Glycopegylated Factor Ix

Abstract

The present invention provides conjugates between Factor Ix and PEG moieties. The conjugates are linked via a glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. 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 Factor IX peptide sequence and at least one conjugated moiety 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  is a moiety comprising a member selected a straight-chain or branched poly(ethylene glycol) residue; and 
 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 peptide having a recovery value that is at least 15% greater 24 hours after in vivo administration than said peptide sequence without said conjugated moiety. 
 
     
     
         2 . The Factor IX peptide according to  claim 1 , wherein L-R 1  has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 a is an integer from 0 to 20. 
 
     
     
         3 . The Factor IX peptide according to  claim 1 , wherein R 1  has a structure that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 e and f are integers independently selected from 1 to 2500; and 
 q is an integer from 0 to 20. 
 
     
     
         4 . The Factor IX peptide according to  claim 1 , 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. 
 
     
     
         5 . The Factor IX peptide according to  claim 1 , 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, q′ and q″ are integers independently selected from 1 to 20. 
 
     
     
         6 . The Factor IX peptide according to  claim 1  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 Factor IX peptide according to  claim 1 , wherein said moiety has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The Factor IX peptide according to  claim 1 , wherein said moiety has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The Factor IX peptide according to  claim 1 , wherein said moiety has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 AA is an amino acid residue of said peptide. 
 
     
     
         10 . The Factor IX peptide according to  claim 9 , wherein said amino acid residue is a member selected from serine or threonine. 
     
     
         11 . The Factor IX peptide according to  claim 1 , wherein said peptide has the amino acid sequence of SEQ. ID. NO:1. 
     
     
         12 . The Factor IX peptide according to  claim 11 , wherein said amino acid residue is serine at position 61 of SEQ. ID. NO:1. 
     
     
         13 . The Factor IX peptide according to  claim 1 , wherein said moiety has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 a, b, c, d, i, r, s, t, and u are integers independently selected from 0 and 1; 
 q is 1; 
 e, f, g, and h are members independently selected from the integers from 0 to 6; 
 j, k, l, and m are members independently selected from the integers from 0 and 100; 
 v, w, x, and y are independently selected from 0 and 1, and least one of v, w, x and y is 1; 
 AA is an amino acid residue of said Factor IX peptide; 
 Sia-(R) has 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  is a moiety comprising a member selected a straight-chain or branched poly(ethylene glycol) residue; and 
 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—. 
 
       
     
     
         14 . The Factor IX peptide according to  claim 7 , wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof. 
     
     
         15 . A pharmaceutical formulation comprising the Factor IX according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         16 . A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal said Factor IX peptide according to  claim 1 . 
     
     
         17 . A method of treating hemophilia in a subject, said method comprising administering to said subject said Factor IX peptide according to  claim 1 . 
     
     
         18 . A method of making a Factor IX peptide conjugate comprising the moiety: 
       
         
           
           
               
               
           
         
       
       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  is a moiety comprising a member selected a straight-chain or branched poly(ethylene glycol) residue; and 
 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 substrate Factor IX peptide with a PEG-sialic acid donor moiety having the formula: 
 
       
         
           
           
               
               
           
         
         
           and an enzyme that transfers said PEG-sialic acid onto an amino acid or glycosyl residue of said Factor IX peptide, under conditions appropriate for the transfer. 
         
       
     
     
         19 . The method according to  claim 18 , wherein L-R 1  has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 a is an integer from 0 to 20. 
 
     
     
         20 . The method according to  claim 18 , wherein R 1  has a structure that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 e and f are integers independently selected from 1 to 2500; and 
 q is an integer from 0 to 20. 
 
     
     
         21 . The method according to  claim 18 , 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. 
 
     
     
         22 . The method according to  claim 18 , 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, q′ and q″ are integers independently selected from 1 to 20. 
 
     
     
         23 . The method according to  claim 18  wherein R 1  has a structure that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 e and f are integers independently selected from 1 to 2500. 
 
     
     
         24 . The method according to  claim 18 , wherein said Factor IX peptide conjugate comprises a moiety having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         25 . The method according to  claim 18 , wherein said Factor IX peptide conjugate comprises a moiety having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         26 . The method according to  claim 18 , wherein said factor IX peptide conjugate comprises a moiety having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 AA is an amino acid residue of said Factor IX peptide. 
 
     
     
         27 . The method according to  claim 26 , wherein said amino acid residue is a member selected from serine or threonine. 
     
     
         28 . The method according to  claim 18 , wherein said factor IX substrate peptide has the amino acid sequence of SEQ. ID. NO:1. 
     
     
         29 . The Factor IX peptide according to  claim 28 , wherein said amino acid residue is serine at position 61 of SEQ. ID. NO:1. 
     
     
         30 . The method according to  claim 18 , wherein said Factor IX conjugate comprises a glycosyl residue having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 a, b, c, d, i, r, s, t, and u are integers independently selected from 0 and 1; 
 q is 1; 
 e, f, g, and h are members independently selected from the integers from 0 to 6; 
 j, k, l, and m are members independently selected from the integers from 0 and 100; 
 v, w, x, and y are independently selected from 0 and 1, and at least one of v, w, x, and y is 1; 
 AA is an amino acid residue of said Factor IX peptide; 
 Sia-(R) has 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  is a moiety comprising a member selected a straight-chain or branched poly(ethylene glycol) residue; and 
 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—. 
 
       
     
     
         31 . The method according to  claim 30 , wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof. 
     
     
         32 . The method of  claim 18 , further comprising, prior to step (a):
 (b) expressing said substrate Factor IX peptide in a suitable host cell.   
     
     
         33 . The method of  claim 32 , wherein said host is selected from an insect cell and a mammalian cell. 
     
     
         34 . A Factor IX peptide conjugate comprising a water-soluble polymer conjugated to a Factor IX peptide sequence, said peptide having a recovery at least about 15% greater 24 hours after in vivo administration than said peptide conjugate without said water-soluble polymer conjugated thereto. 
     
     
         35 . A Factor IX peptide conjugate comprising a Factor IX peptide sequence and a conjugated 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. 
 
     
     
         36 . The Factor IX peptide conjugate according to  claim 35  wherein said peptide conjugate has a recovery at least about 15% greater 24 hours after in vivo administration than said peptide conjugate without said conjugated glycosyl linking group conjugated thereto.