Humanized antibodies to LIV-1 and use of same to treat cancer

Abstract

The invention provides humanized antibodies that specifically bind to LIV-1. The antibodies are useful for treatment and diagnoses of various cancers as well as detecting LIV-1.

What is claimed is: 
     
       1. A humanized antibody, that specifically binds human LIV-1 comprising a mature heavy chain variable region comprising three CDRs of SEQ ID NO:53 and having an amino acid sequence at least 95% identical to SEQ ID NO:53 provided that position H27 is occupied by L, position H29 is occupied by I, H30 by E and H94 by V and a mature light chain variable region comprising three CDRs of SEQ ID NO:60 at least 95% identical to SEQ ID NO:60 provided position L36 is occupied by Y and position L46 by P. 
     
     
       2. The humanized antibody of  claim 1 , further provided position H76 is occupied by N. 
     
     
       3. The humanized antibody of  claim 1  wherein the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain variable region is fused to a light chain constant region. 
     
     
       4. The humanized antibody of  claim 3 , wherein the heavy chain constant region is a mutant form of a natural human constant region which has reduced binding to an Fcgamma receptor relative to the natural human constant region. 
     
     
       5. The humanized antibody of  claim 3 , wherein the heavy chain constant region is of IgG1 isotope. 
     
     
       6. The humanized antibody of  claim 3 , wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:44 and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42. 
     
     
       7. The humanized antibody of  claim 3 , wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:46 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42. 
     
     
       8. The humanized antibody of  claim 1 , wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:52 or 53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO: 59 or 60. 
     
     
       9. The humanized antibody of  claim 1 , wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO:60. 
     
     
       10. The humanized antibody of  claim 1 , wherein the antibody is conjugated to a cytotoxic or cytostatic agent. 
     
     
       11. The humanized antibody of  claim 1 , having an association constant for human or cynomolgus monkey LIV-1 of 0.5 to 2×10 9  M −1 . 
     
     
       12. A nucleic acid encoding a the mature heavy chain variable region and/or a the mature light chain variable region as defined by  claim 1 . 
     
     
       13. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of a the humanized antibody of  claim 1 . 
     
     
       14. The method of  claim 13 , wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma. 
     
     
       15. A pharmaceutical composition comprising a the humanized antibody of  claim 1 . 
     
     
       16. The method of  claim 13 , wherein the antibody is conjugated to a cytotoxic or cytostatic agent. 
     
     
       17. The method of  claim 13 , wherein the isotype of the antibody is human IgG1. 
     
     
       18. A vector comprising the nucleic acid of claim 12.   
     
     
       19. The vector of claim 18, wherein the vector is an expression vector.  
     
     
       20. A host cell comprising the nucleic acid of claim 12.   
     
     
       21. The host cell of claim 20, wherein the host cell is a Chinese hamster ovary (CHO) cell.  
     
     
       22. A method of producing an anti-LIV-1 antibody comprising culturing the host cell of claim 20 under a condition suitable for production of the anti-LIV-1 antibody.  
     
     
       23. The method of claim 22, further comprising isolating the anti-LIV-1 antibody produced by the host cell.  
     
     
       24. A method of producing an anti-LIV-1 antibody-drug conjugate comprising culturing the host cell of claim 20 under a condition suitable for production of an anti-LIV-1 antibody; isolating the anti-LIV-1 antibody produced from the host cell; and conjugating the anti-LIV-1 antibody to a cytotoxic or cytostatic agent.  
     
     
       25. The method of claim 24, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       26. The method of claim 24, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       27. The method of claim 26, wherein the auristatin is monomethyl auristatin E.  
     
     
       28. The method of claim 26, wherein the auristatin is monomethyl auristatin F.  
     
     
       29. The method of claim 24, wherein the anti-LIV-1 antibody is conjugated to the cytotoxic or cytostatic agent via a linker.  
     
     
       30. The method of claim 29, wherein the linker is a cleavable peptide linker.  
     
     
       31. The method of claim 30, wherein the cleavable peptide linker has a formula:
   -A a -W w —Y y —,
   wherein:   -A- is a stretcher unit;   a is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   −Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       32. The method of claim 29, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       33. The method of claim 29, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       34. The method of claim 33, wherein the anti-LIV-1 antibody-drug conjugate has the following structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the anti-LIV-1 antibody.  
       
