Antibodies with engineered ch2 domains, compositions thereof and methods of using the same

Abstract

The present disclosure relates to antibodies and antibody conjugates having one or more site-specific mutations in the CH2 domain of the heavy chain. The antibody variants disclosed herein can have improved characteristics (e.g., thermal stability, antibody yields, antibody titers, cell-killing) relative to a parent or wild type antibody, including aglycosylated parent or wild type antibodies. Pharmaceutical compositions, diagnostic compositions and kits comprising the same, as well as methods of using these compositions and kits for therapeutic and diagnostic purposes, are also described.

1 - 80 . (canceled) 
     
     
         81 . An antibody comprising a light chain, wherein said light chain comprises a non-natural amino acid substitution at an amino position corresponding to K42 according to the Kabat or Chothia numbering scheme. 
     
     
         82 . The antibody of  claim 81 , wherein the non-natural amino acid comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine. 
     
     
         83 . The antibody of  claim 81 , wherein the non-natural amino acid is selected from the group consisting of para-azido phenylalanine and para-azido methyl phenylalanine. 
     
     
         84 . The antibody of  claim 81 , wherein the antibody is a monoclonal antibody. 
     
     
         85 . The antibody of  claim 81 , wherein the antibody is an IgA, an IgD, an IgE, an IgG, or an IgM antibody. 
     
     
         86 . The antibody of  claim 81 , wherein the antibody is humanized or human. 
     
     
         87 . The antibody of  claim 81 , wherein the antibody is aglycosylated. 
     
     
         88 . The antibody of  claim 81 , wherein the antibody is an antibody fragment. 
     
     
         89 . The antibody of  claim 88 , wherein the antibody fragment is selected from an Fv fragment, a Fab fragment, a F(ab′)2 fragment, a Fab′ fragment, an scFv (sFv) fragment, and an scFv-Fc fragment. 
     
     
         90 . The antibody of  claim 81 , wherein the antibody is a bispecific, trispecific, or multispecific antibody. 
     
     
         91 . A pharmaceutical composition comprising the antibody of  claim 81  and a pharmaceutically acceptable carrier. 
     
     
         92 . A polynucleotide encoding the antibody of  claim 81 . 
     
     
         93 . A vector comprising the polynucleotide of  claim 92 . 
     
     
         94 . A host cell comprising the vector of  claim 93 . 
     
     
         95 . A method for producing an antibody, comprising:
 generating the antibody, wherein the antibody comprises a non-natural amino acid substitution at a position corresponding to light chain K42 according to the Kabat, Chothia, or EU numbering scheme.   
     
     
         96 . The method of  claim 95 , wherein the non-natural amino acid comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine. 
     
     
         97 . The method of  claim 95 , wherein the non-natural amino acid is selected from the group consisting of para-azido phenylalanine and para-azido methyl phenylalanine. 
     
     
         98 . The method of  claim 95 , wherein the antibody is produced in a cell-free system. 
     
     
         99 . The method of  claim 98 , wherein the cell-free system utilizes a cell-free extract from  Escherichia coli.    
     
     
         100 . The method of  claim 95 , wherein said antibody is substantially pure.