Glycopegylated factor ix

Abstract

The present invention provides conjugates between Factor IX and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

1 . A Factor IX peptide conjugate comprising a glycosyl linking group attached to an amino acid residue of said peptide, said glycosyl linking group comprising a modified sialyl residue having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 2  is H, CH 2 OR 7 , COOR 7  or OR 7    
 wherein
 R 7  represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; 
 
 R 3  and R 4  are members independently selected from H, substituted or unsubstituted alkyl, OR 8 , NHC(O)R 9    
 wherein
 R 8  and R 9  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid; 
 
 L a  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl 
 R 16  and R 17  are independently selected polymeric arms; 
 X 2  and X 4  are independently selected linkage fragments joining polymeric moieties R 16  and R 17  to C; and 
 X 5  is a non-reactive group. 
 
     
     
         2 . The peptide conjugate according to  claim 1 , wherein the moiety: 
       
         
           
           
               
               
           
         
       
       has a formula that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 Q is selected from H and substituted or unsubstituted C 1 -C 6  alkyl; 
 e and f are integers independently selected from 1 to 2500; and 
 q is an integer from 0 to 20. 
 
     
     
         3 . The peptide conjugate according to  claim 2 , wherein said moiety has a formula that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 Q is selected from H and substituted or unsubstituted C 1 -C 6  alkyl; 
 e, f and f′ are integers independently selected from 1 to 2500; and 
 q and q′ are integers independently selected from 1 to 20. 
 
     
     
         4 . The peptide conjugate according to  claim 1 , wherein said glycosyl linking group has a formula selected from: 
       
         
           
           
               
               
           
         
       
     
     
         5 . The peptide conjugate according to  claim 4 , wherein said glycosyl linking group has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         6 . The peptide conjugate according to  claim 5 , wherein said glycosyl linking group attached to said amino acid residue has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 AA is said amino acid residue of said peptide. 
 
     
     
         7 . The peptide conjugate according to  claim 6 , wherein said amino acid residue is a member selected from serine or threonine. 
     
     
         8 . The peptide conjugate according to  claim 1 , wherein said peptide has the amino acid sequence of SEQ. ID. NO: 1. 
     
     
         9 . The peptide according to  claim 8 , wherein said amino acid residue is a serine at position 61 of SEQ. ID. NO:1. 
     
     
         10 . The peptide conjugate according to  claim 1 , wherein said peptide comprises at least one glycosyl linking group comprising a substructure having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 15  is said modified sialyl residue; and 
 p is an integer from 1 to 10. 
 
     
     
         11 . The peptide conjugate according to  claim 10 , wherein said at least one glycosyl linking group attached to an amino acid of said peptide has a formula selected from: 
       
         
           
           
               
               
           
         
       
       and combinations thereof 
       wherein
 AA is said amino acid residue of said peptide; 
 t is an integer equal to 0 or 1; 
 p is an integer from 1 to 10; and 
 R 15′  is a member selected from H, OH, sialic acid, said modified sialyl residue and Sia-Sia p    
 wherein
 Sia p  is said modified sialyl residue, 
 
 wherein at least one R 15′  is selected from said modified sialyl residue and Sia-Sia p . 
 
     
     
         12 . The peptide conjugate according to  claim 11 , wherein said amino acid residue is an asparagine residue. 
     
     
         13 . The peptide conjugate according to  claim 12 , wherein said peptide has the amino acid sequence of SEQ ID NO: 1. 
     
     
         14 . The peptide conjugate according to  claim 13 , wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof. 
     
     
         15 . A method of preparing a peptide conjugate according to  claim 1 , said method comprising:
 (a) contacting a substrate Factor IX peptide comprising a glycosyl moiety selected from:   
       
         
           
           
               
               
           
         
       
       with a PEG-sialic acid donor having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 c is 0 or 1; and 
 (b) contacting said Factor IX and said PEG-sialic acid donor with an enzyme that transfers PEG-sialic acid from said donor onto said glycosyl moiety, under conditions appropriate for said transfer. 
 
     
     
         16 . The method of  claim 15 , further comprising, prior to step (a):
 (b) expressing said substrate Factor IX peptide in a suitable host.   
     
     
         17 . The method of  claim 16 , wherein said host is selected from an insect cell and a mammalian cell. 
     
     
         18 . The method of  claim 17 , wherein said insect cell is a  Spodoptera frugiperda  cell line. 
     
     
         19 . A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal a peptide conjugate according to  claim 1  in an amount sufficient to stimulate said blood coagulation. 
     
