Methods for making conjugates from disulfide-containing proteins

Abstract

The invention provides methods to prepare protein conjugates from proteins having at least four accessible cysteine residues. In one embodiment, an antibody with four reducible disulfide linkages is reduced to provide four pairs of free cysteines. Each pair of free cysteines is reacted with a 1,3-dihaloacetone or similar reactant, linking the sulfur atoms of each pair together though a 3-carbon tether with a reactive ketone. A pair of these reactive ketones are linked together with and used to attach a single payload molecule, thus conjugating the antibody to two payload groups. This method gives a stabilized antibody conjugate with high selectivity for a ratio of two payloads per antibody.

1 . A compound of the formula (IIIa) 
       
         
           
           
               
               
           
         
         wherein:
 R is a therapeutic agent, a detectable label, or a functional group selected from N 3 , COOH, NH 2 , SH, —S—Ar, maleimide, and HC≡C—, where Ar represents phenyl or pyridyl; 
 L 1 , L 2  and L 3  are linking groups; and 
 A is N, CH, phenyl, C 3-6  cycloalkyl, or a 5-6 membered heterocyclic or heteroaryl group having up to two heteroatoms selected from N, O and S as ring members; 
 
         or a salt thereof. 
       
     
     
         2 . The compound of  claim 1 , wherein L 1  and L 2  are the same. 
     
     
         3 . The compound of  claim 2 , wherein R is a cytotoxin. 
     
     
         4 . The compound of  claim 2 , wherein L 3  comprises at least one group selected from —(CH 2 ) 1-6 —, —(CH 2 CH 2 O) 1-6 — and —(OCH 2 CH 2 ) 1-6 —. 
     
     
         5 . The compound of  claim 2 , wherein L 3  comprises at least one group selected from —C(O)—, —NHC(O)—, —C(O)NH— and —S—. 
     
     
         6 . The compound of claim  13 - 17 , which is of the formula 
       
         
           
           
               
               
           
         
       
       and R 3  is H or (C 1 -C 6 )alkyl. 
     
     
         7 . The compound of  claim 6 , wherein R-L 3 - is of the formula R—Z—C(═O)—, wherein Z comprises at least one group selected from —(CH 2 ) 1-6 —, —(CH 2 CH 2 O) 1-6 — and —(OCH 2 CH 2 ) 1-6 —. 
     
     
         8 . A method to prepare a polypeptide-payload conjugate, which comprises contacting an activated polypeptide or polypeptide complex of formula (IIIa) or (IIIb) with the compound of  claim 7 , 
       
         
           
           
               
               
           
         
         wherein the circle represents a polypeptide or polypeptide complex, which is activated by the inserted carbonyl groups, and each S represents a sulfur atom of a cysteine residue comprised in the polypeptide or polypeptide complex. 
       
     
     
         9 . The method of  claim 8 , wherein the polypeptide or polypeptide complex is an antibody or an Fc portion of an antibody. 
     
     
         10 . The method of  claim 9 , which further comprises a step of preparing the activated polypeptide or polypeptide complex by a method that comprises reducing at least two disulfide bonds of an antibody or antibody Fc to make a reduced polypeptide or polypeptide complex,
 and contacting the reduced polypeptide or polypeptide complex with dichloroacetone to provide the activated polypeptide or polypeptide complex.