Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity

• US 20040136952A1
• Filed: 12/23/2003
• Published: 07/15/2004
• Est. Priority Date: 12/26/2002
• Status: Abandoned Application

• Current Assignee: Mountain View Pharmaceuticals, Inc.
• Original Assignee: Mountain View Pharmaceuticals, Inc.
• Inventors: Bhaskaran, Shyam S., Sherman, Merry R., Williams, L. David, Saifer, Mark G.P.
• Application Number:
  US10/743,295
• Publication Number:
  US 20040136952A1
• Time in Patent Office: Days
• US Class Current: 424/85.1
• CPC Class Codes: A61K 38/1808 Epidermal growth factor [EGF] urogastrone
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/2013 IL-2
• CPC Class Codes: A61K 38/212 IFN-alpha
• CPC Class Codes: A61K 38/30 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 insulin-like growth factor binding protein A61K38/1754
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: C07K 14/52 : Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/521 : Chemokines

Abstract

Methods are provided for the synthesis of polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and receptor-binding antagonists thereof, which conjugates retain unusually high receptor-binding activity. Preparation of polymer conjugates according to the methods of the present invention diminishes or avoids steric inhibition of receptor-ligand interactions that commonly results from the attachment of polymers to receptor-binding regions of cytokines, chemokines, growth factors and polypeptide hormones, as well as to agonistic and antagonistic analogs thereof. The invention also provides conjugates and compositions produced by such methods. The conjugates of the present invention retain a higher level of receptor-binding activity than those produced by traditional polymer coupling methods that are not targeted to avoid receptor-binding domains of cytokines, chemokines, growth factors and polypeptide hormones. The conjugates of the present invention also exhibit an extended half-life in vivo and in vitro compared to unconjugated cytokines, chemokines, growth factors and polypeptide hormones. The present invention also provides kits comprising such conjugates and/or compositions, and methods of use of such conjugates and compositions in a variety of diagnostic, prophylactic, therapeutic and bioprocessing applications.

Claims

1. A method for synthesizing conjugates of one or more synthetic water-soluble polymers with a cytokine, a chemokine, a growth factor or a polypeptide hormone, or an antagonist thereof, that preserves more of the receptor-binding potency of said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, than is preserved when such polymers are randomly coupled, comprising:

(a) selecting a cytokine, chemokine, growth factor or polypeptide hormone in which the amino-terminal amino acid is located remotely from one or more receptor-binding domains of said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof; and

(b) coupling said one or more polymers selectively to said amino-terminal amino acid.

2. The method of claim 1, wherein said one or more polymers is/are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylenes), one or more poly(amino acids), one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

3. The method of claim 1, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a four helix bundle structure.

4. The method of claim 3, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), thrombopoietin (Tpo), an erythropoietin (EPO), stem cell factor (SCF), Flt3 ligand, oncostatin M (OSM), an interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, IL-17, an interferon-alpha (IFN-α

), an interferon-beta (IFN-β

), consensus interferon, prolactin and growth hormone, and muteins, antagonists, variants, analogs and derivatives thereof.

5. The method of claim 1, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a β

-sheet or β

-barrel structure.

6. The method of claim 5, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of tumor necrosis factor-alpha (TNF-α

), IL-1α

, IL-1β

, IL-12 (p40 subunit), IL-16, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), acidic FGF, FGF-4 and keratinocyte growth factor (KGF;

FGF-7), and muteins, antagonists, variants, analogs and derivatives thereof.

7. The method of claim 1, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a mixed α

/β

structure.

8. The method of claim 7, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of neutrophil activating peptide-2 (NAP-2), stromal cell-derived factor-1α

(SDF-1α

), IL-8, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, eotaxin-1, eotaxin-2, eotaxin-3, RANTES, myeloid progenitor inhibitory factor-1 (MPIF-1), neurotactin, macrophage migration inhibitory factor (MIF) and growth-related oncogene/melanoma growth stimulatory activity (GRO-α

/MGSA), and muteins, antagonists, variants, analogs and derivatives thereof.