     
         20 . A method of treating hemophilia in a subject, said method comprising administering to said subject a peptide conjugate according to  claim 1  in an amount effective to treat said hemophilia. 
     
     
         21 . A pharmaceutical formulation comprising the Factor IX peptide conjugate according to  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         22 . A Factor IX peptide conjugate comprising a glycosyl linking group attached to an amino acid residue of said peptide, said glycosyl linking group comprising a modified sialyl residue having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 2  is H, CH 2 OR 7 , COOR 7 , COO −  or OR 7    
 wherein
 R 7  represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; 
 
 R 3  and R 4  are members independently selected from H, substituted or unsubstituted alkyl, OR 8 , NHC(O)R 9    
 wherein
 R 8  and R 9  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid; 
 
 s is an integer from 1 to 20; 
 f is an integer from 1 to 2500; and 
 Q is a member selected from H and substituted or unsubstituted C 1 -C 6  alkyl. 
 
     
     
         23 . The peptide conjugate according to  claim 22 , wherein said modified sialyl residue has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         24 . The peptide conjugate according to  claim 23 , wherein Q is selected from H and CH 3 . 
     
     
         25 . The peptide conjugate according to  claim 22 , wherein said peptide comprises at least one glycosyl linking group comprising a substructure having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 15  is said modified sialyl residue; and 
 p is an integer from 1 to 10. 
 
     
     
         26 . The peptide conjugate according to  claim 25 , wherein said at least one glycosyl linking group attached to an amino acid of said peptide has a formula selected from: 
       
         
           
           
               
               
           
         
       
       and combinations thereof 
       wherein
 AA is said amino acid residue of said peptide; 
 t is an integer equal to 0 or 1; 
 p is an integer from 1 to 10; and 
 R 15′  is a member selected from H, OH, sialic acid, said modified sialyl residue and Sia-Sia p    
 wherein
 Sia p  is said modified sialyl residue, 
 wherein at least one R 15′  is selected from said modified sialyl residue and Sia-Sia p . 
 
 
     
     
         27 . The peptide conjugate according to  claim 22 , wherein said amino acid residue is asparagine. 
     
     
         28 . The peptide conjugate according to  claim 27 , wherein said peptide has the amino acid sequence of SEQ ID NO: 1. 
     
     
         29 . The peptide conjugate according to  claim 28 , wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof. 
     
     
         30 . The peptide conjugate according to  claim 22 , wherein said glycosyl linking group comprises the formula: 
       
         
           
           
               
               
           
         
       
       in which
 b is 0 or 1. 
 
     
     
         31 . The peptide conjugate according to  claim 30 , wherein s is 1; and f is an integer from about 200 to about 300. 
     
     
         32 . The peptide conjugate according to  claim 22 , wherein said amino acid residue is a member selected from serine or threonine. 
     
     
         33 . The peptide conjugate according to  claim 32 , wherein said peptide has the amino acid sequence of SEQ. ID. NO:1. 
     
     
         34 . The peptide conjugate according to  claim 33 , wherein said amino acid residue is a serine at position 61 of SEQ. ID. NO:1. 
     
     
         35 . A method of preparing the peptide conjugate according to  claim 22 , said method comprising:
 (a) contacting a substrate Factor IX peptide comprising a glycosyl moiety selected from:   
       
         
           
           
               
               
           
         
         with a PEG-sialic acid donor having the formula: 
       
       
         
           
           
               
               
           
         
       
       wherein
 c is 0 or 1; 
 r is 0 or 1; and 
 (b) contacting Factor IX and said PEG-sialic acid donor with an enzyme that transfers PEG-sialic acid from said donor onto said glycosyl moiety, under conditions appropriate for said transfer. 
 
     
     
         36 . The method of  claim 35 , further comprising, prior to step (a):
 (b) expressing said substrate Factor IX peptide in a suitable host.   
     
     
         37 . The method of  claim 36 , wherein said host an insect cell. 
     
     
         38 . The method of  claim 37 , wherein said insect cell is a  Spodoptera frugiperda  cell line. 
     
     
         39 . A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal a peptide conjugate according to  claim 22  in an amount sufficient to stimulate said blood coagulation. 
     
     
         40 . A method of treating hemophilia in a subject, said method comprising administering to said subject a peptide conjugate according to  claim 22  in an amount effective to treat said hemophilia. 
     
     
         41 . A pharmaceutical formulation comprising the Factor IX peptide conjugate according to  claim 22 , and a pharmaceutically acceptable carrier.