Source: http://www.google.com/patents/US7226903?ie=ISO-8859-1&dq=Xerox+%2B+%22centroid
Timestamp: 2015-11-29 16:15:21
Document Index: 719421537

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60']

Patent US7226903 - Interferon beta: remodeling and glycoconjugation of interferon beta - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe 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....http://www.google.com/patents/US7226903?utm_source=gb-gplus-sharePatent US7226903 - Interferon beta: remodeling and glycoconjugation of interferon betaAdvanced Patent SearchPublication numberUS7226903 B2Publication typeGrantApplication numberUS 10/410,930Publication dateJun 5, 2007Filing dateApr 9, 2003Priority dateOct 10, 2001Fee statusPaidAlso published asUS20040115168Publication number10410930, 410930, US 7226903 B2, US 7226903B2, US-B2-7226903, US7226903 B2, US7226903B2InventorsShawn DeFrees, David Zopf, Robert Bayer, Caryn Bowe, David Hakes, Xi ChenOriginal AssigneeNeose Technologies, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (54), Non-Patent Citations (101), Referenced by (43), Classifications (18), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetInterferon beta: remodeling and glycoconjugation of interferon beta
US 7226903 B2Abstract
Images(497) Claims(113)
1. A cell-free, in vitro method of forming a covalent conjugate of an interferon beta peptide, said peptide having the formula:
X1–X2 is a saccharide covalently linked to said AA, wherein
(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 water-soluble polymer,
thereby forming said covalent conjugate of said interferon beta peptide.
4. The method of claim 1, wherein the peptide has the formula:
5. The method of claim 1, wherein said water soluble polymer comprises poly(ethylene glycol).
6. The method of claim 5, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
7. The method of claim 1, said peptide having the formula:
8. The method of claim 1, said peptide havin the formula:
9. The method of claim 8, wherein X11 and X12 are (mannose)q, wherein
X14 and X15 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.
12. The method of claim 1, wherein said peptide has the formula:
s and i are integers independently selected from 0 and 1.
13. The method of claim 1, wherein said removing utilizes a glycosidase.
14. A cell-free, in vitro method of forming a covalent conjugate of an interferon beta peptide, said peptide having the formula:
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 water-soluble polymer,
15. The method of claim 14, wherein said water soluble polymer comprises poly(ethylene glycol).
16. The method of claim 15, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
17. A cell-free, in vitro method of forming a covalent conjugate between a water-soluble polymer and a glycosylated or non-glycosylated interferon beta peptide, wherein said water-soluble polymer is conjugated to said interferon beta peptide via an intact glycosyl linking group interposed between and covalently linked to both said interferon beta peptide and said water-soluble polymer, said method comprising:
contacting said interferon beta peptide with a mixture comprising a nucleotide sugar covalently linked to said water-soluble polymer, 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 interferon beta peptide, wherein said modified sugar moiety comprises at least one modifying group which is a water-soluble polymer, thereby forming said covalent conjugate.
18. The method of claim 17, wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.
19. The method of claim 17, wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.
20. The method of claim 17, wherein said water-soluble polymer comprises a polyalkylene oxide.
21. The method of claim 20, wherein said polyalkylene oxide is poly(ethylene glycol).
22. The method of claim 21, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 20,000.
23. The method of claim 22, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 5,000.
24. The method of claim 23, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 1,000.
25. The method of claim 17, wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, and mannosyltransferase.
26. The method of claim 17, wherein said glycosyltransferase is recombinantly produced.
27. The method of claim 26, wherein said glycosyltransferase is a recombinant prokaryotic enzyme.
28. The method of claim 26, wherein said glycosyltransferase is a recombinant eukaryotic enzyme.
29. The method of claim 17, wherein said nucleotide sugar is selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.
30. The method of claim 29, 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, and CMP-NeuAc.
31. The method of claim 17, wherein said glycosylated peptide is partially deglycosylated prior to said contacting.
32. The method of claim 17, wherein said intact glycosyl linking group is a sialic acid residue.
33. The method of claim 17, wherein said method is performed in a cell-free environment.
34. The method of claim 17, wherein said covalent conjugate is isolated.
35. The method of claim 34, wherein said covalent conjugate is isolated by membrane filtration.
36. A cell-free, in vitro method of forming a covalent conjugate of an interferon beta peptide, said peptide having, the formula:
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 water-soluble polymer,
37. A cell-free, in vitro method of forming a covalent conjugate between an interferon beta peptide and a modifying group, wherein said modifying group is covalently attached to said interferon beta peptide through an intact glycosyl linking group, said interferon beta peptide comprising a glycosyl residue having the formula:
a, b, c, d, i, n, p, q, r, s, t, and u 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 100;
v, w, x, and y are 0;
each R is a water-soluble polymer; and
R′ is a member selected from H, a glycosyl, a modifying group and a glycoconjugate group,
(a) contacting said interferon beta 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 interferon beta peptide, wherein said modified sugar moiety comprises at least one modifying group which is a water soluble polymer, such that, following said contacting, at least one of v, w, x or y is 1,
(b) prior to step (a), contacting said interferon beta peptide with a sialidase under conditions appropriate to remove sialic acid from said interferon beta peptide.
