Glycoengineered antibody drug conjugates

Abstract

The current disclosure provides binding polypeptides (e.g., antibodies), and targeting moiety conjugates thereof, comprising a site-specifically engineered glycan linkage within native or engineered glycans of the binding polypeptide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

1 - 16 . (canceled) 
     
     
         17 . A method of making an effector moiety conjugated binding polypeptide comprising the steps of:
 (a) reacting a CMP-sialic acid derivative with a glycan of a binding polypeptide to form a sialic acid derivative-conjugated binding polypeptide;   (b) reacting the sialic acid derivative-conjugated binding polypeptide with an effector moiety to form the effector moiety conjugated binding polypeptide, wherein a thioether bond is formed.   
     
     
         18 . The method of  claim 17 , wherein neither the binding polypeptide nor the sialic acid derivative-conjugated binding polypeptide are treated with an oxidizing agent. 
     
     
         19 . The method of  claim 17 , wherein the sialic acid derivative comprises a terminal thiol moiety. 
     
     
         20 . The method of  claim 17 , wherein the effector moiety comprises a maleimide moiety. 
     
     
         21 . The method of  claim 17 , wherein the effector moiety is bis-mannose-6-phosphate hexamannose maleimide, lactose maleimide, or any other component comprising at least one maleimide moiety of the following structural formula: 
       
         
           
           
               
               
           
         
       
     
     
         22 . The method of  claim 17 , wherein the effector moiety comprises one or more proteins, nucleic acids, lipids, carbohydrates, or combinations thereof. 
     
     
         23 . The method of  claim 17 , wherein the effector moiety comprises a glycan. 
     
     
         24 . The method of  claim 17 , wherein the effector moiety comprises one or more glycoproteins, glycopeptides, or glycolipids. 
     
     
         25 . The method of  claim 17 , wherein the binding protein has one or more native or engineered glycosylation sites. 
     
     
         26 . The method of  claim 17 , further comprising achieving or modifying the glycosylation of the binding protein using one or more glycosyltransferases, one or more glycosidases, or a combination thereof. 
     
     
         27 . The method of  claim 26 , wherein step (a) occurs in a reaction with sialyltransferase. 
     
     
         28 . The method of  claim 27 , wherein the sialyltransferase is a mammalian sialyltransferase. 
     
     
         29 . The method of  claim 28 , wherein the sialyltransferase is beta-galactoside alpha-2,6-sialyltransferase 1. 
     
     
         30 . The method of  claim 17 , wherein the CMP-sialic acid derivative has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The method of  claim 17 , wherein the binding polypeptide comprises a S298N mutant. 
     
     
         32 . The method of  claim 17 , wherein the binding polypeptide comprises an A114N mutant in the CH1 domain. 
     
     
         33 . The method of  claim 17 , wherein the binding polypeptide comprises a Y300S mutant. 
     
     
         34 . The method of  claim 17 , wherein the binding polypeptide comprises a heavy chain of SEQ ID NO:14 and a light chain of SEQ ID NO:12. 
     
     
         35 . The method of  claim 17 , wherein the effector moiety comprises polyethylene glycol (PEG). 
     
     
         36 . The method of  claim 17 , wherein the effector moiety comprises one of the following structures: