US2008305991A1PendingUtilityA1
Factor IX: remodeling and glycoconjugation of factor IX
Est. expiryOct 10, 2021(expired)· nominal 20-yr term from priority
A61P 9/00C07K 14/505A61K 38/37C07K 1/1077A61K 47/60A61K 38/00C07K 1/13C07K 14/755C12N 9/644C12N 9/96C07K 9/00C07K 1/006
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Claims
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.
Claims
exact text as granted — not AI-modified1 . A cell-free, in vitro method of remodeling a Factor IX 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;
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 glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said truncated glycan, thereby remodeling said Factor IX peptide.
2 . The method of claim 1 , further comprising:
(c) removing X 1 , thereby exposing said AA; and (d) contacting said AA with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer said at least one glycosyl donor to said AA, thereby remodeling said Factor IX peptide.
3 . The method of claim 1 , further comprising:
(e) prior to step (b), removing a group added to said saccharide during post-translational modification.
4 . The method of claim 3 , wherein said group is a member selected from phosphate, sulfate, carboxylate and esters thereof.
5 . The method of claim 1 , wherein said peptide has the formula:
wherein
Z is a member selected from O, S, NH and a crosslinker.
6 . The method of claim 1 , wherein at least one of said glycosyl donors comprises a modifying group.
7 . The method of claim 1 , wherein said modifying group is a member selected from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a targeting moiety, and a peptide.
8 . The method of claim 7 , wherein said modifying group is a water soluble polymer.
9 . The method of claim 8 , wherein said water soluble polymer comprises poly(ethylene glycol).
10 . The method of claim 9 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
11 . A cell-free in vitro method of remodeling a Factor IX peptide, said peptide having the formula:
wherein
X 3 , X 4 , X 5 , X 6 , X 7 , and X 17 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 X 3 , X 4 , X 5 , X 6 , X 7 , or X 17 , 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 Factor IX peptide.
12 . The method of claim 11 , wherein said removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.
13 . The method of claim 11 , wherein X 3 , X 5 , and X 7 , are selected from the group consisting of (mannose) z and (mannose) z -(X 8 ) y wherein
X 8 is a glycosyl moiety selected from mono- and oligo-saccharides;
y is an integer selected from 0 and 1; and
z is an integer between 1 and 20, wherein
when z is 3 or greater, (mannose) z is selected from linear and branched structures.
14 . The method of claim 11 , wherein X 4 is selected from the group consisting of GlcNAc and xylose.
15 . The method of claim 11 , wherein X 3 , X 5 , and X 7 are (mannose) u , wherein u is selected from the integers between 1 and 20, and when u is 3 or greater, (mannose) u is selected from linear and branched structures.
16 . The method of claim 11 , wherein at least one of said glycosyl donors comprises a modifying group.
17 . The method of claim 16 , wherein said modifying group is a member selected from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a targeting moiety, and a peptide.
18 . The method of claim 17 wherein said modifying group is a water soluble polymer.
19 . The method of claim 18 , wherein said water soluble polymer comprises poly(ethylene glycol).
20 . The method of claim 19 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
21 . A cell-free in vitro method of remodeling a Factor IX 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 Factor IX peptide.
22 . The method of claim 21 , wherein at least one of said glycosyl donors comprises a modifying group.
23 . The method of claim 22 , wherein said modifying group is a member selected from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a targeting moiety, and a peptide.
24 . The method of claim 23 wherein said modifying group is a water soluble polymer.
25 . The method of claim 24 , wherein said water soluble polymer comprises poly(ethylene glycol).
26 . The method of claim 25 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
27 . The method of claim 1 , wherein said peptide has the formula:
wherein
X 9 and X 10 are independently selected monosaccharyl or oligosaccharyl residues; and
m, n and f are integers selected from 0 and 1.
28 . The method of claim 1 , 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.
29 . The method of claim 28 , wherein X 11 and X 12 are (mannose) q , wherein q is selected from the integers between 1 and 20, and when q is three or greater, (mannose) q is selected from linear and branched structures.
30 . The method of claim 1 , 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.
31 . The method of claim 30 , 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.
32 . The method of claim 1 , wherein said peptide has the formula:
wherein
X 16 is a member selected from:
wherein
and i are integers independently selected from 0 and 1.
33 . The method of claim 1 , wherein said removing utilizes a glycosidase.
34 . A cell-free, in vitro method of remodeling a Factor IX 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:
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 Factor IX peptide.
35 . The method of claim 34 , wherein said modifying group is a member selected from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a targeting moiety, and a peptide.
36 . The method of claim 35 wherein said modifying group is a water soluble polymer.