(c) contacting the product from step (a) with a moiety that reacts with said modifying group, thereby forming a covalent conjugate between said intact glycosyl linking group and said moiety.
(d) prior to step (a) contacting said interferon beta peptide with a combination of a glycosidase and a sialidase.
(e) prior to step (a), contacting said interferon beta peptide with an endoglycanase under conditions appropriate to cleave a glycosyl moiety from said interferon beta peptide.
(f) prior to step (a), contacting said interferon beta peptide with N-acetylglucosamine transferase and a GlcNAc donor under conditions appropriate to transfer GlcNAc to said interferon beta peptide.
(g) prior to step (a), contacting said interferon beta peptide with a galactosyl transferase and a galactose donor under conditions appropriate to transfer galactose to said product.
(h) prior to step (b), contacting said interferon beta peptide with endoglycanase under conditions appropriate to cleave a glycosyl moiety from said interferon beta peptide.
(i) prior to step (a), contacting said interferon beta peptide with a mannosidase under conditions appropriate to remove mannose from said interferon beta peptide.
(j) contacting the product of step (a) with a sialyltransferase and a sialic acid donor under conditions appropriate to transfer sialic acid to said product.
h is a member independently selected from the integers between 1 and 3;
a, b, c, d, e, f, g, i, j, k, l, m, r, s, t, and u are members independently selected from 0 and 1;
n, v, w, x, and y are 0; and
q, p are 1.
48. The method of claim 37, wherein
a, b, c, d, f, h, j, k, l, m, n, s, u, v, w, x, and y are 0;
e, g, i, r, and t are members independently selected from 0 and 1; and
49. The method of claim 37, wherein
a, b, c, d, e, f, g, h, j, k, l, m, n, r, s, t, u, v, w, x, and y are 0;
q, p are 1; and
i is independently selected from 0 and 1.
50. The method of claim 37, wherein
a, b, c, d, e, f, g, h, i, j, k, l, m, n, r, s, t, u, v, w, x, and y are 0; and
p, q are 1.
51. The method of claim 37, wherein
a, b, c, d, e, f, g, h, i, j, k, l, m, and n are 0;
r, s, t, u, v, w, x, and y are members independently selected from 0 and 1.
52. The method of claim 37, wherein
a, b, c, d, e, f, g, h, i, r, s, t, and u are members independently selected from 0 and 1;
j, k, l, m, n, v, w, x, and y are 0; and
53. The method of claim 37, wherein
a, b, c, d, h, i, j, k, l, m, r, s, t, and u are members independently selected from 0 and 1;
e, f, g, are members selected from the integers between 0 and 3;
54. The method of claim 37, wherein
a, b, c, d, i, j, k, l, m, r, s, t, u, p and q are members independently selected from 0 and 1;
e, f, g, and h are 1; and
n, v, w, x, and y are 0.
55. A cell-free, in vitro method of forming a covalent conjugate between an interferon beta peptide and a water-soluble polymer modifying group, wherein said water-soluble polymer modifying group is covalently attached to said interferon beta peptide through an intact glycosyl linking group, said interferon beta peptide having the formula:
each R is said water-soluble polymer modifying group;
a, b, c, d, e, f, g and h are independently selected from 0 and 1;
j, k, l and m are independently selected from 0 and 1;
i is 0 or 1; and
n, v, w, x and y are 0,
(a) contacting said interferon beta peptide with a sialyltransferase and cytidine monophosphoryl sialic acid modified with poly(ethylene glycol) under conditions suitable for said sialyltransferase to transfer said sialic acid modified with poly(ethylene glycol) to said interferon beta peptide, such that, following said contacting, at least one of v, w, x and y is 1.
56. The method according to claim 55, wherein said glycosyltransferase is ST3Gal3 and said cytidine monophosphoryl sialic acid modified with poly(ethylene glycol) has the formula:
R is a poly(ethylene glycol) moiety.
57. The method of claim 1 wherein, following said forming said covalent conjugate, said peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto X1.
