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.

What is claimed is: 
     
         1 . An immunoconjugate comprising a modified antibody or antibody fragment thereof and a payload, wherein the modified antibody or antibody fragment comprises a cysteine substitution at amino acid position 152 of a heavy chain constant region of the antibody or antibody fragment, wherein the amino acid position is numbered according to the EU system, and wherein the drug antibody ratio is 2, 4, 6 or 8. 
     
     
         2 . The immunoconjugate of  claim 1 , further comprising one or more additional cysteine amino acid substitutions of the heavy chain constant region of the antibody or antibody fragment. 
     
     
         3 . The immunoconjugate of  claim 1 , further comprising a cysteine substitution at amino acid position 375 of the heavy chain constant region of the antibody or antibody fragment. 
     
     
         4 . The immunoconjugate of  claim 1 , wherein the drug antibody ratio is 2. 
     
     
         5 . The immunoconjugate of  claim 1 , wherein the drug antibody ratio is 4. 
     
     
         6 . The immunoconjugate of  claim 1 , wherein the drug antibody ratio is 6. 
     
     
         7 . The immunoconjugate of  claim 1 , wherein the drug antibody ratio is 8. 
     
     
         8 . The immunoconjugate of  claim 1 , wherein the payload is attached to the modified antibody or antibody fragment indirectly or directly through a sulfur of the cysteine at amino acid position 152. 
     
     
         9 . The immunoconjugate of  claim 1 , wherein the payload is attached to the modified antibody or antibody fragment indirectly or directly through a linker. 
     
     
         10 . The immunoconjugate of  claim 9 , wherein the linker is a cleavable linker, a non-cleavable linker, a thiol-maleimide linkage, a —S—CH 2 —C(═O)— linkage, or a disulfide linkage. 
     
     
         11 . The immunoconjugate of  claim 1 , wherein the payload is a cytotoxic agent. 
     
     
         12 . The immunoconjugate of  claim 1 , wherein the payload is selected from the group consisting of taxanes, DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, and maytansinoids. 
     
     
         13 . The immunoconjugate of  claim 1 , wherein the antibody is an IgG antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully humanized antibody, a bispecific antibody, or a multi-specific antibody. 
     
     
         14 . The immunoconjugate of  claim 1 , wherein the antibody or antibody fragment specifically binds to a cell surface marker characteristic of a tumor. 
     
     
         15 . A composition comprising the immunoconjugate of  claim 1 . 
     
     
         16 . A method of producing the immunoconjugate of  claim 1 , wherein the method comprises attaching a Linker Unit (LU) or a Linker Unit-Payload combination (-LU-X) to the cysteine at amino acid position 152 of the antibody or antibody fragment. 
     
     
         17 . An immunoconjugate comprising a modified antibody or antibody fragment thereof and a payload, wherein the modified antibody or antibody fragment comprises a cysteine substitution at amino acid position 152 of the heavy chain constant region of the antibody or antibody fragment, wherein the amino acid position is numbered according to the EU system, and wherein the drug antibody ratio is 2. 
     
     
         18 . An immunoconjugate comprising a modified antibody or antibody fragment thereof wherein the modified antibody or antibody fragment comprises a combination of cysteine substitutions at amino acid positions 152 and 375 of a heavy chain constant region of the antibody or antibody fragment, wherein the amino acid positions are numbered according to the EU system, and wherein the drug antibody ratio is 4. 
     
     
         19 . An immunoconjugate comprising a modified antibody or antibody fragment thereof wherein said modified antibody or antibody fragment comprises a combination of cysteine substitutions at amino acid positions 152 and 375 of a heavy chain constant region of said antibody or antibody fragment, wherein said amino acid positions are numbered according to the EU system, and wherein the drug antibody ratio is 8. 
     
     
         20 . The immunoconjugate of  claim 19 , further comprising one or more additional cysteine amino acid substitutions of the heavy chain constant region of the antibody or antibody fragment.