9. The method of claim 1, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of an interferon-alpha, an interferon-beta, an IL-2, IL-4, IL-10, TNF-alpha, IGF-1, EGF, bFGF, insulin, a TNF-alpha antagonist, an hGH antagonist and a prolactin antagonist.

10. The method of claim 9, wherein said cytokine is an IL-2.

11. The method of claim 9, wherein said cytokine is an interferon-alpha.

12. The method of claim 9, wherein said cytokine is TNF-alpha.

13. The method of claim 9, wherein said cytokine antagonist is a TNF-alpha antagonist.

14. The method of claim 9, wherein said growth factor is EGF.

15. The method of claim 9, wherein said growth factor is IGF-1.

16. The method of claim 1, wherein said polymer is covalently coupled to the alpha amino group of said amino-terminal amino acid.

17. The method of claim 16, wherein said covalent coupling of said polymer to said alpha amino group is via a secondary amine linkage.

18. The method of claim 1, wherein said polymer is coupled to a chemically reactive side chain group of said amino-terminal amino acid.

19. The method of claim 18, wherein said reactive side chain is selected from the group consisting of a hydroxyl group, a sulfhydryl group, a guanidino group, an imidazole group, an amino group, a carboxyl group and an aldehyde group.

20. The method of claim 1, wherein said water-soluble polymer is a polyalkylene glycol.

21. The method of claim 20, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a monomethoxypoly(ethylene glycol) and a monohydroxypoly(ethylene glycol).

22. The method of claim 21, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

23. The method of claim 21, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

24. The method of claim 20, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

25. The method of claim 24, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 5 kDa, inclusive.

26. The method of claim 24, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 20 kDa, inclusive.

27. The method of claim 24, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 60 kDa, inclusive.

28. The method of claim 24, wherein said polyalkylene glycol has a molecular weight of between about 12 kDa and about 30 kDa, inclusive.

29. The method of claim 28, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

30. The method of claim 24, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

31. The method of claim 4, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of prolactin and prolactin analogs that mimic or antagonize the biological effects of prolactin that are mediated by prolactin receptors.

32. The method of claim 4, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of growth hormone and growth hormone analogs that mimic or antagonize the biological effects of growth hormone that are mediated by growth hormone receptors.

33. The method of claim 4, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of nonglycosylated erythropoietin and erythropoietin analogs that mimic or antagonize the biological effects of erythropoietin that are mediated by erythropoietin receptors.

34. The method of claim 1, wherein the coupling of said polymer to said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, at said amino-terminal amino acid mimics the beneficial effects of glycosylation or hyperglycosylation of said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof.

35. The method of claim 1, wherein said receptor-binding potency is measured by one or more methods selected from the group including ultracentrifugation, cell-based assays, competitive binding assays, radioreceptor assays, surface plasmon resonance and dynamic light scattering.

36. A conjugate produced by the method of claim 1.

37. A pharmaceutical composition comprising one or more of the conjugates of claim 36 and one or more pharmaceutically acceptable excipients or carriers.

73. A kit comprising the pharmaceutical composition of claim 37.

38. A conjugate comprising a cytokine, a chemokine, a growth factor or a polypeptide hormone, or antagonist thereof, coupled to one or more synthetic water-soluble polymers, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected for its membership in the group of receptor-binding proteins and polypeptides in which the amino-terminal amino acid is located remotely from one or more receptor-binding domains and wherein said one or more polymers is/are coupled to said amino-terminal amino acid.

39. The conjugate of claim 38, wherein said one or more polymers is/are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylenes), one or more poly(amino acids), one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

40. The conjugate of claim 38, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a four helix bundle structure.

41. The conjugate of claim 40, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), thrombopoietin (Tpo), an erythropoietin (EPO), stem cell factor (SCF), Flt3 ligand, oncostatin M (OSM), an interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, IL-17, an interferon alpha (IFN-α

), an interferon beta (IFN-β

), consensus interferon, prolactin and growth hormone, and muteins, antagonists, variants, analogs and derivatives thereof.

42. The conjugate of claim 38, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a β

-sheet or β

-barrel structure.