58. The method of claim 5, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
59. The method of claim 5, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
60. The method of claim 1, wherein said at least one glycosyltransferase is CST-II.
61. The method of claim 14 wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto X1.
62. The method of claim 15, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
63. The method of claim 15, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
64. The method of claim 14, wherein said at least one glycosyltransferase is CST-II.
65. The method of claim 17, wherein, following said forming said covalent conjugate, said interferon beta peptide 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.
66. The method of claim 17, wherein said water-soluble polymer is poly(ethylene glycol).
67. The method of claim 66, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
68. The method of claim 66, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
69. The method of claim 17, wherein said glycosyltransferase is CST-II.
70. The method of claim 36, wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
71. The method of claim 37, wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
72. The method of claim 37, wherein said water-soluble polymer is poly(ethylene glycol).
73. The method of claim 72, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
74. The method of claim 72, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
75. The method of claim 37, wherein said glycosyltransferase is CST-II.
76. A cell-free in vitro method of forming a covalent conjugate an interferon-beta peptide, said peptide having the formula:
(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 water-soluble polymer, thereby forming said covalent conjugate of said interferon beta peptide.
77. The method of claim 76, wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
78. The method according to claim 76 wherein said water-soluble polymer is poly(ethylene glycol).
79. The method according to claim 78 wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
80. The method of claim 78, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
81. The method of claim 78, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
82. The method of claim 76, wherein said glycosyltransferase is CST-II.
83. A cell-free, in vitro method of forming a covalent conjugate of an interferon beta peptide, said peptide having the formula:
(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 water-soluble polymer,
84. The method of claim 83 wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
85. The method according to claim 83 wherein said water-soluble polymer is poly(ethylene glycol).
86. The method according to claim 85 wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
87. The method of claim 85 wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
88. The method of claim 85 wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
89. The method of claim 83 wherein said glycosyltransferase is CST-II.
90. A cell-free, in vitro method of forming a covalent conjugate of an interferon beta peptide, said peptide having the formula:
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:
(b) contacting said truncated glycan with at least one glycosyltransferase and at least one modified sugar donor under conditions suitable 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 water-soluble polymer,
91. The method of claim 90 wherein said removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.
92. The method of claim 90 wherein X3, X5, and X7, are selected from the group consisting of (mannose)z and (mannose)z-(X8)y wherein
93. The method of claim 90 wherein X4 is selected from the group consisting of GlcNAc and xylose.
94. The method of claim 90 wherein X3, X5, and X7 are (mannose)u, wherein
95. The method of claim 90 wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
96. The method of claim 90 wherein said water soluble polymer comprises poly(ethylene glycol).
97. The method of claim 96 wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
98. The method of claim 96 wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
99. The method of claim 96 wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
100. The method of claim 90 wherein said glycosyltransferase is CST-II.
101. The method of claim 55 wherein, following said forming said covalent conjugate, said interferon beta peptide is contacted with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said interferon beta peptide.
102. The method according to claim 55 wherein said water-soluble polymer is poly(ethylene glycol).
103. The method according to claim 102 wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
104. The method of claim 102, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
105. The method of claim 102, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
106. The method of claim 55, wherein said glycosyltransferase is CST-II.
107. The method of claim 66, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
108. The method of claim 36, wherein said water-soluble polymer is poly(ethylene glycol).
109. The method of claim 108, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
110. The method of claim 108, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
111. The method of claim 108, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
112. The method of claim 36, wherein said glycosyltransferase is CST-II.
113. The method of claim 72, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse. Description
This application is a continuation-in-part of U.S. patent application Ser. No. 10/360,779, filed Feb. 19, 2003 now abandoned; U.S. patent application Ser. No. 10/360,770, filed Jan. 6, 2003 now abandoned; U.S. patent application Ser. No. 10/287,994, filed Nov. 5, 2002, which is a Continuation of prior Application No. PCT/US02/32263, filed Oct. 9, 2002; and prior Application No. PCT/US02/32263, filed Oct. 9, 2002; and prior application No. PCT/US02/32263, filed Oct. 9, 2002; all of which claim priority under 35 U.S.C. � 119(e) to Provisional Patent Application No. 60/407,527, filed Aug. 28, 2002, Provisional Patent Application No. 60/404,249, filed Aug. 16, 2002, Provisional Patent Application No. 60/396,594, filed Jul. 17, 2002, Provisional Patent Application No. 60/391,777, filed Jun. 25, 2002, Provisional Patent Application No. 60/387,292, filed Jun. 7, 2002, Provisional Patent Application No. 60/334,301, filed Nov. 28, 2001, Provisional Patent Application No. 60/334,233, filed Nov. 28, 2001, Provisional Patent Application No. 60/344,692, filed Oct. 19, 2001, and Provisional Patent Application No. 60/328,523, filed Oct. 10, 2001. Each of the above-referenced patent applications is hereby incorporated in its entirety by reference herein.
