GLYCOPEGYLATION METHODS AND PROTEINS/PEPTIDES PRODUCED BY THE METHODS
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.
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.