     
     
       35. The method of claim 34, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       36. A nucleic acid encoding the mature heavy chain variable region and/or the mature light chain variable region as defined by claim 9.   
     
     
       37. A vector comprising the nucleic acid of claim 36.   
     
     
       38. The vector of claim 37, wherein the vector is an expression vector.  
     
     
       39. A host cell comprising the nucleic acid of claim 36.   
     
     
       40. The host cell of claim 39, wherein the host cell is a Chinese hamster ovary (CHO) cell.  
     
     
       41. A method of producing an anti-LIV-1 antibody comprising culturing the host cell of claim 39 under a condition suitable for production of the anti-LIV-1 antibody.  
     
     
       42. The method of claim 41, further comprising isolating the anti-LIV-1 antibody produced by the host cell.  
     
     
       43. A method of producing an anti-LIV-1 antibody-drug conjugate comprising culturing the host cell of claim 39 under a condition suitable for production of an anti-LIV-1 antibody; isolating the anti-LIV-1 antibody produced from the host cell; and conjugating the anti-LIV-1 antibody to a cytotoxic or cytostatic agent.  
     
     
       44. The method of claim 43, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       45. The method of claim 43, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       46. The method of claim 45, wherein the auristatin is monomethyl auristatin E.  
     
     
       47. The method of claim 45, wherein the auristatin is monomethyl auristatin F.  
     
     
       48. The method of claim 43, wherein the anti-LIV-1 antibody is conjugated to the cytotoxic or cytostatic agent via a linker.  
     
     
       49. The method of claim 48, wherein the linker is a cleavable peptide linker.  
     
     
       50. The method of claim 49, wherein the cleavable peptide linker has a formula:
   -A a -W w —Y y —
   wherein:   -A- is a stretcher unit;   a is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   —Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       51. The method of claim 48, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       52. The method of claim 48, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       53. The method of claim 52, wherein the anti-LIV-1 antibody-drug conjugate has the following structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the anti-LIV-1 antibody.  
       
     
     
       54. The method of claim 53, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       55. The humanized antibody of claim 6, wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO:60.  
     
     
       56. The humanized antibody of claim 10, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       57. The humanized antibody of claim 10, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       58. The humanized antibody of claim 57, wherein the auristatin is monomethyl auristatin E.  
     
     
       59. The humanized antibody of claim 57, wherein the auristatin is monomethyl auristatin F.  
     
     
       60. The humanized antibody of claim 10, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.  
     
     
       61. The humanized antibody of claim 60, wherein the linker is a cleavable peptide linker.  
     
     
       62. The humanized antibody of claim 61, wherein the cleavable peptide linker has a formula:
   -A a -W w —Y y —,
   wherein:   -A- is a stretcher unit;   a is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   —Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       63. The humanized antibody of claim 60, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       64. The humanized antibody of claim 60, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       65. The humanized antibody of claim 64, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       66. The humanized antibody of claim 65, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       67. The humanized antibody of claim 6, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.  
     
     
       68. The humanized antibody of claim 67, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       69. The humanized antibody of claim 67, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       70. The humanized antibody of claim 69, wherein the auristatin is monomethyl auristatin E.  
     
     
       71. The humanized antibody of claim 69, wherein the auristatin is monomethyl auristatin F.  
     
     
       72. The humanized antibody of claim 67, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.  
     
     
       73. The humanized antibody of claim 72, wherein the linker is a cleavable peptide linker.  
     
     
       74. The humanized antibody of claim 73, wherein the cleavable peptide linker has a formula:
   -A a -W w —Y y —,
   wherein:   -A- is a stretcher unit;   α is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   —Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       75. The humanized antibody of claim 72, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       76. The humanized antibody of claim 72, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       77. The humanized antibody of claim 76, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       78. The humanized antibody of claim 77, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       79. The humanized antibody of claim 9, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.  
     
     
       80. The humanized antibody of claim 79, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       81. The humanized antibody of claim 79, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       82. The humanized antibody of claim 81, wherein the auristatin is monomethyl auristatin E.  
     
     
       83. The humanized antibody of claim 81, wherein the auristatin is monomethyl auristatin F.  
     
     
       84. The humanized antibody of claim 79, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.  
     
     
       85. The humanized antibody of claim 84, wherein the linker is a cleavable peptide linker.  
     
     
       86. The humanized antibody of claim 85, wherein the cleavable peptide linker has a formula:
   -A a -W w —Y y —,
   wherein:   -A- is a stretcher unit;   a is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   —Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       87. The humanized antibody of claim 84, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       88. The humanized antibody of claim 84, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       89. The humanized antibody of claim 88, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       90. The humanized antibody of claim 89, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       91. The humanized antibody of claim 55, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.  
     
