Factor VII: remodeling and glycoconjugation of Factor VII
AssigneeNovo Nordisk A/S
InventorsShawn DeFrees, David Zopf, Robert Bayer, Caryn Bowe, David Hakes, Xi Chen
Life of this patent
Estimating the term…
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.
Claims87
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa 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 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 a urethane linker attached to at least one modifying group which is a poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said 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:
- (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 a urethane linker attached to at least one poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
- wherein
- a, b, 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 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 a urethane linker attached to at least one poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa 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 X1 and X2, exposing said AA;
- and
- (b) 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 a urethane linker attached to at least one poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
- wherein
- X3, X4, X5, X6, X7, and X17 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, and e and x is 1 or 2;
- said method comprising:
- (a) removing at least one of X3, X4, X5, X6, X7, or X17, 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, 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 a urethane linker attached to at least one modifying group which is a poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate between a poly(ethylene) glycol and a glycosylated or non-glycosylated Factor VIIa peptide, wherein said poly(ethylene) glycol is conjugated to said Factor VIIa peptide via a glycosyl linking group interposed between and covalently linked to both said Factor VIIa peptide and said poly(ethylene) glycol, said method comprising:
- (a) contacting said Factor VIIa peptide with a mixture comprising a nucleotide sugar having a urethane linker attached to said poly(ethylene) glycol, 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 onto said Factor VIIa peptide, wherein said modified sugar moiety comprises a urethane linker attached to at least one poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
- wherein
- AA is a terminal or internal amino acid residue of said peptide,
- said 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 amino acid residue, wherein said modified sugar moiety comprises a urethane linker attached to at least one poly(ethylene) glycol,
- thereby forming said covalent conjugate of said Factor VIIa peptide.
A method of forming a covalent conjugate between a Factor VIIa peptide and a poly(ethylene) glycol, wherein said poly(ethylene) glycol is covalently attached to said Factor VIIa peptide through an intact glycosyl linking group, said Factor VIIa peptide comprising a glycosyl residue having a formula which is a member selected from:
- wherein
- a, b, c, d, i, o, p, q, r, s, t, and u, are members independently selected from 0 and 1;
- e, f, g, h and n are members independently selected from the integers from 0 to 6;
- j, k, l and m are members independently selected from the integers from 0 to 20;
- v, w, x and y are 0;
- and
- R is a member selected from poly(ethylene) glycol, a mannose, an oligomannose, SialylLewisx and SialylLewisa;
- said method comprising:
- (a) contacting said Factor VIIa 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 Factor VIIa peptide, wherein said modified sugar moiety comprises a urethane linker attached to at least one poly(ethylene) glycol, such that, following said contacting, at least one of v, w, x or y is 1,
- thereby forming said covalent conjugate.
Dependencies are inferred from the claim text.