Source: http://www.google.com/patents/US8008252?dq=6,373,753
Timestamp: 2015-11-25 18:41:11
Document Index: 565975973

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 US8008252 - Factor VII: remodeling and glycoconjugation of Factor VII - 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/US8008252?utm_source=gb-gplus-sharePatent US8008252 - Factor VII: remodeling and glycoconjugation of Factor VIIAdvanced Patent SearchPublication numberUS8008252 B2Publication typeGrantApplication numberUS 10/411,044Publication dateAug 30, 2011Filing dateApr 9, 2003Priority dateOct 10, 2001Fee statusLapsedAlso published asUS20100015684Publication number10411044, 411044, US 8008252 B2, US 8008252B2, US-B2-8008252, US8008252 B2, US8008252B2InventorsShawn DeFrees, David Zopf, Robert Bayer, Caryn Bowe, David Hakes, Xi ChenOriginal AssigneeNovo Nordisk A/SExport CitationBiBTeX, EndNote, RefManPatent Citations (316), Non-Patent Citations (423), Referenced by (18), Classifications (9), Legal Events (6) External Links: USPTO, USPTO Assignment, EspacenetFactor VII: remodeling and glycoconjugation of Factor VII
US 8008252 B2Abstract
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.
Images(497) Claims(87)
1. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
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;
(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.
(c) prior to step (b), removing a group added to said saccharide during post-translational modification.
3. The method of claim 2, wherein said group is a member selected from phosphate, sulfate, carboxylate and esters thereof.
4. The method of claim 1, wherein said peptide has the formula:
Z is a member selected from O, S, NH or a cross linker.
5. The method of claim 1, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).
6. The method of claim 1, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
7. The method of claim 1, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
8. The method of claim 1, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI CST-II and combinations thereof.
(d) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.
10. The method of claim 1, wherein said peptide has the formula:
X9 and X10 are independently selected from monosaccharyl and oligosaccharyl residues; and
m, n and f are integers selected from 0 and 1.
11. The method of claim 1, wherein said peptide has the formula:
X11 and X12 are independently selected glycosyl moieties; and
r and x are integers independently selected from 0 and 1.
12. The method of claim 11, wherein X11 and X12 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.
13. The method of claim 1, wherein said peptide has the formula:
X13, X14, and X15 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.
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 when k is 1, g, h, and j are 0.
15. The method of claim 1, wherein said peptide has the formula:
X16 is a member selected from:
and i are integers independently selected from 0 and 1.
16. The method of claim 1, wherein said removing of step (a) utilizes a glycosidase.
17. The method of claim 1, wherein said removing of step (a) involves removal of a sialic acid moiety using a sialidase.
18. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
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,
(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,
19. The method of claim 18, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).
20. The method of claim 18, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
21. The method of claim 18, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
22. The method of claim 18, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI, CST-II and combinations thereof.
(b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.
24. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
a, b, r, s, and t are integers independently selected from 0 and 1,
26. The method according to claim 24, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
27. The method of claim 24, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
28. The method of claim 24, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
29. The method of claim 24, wherein said at least one glycosyltransferase is a member selected from the group consisting of GalT, ST3Gal3, CST-II and combinations thereof.
30. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
(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,
(c) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.
32. The method according to claim 30, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
33. The method of claim 30, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
34. The method of claim 30, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
35. The method of claim 30, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof.
36. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
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,
37. The method of claim 36, wherein said removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.
38. The method of claim 36, wherein X3, X5, and X7, are selected from the group consisting of (mannose)z and (mannose)z-(X8)y wherein
X8 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.
39. The method of claim 36, wherein X4 is selected from the group consisting of GlcNAc and xylose.
40. The method of claim 36, wherein 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.
42. The method of claim 36, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
43. The method of claim 36, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
44. The method of claim 36, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
45. The method of claim 36, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI CST-II and combinations thereof.
46. 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,
47. The method of claim 46, wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.
48. The method of claim 46, wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.
49. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 40,000.
50. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 80,000, and further wherein said poly(ethylene glycol) is a branched structure.
51. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 30,000, and further wherein said poly(ethylene glycol) is a linear structure.
52. The method of claim 49, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 5,000.
53. The method of claim 52, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 1,000.
54. The method of claim 46, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
55. The method of claim 46, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
56. The method of claim 46, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
57. The method of claim 46, wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof.
58. The method of claim 49, further comprising:
59. The method of claim 46, wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, mannosyltransferase and xylosyltransferase.
