Interleukin-2:remodeling and glycoconjugation of interleukin-2
AssigneeNeose Technologies, Inc.
InventorsShawn DeFrees, David Zopf, Robert Bayer, Caryn Bowe, David Hakes, Xi Chen
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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.
Claims111
A cell-free, in vitro method of remodeling an interleukin-2 (IL-2) peptide, said peptide having the formula:
- wherein
- AA is a terminal or internal amino acid residue of said peptide;
- X1-X2 is a saccharide covalently linked to said AA, wherein
- X1 is a first glycosyl residue;
- and
- X2 is a second glycosyl residue covalently linked to X1, wherein X1 and X2 are selected from monosaccharyl and oligosaccharyl residues;
- said method comprising:
- (a) removing X2 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 glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said truncated glycan, thereby remodeling said IL-2 peptide.
A cell-free in vitro method of remodeling an IL-2 peptide, said peptide having the formula:
- wherein
- X3, X4, X5, X6, X7, and X17 are independently selected monosaccharyl or 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, and e and x are 1 or 2;
- said method comprising:
- (a) removing at least one of X3, X4, X5, X6, X7, or X17, 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 glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said truncated glycan, thereby remodeling said IL-2 peptide.
A cell-free in vitro method of remodeling an IL-2 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 glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said glycan, thereby remodeling said IL-2 peptide.
A cell-free, in vitro method of remodeling an IL-2 peptide having the formula:
- wherein
- AA is a terminal or internal amino acid residue of said peptide;
- X1 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:
- contacting said peptide with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said truncated glycan, wherein said glycosyl donor comprises a modifying group, thereby remodeling said IL-2 peptide.
A covalent conjugate between a IL-2 peptide and a modifying group that alters a property of said peptide, wherein said modifying group is covalently attached to said peptide at a preselected glycosyl or amino acid residue of said peptide via an intact glycosyl linking group.
A method of forming a covalent conjugate between a polymer and a glycosylated or non-glycosylated peptide, wherein said polymer is conjugated to said peptide via an intact glycosyl linking group interposed between and covalently linked to both said peptide and said polymer, said method comprising:
- contacting said peptide with a mixture comprising a nucleotide sugar covalently linked to said polymer and a glycosyltransferase for which said nucleotide sugar is a substrate under conditions sufficient to form said conjugate, wherein said peptide is IL-2.
A composition for forming a 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 member selected from a polymer, a therapeutic moiety and a biomolecule, wherein said peptide is IL-2.
A cell-free, in vitro method of remodeling a peptide having the formula:
- wherein
- AA is a terminal or internal amino acid residue of said peptide, said method comprising:
- contacting said peptide with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said amino acid residue, wherein said glycosyl donor comprises a modifying group, thereby remodeling said peptide, wherein said peptide is IL-2.
A method of forming a conjugate between a IL-2 peptide and a modifying group, wherein said modifying group is covalently attached to said IL-2 peptide through an intact glycosyl linking group, said IL-2 peptide comprising a glycosyl residue having a formula which is a member selected from:
- wherein
- a, b, c, and e are members independently selected from 0 and 1;
- d is 0;
- and
- R is a modifying group,
- said method comprising:
- (a) contacting said IL-2 peptide with a glycosyltransferase and a modified glycosyl donor, comprising a glycosyl moiety which is a substrate for said glycosyltransferase covalently linked to said modifying group, under conditions appropriate for the formation of said intact glycosyl linking group.
An IL-2 peptide comprising one or more glycans, having a glycoconjugate molecule covalently attached to said peptide.
A method of stimulating cytotoxic T cells in a mammal, said method comprising administering to said mammal an IL-2 peptide having one or more glycans having a glycoconjugate molecule attached to said peptide.
Dependencies are inferred from the claim text.