     
       92. The humanized antibody of claim 91, wherein the cytotoxic or cytostatic agent is an antitubulin agent.  
     
     
       93. The humanized antibody of claim 91, wherein the cytotoxic or cytostatic agent is an auristatin.  
     
     
       94. The humanized antibody of claim 93, wherein the auristatin is monomethyl auristatin E.  
     
     
       95. The humanized antibody of claim 93, wherein the auristatin is monomethyl auristatin F.  
     
     
       96. The humanized antibody of claim 91, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.  
     
     
       97. The humanized antibody of claim 96, wherein the linker is a cleavable peptide linker.  
     
     
       98. The humanized antibody of claim 97, wherein the cleavable peptide linker has a formula:
   -A a -W W —Y y —,
   wherein:   -A- is a stretcher unit;   a is 0 or 1;   each —W— is independently an amino acid unit;   w is independently an integer ranging from 0 to 12;   —Y— is a spacer unit; and   y is 0, 1 or 2.    
     
     
       99. The humanized antibody of claim 96, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.  
     
     
       100. The humanized antibody of claim 96, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.  
     
     
       101. The humanized antibody of claim 100, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       102. The humanized antibody of claim 101, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       103. A pharmaceutical composition comprising the humanized antibody of claim 6.   
     
     
       104. A pharmaceutical composition comprising the humanized antibody of claim 9.   
     
     
       105. A pharmaceutical composition comprising the humanized antibody of claim 55.   
     
     
       106. A pharmaceutical composition comprising the antibody-drug conjugate of claim 65.   
     
     
       107. A pharmaceutical composition comprising the antibody-drug conjugate of claim 101.   
     
     
       108. The method of claim 14, wherein the breast cancer is triple negative breast cancer.  
     
     
       109. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the humanized antibody of claim 55.   
     
     
       110. The method of claim 109, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.  
     
     
       111. The method of claim 110, wherein the breast cancer is triple negative breast cancer.  
     
     
       112. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 65.   
     
     
       113. The method of claim 112, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.  
     
     
       114. The method of claim 113, wherein the breast cancer is triple negative breast cancer.  
     
     
       115. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 101.   
     
     
       116. The method of claim 115, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.  
     
     
       117. The method of claim 116, wherein the breast cancer is triple negative breast cancer.  
     
     
       118. A pharmaceutical composition comprising an antibody-drug conjugate, wherein the antibody-drug conjugate comprises a humanized antibody conjugated to monomethyl auristatin E, wherein the humanized antibody specifically binds to human LIV-1 and comprises a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO:53 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO:60, wherein the mature heavy chain variable region is fused to an IgG1 isotype heavy chain constant region comprising the amino acid sequence of SEQ ID NO:44 and the mature light chain variable region is fused to a light chain constant region comprising the amino acid sequence of SEQ ID NO:42, wherein the antibody-drug conjugate further comprises a linker between the humanized antibody and the monomethyl auristatin E, wherein the antibody-drug conjugate has the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       119. The pharmaceutical composition of claim 118, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       120. An antibody-drug conjugate comprising a humanized antibody conjugated to monomethyl auristatin E, wherein the humanized antibody specifically binds to human LIV-1 and comprises a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO:53 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO:60, wherein the mature heavy chain variable region is fused to an IgG1 isotype heavy chain constant region comprising the amino acid sequence of SEQ ID NO:44 and the mature light chain variable region is fused to a light chain constant region comprising the amino acid sequence of SEQ ID NO:42, wherein the antibody-drug conjugate further comprises a linker between the humanized antibody and the monomethyl auristatin E, wherein the antibody-drug conjugate has the structure: 
       
         
           
           
               
               
           
         
         wherein p denotes a number from 1 to 8 and Ab designates the humanized antibody.  
       
     
     
       121. The antibody-drug conjugate of claim 120, wherein the average value of p in a population of the antibody-drug conjugate is about 4.  
     
     
       122. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 120.   
     
     
       123. The method of claim 122, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.  
     
     
       124. The method of claim 123, wherein the breast cancer is triple negative breast cancer.