Peptides may also be modified by addition of O-linked glycans, also called mucin-type glycans because of their prevalence on mucinous glycopeptide. Unlike N-glycans that are linked to asparagine residues and are formed by en bloc transfer of oligosaccharide from lipid-bound intermediates, O-glycans are linked primarily to serine and threonine residues and are formed by the stepwise addition of sugars from nucleotide sugars (Tanner et al., Biochim. Biophys. Acta. 906:81–91 (1987); and Hounsell et al., Glycoconj. J. 13:19–26 (1996)). Peptide function can be affected by the structure of the O-linked glycans present thereon. For example, the activity of P-selectin ligand is affected by the O-linked glycan structure present thereon. For a review of O-linked glycan structures, see Schachter and Brockhausen, The Biosynthesis of Branched O-Linked Glycans, 1989, Society for Experimental Biology, pp. 1–26 (Great Britain). Other glycosylation patterns are formed by linking glycosylphosphatidylinositol to the carboxyl-terminal carboxyl group of the protein (Takeda et al., Trends Biochem. Sci. 20:367–371 (1995); and Udenfriend et al., Ann. Rev. Biochem. 64:593–591 (1995).
Methods combining both chemical and enzymatic synthetic elements are also known in the art. For example, Yamamoto and coworkers (Carbohydr. Res. 305: 415–422 (1998)) reported the chemoenzymatic synthesis of the glycopeptide, glycosylated Peptide T, using an endoglycosidase. The N-acetylglucosaminyl peptide was synthesized by purely chemical means. The peptide was subsequently enzymatically elaborated with the oligosaccharide of human transferrin peptide. The saccharide portion was added to the peptide by treating it with an endo-β-N-acetylglucosaminidase. The resulting glycosylated peptide was highly stable and resistant to proteolysis when compared to the peptide T and N-acetylglucosaminyl peptide T.
Glycosyl residues have also been modified to contain ketone groups. For example, Mahal and co-workers (Science 276: 1125 (1997)) have prepared N-levulinoyl mannosamine (“ManLev”), which has a ketone functionality at the position normally occupied by the acetyl group in the natural substrate. Cells were treated with the ManLev, thereby incorporating a ketone group onto the cell surface. See, also Saxon et al., Science 287: 2007 (2000); Hang et al., J. Am. Chem. Soc. 123: 1242(2001); Yarema et al., J. Biol. Chem. 273: 31168 (1998); and Charter et al., Glycobiology 10:1049 (2000).
The invention includes a cell-free, in vitro method of remodeling an interferon beta peptide, the peptide having the formula:
(a) removing X2 or a saccharyl subunit thereof from the peptide, thereby forming a truncated glycan; and (b) contacting the truncated glycan with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to the truncated glycan, thereby remodeling the interferon beta peptide. The method further comprises:
(c) removing X1, thereby exposing the AA; and (d) contacting the AA with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to the AA, thereby remodeling the interferon beta peptide. Additionally, the method comprises:
In another aspect of the method of the invention, the peptide has the formula:
Z is a member selected from O, S, NH and a crosslinker. In one embodiment, at least one of the glycosyl donors comprises a modifying group. On another embodiment, the 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. In a further embodiment, the modifying group is a water soluble polymer, which preferably comprises poly(ethylene glycol). In one embodiment, the poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
There is also included a cell-free in vitro method of remodeling an interferon beta peptide, the peptide having the formula:
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; the method comprising: (a) removing at least one of X3, X4, X5, X6, X7, or X17, a saccharyl subunit thereof from the peptide, thereby forming a truncated glycan; and (b) contacting the truncated glycan with at least one glycosyltransferase and at least one glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to the truncated glycan, thereby remodeling the interferon beta peptide. In one aspect, the removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.
In another aspect, X3, X5, and X7, are selected from the group consisting of (mannose)z and (mannose)z—(X8)y wherein
when z is 3 or greater, (mannose), is selected from linear and branched structures.
In a further aspect, wherein X4 is selected from the group consisting of GlcNAc and xylose.
Additionally, X3, X5, and X7 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. In a further aspect, at least one of the glycosyl donors comprises a modifying group, where the 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, preferably, the modifying group is a water soluble polymer, and preferably, the water soluble polymer comprises poly(ethylene glycol), where preferably, the poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
Also included is a cell-free in vitro method of remodeling an interferon beta peptide comprising a glycan having the formula:
r, s, and t are integers independently selected from 0 and 1, the method co