Site-specific covalent chemical ligation to monoclonal and polyclonal immunoglobulin

Abstract

Methods and compositions are described herein for covalently linking an antibody to a molecular payload. Compositions are described herein containing an antibody covalently linked to a molecular payload.

1 . A compound comprising:
 i) a targeting moiety that specifically binds a nucleotide binding pocket of an antibody;   ii) a cross-linking agent;   iii) an active agent or a conjugating agent; and   iv) a linker, wherein the linker covalently links:
 a) the targeting moiety, 
 b) the cross-linking agent, and 
 c) the active agent or conjugating agent. 
   
     
     
         2 . The compound of  claim 1 , wherein the cross-linking agent comprises 5-fluoro-2,4-dinitrobenzene (DNFB). 
     
     
         3 . The compound of  claim 1 , wherein the cross-linking agent comprises maleimide. 
     
     
         4 . The compound of  claim 1 , wherein the linker comprises an ethylene glycol dimer, or a PEG polymer. 
     
     
         5 . The compound of  claim 1 , wherein the linker comprises an amino acid sequence. 
     
     
         6 .- 25 . (canceled) 
     
     
         26 . A method for covalently conjugating an antibody to a molecular payload, wherein the molecular payload comprises a compound of  claim 1 , the method comprising:
 a) forming a reaction mixture comprising the antibody and the molecular payload under conditions suitable to form a non-covalent binding interaction between a nucleotide binding pocket of the antibody and a targeting moiety of the molecular payload, wherein the reaction mixture is an aqueous solution having a pH of less than about 7.5; and   b) raising the pH of the reaction mixture above about 8.0, under conditions suitable to form a covalent bond between the antibody and the cross-linking agent.   
     
     
         27 . The method of  claim 26 , wherein the cross-linking comprises forming a covalent bond between the cross-linking agent and a primary amine, a sulfhydryl, or a guanidinium group of the antibody. 
     
     
         28 . The method of  claim 26 , wherein the primary amine, sulfhydryl, or guanidinium group is within 8 Å of the nucleotide binding pocket. 
     
     
         29 . The method of  claim 26 , wherein a) comprises incubating the reaction mixture for at least about 0.25 h. 
     
     
         30 . The method of  claim 26 , wherein the conditions suitable to form the non-covalent binding interaction between the nucleotide binding pocket of the antibody and the targeting moiety of the molecular payload comprise a reaction mixture temperature of about 20° C. 
     
     
         31 . The method of  claim 26 , wherein the reaction mixture of a) comprises phosphate buffered saline at a pH of about 7.0 (PBS 7.0) or phosphate buffered saline at a pH of about 7.5 (PBS 7.5). 
     
     
         32 . The method of  claim 26 , wherein the reaction mixture of a) comprises a molar excess of molecular payload relative to nucleotide binding pockets and the method further comprises removing unbound molecular payload from the reaction mixture after a) and before b). 
     
     
         33 . The method of  claim 26 , wherein the removing comprises dialysis or size-exclusion chromatography. 
     
     
         34 . The method of  claim 26 , wherein the molar excess of molecular payload relative to nucleotide binding pockets is a 5-fold molar excess. 
     
     
         35 . The method of  claim 26 , wherein b) comprises raising the pH of the reaction mixture to at least about 8.5. 
     
     
         36 . The method of  claim 26 , wherein b) comprises adding a basic solution to the reaction mixture of a) and incubating the reaction mixture for at least 0.25 h. 
     
     
         37 . The method of  claim 26 , wherein the basic solution comprises 0.1 M sodium bicarbonate pH 8.5, 0.1 N NaOH or 0.1 N NH 4 OH. 
     
     
         38 . The method of  claim 26 , wherein the compound of  claim 1  comprises a conjugating agent comprising an azide or alkyne, and after forming the covalent bond between the antibody and the cross-linking agent, the method further comprises conjugating an active agent to the azide or alkyne via azide alkyne Huisgen cycloaddition or Copper (I)-catalyzed azide-alkyne cycloaddition. 
     
     
         39 . The method of  claim 26 , wherein the compound of  claim 1  comprises a conjugating agent comprising a 1,2-dihydroxybenzene moiety, and after forming the covalent bond between the antibody and the cross-linking agent, the method further comprises conjugating a boronic acid containing active agent to the 1,2-dihydroxybenzene moiety to form a boronic ester. 
     
     
         40 .- 48 . (canceled)