43. The conjugate of claim 42, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of tumor necrosis factor alpha (TNF-α

), IL-1α

, IL-1β

, IL-12 (p40 subunit), IL-16, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), acidic FGF, FGF-4 and keratinocyte growth factor (KGF;

FGF-7), and muteins, antagonists, variants, analogs and derivatives thereof.

44. The conjugate of claim 38, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a mixed α

/β

structure.

45. The conjugate of claim 44, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of neutrophil activating peptide-2 (NAP-2), stromal cell-derived factor-1α

(SDF-1α

), IL-8, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, eotaxin-1, eotaxin-2, eotaxin-3, RANTES, myeloid progenitor inhibitory factor-1 (MPIF-1), neurotactin, macrophage migration inhibitory factor (MIF) and GRO/melanoma growth stimulatory activity (GRO-α

/MGSA), and muteins, variants, analogs and derivatives thereof.

46. The conjugate of claim 38, wherein said cytokine, chemokine, growth factor or polypeptide hormone or antagonist thereof, is selected from the group consisting of an interferon-alpha, an interferon-beta, an IL-2, IL-4, IL-10, TNF-alpha, IGF-1, EGF, bFGF, hGH, insulin and prolactin, and antagonists thereof.

47. The conjugate of claim 46, wherein said cytokine is an IL-2.

48. The conjugate of claim 46, wherein said cytokine is an interferon-alpha.

49. The conjugate of claim 46, wherein said cytokine is TNF-alpha.

50. The conjugate of claim 46, wherein said cytokine antagonist is a TNF-alpha antagonist.

51. The conjugate of claim 46, wherein said growth factor is EGF.

52. The conjugate of claim 46, wherein said growth factor is IGF-1.

53. The conjugate of claim 38, wherein said polymer is covalently coupled to the alpha amino group of said amino-terminal amino acid.

54. The conjugate of claim 53, wherein said covalent coupling of said polymer to said alpha amino group is via a secondary amine linkage.

55. The conjugate of claim 38, wherein said polymer is coupled to a chemically reactive side chain group of said amino-terminal amino acid.

56. The conjugate of claim 55, wherein said reactive side chain is selected from the group consisting of a hydroxyl group, a sulfhydryl group, a guanidino group, an imidazole group, an amino group, a carboxyl group and an aldehyde group.

57. The conjugate of claim 38, wherein said water-soluble polymer is a polyalkylene glycol.

58. The conjugate of claim 57, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a monomethoxypoly(ethylene glycol) and a monohydroxypoly(ethylene glycol).

59. The conjugate of claim 58, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

60. The conjugate of claim 58, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

61. The conjugate of claim 57, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

62. The conjugate of claim 61, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 5 kDa, inclusive.

63. The conjugate of claim 61, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 20 kDa, inclusive.

64. The conjugate of claim 61, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 60 kDa, inclusive.

65. The conjugate of claim 61, wherein said polyalkylene glycol has a molecular weight of between about 12 kDa and about 30 kDa, inclusive.

66. The conjugate of claim 65, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

67. The conjugate of claim 61, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

68. The conjugate of claim 40, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of prolactin and prolactin analogs that mimic or antagonize the biological effects of prolactin that are mediated by prolactin receptors.

69. The conjugate of claim 40, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of growth hormone and growth hormone analogs that mimic or antagonize the biological effects of growth hormone that are mediated by growth hormone receptors.

70. The conjugate of claim 41, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of nonglycosylated erythropoietin and erythropoietin analogs that mimic or antagonize the biological effects of erythropoietin that are mediated by erythropoietin receptors.

71. The conjugate of claim 38, wherein the coupling of said polymer to said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, at said amino-terminal amino acid mimics the beneficial effects of glycosylation or hyperglycosylation of said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof.

72. A pharmaceutical composition comprising the conjugate of claim 38 and a pharmaceutically acceptable carrier or excipient.