37 . The method of claim 36 , wherein said water soluble polymer comprises poly(ethylene glycol).
38 . The method of claim 37 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
39 . A covalent conjugate between a Factor IX 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.
40 . The covalent conjugate of claim 39 , wherein said modifying group is a member selected from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a targeting moiety, and a peptide.
41 . The covalent conjugate of claim 39 , wherein said 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 conjugate.
42 . The covalent conjugate of claim 39 comprising:
a first modifying group covalently linked to a first residue of said peptide via a first intact glycosyl linking group, and a second glycosyl linking group linked to a second residue of said peptide via a second intact glycosyl linking group.
43 . The covalent conjugate of claim 42 , wherein said first residue and said second residue are structurally identical.
44 . The covalent conjugate of claim 42 , wherein said first residue and said second residue have different structures.
45 . The covalent conjugate of claim 42 wherein said first residue and said second residue are glycosyl residues.
46 . The covalent conjugate of claim 42 , wherein said first residue and said second residue are amino acid residues.
47 . The covalent conjugate of claim 39 , wherein said peptide is remodeled prior to forming said conjugate.
48 . The covalent conjugate of claim 47 , wherein the remodeled peptide is remodeled to introduce an acceptor moiety for said intact glycosyl linking group.
49 . The covalent conjugate of claim 39 , wherein said modifying group is a water-soluble polymer.
50 . The covalent conjugate of claim 49 , wherein said water-soluble polymer comprises poly(ethylene glycol).
51 . The covalent conjugate of claim 39 , wherein said intact glycosyl linking unit is a member selected from the group consisting of a sialic acid residue, a Gal residue, a GlcNAc residue, a GalNAc residue, a glucose residue, a xylose residue, a fucose residue.
52 . The covalent conjugate of claim 50 , wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
53 . 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 Factor IX.
54 . The method of claim 53 , wherein said polymer is a water-soluble polymer.
55 . The method of claim 53 , wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.
56 . The method of claim 53 , wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.
57 . The method of claim 53 , wherein said polymer comprises a member selected from the group consisting of a polyalkylene oxide and a polypeptide.
58 . The method of claim 57 , wherein said polyalkylene oxide is poly(ethylene glycol).
59 . The method of claim 58 , wherein said poly(ethylene glycol) has a degree of polymerization of from about 1 to about 20,000.
60 . The method of claim 59 , wherein said polyethylene glycol has a degree of polymerization of from about 1 to about 5,000.
61 . The method of claim 60 , wherein said polyethylene glycol has a degree of polymerization of from about 1 to about 1,000.
62 . The method of claim 53 , wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, mannosyltransferase, xylose transferase.
63 . The method of claim 53 , wherein said glycosyltransferase is recombinantly produced.
64 . The method of claim 63 , wherein said glycosyltransferase is a recombinant prokaryotic enzyme.
65 . The method of claim 63 , wherein said glycosyltransferase is Recombinant eukaryotic enzyme.
66 . The method of claim 53 , wherein said nucleotide sugar is selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.
67 . The method of claim 66 , 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, CMP-NeuAc.
68 . The method of claim 53 , wherein said glycosylated peptide is partially deglycosylated prior to said contacting.
69 . The method of claim 53 , wherein said intact glycosyl linking group is a sialic acid residue.
70 . The method of claim 53 , wherein said method is performed in a cell-free environment.
71 . The method of claim 53 , wherein said covalent conjugate is isolated.
72 . The method of claim 71 , wherein said covalent conjugate is isolated by membrane filtration.
73 . 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 Factor IX.
74 . A Factor IX peptide remodeled by the method of claim 1 .
75 . A pharmaceutical composition comprising the Factor IX peptide of claim 74 .
76 . A Factor IX peptide remodeled by the method of claim 11 .
77 . A pharmaceutical composition comprising the Factor IX peptide of claim 76 .
78 . A Factor IX peptide remodeled by the method of claim 21 .
79 . A pharmaceutical composition comprising the Factor IX peptide of claim 78 .
80 . A Factor IX peptide remodeled by the method of claim 27 .
81 . A pharmaceutical composition comprising the Factor IX peptide of claim 80 .
82 . A Factor IX peptide remodeled by the method of claim 28 .
83 . A pharmaceutical composition comprising the Factor IX peptide of claim 82 .
84 . A Factor IX peptide remodeled by the method of claim 34 .
85 . A pharmaceutical composition comprising the Factor IX peptide of claim 84 .
86 . 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 Factor IX.
