Specific sites for modifying antibodies to make immunoconjugates

Abstract

The present invention provides specific sites for modifying antibodies or antibody fragments by replacing at least one native amino acid in the constant region of a parental antibody or antibody fragment with cysteine, which can be used as a site of attachment for a payload or linker-payload combination.

1 - 57 . (canceled) 
     
     
         58 . An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine:
 a. on its constant region at a site selected from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system;   b. on its constant region at a site selected from positions 107, 108, 142, 145, 159, 161, and 165 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain;   c. on its constant region at a site selected from positions 143, 147, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is human a lambda light chain;   d. on its constant region selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 and 422 of the heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system;   e. on its constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is a human kappa light chain; or   f. selected from positions 143, 145, 147, 156, 159, 163, 168 on its constant region of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.   
     
     
         59 . The immunoconjugate of  claim 58 , wherein said antibody or antibody fragment comprises a sequence selected from:
 a. the group consisting of SEQ ID NOs: 4, 5, 10, 17, 18, 29, 35, 42, 43, 48, 50, 54, 290, 291, 292, 293, 294, and 295;   b. the group consisting of SEQ ID NOs: 61, 62, 69, 71, 75, 76, and 77; or   c. the group consisting of SEQ ID NOs: 92, 94, 96, 97, and 98.   
     
     
         60 . The immunoconjugate according to  claim 58 , wherein said modified antibody or antibody fragment comprises a combination of substitutions of two or more amino acids with cysteine:
 a. on a constant region of a heavy chain at positions 152 and 375, wherein said positions are numbered according to the EU system;   b. on a constant region of a heavy chain at positions 327 and 375, wherein said positions are numbered according to the EU system;   c. on a constant region at position 107 of a light chain and position 360 of a heavy chain, wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system;   d. on a constant region at position 375 of a heavy chain and position 165 of a light chain, wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system; or   e. on a constant region at position 334 of a heavy chain and at position 165 of a light chain, wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system.   
     
     
         61 . The immunoconjugate of  claim 58  comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of three or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions selected from:
 a. positions 375 and 392 of an antibody heavy chain and position 165 of an antibody light chain; 
 b. positions 334 and 375 of an antibody heavy chain and position 165 of an antibody light chain; and 
 c. positions 334 and 392 of an antibody heavy chain and position 165 of an antibody light chain; 
 d. positions 152, 375 and 392 of the an antibody heavy chain, 
 e. positions 152, 334 and 375 of an antibody heavy chain, and 
 f. positions 152, 334 and 392 of an antibody heavy chain,
 wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system. 
 
 
     
     
         62 . The immunoconjugate of  claim 58  comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment thereof comprises a combination of substitution of four or more amino acids with cysteine:
 a. on its constant region at positions 334, 375, and 392 of a heavy chain and position 165 of a light chain, 
 b. on its constant region at positions 333, 375, and 392 of a heavy chain and at position 165 of a light chain; 
 c. on its constant region at positions 152, 334, 375, and 392 of a heavy chain; or 
 d. on its constant region at positions 152, 333, 375, and 392 of a heavy chain;
 wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system. 
 
 
     
     
         63 . The immunoconjugates of  claim 58  wherein the drug antibody ratio is about 4, about 6, or about 8. 
     
     
         64 . The immunoconjugate of  claim 58  further comprising a drug moiety. 
     
     
         65 . The immunoconjugate of  claim 64 , wherein the drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker. 
     
     
         66 . The immunoconjugate of  claim 65 , wherein said drug moiety is connected to said sulfur of said cysteine through a cleavable or non-cleavable linker. 
     
     
         67 . The immunoconjugate of  claim 65 , wherein said immunoconjugate comprises a thiol-maleimide linkage, a —S—CH 2 —C(═O)— linkage or a disulfide linkage. 
     
     
         68 . The immunoconjugate of  claim 65 , wherein said drug moiety is a cytotoxic agent. 
     
     
         69 . The immunoconjugate of  claim 68 , wherein said drug moiety is selected from the group consisting of taxanes, DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatins, and maytansinoids. 
     
     
         70 . The immunoconjugate of  claim 58 , wherein said antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully humanized antibody, a bispecific antibody, or a multi-specific antibody. 
     
     
         71 . The immunoconjugate of  claim 58 , wherein said antibody or antibody fragment specifically binds to a cell surface marker characteristic of a tumor. 
     
     
         72 . A pharmaceutical composition comprising the immunoconjugate of claim  1 . 
     
     
         73 . A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with cysteine on its constant region selected from:
 a. positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400, and 422 of a heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system;   b. positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the EU system, and wherein said light chain is human kappa light chain;   c. positions 143, 145, 147, 156, 159, 163, 168 on its constant region of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is human lambda light chain.   
     
     
         74 . The modified antibody or antibody fragment of  claim 73 , wherein the substitution is selected from:
 a. positions 152 and 375 of a heavy chain;   b. positions 327 and 375 of a heavy chain;   c. position 107 of a light chain and position 360 of a heavy chain;   d. position 375 of a heavy chain and position 165 of a light chain;   e. at position 334 of a heavy chain and position 165 of a light chain;   f. positions 375 and 392 of a heavy chain and position 165 of a light chain;   g. positions 334 and 375 of a heavy chain and position 165 of a light chain;   h. positions 334 and 392 of a heavy chain and position 165 of a light chain;   i. positions 152, 375 and 392 of a heavy chain;   j. positions 152, 334 and 375 of a heavy chain;   k. positions 152, 334 and 392 of an antibody heavy chain;   l. positions 334, 375, and 392 of a heavy chain and position 165 of a light chain,   m. positions 333, 375, and 392 of a heavy chain and at position 165 of a light chain;   n. at positions 152, 334, 375, and 392 of a heavy chain; or   o. at positions 152, 333, 375, and 392 of a heavy chain;
 wherein said light chain is a kappa chain, and wherein said positions are numbered according to the EU system. 
   
     
     
         75 . A nucleic acid encoding the modified antibody or antibody fragment of  claim 73 . 
     
     
         76 . A host cell comprising the nucleic acid of  claim 75 . 
     
     
         77 . A method of producing a modified antibody or antibody fragment comprising incubating the host cell of  claim 76  under suitable conditions for expressing the antibody or antibody fragment, and isolating said antibody or antibody fragment.