Glycopegylation methods and proteins/peptides produced by the methods

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.

What is claimed is: 
     
         1 . A covalent conjugate between a peptide and a water-soluble polymer, wherein the water-soluble polymer is not a naturally occurring sugar and is covalently attached to the peptide through an intact glycosyl linking group. 
     
     
         2 . The covalent conjugate according to  claim 1 , wherein the water-soluble polymer is a poly(ether) 
     
     
         3 . The covalent conjugate according to  claim 2 , wherein the poly(ether) is a poly(alkylene oxide). 
     
     
         4 . The covalent conjugate according to  claim 3 , wherein the poly(alkylene oxide) is a poly(ethylene glycol). 
     
     
         5 . The covalent conjugate according to  claim 4 , wherein the poly(ethylene glycol) is a linear poly(ethylene glycol) or a branched poly(ethylene glycol). 
     
     
         6 . The covalent conjugate according to  claim 4 , wherein the poly(ethylene glycol) has a degree of polymerisation of from about 1 to about 5,000. 
     
     
         7 . The covalent conjugate according to  claim 6 , wherein the poly(ethylene glycol) has a degree of polymerisation of from about 1 to about 1,000. 
     
     
         8 . The covalent conjugate according to  claim 1 , wherein the intact glycosyl linking group is attached to a member selected from the group consisting of a carbohydrate moiety, an amino acid moiety, and combinations thereof. 
     
     
         9 . The covalent conjugate according to  claim 8 , wherein the intact glycosyl linking group is attached to the carbohydrate moiety, which is a member selected from the group consisting of an O-linked glycan, an N-linked glycan, and combinations thereof. 
     
     
         10 . The covalent conjugate according to  claim 8 , wherein the intact glycosyl linking group is attached to hydroxyl or amino groups or combinations thereof of the amino acid moiety. 
     
     
         11 . The covalent conjugate according to  claim 8 , wherein the intact glycosyl linking group comprises a sialic acid, galactose, N-acetylglucosamine, or N-acetylgalactosamine residue. 
     
     
         12 . The covalent conjugate according to  claim 1 , wherein the water-soluble polymer is attached to a sialic acid residue at the 5- or the 9-position of the sialic acid. 
     
     
         13 . The covalent conjugate according to  claim 1 , wherein the peptide is a therapeutic agent. 
     
     
         14 . The covalent conjugate according to  claim 1 , wherein the peptide is selected from the group consisting of granulocyte colony stimulating factor, interferon-alpha, interferon-beta, Factor VIIa, Factor VIII, Factor IX, follicle stimulating hormone, erythropoietin, granulocyte macrophage colony stimulating factor, interferon-gamma, alpha-1-protease inhibitor, beta-glucosidase, tissue plasminogen activator protein, interleukin-2, chimeric tumor necrosis factor receptor, urokinase, chimeric anti-glycoprotein IIb/IIIa antibody, chimeric anti-HER2 antibody, chimeric anti-respiratory syncytial virus antibody, chimeric anti-CD20 antibody, DNase, chimeric anti-tumor necrosis factor antibody, human insulin, hepatitis B sAg, and human growth hormone. 
     
     
         15 . A pharmaceutical composition comprising the covalent conjugate according to  claim 1  and a pharmaceutically acceptable diluent. 
     
     
         16 . A cell-free, in vitro method of forming a covalent conjugate between a peptide and a water-soluble polymer, wherein the water-soluble polymer is covalently attached to the peptide through an intact glycosyl linking group, the method comprising:
 contacting the peptide with a mixture comprising at least one nucleotide sugar covalently linked to the water soluble polymer and at least one glycosyltransferase for which the nucleotide sugar is a substrate, thereby forming the covalent conjugate of the peptide.   
     
     
         17 . The method according to  claim 16 , wherein the water-soluble polymer is a poly(ether). 
     
     
         18 . The method according to  claim 17 , wherein the poly(ether) is a poly(ethylene glycol). 
     
     
         19 . The method of  claim 16 , wherein the glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, and mannosyltransferase. 
     
     
         20 . The method of  claim 16 , wherein the peptide is selected from the group consisting of granulocyte colony stimulating factor, interferon-alpha, interferon-beta, Factor VIIa, Factor VIII, Factor IX, follicle stimulating hormone, erythropoietin, granulocyte macrophage colony stimulating factor, interferon-gamma, alpha-1-protease inhibitor, beta-glucosidase, tissue plasminogen activator protein, interleukin-2, chimeric tumor necrosis factor receptor, urokinase, chimeric anti-glycoprotein IIb/IIIa antibody, chimeric anti-HER2 antibody, chimeric anti-respiratory syncytial virus antibody, chimeric anti-CD20 antibody, DNase, chimeric anti-tumor necrosis factor antibody, human insulin, hepatitis B sAg, and human growth hormone.