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.

1 - 77 . (canceled) 
     
     
         78 . A peptide conjugate comprising a polymeric modifying group made by a cell-free, in vitro method of conjugating said polymeric modifying group to a precursor peptide, using a glycosyltransferase, said precursor 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; 
 
         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 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 peptide conjugate comprising said polymeric modifying group. 
       
     
     
         79 . The peptide conjugate of  claim 78 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         80 . The peptide conjugate of  claim 79 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         81 . The peptide conjugate of  claim 80 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         82 . The peptide conjugate of  claim 80 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         83 . The peptide conjugate of  claim 80 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         84 . The peptide conjugate of  claim 78 , wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI CST-II and combinations thereof. 
     
     
         85 . The peptide conjugate of  claim 78 , wherein,
 (c) said peptide conjugate is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said peptide conjugate, thereby transferring a sialic acid moiety onto said peptide conjugate.   
     
     
         86 . A pharmaceutical composition comprising the peptide conjugate of  claim 78 . 
     
     
         87 . A peptide conjugate comprising a polymeric modifying group made by a cell-free, in vitro method of conjugating said polymeric modifying group to a precursor peptide, using a glycosyltransferase, said precursor peptide having the formula: 
       
         
           
           
               
               
           
         
         wherein
 X 3 , X 4 , X 5 , X 6 , X 7 , and X 17 , are independently selected from monosaccharyl and oligosaccharyl residues; and 
 a, b, c, d, e, and x are independently selected from the integers 0, 1 and 2, with the proviso that at least one member selected from a, b, c, d, e and x is 1 or 2; 
 said method comprising: 
 (a) removing at least one of X 3 , X 4  X 5 , X 6 , X 7 , X 17 , 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 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 peptide conjugate comprising said polymeric modifying group. 
       
     
     
         88 . The peptide conjugate of  claim 87 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         89 . The peptide conjugate of  claim 88 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         90 . The peptide conjugate of  claim 89 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         91 . The peptide conjugate of  claim 89 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         92 . The peptide conjugate of  claim 89 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         93 . The peptide conjugate of  claim 87 , wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI CST-II and combinations thereof. 
     
     
         94 . The peptide conjugate of  claim 87 , wherein,
 (c) said peptide conjugate is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said peptide conjugate, thereby transferring a sialic acid moiety onto said peptide conjugate.   
     
     
         95 . A pharmaceutical composition comprising the peptide conjugate of  claim 87 . 
     
     
         96 . A peptide conjugate comprising a polymeric modifying group made by a cell-free, in vitro method of conjugating said polymeric modifying group to a precursor peptide, using a glycosyltransferase, said precursor peptide comprising a glycan having the formula: 
       
         
           
           
               
               
           
         
         wherein
 r, s, and t are integers independently selected from 0 and 1, 
 
         said method comprising: 
         (a) contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable to transfer a modified sugar moiety of said at least one modified sugar donor to said glycan, wherein said modified sugar moiety comprises at least one polymeric modifying group, 
         thereby forming said peptide conjugate comprising said polymeric modifying group. 
       
     
     
         97 . The peptide conjugate of  claim 96 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         98 . The peptide conjugate of  claim 97 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         99 . The peptide conjugate of  claim 98 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         100 . The peptide conjugate of  claim 98 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         101 . The peptide conjugate of  claim 98 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         102 . The peptide conjugate of  claim 96 , wherein said at least one glycosyltransferase is a member selected from the group consisting of GalT, ST3Gal3, CST-II and combinations thereof. 
     
     
         103 . The peptide conjugate of  claim 96 , wherein,
 (b) said peptide conjugate is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said peptide conjugate, thereby transferring a sialic acid moiety onto said peptide conjugate.   
     
     
         104 . A pharmaceutical composition comprising the peptide conjugate of  claim 96 . 
     
     
         105 . A peptide conjugate comprising a polymeric modifying group made by a cell-free, in vitro method of modifying a precursor peptide comprising said polymeric modifying group, using a glycosyltransferase, said precursor 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, 
 
         said method comprising: 
         (a) contacting said peptide with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable 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 peptide conjugate comprising said polymeric modifying group. 
       
     
     
         106 . The peptide conjugate of  claim 105 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         107 . The peptide conjugate of  claim 106 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         108 . The peptide conjugate of  claim 107 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         109 . The peptide conjugate of  claim 107 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         110 . The peptide conjugate of  claim 107 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         111 . The peptide conjugate of  claim 105 , wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI, CST-II and combinations thereof. 
     
     
         112 . The peptide conjugate of  claim 105 , wherein,
 (b) said peptide conjugate is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said peptide conjugate, thereby transferring a sialic acid moiety onto said peptide conjugate.   
     
     
         113 . A pharmaceutical composition comprising the peptide conjugate of  claim 105 . 
     
     
         114 . A covalent conjugate formed between a peptide and a polymeric modifying group, wherein said polymeric modifying group is covalently attached to said peptide at a first glycosyl or amino acid residue of said peptide via an first intact glycosyl linking group covalently attached to said glycosyl or amino acid residue by a glycosyltransferase. 
     
