Erythropoietin: remodeling and glycoconjugation of erythropoietin

Abstract

The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.

1 - 52 . (canceled)  
     
     
         53 . A method of forming a covalent conjugate between a polymer and a glycosylated or non-glycosylated EPO peptide, wherein said polymer is conjugated to said peptide via an intact glycosyl linking group interposed between and covalently linked to both said peptide and said polymer, said method comprising: 
 (a) contacting said peptide with a mixture comprising a nucleotide sugar covalently linked to said polymer and a glycosyltransferase for which said nucleotide sugar is a substrate under conditions sufficient to form said conjugate.    
     
     
         54 . The method of  claim 53 , wherein said polymer is a water-soluble polymer.  
     
     
         55 . The method of  claim 53 , wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.  
     
     
         56 . The method of  claim 53 , wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.  
     
     
         57 . The method of  claim 53 , wherein said polymer comprises a member selected from the group consisting of a polyalkylene oxide and a polypeptide.  
     
     
         58 . The method of  claim 57 , wherein said polyalkylene oxide is poly(ethylene glycol).  
     
     
         59 . The method of  claim 58 , wherein said poly(ethylene glycol) has a degree of polymerization of from about 1 to about 20,000.  
     
     
         60 . The method of  claim 59 , wherein said polyethylene glycol has a degree of polymerization of from about 1 to about 5,000.  
     
     
         61 . The method of  claim 60 , wherein said polyethylene glycol has a degree of polymerization of from about 1 to about 1,000.  
     
     
         62 . The method of  claim 53 , wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, and a GlcNAc transferase.  
     
     
         63 . The method of  claim 53 , wherein said glycosyltransferase is recombinantly produced.  
     
     
         64 . The method of  claim 63 , wherein said glycosyltransferase is a recombinant prokaryotic enzyme.  
     
     
         65 . The method of  claim 63 , wherein said glycosyltransferase is a recombinant eukaryotic enzyme.  
     
     
         66 . The method of  claim 53 , wherein said nucleotide sugar is selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.  
     
     
         67 . The method of  claim 66 , wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, CMP-NeuAc.  
     
     
         68 . The method of  claim 53 , wherein said glycosylated peptide is partially deglycosylated prior to said contacting.  
     
     
         69 . The method of  claim 53 , wherein said intact glycosyl linking group is a sialic acid residue.  
     
     
         70 . The method of  claim 53 , wherein said method is performed in a cell-free environment.  
     
     
         71 . The method of  claim 53 , wherein said covalent conjugate is isolated.  
     
     
         72 . The method of  claim 71 , wherein said covalent conjugate is isolated by membrane filtration.  
     
     
         73 - 178 . (canceled)  
     
     
         179 . The method of  claim 53 , wherein said nucleotide sugar covalently linked to said polymer has the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 R is said polymer or is said polymer attached to said nucleotide sugar through a linker.  
 
     
     
         180 . The method according to  claim 179 , wherein said polymer is poly(ethylene glycol).  
     
     
         181 . The method according to  claim 180 , wherein said poly(ethylene glycol) is selected from linear and branched poly(ethylene glycol).  
     
     
         182 . The method according to  claim 179 , wherein said nucleotide sugar has the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 n is an integer from 1 to 2000.  
 
     
     
         183 . The method according to  claim 53 , wherein said EPO peptide is a glycosylated peptide.  
     
     
         184 . The method according to  claim 183 , wherein said EPO peptide comprises a glycosyl residue having a formula which is a member selected from:  
       
         
           
           
               
               
           
         
       
       wherein 
 a, b, c, d, i, n, o, p, q, r, s, t, and u are members independently selected from 0 and 1;  
 e, f, g, and h are members independently selected from the integers between 0 and 4;  
 j, k, l, and m are members independently selected from the integers between 0 and 20;  
 v, w, x, y, and z are 0; and  
 R is said polymer.  
 
     
     
         185 . The method of  claim 183 , further comprising: 
 (b) prior to step (a), contacting said EPO peptide with a sialidase under conditions appropriate to remove sialic acid from said EPO peptide.    
     
     
         186 . The method of  claim 183 , further comprising: 
 (c) contacting the product of step (a) with a sialyltransferase and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.    
     
     
         187 . The method of  claim 183 , further comprising: 
 (d) prior to step (a), contacting said EPO peptide with a galactosidase operating synthetically under conditions appropriate to add a galactose to said EPO peptide.    
     
     
         188 . The method of  claim 183 , further comprising: 
 (e) prior to step (a), contacting said EPO peptide with a galactosyl transferase and a galactose donor under conditions appropriate to transfer said galactose to said EPO peptide.    
     
     
         189 . The method of  claim 187 , further comprising: 
 (f) contacting the product from step (e) with ST3Gal3 and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.    
     
     
         190 . The method of  claim 183 , further comprising: 
 (g) contacting the product from step (a) with a moiety that reacts with said modifying group, thereby forming a conjugate between said intact glycosyl linking group and said moiety.    
     
     
         191 . The method of  claim 183 , further comprising: 
 (h) prior to step (a), contacting said EPO peptide with N-acetylglucosamine transferase and a GlcNAc donor under conditions appropriate to transfer GlcNAc to said EPO peptide.    
     
     
         192 . The method of  claim 183 , wherein 
 a, b, c, d, e, f, g, n, and q are 1;    h is a member selected from the integers between 1 and 3;    i, j, k, l, m, o, p, r, s, t, and u are members independently selected from 0 and 1; and    v, w, x, y and z are 0.    
     
     
         193 . The method of  claim 183 , wherein 
 a, b, c, d, f, h, j, k, l, m, q, s, u, v, w, x, y, and z are 0; and    e, g, i, r, and t are members independently selected from 0 and 1.    
     
     
         194 . The method of  claim 183 , wherein 
 a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, and u are members independently selected from 0 and 1; and    v, w, x, y, and z are 0.    
     
     
         195 . The method of  claim 183 , wherein 
 a, b, c, d, e, f, g, n, and q are 1;    h is a member selected from the integers between 1 and 3;    i, j, k, l, m, o, p, r, s, t, and u are members independently selected from 0 and 1; and    v, w, x, y and z are 0.    
     
     
         196 . The method of  claim 183 , wherein 
 a, b, c, d, f, h, j, k, l, m, o, p, s, u, v, w, x, y, and z are 0; and    e, g, i, n, q, r, and t are independently selected from 0 and 1.    
     
     
         197 . The method of  claim 183 , wherein 
 a, b, c, d, f, h, j, k, l, m, n, o, p, s, u, v, w, x, y, and z are 0; and    e, g, i, q, r, and t are members independently selected from 0 and 1.    
     
     
         198 . The method of  claim 183 , wherein 
 q is 1;    a, b, c, d, e, f, g, h, i, n, r, s, t, and u are members independently selected from 0 and 1; and    j, k, l, m, o, p, v, w, x, y, and z are 0.