Glycoconjugation 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.

1 - 88 . (canceled) 
     
     
         89 . An O-linked covalent conjugate of a peptide, said peptide having the formula: 
       
         
           
           
               
               
           
         
         wherein
 AA is a terminal or internal amino acid residue of said peptide; 
 X 1 -X 2  is a saccharide covalently linked to said AA, wherein 
 X 1  is a first glycosyl residue; and 
 X 2  is a second glycosyl residue covalently linked to X 1 , wherein X 1  and X 2  are selected from monosaccharyl and oligosaccharyl residues; 
 
       
       wherein said covalent conjugate is obtained by a cell-free, in vitro method, said method comprising:
 (a) removing X 2  or a saccharyl subunit thereof from said peptide, thereby forming a truncated glycan; and 
 (b) contacting said truncated glycan with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said truncated glycan, wherein said modified sugar moiety comprises at least one polymeric modifying group, thereby forming said covalent conjugate of said peptide. 
 
     
     
         90 . The covalent conjugate of  claim 89 , wherein said amino acid residue of said peptide is a member selected from serine (Ser) and threonine (Thr). 
     
     
         91 . The covalent conjugate of  claim 89 , wherein said method further comprises:
 (b) prior to step (a), removing a group added to said saccharide during post-translational modification.   
     
     
         92 . The covalent conjugate of  claim 91 , wherein said group is a member selected from phosphate, sulfate, carboxylate and esters thereof. 
     
     
         93 . The covalent conjugate of  claim 89 , wherein said peptide has the formula: 
       
         
           
           
               
               
           
         
         wherein
 X 9  and X 10  are independently selected from monosaccharyl and oligosaccharyl residues; and 
 
         m, n and f are integers selected from 0 and 1. 
       
     
     
         94 . The covalent conjugate of  claim 89 , wherein said peptide has the formula: 
       
         
           
           
               
               
           
         
         wherein
 X 13 , X 14 , and X 15  are independently selected glycosyl residues; and 
 g, h, i, j, k, and p are independently selected from the integers 0 and 1, with the proviso that at least one of g, h, i, j, k and p is 1. 
 
       
     
     
         95 . The covalent conjugate of  claim 94 ,
 wherein
 X 14  and X 15  are members independently selected from GlcNAc and Sia; and 
 i and k are independently selected from the integers 0 and 1, with the proviso that at least one of i and k is 1, and if k is 1, g, h, and j are 0. 
   
     
     
         96 . The covalent conjugate of  claim 89 , wherein said peptide has the formula: 
       
         
           
           
               
               
           
         
         wherein
 X 16  is a member selected from: 
 
       
       
         
           
           
               
               
           
         
         wherein
 s, u and i are independently selected from the integers 0 and 1. 
 
       
     
     
         97 . The covalent conjugate of  claim 89 , wherein said peptide has the formula: 
       
         
           
           
               
               
           
         
         wherein
 X 11  and X 12  are independently selected glycosyl moieties; and 
 r and x are integers independently selected from 0 and 1. 
 
       
     
     
         98 . The covalent conjugate of  claim 97 , wherein X 11  and X 12  are (mannose) q , wherein q is an integer selected from 1 to 20, and when q is three or greater, (mannose) q  is selected from linear and branched structures. 
     
     
         99 . The covalent conjugate of  claim 98  having a formula, which is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 R is a polymeric modifying group; and 
 a1, a2, a3, a4 and a5 are integers selected from 0 and 1 with the proviso that
 (i) when a2 is 1, then a1 is also 1; 
 (ii) when a5 is 1 and a4 is 0, then a1 is 1. 
 
 
     
     
         100 . The covalent conjugate of  claim 99  having a formula, which is a member selected from: 
       
         
           
           
               
               
           
         
       
     
     
         101 . The covalent conjugate of  claim 89 , wherein said modified sugar donor is a nucleotide sugar selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside. 
     
     
         102 . The covalent conjugate of  claim 101 , wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid and CMP-NeuAc. 
     
     
         103 . An O-linked covalent conjugate of a peptide, said peptide having the formula: 
       
         
           
           
               
               
           
         
         wherein
 AA is a terminal or internal amino acid residue of said peptide; 
 X 1  is a glycosyl residue covalently linked to said AA, selected from monosaccharyl and oligosaccharyl residues; and 
 u is an integer selected from 0 and 1, 
 
       
       wherein said covalent conjugate is obtained by a cell-free, in vitro method comprising:
 (a) contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said peptide, wherein said modified sugar moiety comprises at least one polymeric modifying group, 
 thereby forming said covalent conjugate of said peptide. 
 
     
     
         104 . The covalent conjugate of  claim 103 , wherein said amino acid residue of said peptide is a member selected from serine (Ser) and threonine (Thr). 
     
     
         105 . The covalent conjugate of  claim 103 , wherein said method further comprises following said forming said covalent conjugate:
 (b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.   
     
     
         106 . The covalent conjugate of  claim 103 , wherein said modified sugar donor is a nucleotide sugar selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside. 
     
     
         107 . The covalent conjugate of  claim 106 , wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid and CMP-NeuAc. 
     
     
         108 . The covalent conjugate of  claim 103 , wherein X 1  is a member selected from GalNAc, GalNAc-Gal, GalNAc-Gal-Sia, GlcNAc and GlcNAc-Gal. 
     
     
         109 . The covalent conjugate of  claim 103  having a formula, which is a member selected from: 
       
         
           
           
               
               
           
         
         wherein
 n is an integer selected from 0 to 6; 
 m is an integer selected from 0 and 1; and 
 R is a polymeric modifying group. 
 