74. A kit comprising the conjugate of claim 38.

75. A kit comprising the conjugate of claim 40.

76. A kit comprising the pharmaceutical composition of claim 72.

77. A method for synthesizing conjugates of one or more synthetic water-soluble polymers with a cytokine, a chemokine, a growth factor or a polypeptide hormone, or antagonist thereof, that preserves more of the receptor-binding potency of said cytokine, chemokine, growth factor or polymers are randomly coupled, comprising:

(a) selecting a cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, in which a naturally occurring or genetically engineered glycosylation site is located remotely from one or more receptor-binding domains of said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof; and

(b) coupling said one or more polymers selectively to said glycosylation site or to a carbohydrate moiety attached thereto.

78. The method of claim 77, wherein said one or more polymers is/are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylene), one or more poly(amino acids) one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

79. The method of claim 77, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a four helix bundle structure.

80. The method of claim 79, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), thrombopoietin (Tpo), an erythropoietin (EPO), stem cell factor (SCF), Flt3 ligand, oncostatin M (OSM), an interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, IL-17, an interferon alpha (IFN-α

), an interferon beta (IFN-β

), consensus interferon, prolactin and growth hormone, and muteins, antagonists, variants, analogs and derivatives thereof.

81. The method of claim 77, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a β

-sheet or β

-barrel structure.

82. The method of claim 81, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of tumor necrosis factor alpha (TNF-α

), IL-1α

, IL-1β

, IL-12 (p40 subunit), IL-16, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), acidic FGF, FGF-4 and keratinocyte growth factor (KGF;

FGF-7), and muteins, antagonists, variants, analogs and derivatives thereof.

83. The method of claim 77, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a mixed α

/β

structure.

84. The method of claim 83, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of neutrophil activating peptide-2 (NAP-2), stromal cell-derived factor-1α

(SDF-1α

), IL-8, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, eotaxin-1, eotaxin-2, eotaxin-3, RANTES, myeloid progenitor inhibitory factor-1 (MPIF-1), neurotactin, macrophage migration inhibitory factor (MIF) and GRO/melanoma growth stimulatory activity (GRO-α

/MGSA), and muteins, variants, analogs and derivatives thereof.

85. The method of claim 77, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of an interferon-alpha, an interferon-beta, an IL-2, IL-4, IL-10, TNF-alpha, IGF-1, EGF, bFGF, hGH, prolactin, insulin, and antagonists thereof.

86. The method of claim 85, wherein said cytokine is an IL-2.

87. The method of claim 85, wherein said cytokine is an interferon-alpha.

88. The method of claim 85, wherein said cytokine is TNF-alpha.

89. The method of claim 85, wherein said cytokine antagonist is a TNF-alpha antagonist.

90. The method of claim 85, wherein said growth factor is EGF.

91. The method of claim 85, wherein said growth factor is IGF-1.

92. The method of claim 77, wherein said water-soluble polymer is a polyalkylene glycol.

93. The method of claim 92, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a monomethoxypoly(ethylene glycol) and a monohydroxypoly(ethylene glycol).

94. The method of claim 93, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

95. The method of claim 93, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

96. The method of claim 92, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

97. The method of claim 96, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 5 kDa, inclusive.

98. The method of claim 96, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 20 kDa, inclusive.

99. The method of claim 96, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 60 kDa, inclusive.

100. The method of claim 96, wherein said polyalkylene glycol has a molecular weight of between about 12 kDa and about 30 kDa, inclusive.

101. The method of claim 100, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

102. The method of claim 96, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

103. The method of claim 77, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of prolactin and prolactin analogs that mimic or antagonize the biological effects of prolactin that are mediated by prolactin receptors.

104. The method of claim 77, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of growth hormone and growth hormone analogs that mimic or antagonize the biological effects of growth hormone that are mediated by growth hormone receptors.

105. The method of claim 77, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of nonglycosylated erythropoietin and erythropoietin analogs that mimic or antagonize the biological effects of erythropoietin that are mediated by erythropoietin receptors.

106. The method of claim 77, wherein the coupling of said polymer to said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, at or near said glycosylation site or sites mimics the beneficial effects of glycosylation or hyperglycosylation of said cytokine, chemokine, growth factor or polypeptide hormone.