     
         115 . The covalent conjugate of  claim 114 , wherein said polymeric modifying group and an intact glycosyl linking group precursor are linked as a covalently attached unit to said peptide via the action of an enzyme, said enzyme converting said precursor to said intact glycosyl linking group, thereby forming said covalent conjugate. 
     
     
         116 . The covalent conjugate of  claim 115 , further comprising:
 a polymeric modifying group linked to a second glycosyl or amino acid residue of said peptide via a second intact glycosyl linking group covalently attached to said glycosyl or amino acid residue by a glycosyltransferase.   
     
     
         117 . The covalent conjugate of  claim 116 , wherein said first residue and said second residue are structurally identical. 
     
     
         118 . The covalent conjugate of  claim 116 , wherein said first residue and said second residue have different structures. 
     
     
         119 . The covalent conjugate of  claim 116  wherein said first residue and said second residue are glycosyl residues. 
     
     
         120 . The covalent conjugate of  claim 116 , wherein said first residue and said second residue are amino acid residues. 
     
     
         121 . The covalent conjugate of  claim 116 , wherein said peptide is modified to introduce an acceptor moiety for said intact glycosyl linking group prior to forming said covalent conjugate. 
     
     
         122 . The covalent conjugate of  claim 114 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         123 . The covalent conjugate of  claim 122 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         124 . The covalent conjugate of  claim 123 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         125 . The covalent conjugate of  claim 123 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         126 . The covalent conjugate of  claim 123 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         127 . The covalent conjugate of  claim 114 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof. 
     
     
         128 . The covalent conjugate of  claim 114 , wherein, said covalent conjugate is contacted 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. 
     
     
         129 . A pharmaceutical composition comprising a pharmaceutically acceptable diluent and a covalent conjugate between a polymeric modifying group and a glycosylated or non-glycosylated peptide, wherein said polymeric modifying group is conjugated to said peptide via an intact glycosyl linking group covalently attached to said peptide by a glycosyltransferase and interposed between and covalently linked to both said peptide and said polymeric modifying group. 
     
     
         130 . The pharmaceutical composition of  claim 129 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         131 . The pharmaceutical composition of  claim 130 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         132 . The pharmaceutical composition of  claim 131 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         133 . The pharmaceutical composition of  claim 131 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         134 . The pharmaceutical composition of  claim 131 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         135 . The pharmaceutical composition of  claim 129 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof. 
     
     
         136 . The pharmaceutical composition of  claim 129 , wherein, said covalent conjugate is contacted 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. 
     
     
         137 . A composition for forming a covalent conjugate between a peptide and a modified sugar, said composition comprising: an admixture of a modified sugar, a glycosyltransferase, and a peptide acceptor substrate, wherein said modified sugar has covalently attached thereto a polymeric modifying group. 
     
     
         138 . The composition of  claim 137 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         139 . The composition of  claim 138 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         140 . The composition of  claim 139 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         141 . The composition of  claim 139 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         142 . The composition of  claim 139 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         143 . The composition of  claim 137 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof. 
     
     
         144 . The composition of  claim 137 , wherein, said covalent conjugate is contacted 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. 
     
     
         145 . A peptide conjugate comprising one or more glycans, having a polymeric modifying group covalently attached to said peptide, wherein said polymeric modifying group is added to said one or more glycans using a glycosyltransferase. 
     
     
         146 . The peptide conjugate of  claim 145 , wherein said one or more glycans is a monoantennary glycan. 
     
     
         147 . The peptide conjugate of  claim 145 , wherein said one or more glycans is a biantennary glycan. 
     
     
         148 . The peptide conjugate of  claim 145 , wherein said one or more glycans is a triantennary glycan. 
     
     
         149 . The peptide conjugate of  claim 148 , wherein said one or more glycans is at least a triantennary glycan. 
     
     
         150 . The peptide conjugate of  claim 148 , wherein said one or more glycans comprises at least two glycans comprising a mixture of mono and multiantennary glycans. 
     
     
         151 . The peptide conjugate of  claim 148 , wherein said one or more glycans is selected from an N-linked glycan and an O-linked glycan. 
     
     
         152 . The peptide conjugate of  claim 148 , wherein said one or more glycans is at least two glycans selected from an N-linked and an O-linked glycan. 
     
     
         153 . The peptide conjugate of  claim 148 , wherein said peptide is expressed in a cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell. 
     
     
         154 . The peptide conjugate of  claim 153 , wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell and a yeast cell. 
     
     
         155 . The peptide conjugate of  claim 145 , wherein said polymeric modifying group is a water-soluble polymer. 
     
     
         156 . The peptide conjugate of  claim 155 , wherein said water-soluble polymer comprises poly(ethylene glycol). 
     
     
         157 . The peptide conjugate of  claim 156 , wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol). 
     
     
         158 . The peptide conjugate of  claim 156 , wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol). 
     
     
         159 . The peptide conjugate of  claim 156 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. 
     
     
         160 . The peptide conjugate of  claim 145 , wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof. 
     
     
         161 . The peptide conjugate of  claim 145 , wherein, said peptide conjugate is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said peptide conjugate, thereby transferring a sialic acid moiety onto said peptide conjugate.