Factor VIII: remodeling and glycoconjugation of factor VIII
AssigneeNovo Nordisk A/S
InventorsShawn DeFrees, David A. Zopf, Robert J. Bayer, Caryn Bowe, David James 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.
Claims110
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII 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;
- wherein said covalent conjugate is prepared by a cell-free in vitro 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 at least one modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII 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,
- wherein said covalent conjugate is prepared 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 modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII peptide, said peptide having the formula:
- wherein
- r, s, and t are integers independently selected from 0 and 1,
- wherein said covalent conjugate is prepared 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 modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII 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;
- wherein said covalent conjugate is prepared by a cell-free in vitro 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 at least one modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII 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;
- wherein said covalent conjugate is prepared by a cell-free in vitro 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 at least one modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a glycosylated or non-glycosylated Factor VIII peptide, wherein said polymeric modifying group is conjugated to said Factor VIII peptide via an intact glycosyl linking group interposed between and covalently linked to both said Factor VIII peptide and said poly(ethylene glycol), wherein said covalent conjugate is prepared by a cell-free in vitro method comprising:
- (a) contacting said Factor VIII peptide with a mixture comprising a nucleotide sugar covalently linked 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 to said Factor VIII peptide, wherein said modified sugar moiety comprises at least one modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a poly(ethylene glycol) and a Factor VIII peptide, said peptide having the formula:
- wherein
- AA is a terminal or internal amino acid residue of said peptide, wherein said covalent conjugate is prepared 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 amino acid residue, wherein said modified sugar moiety comprises at least one modifying group which is a poly(ethylene glycol),
- thereby forming said covalent conjugate of said Factor VIII peptide.
A covalent conjugate formed between a Factor VIII peptide and a poly(ethylene glycol), wherein said poly(ethylene glycol) is covalently attached to said Factor VIII peptide through an intact glycosyl linking group, said Factor VIII peptide comprising a glycosyl residue having a formula which is a member selected from:
- wherein
- a, b, c, d, i, n, o, p, q, r, s, t, u, aa, cc, and dd are members independently selected from 0 and 1;
- e, f, g, and h are members independently selected from the integers between 0 and 6;
- j, k, l, and m are members independently selected from the integers between 0 and 20;
- v, w, x, y and z are 0;
- and
- each R is a poly(ethylene glycol);
- and
- R′ is a member selected from H, a glycosyl residue, a modifying group and a glycoconjugate,
- wherein said covalent conjugate is prepared by a cell-free in vitro method comprising:
- (a) contacting said Factor VIII 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 VIII peptide, wherein said modified sugar moiety comprises at least one modifying group which is a 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.