixed and Parrffin-Embedded tissue was referred as FFPE sample) and IHC (Immunohistochemistry); the other for Frozen in liquid nitrogen.

[0402] (2) Observation and recording: The tumor volume was measured 2-3 times per week, the mice were weighed, and the data were recorded.

[0403] (3) Tumor measurement and endpoint

[0404] The endpoint was mainly dependent on whether the tumor growth was delayed or whether the mouse could be cured. The tumor volume was measured twice a week with a caliper in two dimensions (in mm.sup.3);

[0405] The tumor volume (V) was calculated as:

[0406] V=0.5ab.sup.2, wherein a and b represent length and width, respectively;

[0407] The tumor volume was used to calculate T-C value, T/C value, T-C value by T (the mean time required for the tumor in treatment group to reach 1000 mm.sup.3, in days) and C (the mean time required for the tumor in control group to reach the same size, in days); the T/C value (percentage) was used as an indicator of antitumor efficacy, in particular T=T.sub.i/T.sub.0, C=C.sub.i/C.sub.0, T.sub.i is the mean tumor volume of the treatment group on certain day, T.sub.0 is the mean tumor volume of the treatment group at the beginning of treatment, C.sub.i is the mean tumor volume of the solvent control group at the same time as T.sub.i, and V.sub.0 is the mean tumor volume of the solvent group at the beginning of treatment.

[0408] (4) Data analysis: Summary statistics, including mean and standard error (SEM), statistic analysis of differences in tumor volume between different groups, and data analysis of drug interactions after the last administration (day 17 after grouping) performed by the data obtained at optimal treatment time point, one-way variance analysis was performed to compare tumor volume and tumor weight between groups; when non-significant F-statistic was obtained (p<0.001, treatment variance vs. error variance), Games-Howell inter-group comparison was performed; all data were analyzed using SPSS 17.0, P<0.05 was considered statistically significant.

[0409] Experimental Results:

TABLE-US-00026 TABLE 11-1 the change of tumor volume over time tumor volume (mm.sup.3).sup.a ADC-12 ADC-12 ADC-12 injection injection injection solution solution solution Ab-10 Day.sup.b solvent 1.0 mg/kg 3.0 mg/kg 10 mg/kg 10 mg/kg 0 151 20 152 10 151 17 152 27 152 16 4 268 47 189 10 121 17 129 27 259 22 7 425 74 140 10 60 9 63 19 384 43 11 677 105 96 10 41 8 43 16 574 91 14 972 162 76 9 31 5 31 11 815 141 17 1,320 201 60 7 22 4 24 9 1,159 221 20 53 6 20 4 20 8 1,556 280 24 43 7 16 4 15 6 27 41 6 8 2 14 5 31 50 11 3 2 9 5 34 75 28 2 1 7 4 38 114 56 0 0 4 3 41 177 84 0 0 2 2 45 248 130 0 0 1 1 .sup.ais mean standard error; .sup.bis the number of days after beginning the treatment; .sup.cmeans n = 5; short line, means that the animals in corresponding group were sacrificed at this time, no data were obtained.

TABLE-US-00027 TABLE 11-2 Analysis of the inhibitory effect of molecules of the present invention on tumor growth of human hepatic carcinoma subcutaneous xenograft tumor in BALB/c nude mice Day 17 Day 21 tumor T-C (day) tumor size T/C.sup.b value p size p sample (mm.sup.3).sup.a (%) at 1000 mm.sup.3 value.sup.c (mm.sup.3).sup.a value.sup.c solvent 1,320 201 ADC-12 injection 60 7 4.51 >31 0.008 53 6 0.011 solution (1 mg/kg) ADC-12 injection 22 4 1.66 >31 0.007 20 4 0.010 solution (3 mg/kg) ADC-12 injection 24 9 1.84 >31 0.007 20 8 0.010 solution (10 mg/kg) Ab-10 (10 mg/kg) 1,159 221 87.75 1 0.980 1,556 280 .sup.ais mean standard error; .sup.bis tumor growth inhibition, calculated by dividing the mean tumor volume of the treatment group by the mean tumor volume of the control group, T/C must be less than or equal to 50%; .sup.cis the p value calculated on the basis of tumor size; .sup.dmeans 3-5 tumor-bearing animals in each group.

TABLE-US-00028 TABLE 11-3 Immunohistochemistry (IHC) of c-Met staining No. PDX model IHC score 1 LI-03-0010 (negative control) 0 2 LI-03-0240 3+

[0410] The particular staining area is shown in FIG. 5 and FIG. 6 of the specification.

[0411] Note: 0 means unstained, + means light staining, ++ means medium staining, +++ means dark staining; the entire section was read under microscope, the percentage of different staining intensity in cells was determined by visual evaluation, and the H score was calculated as 1(% of +cells)+2(% of ++cells)+3(% of +++cells); then the scoring standard was used to evaluate the figure: the score of 0-0.3 is weakly positive, 0.3-1.5 is moderate positive and 1.5-3 is strongly positive.

[0412] Experimental Conclusion:

[0413] The results of the above table demonstrate that the ADC drug of the present invention is significantly stronger than the antibody for inhibiting the proliferation of hepatic carcinoma cells; treatment with ADC-12 injection solution resulted in significant antitumor activity with a mean tumor volume of 52.3 mm.sup.3 (T/C value=3.25%, p=0.001), when compared with the solvent group; the tumor growth volume was controlled within 1000 mm.sup.3 and delayed by 32 days; however, treatment with Ab-10 antibody solution resulted in only minimal antitumor activity, the mean tumor size was controlled within 1,067 mm.sup.3 (T/C value=74.47%) and delayed by 3 days; there was no statistically significant difference when compared with the solvent group (p=0.252). The LI-03-0240 PDX model had an impression score of 3+ for c-Met protein expression, indicating that the expression level of c-Met protein in the model is strongly positive and can be used for further research in vivo. Therefore, the ADC drug of the present invention had significant antitumor activity in a LI-03-0240 HCC patient-derived tumor transplantation (PDX) model study, and was well tolerated in tumor-bearing animals.