One pot desialylation and glycopegylation of therapeutic peptides

Abstract

The present invention provides conjugates between peptides and PEG moieties. The conjugates are linked via an intact glycosyl linking group that is interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from both glycosylated and unglycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto either an amino acid or glycosyl residue on the peptide. Also provided are pharmaceutical formulations including the conjugates. Methods for preparing the conjugates are also within the scope of the invention.

1 . A method of altering glycosylation of a peptide, said method comprising: 
 (a) contacting said peptide with a sialidase, thereby removing at least one sialic acid moiety from said peptide;    (b) contacting product from step (a) with at least one glycosyltransferase and a sugar donor which is a substrate for said at least one glycosyltransferase, thereby transferring at least one sugar moiety from said donor a glycosyl moiety or amino acid of said said product from step (a);    (c) separating product from step (b) from sialidase; and    (d) contacting product from step (c) with a sialic acid donor and a sialyltransferase, thereby forming a peptide with altered glycosylation.    
     
     
         2 . The method according to  claim 1  wherein said peptide with altered glycosylation comprises a modified sialyl residue having the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 D is a member selected from —OH and R 1 -L-HN—;  
 G is a member selected from R 1 -L- and —C(O)(C 1 -C 6 )alkyl-R 1 ;  
 R 1  is a moiety comprising a member selected from a straight-chain poly(ethylene glycol) residue and branched poly(ethylene glycol) residue; and  
 M is a member selected from H, a metal and a single negative charge;  
 L is a linker which is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl,  
 such that when D is OH, G is R 1 -L-, and when G is —C(O)(C 1 -C 6 )alkyl, D is R 1 -L-NH— 
 said method comprising:  
 (a) contacting a peptide comprising the glycosyl moiety:  
                     with a PEG-sialic acid donor moiety having the formula:                          
 and an enzyme that transfers said PEG-sialic acid onto the Gal of said glycosyl moiety, under conditions appropriate for said transfer.  
 
     
     
         3 . The method according to  claim 2 , wherein L-R 1  has the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 a is an integer selected from 0 to 20.  
 
     
     
         4 . The method according to  claim 2 , wherein R 1  has a structure that is a member selected from:  
       
         
           
           
               
               
           
         
       
       wherein 
 e, f, m and n are integers independently selected from 1 to 2500; and  
 q is an integer selected from 0 to 20.  
 
     
     
         5 . The method according to  claim 2 , wherein R 1  has a structure that is a member selected from:  
       
         
           
           
               
               
           
         
       
       wherein 
 e, f and f′ are integers independently selected from 1 to 2500; and  
 q and q′ are integers independently selected from 1 to 20.  
 
     
     
         6 . The method according to  claim 2 , wherein R 1  has a structure that is a member selected from:  
       
         
           
           
               
               
           
         
       
       wherein 
 e and f are integers independently selected from 1 to 2500.  
 
     
     
         7 . The method according to  claim 2 , wherein said glycosyl linker has the formula:  
       
         
           
           
               
               
           
         
       
     
     
         8 . The method according to  claim 2 , wherein said peptide conjugate comprises at least one of said glycosyl linker according to a formula selected from:  
       
         
           
           
               
               
           
         
         wherein AA is an amino acid residue of said peptide conjugate and t is an integer selected from 0 and 1.  
       
     
     
         9 . The method according to  claim 2  wherein said peptide conjugate comprises at least one of said glycosyl linker wherein each of said glycosyl linker has a structure which is a member independently selected from the following formulae:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein AA is an amino acid residue of said peptide conjugate and t is an integer selected from 0 and 1.  
       
     
     
         10 . The method of  claim 2 , wherein said peptide conjugate comprises at least one of said glycosyl linker according to a formula selected from:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein AA is an amino acid residue of said peptide conjugate and t is an integer selected from 0 and 1  
         and wherein a member selected from 0 and 2 of the sialyl moieties which do not comprise G are absent.  
       
     
     
         11 . The method according to  claim 2  wherein said peptide conjugate comprises at least one said glycosyl linker according to a formula selected from:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein AA is an amino acid residue of said peptide conjugate and t is an integer selected from 0 and 1  
         and wherein a member selected from 0 and 2 of the sialyl moieties which do not comprise G are absent.  
       
     
     
         12 . The method according to  claim 2 , wherein said peptide has the amino acid sequence of SEQ. ID. NO:1.  
     
     
         13 . The method according to  claim 2 , wherein said glycosyl linker is attached to said peptide through an amino acid residue selected from serine and threonine.  
     
     
         14 . The method according to  claim 2 , wherein said glycosyl linker is attached to said peptide through an amino acid residue which is an asparagine residue.  
     
     
         15 . The method according to  claim 14 , wherein said asparagine residue is a member selected from N152, N322 and combinations thereof.  
     
     
         16 . The method according to  claim 2 , wherein said peptide is a bioactive peptide.  
     
     
         17 . The method of  claim 2 , further comprising, prior to step (a): (b) expressing said peptide in a suitable host.  
     
     
         18 . The method of  claim 17 , wherein said host is a mammalian expression system.  
     
     
         19 . A method of treating a condition in a subject in need thereof, said condition characterized by compromised clotting potency in said subject, said method comprising the step of administering to the subject an amount of the peptide conjugate produced according to the method of  claim 2 , effective to ameliorate said condition in said subject.  
     
     
         20 . A method of enhancing clotting potency in a mammal, said method comprising administering to said mammal an amount of the peptide conjugate produced according to the method of  claim 2.