107. A conjugate produced by the method of claim 77.

108. A pharmaceutical composition comprising one or more of the conjugates of claim 107 and one or more pharmaceutically acceptable excipients or carriers.

140. A kit comprising the conjugate of claim 107.

141. A kit comprising the pharmaceutical composition of claim 108.

144. A method for preventing, diagnosing, or treating a physical disorder in an animal suffering from or predisposed to said physical disorder, comprising administering to said animal an effective amount of the conjugate of any one of claims 36, 38, 107 and 109.

145. A method for preventing, diagnosing, or treating a physical disorder in an animal suffering from or predisposed to said physical disorder, comprising administering to said animal an effective amount of the pharmaceutical composition of any one of claims 37, 72, 108 and 139.

146. The method of claim 144, wherein said animal is a mammal.

147. The method of claim 145, wherein said animal is a mammal.

148. The method of claim 146 or claim 147, wherein said mammal is a human.

149. The method of claim 144, wherein said physical disorder is selected from the group consisting of a cancer, an infectious disease, a neurodegenerative disorder, an autoimmune disorder, and a genetic disorder.

150. The method of claim 149, wherein said cancer is selected from the group consisting of a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a lung cancer, a leukemia, a lymphoma, a colon cancer, a gastrointestinal cancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a bone cancer, a neurological cancer, a head and neck cancer, a skin cancer, a sarcoma, a carcinoma, an adenoma and a myeloma.

151. The method of claim 149, wherein said infectious disease is selected from the group consisting of a bacterial disease, a fungal disease, a viral disease and a parasitic disease.

152. The method of claim 151, wherein said viral disease is selected from the group consisting of hepatitis B, hepatitis C, a disease caused by a cardiotropic virus and HIV/AIDS.

153. The method of claim 149, wherein said autoimmune disorder is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis and psoriasis.

154. The method of claim 149, wherein said genetic disorder is selected from the group consisting of anemia, neutropenia, thrombocytopenia, hemophilia, dwarfism and severe combined immunodeficiency disease (“

SCID”

).

155. The method of claim 149, wherein said neurodegenerative disorder is multiple sclerosis.

156. The method of claim 149, wherein said neurodegenerative disease is Creutzfeldt-Jakob disease or Alzheimer'"'"'s disease.

157. The method of claim 145, wherein said physical disorder is selected from the group consisting of a cancer, an infectious disease, a neurodegenerative disorder, an autoimmune disorder, and a genetic disorder.

158. The method of claim 157, wherein said cancer is selected from the group consisting of a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a lung cancer, a leukemia, a lymphoma, a colon cancer, a gastrointestinal cancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a bone cancer, a neurological cancer, a head and neck cancer, a skin cancer, a sarcoma, a carcinoma, an adenoma and a myeloma.

159. The method of claim 157, wherein said infectious disease is selected from the group consisting of a bacterial disease, a fungal disease, a viral disease and a parasitic disease.

160. The method of claim 159, wherein said viral disease is selected from the group consisting of hepatitis B, hepatitis C, a disease caused by a cardiotropic virus and HIV/AIDS.

161. The method of claim 157, wherein said autoimmune disorder is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis and psoriasis.

162. The method of claim 157, wherein said genetic disorder is selected from the group consisting of anemia, neutropenia, thrombocytopenia, hemophilia, dwarfism and severe combined immunodeficiency disease (“

SCID”

).

163. The method of claim 157, wherein said neurodegenerative disorder is multiple sclerosis.

164. The method of claim 157, wherein said neurodegenerative disease is Creutzfeldt-Jakob disease or Alzheimer'"'"'s disease.

109. A conjugate comprising a cytokine, a growth factor, a chemokine or a polypeptide hormone, or antagonist thereof, coupled to one or more synthetic water-soluble polymers, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected for its membership in the group of receptor-binding proteins and polypeptides in which a glycosylation site is located remotely from one or more receptor-binding domains and wherein said one or more polymers is/are coupled at or near one or more glycosylation sites or to a carbohydrate moiety attached thereto.

