Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use

Abstract

Provided herein are antibodies comprising multiple non-natural amino acid residues at site-specific positions, compositions comprising the antibodies, methods of their production and methods of their use. The antibodies are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

1 . An antibody comprising
 a first site-specific non-natural amino acid residue and a second site-specific non-natural amino acid residue;   wherein the first site-specific non-natural amino acid residue and the second site-specific non-natural amino acid residue have different reactive groups that are capable of mutually non-overlapping site-specific conjugations in the same reaction; and wherein the first and the second site specific non-natural amino acid residues comprise a reactive pair selected from the group consisting of amino, carboxyl, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, tetrazine, and strained alkene.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The antibody of  claim 1  comprising at least one site-specific non-natural amino acid in a light chain polypeptide and at least one site-specific non-natural amino acid in a heavy chain polypeptide. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The antibody of  claim 1  comprising a heavy chain of a type selected from the group consisting of α, δ, ε and μ. 
     
     
         14 . The antibody of  claim 1  comprising a light chain of a type selected from and x. 
     
     
         15 . The antibody of  claim 1 , wherein said antibody is of a class or subclass selected from the group consisting of IgA, IgA, IgA1, IgA2, IgD, IgE, IgG, IgG1, IgG2, IgG3 and IgM. 
     
     
         16 . The antibody of  claim 1 , wherein said antibody is in a form selected from the group consisting of Fv, Fc, Fab, (Fab′) 2 , single chain Fv (scFv) and full-length antibody. 
     
     
         17 . The antibody of  claim 1 , wherein at least one of said non-natural amino acid residues comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine. 
     
     
         18 . The antibody of  claim 1 , wherein at least one of said non-natural amino acid residues comprises an azide moiety. 
     
     
         19 . The antibody of  claim 1 , wherein at least one of said non-natural amino acid residues comprises an acetyl moiety. 
     
     
         20 . The antibody of  claim 1 , wherein at least one of said non-natural amino acid residues comprises an azide moiety and at least one of said non-natural amino acid residues comprises an acetyl moiety. 
     
     
         21 . The antibody of  claim 1 , wherein each non-natural amino acid residue is according to the formula 
       
         
           
           
               
               
           
         
         wherein each L is independently a divalent linker; and 
         each R is independently a functional group. 
       
     
     
         22 . The antibody of  claim 21  wherein R is a reactive group, a therapeutic moiety, or a labeling moiety. 
     
     
         23 . The antibody of  claim 21 , wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine. 
     
     
         24 . The antibody  claim 21 , wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene, and substituted heteroarylene. 
     
     
         25 . An antibody conjugate comprising the antibody of  claim 1  linked to one or more therapeutic moieties or labeling moieties. 
     
     
         26 . The antibody conjugate of  claim 25 , wherein said antibody is linked to one or more drugs or polymers. 
     
     
         27 . The antibody conjugate of  claim 25 , wherein said antibody is linked to one or more labeling moieties. 
     
     
         28 . The antibody conjugate of  claim 25 , wherein said antibody is linked to one or more single-chain binding domains (scFv). 
     
     
         29 . The antibody conjugate of  claim 25 , wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a non-natural amino acid residue comprising an azide moiety. 
     
     
         30 . The antibody conjugate of  claim 25 , wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a non-natural amino acid residue comprising an acetyl moiety. 
     
     
         31 . The antibody conjugate of  claim 25 , wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a non-natural amino acid residue comprising an azide moiety and at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a non-natural amino acid residue comprising an acetyl moiety. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The antibody conjugate of  claim 25 , wherein said antibody is linked to said one or more therapeutic moieties or labeling moieties via one or more linkers. 
     
     
         37 . The antibody conjugate of  claim 25 , wherein said antibody conjugate has a melting temperature within about five degrees Celsius of a parent antibody. 
     
     
         38 . The antibody conjugate of  claim 25 , wherein said antibody conjugate has a melting temperature that is at least about three degrees Celsius greater than that of a parent antibody. 
     
     
         39 . A composition comprising the antibody of  claim 1 , wherein said antibody is substantially pure. 
     
     
         40 . A composition comprising the antibody of  claim 1 , wherein said antibody is at least 95% by mass of the total antibody of said composition. 
     
     
         41 . A method of treating a subject in need thereof comprising administering to said subject an effective amount of an antibody conjugate of  claim 25 . 
     
     
         42 . The method of  claim 41 , wherein said subject is afflicted with cancer. 
     
     
         43 . The method of  claim 42 , wherein said cancer is breast cancer. 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 25 , wherein the therapeutic moiety is an immunomodulatory agent.