Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins

Abstract

Methods to introduce highly phosphorylated mannopyranosyl oligosaccharide derivatives containing mannose 6-phosphate (M6P), or other oligosacharides bearing other terminal hexoses, to carbonyl groups on oxidized glycans of glycoproteins while retaining their biological activity are described. The methods are useful for modifying glycoproteins, including those produced by recombinant protein expression systems, to increase uptake by cell surface receptor-mediated mechanisms, thus improving their therapeutic efficacy in a variety of applications.

1 - 21 . (canceled)  
     
     
         22 . A method for coupling an oligosaccharide comprising a hexose to a therapeutic glycoprotein, the method comprising the steps of: 
 (a) derivatizing the oligosaccharide to generate a carbonyl-reactive group;    (b) oxidizing the therapeutic glycoprotein to generate at least one carbonyl group on the therapeutic glycoprotein; and    (c) reacting the derivatized oligosaccharide with the oxidized therapeutic glycoprotein, thereby coupling the oligosaccharide to the therapeutic glycoprotein.    
     
     
         23 . The method of  claim 22 , further comprising producing the therapeutic glycoprotein by recombinant protein expression system prior to step (a).  
     
     
         24 . The method of  claim 22 , wherein the expression system is chosen from yeast, mammalian cell, insect cell plant cell, and transgenic animal.  
     
     
         25 . The method of  claim 22 , wherein the hexose is a phosphorylated mannose.  
     
     
         26 . The method according to  claim 25 , wherein the phosphate group is linked to a terminal mannose.  
     
     
         27 . The method according to  claim 25 , wherein the phosphate group is linked to a penultimate mannose.  
     
     
         28 . The method according to  claim 25 , wherein the oligosaccharide comprises two or more mannose-6-phosphate (M6P) groups.  
     
     
         29 . The method according to  claim 25 , wherein the oxidizing step is carried out with periodate or galactose oxidase.  
     
     
         30 . The method according to  claim 25 , wherein the oligosaccharide is chosen from a biantennary mannopyranosyl oligosaccharide and a triantennary mannopyranosyl oligosaccharide.  
     
     
         31 . The method according to  claim 30 , wherein the biantennary mannopyranosyl oligosaccharide comprises bis-M6P.  
     
     
         32 . The method according to  claim 30 , wherein the triantennary mannopyrannosyl oligoscacharide comprises bis-M6P or tri-M6P.  
     
     
         33 . The method according to  claim 22 , wherein the oligosaccharide comprises:  
       
         
           
           
               
               
           
         
         wherein M is mannose or a mannopyranosyl group.  
       
     
     
         34 . The method according to  claim 22 , wherein the derivatized oligosaccharide has a formula chosen from 6-P-M n -R and (6-P-M x ) m L n -R, 
 wherein M is mannose or a mannopyranosyl group,    is a phosphate group linked to the C-6 position of M,    L is a hexose,    R has at least one carbonyl-reactive group,    m is an integer ranging from 2 to 3,    n is an integer ranging from 1 to 15, wherein if n>1, the L n  are linked to one another by alpha (1,2), alpha (1,3), alpha (1,4), or alpha (1, 6), and    x is an integer ranging from 1 to 15.    
     
     
         35 . The method according to  claim 34 , wherein at least one L is mannose.  
     
     
         36 . The method according to  claim 34 , wherein at least one L is chosen from galactose, N-acetylglucosamine, and fucose.  
     
     
         37 . The method according to  claim 22 , wherein the carbonyl-reactive group is chosen from a hydrazine, a hydrazide, an aminooxy, a semicarbazide.  
     
     
         38 . The method according to  claim 22 , further comprising the step of adding a reducing agent to the coupled therapeutic glycoprotein.  
     
     
         39 . The method according to  claim 22 , wherein the reducing agent comprises cyanoborohydride.  
     
     
         40 . The method of  claim 22 , wherein the carbonyl-reactive group is aminooxy.