Glycopegylated erythropoietin

Abstract

The present invention provides conjugates between erythropoietin 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 - 34 . (canceled) 
     
     
         35 . An erythropoietin peptide conjugate comprising a sialic acid moiety covalently linked to an amino acid residue of said peptide, said sialic acid moiety comprising 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 from a linear poly(ethylene glycol) residue and a 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—. 
 
     
     
         36 . The peptide conjugate according to  claim 35 , wherein L-R 1  has the formula: 
       
         
           
           
               
               
           
         
         wherein a is an integer from 0 to 20. 
       
     
     
         37 . The peptide conjugate according to  claim 35 , wherein said branched poly(ethylene glycol) residue has a formula 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. 
 
     
     
         38 . The peptide conjugate according to  claim 35 , wherein said branched poly(ethylene glycol) residue has a formula 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. 
 
     
     
         39 . The peptide conjugate according to  claim 35 , wherein said branched poly(ethylene glycol) residue has a formula 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. 
 
     
     
         40 . The peptide conjugate according to  claim 35 , wherein said linear poly(ethylene glycol) residue has a formula that is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 e and f are integers independently selected from 1 to 2500. 
 
     
     
         41 . The peptide conjugate according to  claim 35 , wherein said peptide has an amino acid sequence comprising SEQ ID NO:1. 
     
     
         42 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety is covalently linked to an asparagine residue of said peptide. 
     
     
         43 . The peptide conjugate according to  claim 42 , wherein said peptide has an amino acid sequence of SEQ ID NO:1 and said asparagine residue is a member selected from N24, N38, N83 and combinations thereof. 
     
     
         44 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety is covalently linked to a serine residue or a threonine residue of said peptide. 
     
     
         45 . The peptide conjugate according to  claim 44 , wherein said peptide has an amino acid sequence of SEQ ID NO:1 and said serine residue is S126. 
     
     
         46 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety comprises the formula: 
       
         
           
           
               
               
           
         
       
     
     
         47 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety comprises the formula: 
       
         
           
           
               
               
           
         
       
     
     
         48 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety has the formula: 
       
         
           
           
               
               
           
         
       
       wherein AA is said amino acid residue of said peptide. 
     
     
         49 . The peptide conjugate according to  claim 35 , wherein said sialic, acid moiety has a formula selected from: 
       
         
           
           
               
               
           
         
       
       wherein AA is said amino acid residue of said peptide; and t is an integer selected from 0 and 1. 
     
     
         50 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety has the formula: 
       
         
           
           
               
               
           
         
       
       wherein AA is said amino acid residue of said peptide; and t is an integer selected from 0 and 1. 
     
     
         51 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety has a formula selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein AA is said amino acid residue of said peptide; and t is an integer equal to 0 or 1. 
       
     
     
         52 . The peptide conjugate according to  claim 35 , wherein said sialic acid moiety has a formula selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein AA is said amino acid residue of said peptide; and t is an integer equal to 0 or 1. 
       
     
     
         53 . The peptide conjugate according to  claim 35 , wherein said peptide is a bioactive erythropoietin peptide. 
     
     
         54 . The peptide conjugate according to  claim 53 , wherein said peptide is erythropoietically active. 
     
     
         55 . The peptide conjugate according to  claim 53 , wherein said peptide is essentially non-erythropoietically active. 
     
     
         56 . The peptide conjugate according to  claim 55 , wherein said peptide is tissue protective. 
     
     
         57 . The peptide conjugate according to  claim 35 , wherein said poly(ethylene glycol) residue has a molecular weight that is essentially homodisperse. 
     
     
         58 . The peptide conjugate of  claim 35 , wherein said poly(ethylene glycol) residue comprises monomethoxy-poly(ethylene glycol). 
     
     
         59 . The peptide conjugate of  claim 35 , wherein said sialic acid moiety is conjugated to a precursor erythropoietin peptide by action of a sialyltransferase. 
     
     
         60 . The peptide conjugate according to  claim 59 , wherein said sialyltransferase is CST-II. 
     
     
         61 . A pharmaceutical formulation comprising a peptide conjugate according to  claim 35 , and a pharmaceutically acceptable carrier. 
     
     
         62 . A method of making a PEG-ylated erythropoietin peptide 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 linear or branched poly(ethylene glycol) residue; and 
 L is a linker, which is a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, 
 
       said method comprising:
 (a) contacting a substrate erythropoietin peptide comprising the glycosyl moiety: 
 
       
         
           
           
               
               
           
         
         with a PEG-sialic acid donor moiety having the formula: 
       
       
         
           
           
               
               
           
         
       
       and an enzyme under conditions sufficient for said enzyme to transfer said PEG-sialic acid onto said Gal of said glycosyl moiety. 
     
     
         63 . The method of  claim 62 , further comprising, prior to step (a):
 (b) expressing said substrate erythropoietin peptide in a host cell.   
     
     
         64 . The method of  claim 63 , wherein said host cell is a member selected from an insect cell and a mammalian cell. 
     
     
         65 . The method of  claim 64 , wherein said insect cell is a  Spodoptera frugiperda  cell. 
     
     
         66 . The method of  claim 64 , wherein said mammalian cell is a CHO cell. 
     
     
         67 . The method according to  claim 62 , wherein said poly(ethylene glycol) residue has a molecular weight that is essentially homodisperse. 
     
     
         68 . The method according to  claim 62 , wherein said poly(ethylene glycol) residue comprises monomethoxy-poly(ethylene glycol). 
     
     
         69 . A method of enhancing red blood cell production in a mammal, said method comprising administering to said mammal a peptide conjugate according to  claim 35 . 
     
     
         70 . A method of treating a tissue injury in a subject in need thereof, said injury resulting from ischemia, trauma, inflammation or contact with a toxic substance, said method comprising the step of administering to said subject an amount of a peptide conjugate according to  claim 35 , effective to ameliorate damage associated with said tissue injury in said subject. 
     
     
         71 . A method of treating anemia in a subject in need thereof, said method comprising administering a peptide conjugate according to  claim 35  to said subject. 
     
     
         72 . The method according to  claim 71 , wherein said anemia is age related anemia, early anemia of prematurity or anemia associated with a member selected from chronic renal failure, cancer chemotherapy treatment, anti-HIV drug treatment, sickle cell disease, beta-thalassemia, cystic fibrosis, pregnancy, menstrual disorders, spinal cord injury, space flight and acute blood loss.