       
     
     
         110 . An O-linked covalent conjugate of a peptide, said conjugate having the formula: 
       
         
           
           
               
               
           
         
         wherein
 AA is a terminal or internal amino acid residue of said peptide; 
 X 1  is a glycosyl residue covalently linked to said AA, selected from monosaccharyl and oligosaccharyl residues; 
 X 3  is a glycosyl linking group; 
 R is a polymeric modifying group; and 
 u is an integer selected from 0 and 1, 
 
       
       wherein said covalent conjugate is obtained by a cell-free, in vitro method comprising:
 (a) contacting said peptide having the formula: 
 
       
         
           
           
               
               
           
         
         
           with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said at least one modified sugar donor to said peptide, wherein said modified sugar moiety comprises at least one polymeric modifying group, 
           thereby forming said covalent conjugate of said peptide. 
         
       
     
     
         111 . The covalent conjugate of  claim 110 , wherein said amino acid residue of said peptide is a member selected from serine (Ser) and threonine (Thr). 
     
     
         112 . The covalent conjugate of  claim 110 , wherein said glycosyl linking group is a sialic acid moiety. 
     
     
         113 . The covalent conjugate of  claim 110 , wherein said modified sugar donor is a nucleotide sugar selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside. 
     
     
         114 . The covalent conjugate of  claim 113 , wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid and CMP-NeuAc. 
     
     
         115 . An O-linked covalent conjugate between a polymeric modifying group and a glycosylated peptide or a non-glycosylated peptide, wherein said polymeric modifying group is conjugated to said peptide via a glycosyl linking group interposed between and covalently linked to both said peptide and said polymeric modifying group,
 wherein said covalent conjugate is obtained by a cell-free, in vitro method comprising:
 (a) contacting said peptide with a mixture comprising a nucleotide sugar covalently linked to said polymeric modifying group, and a glycosyltransferase for which said nucleotide sugar is a substrate under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said nucleotide sugar to said peptide, wherein said modified sugar moiety comprises at least one polymeric modifying group, 
 thereby forming said covalent conjugate. 
   
     
     
         116 . The covalent conjugate of  claim 115 , wherein said glycosylated peptide is partially deglycosylated prior to said contacting. 
     
     
         117 . The covalent conjugate of  claim 115 , wherein said glycosyl linking group is a sialic acid moiety. 
     
     
         118 . The covalent conjugate of  claim 115 , wherein said nucleotide sugar is selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside. 
     
     
         119 . The covalent conjugate of  claim 118 , wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid and CMP-NeuAc. 
     
     
         120 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         121 . The covalent conjugate of  claim 120 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         122 . The covalent conjugate of  claim 121 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         123 . The covalent conjugate of  claim 121 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         124 . The covalent conjugate of  claim 121 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         125 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said peptide is selected from the group consisting of bone morphogenetic protein (BMP), neurotrophin-3 (NT-3), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-alpha, interferon-alpha-2b, interferon-beta, interferon-gamma, α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), interleukin-1 (IL-1), interleukin-2 (IL-2), chimeric IL-2/diphtheria toxin, tumor necrosis factor (TNF) alpha, TNF receptor, TNF receptor-IgG Fc region fusion protein, urokinase, human DNase, insulin, leptin, hirudin, hepatitis B surface antigen (HbsAg), human growth hormone (hGH), human chorionic gonadotropin (hCG), alpha-galactosidase A, alpha-L-iduronidase, alpha-glucosidase, beta-II glucosidase, anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-I (GLP-1), fibroblast growth factor, Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor XIII, prokinetisin, extendin-4, CD4, PSGL-1, monoclonal antibody to RSV, monoclonal antibody to IL-2 receptor, monoclonal antibody to CEA, monoclonal antibody to EGF receptor, monoclonal antibody to CD3, monoclonal antibody to CD4, monoclonal antibody to HER2, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF, and mutants thereof. 
     
     
         126 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said peptide is a therapeutic agent. 
     
     
         127 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said glycosyltransferase is a member selected from sialyltransferases, galactosyltransferases, glucosyltransferases, GalNAc transferases, GlcNAc transferases, fucosyltransferases, xylosyltransferases and mannosyltransferases and combinations thereof. 
     
     
         128 . The covalent conjugate of  claim 127 , wherein said glycosyltransferase is a member selected from GalNAc-T2, GalT1, CST-II, ST3Gal-I, ST3Gal-III, GnT-I, GnT-II and combinations thereof. 
     
     
         129 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said glycosyltransferase is recombinantly produced. 
     
     
         130 . The covalent conjugate of  claim 129 , wherein said glycosyltransferase is a recombinant prokaryotic enzyme. 
     
     
         131 . The covalent conjugate of  claim 129 , wherein said glycosyltransferase is a recombinant eukaryotic enzyme. 
     
     
         132 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said glycosyltransferase is selected from ST3Gal1 and ST6GalNAcI and said modified sugar donor comprises a sialic acid moiety. 
     
     
         133 . The covalent conjugate of  claim 132 , wherein said modified sugar donor has the formula: 
       
         
           
           
               
               
           
         
         wherein
 R is a polymeric modifying group. 
 
       
     
     
         134 . The covalent conjugate of  claim 89 ,  103 ,  110  or  115 , wherein said covalent conjugate is isolated. 
     
     
         135 . The covalent conjugate of  claim 134 , wherein said covalent conjugate is isolated by membrane filtration. 
     
     
         136 . A pharmaceutical composition comprising a covalent conjugate of  claim 89 ,  103 ,  110  or  115 , and a pharmaceutically acceptable diluent.