60. The method of claim 46, wherein said glycosyltransferase is recombinantly produced.
61. The method of claim 60, wherein said glycosyltransferase is a recombinant prokaryotic enzyme.
62. The method of claim 60, wherein said glycosyltransferase is a recombinant eukaryotic enzyme.
63. The method of claim 60, wherein said glycosylated peptide is partially deglycosylated prior to said contacting.
64. The method of claim 46, wherein said intact glycosyl linking group is a sialic acid residue.
65. The method of claim 46, wherein said method is performed in a cell-free environment.
66. The method of claim 46, wherein said covalent conjugate is isolated.
67. The method of claim 66, wherein said covalent conjugate is isolated by membrane filtration.
68. The covalent conjugate of claim 46, wherein said modified sugar donor is a nucleotide sugar selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.
69. The covalent conjugate of claim 68, 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.
70. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:
AA is a terminal or internal amino acid residue of said peptide,
(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,
72. The method according to claim 70, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.
73. The method of claim 70, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
74. The method of claim 70, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).
75. The method of claim 70, wherein said at least one glycosyltransferase is selected from the group consisting of GalNAcT2, ST3Gal1, ST3Gal3, ST6GalNAcI, CST-I, CST-II and combinations thereof.
76. 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:
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;
(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.
(b) prior to step (a), contacting said Factor VIIa peptide with a sialidase under conditions appropriate to remove a sialic acid from said Factor VIIa peptide, thereby removing said sialic acid from said Factor VIIa peptide.
78. The method of claim 76, further comprising:
(c) prior to step (a), contacting said Factor VIIa peptide with a galactosidase under conditions appropriate to remove a galactose from said Factor VIIa peptide, thereby removing said galactose from said Factor VIIa peptide.
(d) prior to step (a), contacting said Factor VIIa peptide with a galactosyl transferase and a galactose donor under conditions appropriate to transfer a galactose to said Factor VIIa peptide, thereby transferring said galactose to said Factor VIIa peptide.
80. The method of claim 76, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).
81. The method of claim 76, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).
82. The method of claim 76, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.
83. The method of claim 76, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, CST-II and combinations thereof.
84. The method of claim 76, further comprising:
(e) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.
85. The method of claim 76, wherein
a, b, c, d, e, g, i, j, l, o, p and q members independently selected from 0 and 1;
r and t are l;
f, h, k, m, s, u, v, w, x and y are 0; and
n is selected from the integers from 0 to 4.
86. The method of claim 76, wherein
a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t and u are members independently selected from 0 and 1;
n is a member selected from the integers from 0 to 4.
87. A method of treating a patient having a condition, said method comprising administering to said patient a covalent conjugate of a Factor VIIa peptide,
said conjugate is formed by a method according to claim 1, 18, 24, 30, 36, 46, 70 or 76; and
said condition is a member selected from hemophilia in combination with a bleeding episode, hemophilia A, hemophilia A in combination with antibodies to Factor III, hemophilia B, hemophilia B in combination with antibodies to Factor IX, liver cirrhosis, cirrhosis in combination with gastrointestinal bleeding, cirrhosis in combination with orthotopic liver transplant, bone marrow transplant and liver resection.
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 now U.S. Pat. No. 7,138,371, 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; 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.
Most naturally occurring peptides contain carbohydrate moieties attached to the peptide via specific linkages to a select number of amino acids along the length of the primary peptide chain. Thus, many naturally occurring peptides are termed “glycopeptides.” The variability of the glycosylation pattern on any given peptide has enormous implications for the function of that peptide. For example, the structure of the N-linked glycans on a peptide can impact various characteristics of the peptide, including the protease susceptibility, intracellular trafficking, secretion, tissue targeting, biological half-life and antigenicity of the peptide in a cell or organism. The alteration of one or more of these characteristics greatly affects the efficacy of a peptide in its natural setting, and also affects the efficacy of the peptide as a therapeutic agent in situations where the peptide has been generated for that purpose.
The carbohydrate structure attached to the peptide chain is known as a “glycan” molecule. The specific glycan structure present on a peptide affects the solubility and aggregation characteristics of the peptide, the folding of the primary peptide chain and therefore its functional or enzymatic activity, the resistance of the peptide to proteolytic attack and the control of proteolysis leading to the conversion of inactive forms of the peptide to active forms. Importantly, terminal sialic acid residues present on the glycan molecule affect the length of the half life of the peptide in the mammalian circulatory system. Peptides whose glycans do not contain terminal sialic acid residues are rapidly removed from the circulation by the liver, an event which negates any potential therapeutic benefit of the peptide.
The glycan structures found in naturally occurring glycopeptides are typically divided into two classes, N-linked and O-linked glycans.