110. The conjugate of claim 109, wherein said one or more polymers is/are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylenes), one or more poly(amino acids), one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

111. The conjugate of claim 109, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a four helix bundle structure.

112. The conjugate of claim 111, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), thrombopoietin (Tpo), an erythropoietin (EPO), stem cell factor (SCF), Flt3 ligand, oncostatin M (OSM), an interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, IL-17, an interferon alpha (IFN-α

), an interferon beta (IFN-β

), consensus interferon, prolactin and growth hormone, and muteins, antagonists, variants, analogs and derivatives thereof.

113. The conjugate of claim 109, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a β

-sheet or β

-barrel structure.

114. The conjugate of claim 113, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of tumor necrosis factor alpha (TNF-α

), IL-1α

, IL-1β

, IL-12 (p40 subunit), IL-16, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), acidic FGF, FGF-4 and keratinocyte growth factor (KGF;

FGF-7), and muteins, antagonists, variants, analogs and derivatives thereof.

115. The conjugate of claim 109, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, has a mixed α

/β

structure.

116. The conjugate of claim 115, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of neutrophil activating peptide-2 (NAP-2), stromal cell-derived factor-1α

(SDF-1α

), IL-8, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, eotaxin-1, eotaxin-2, eotaxin-3, RANTES, myeloid progenitor inhibitory factor-1 (MPIF-1), neurotactin, macrophage migration inhibitory factor (MIF) and growth-related oncogene/melanoma growth stimulatory activity (GRO-α

/MGSA), and muteins, antagonists, variants, analogs and derivatives thereof.

117. The conjugate of claim 109, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of an interferon-alpha, an interferon-beta, an IL-2, IL-4, IL-10, TNF-alpha, IGF-1, EGF, bFGF, hGH, prolactin, insulin, and antagonists thereof.

118. The conjugate of claim 117, wherein said cytokine is an IL-2.

119. The conjugate of claim 117, wherein said cytokine is an interferon-alpha.

120. The conjugate of claim 117, wherein said cytokine is TNF-alpha.

121. The conjugate of claim 117, wherein said cytokine antagonist is a TNF-alpha antagonist.

122. The conjugate of claim 117, wherein said growth factor is EGF.

123. The conjugate of claim 117, wherein said growth factor is IGF-1.

124. The conjugate of claim 109, wherein said water-soluble polymer is a polyalkylene glycol.

125. The conjugate of claim 124, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a monomethoxypoly(ethylene glycol) and a monohydroxypoly(ethylene glycol).

126. The conjugate of claim 125, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

127. The conjugate of claim 125, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

128. The conjugate of claim 124, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

129. The conjugate of claim 128, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 5 kDa, inclusive.

130. The conjugate of claim 128, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 20 kDa, inclusive.

131. The conjugate of claim 128, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 60 kDa, inclusive.

132. The conjugate of claim 128, wherein said polyalkylene glycol has a molecular weight of between about 12 kDa and about 30 kDa, inclusive.

133. The conjugate of claim 132, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

134. The conjugate of claim 128, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

135. The conjugate of claim 109, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of prolactin and prolactin analogs that mimic or antagonize the biological effects of prolactin that are mediated by prolactin receptors.

136. The conjugate of claim 109, wherein said polypeptide hormone, or antagonist thereof, is selected from the group consisting of growth hormone and growth hormone analogs that mimic or antagonize the biological effects of growth hormone that are mediated by growth hormone receptors.

137. The conjugate of claim 109, wherein said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, is selected from the group consisting of nonglycosylated erythropoietin and erythropoietin analogs that mimic or antagonize the biological effects of erythropoietin that are mediated by erythropoietin receptors.

138. The conjugate of claim 109, wherein the coupling of said polymer to said cytokine, chemokine, growth factor or polypeptide hormone, or antagonist thereof, at or near said glycosylation site mimics the beneficial effects of glycosylation or hyperglycosylation of said cytokine, chemokine, growth factor or polypeptide hormone.

139. A pharmaceutical composition comprising the conjugate of claim 109 and a pharmaceutically acceptable carrier or excipient.

142. A kit comprising the conjugate of claim 109.

143. A kit comprising the pharmaceutical composition of claim 139.