87 . A method for forming a conjugate between a Factor IX peptide and a modifying group, wherein said modifying group is covalently attached to said Factor IX peptide through an intact glycosyl linking group, said Factor IX peptide comprising a glycosyl residue having a formula which is a member selected from:
wherein
a, b, c, d, i, n, 0, p, q, r, s, t, u, bb, cc, dd, ee, ff and gg are members independently selected from 0 and 1;
e, f, g, h and aa 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, y and z are 0;
R is a modifying group, a mannose or an oligomannose;
said method comprising:
(a) contacting said Factor IX 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.
88 . The method of claim 87 , further comprising:
(b) prior to step (a), contacting said Factor IX peptide with a sialidase under conditions appropriate to remove sialic acid from said Factor IX peptide.
89 . The method of claim 87 , further comprising:
(c) contacting the product formed in step (a) with a sialyltransferase and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.
90 . The method of claim 88 , further comprising:
(d) contacting the product from step (b) with a galactosyltransferase and a galactose donor under conditions appropriate to transfer said galactose to said product.
91 . The method of claim 90 , further comprising:
(e) contacting the product from step (d) with ST3Gal3 and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.
92 . The method of claim 87 , further comprising:
(d) contacting the product from step (a) with a moiety that reacts with said modifying group, thereby forming a conjugate between said intact glycosyl linking group and said moiety.
93 . The method of claim 87 , wherein said modifying group is a member selected from a polymer, a toxin, a radioisotope, a therapeutic moiety and a glycoconjugate.
94 . The method of claim 87 , wherein
a, b, c, and d are 1; e, f, g and h are members independently selected from the integers from 1 to 4; aa, bb, cc, dd, ee, ff j, k, l, m, i, n, o, p, q, r, s, t and u are members independently selected from 0 and 1; and v, w, x, y, z and gg are 0.
95 . The method of claim 87 , wherein
a, b, c, d, n, q are independently selected from 0 and 1; aa, e, f, g and h are members independently selected from the integers from 1 to 4; bb, cc, dd, ee, ff, j, k, l, m, i, o, p, r, s, t and u are members independently selected from 0 and 1; and v, w, x, y, z and gg are 0.
96 . The method of claim 87 , wherein
a, b, c, d, n, bb, cc, dd and ff are 1; e, f, g, h and aa are members independently selected from the integers from 1 to 4; q, ee, i, j, k, l, m, o, p, r, s, t and u are members independently selected from 0 and 1; and v, w, x, y, z and gg are 0.
97 . The method of claim 87 , wherein
a, b, c, d and q are 1; e, f, g and h are members independently selected from the integers from 1 to 4; aa, bb, cc, dd, ee, ff, j, k, l, m, i, n, o, p, r, s, t and u are members independently selected from 0 and 1; and v, w, x, y, z and gg are 0.
98 . The method of claim 87 , wherein
a, b, c, d, q, bb, cc, dd and ff are 1; aa, e, f, g and h are members independently selected from the integers from 1 to 4; ee, i, j, k, l, m, o, p, r, s, t and u are members independently selected from 0 and 1; and v, w, x, y, z and gg are 0.
99 . A Factor IX peptide conjugate formed by the method of claim 87 .
100 . A Factor IX peptide comprising one or more glycans, having a glycoconjugate molecule covalently attached to said peptide.
101 . The Factor IX peptide of claim 100 , wherein said one or more glycans is a monoantennary glycan.
102 . The Factor IX peptide of claim 100 , wherein said one or more glycans is a biantennary glycan.
103 . The Factor IX peptide of claim 100 , wherein said one or more glycans is a triantennary glycan.
104 . The Factor IX peptide of claim 100 , wherein said one or more glycans is at least a triantennary glycan.
105 . The Factor IX peptide of claim 100 , wherein said one or more glycans comprises at least two glycans comprising a mixture of mono or multiantennary glycans.
106 . The Factor IX peptide of claim 100 , wherein said one or more glycans is selected from an N-linked glycan and an O-linked glycan.
107 . The Factor IX peptide of claim 100 , wherein said one or more glycans is at least two glycans selected from an N-linked and an O-linked glycan.
108 . The Factor IX peptide of claim 100 , wherein said peptide is expressed in a cell selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
109 . The Factor IX peptide of claim 108 , wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell and a yeast cell.
110 . A method of treating a mammal having hemophilia B, said method comprising administering to said mammal a Factor IX peptide having one or more glycans having a glycoconjugate molecule attached to said peptide.
111 . The method of claim 110 , wherein said glyconjugate is poly(ethylene glycol).
112 . The method of claim 110 , wherein said mammal is a human.Cited by (0)
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