Peptides expressed in eukaryotic cells are typically N-glycosylated on asparagine residues at sites in the peptide primary structure containing the sequence asparagine-X-serine/threonine where X can be any amino acid except proline and aspartic acid. The carbohydrate portion of such peptides is known as an N-linked glycan. The early events of N-glycosylation occur in the endoplasmic reticulum (ER) and are identical in mammals, plants, insects and other higher eukaryotes. First, an oligosaccharide chain comprising fourteen sugar residues is constructed on a lipid carrier molecule. As the nascent peptide is translated and translocated into the ER, the entire oligosaccharide chain is transferred to the amide group of the asparagine residue in a reaction catalyzed by a membrane bound glycosyltransferase enzyme. The N-linked glycan is further processed both in the ER and in the Golgi apparatus. The further processing generally entails removal of some of the sugar residues and addition of other sugar residues in reactions catalyzed by glycosidases and glycosyltransferases specific for the sugar residues removed and added.
Typically, the final structures of the N-linked glycans are dependent upon the organism in which the peptide is produced. For example, in general, peptides produced in bacteria are completely unglycosylated. Peptides expressed in insect cells contain high mannose and paunci-mannose N-linked oligosaccharide chains, among others. Peptides produced in mammalian cell culture are usually glycosylated differently depending, e.g., upon the species and cell culture conditions. Even in the same species and under the same conditions, a certain amount of heterogeneity in the glycosyl chains is sometimes encountered. Further, peptides produced in plant cells comprise glycan structures that differ significantly from those produced in animal cells. The dilemma in the art of the production of recombinant peptides, particularly when the peptides are to be used as therapeutic agents, is to be able to generate peptides that are correctly glycosylated, i.e., to be able to generate a peptide having a glycan structure that resembles, or is identical to that present on the naturally occurring form of the peptide. Most peptides produced by recombinant means comprise glycan structures that are different from the naturally occurring glycans.
A variety of methods have been proposed in the art to customize the glycosylation pattern of a peptide including those described in WO 99/22764, WO 98/58964, WO 99/54342 and U.S. Pat. No. 5,047,335, among others. Essentially, many of the enzymes required for the in vitro glycosylation of peptides have been cloned and sequenced. In some instances, these enzymes have been used in vitro to add specific sugars to an incomplete glycan molecule on a peptide. In other instances, cells have been genetically engineered to express a combination of enzymes and desired peptides such that addition of a desired sugar moiety to an expressed peptide occurs within the cell.
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).
Although various techniques currently exist to modify the N-linked glycans of peptides, there exists in the art the need for a generally applicable method of producing peptides having a desired, i.e., a customized glycosylation pattern. There is a particular need in the art for the customized in vitro glycosylation of peptides, where the resulting peptide can be produced at industrial scale. This and other needs are met by the present invention.
The administration of glycosylated and non-glycosylated peptides for engendering a particular physiological response is well known in the medicinal arts. Among the best known peptides utilized for this purpose is insulin, which is used to treat diabetes. Enzymes have also been used for their therapeutic benefits. A major factor, which has limited the use of therapeutic peptides is the immunogenic nature of most peptides. In a patient, an immunogenic response to an administered peptide can neutralize the peptide and/or lead to the development of an allergic response in the patient. Other deficiencies of therapeutic peptides include suboptimal potency and rapid clearance rates. The problems inherent in peptide therapeutics are recognized in the art, and various methods of eliminating the problems have been investigated. To provide soluble peptide therapeutics, synthetic polymers have been attached to the peptide backbone.
Poly(ethylene glycol) (“PEG”) is an exemplary polymer that has been conjugated to peptides. The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides and prolong the clearance time from the circulation. For example, U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenic peptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of polymer are used per mole peptide and at least 15% of the physiological activity is maintained.
WO 93/15189 (Veronese et al.) concerns a method to maintain the activity of polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.
The principal mode of attachment of PEG, and its derivatives, to peptides is a non-specific bonding through a peptide amino acid residue. For example, U.S. Pat. No. 4,088,538 discloses an enzymatically active polymer-enzyme conjugate of an enzyme covalently linked to PEG. Similarly, U.S. Pat. No. 4,496,689 discloses a covalently attached complex of α-1 protease inhibitor with a polymer such as PEG or methoxypoly(ethylene glycol) (“mPEG”). Abuchowski et al. (J. Biol. Chem. 252: 3578 (1977) discloses the covalent attachment of mPEG to an amine group of bovine serum albumin. U.S. Pat. No. 4,414,147 discloses a method of rendering interferon less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethylene succinic anhydride). PCT WO 87/00056 discloses conjugation of PEG and poly(oxyethylated) polyols to such proteins as interferon-β, interleukin-2 and immunotoxins. EP 154,316 discloses and claims chemically modified lymphokines,