Compositions and methods for modifying properties of biologically active polypeptides

• US 20090092582A1
• Filed: 08/15/2008
• Published: 04/09/2009
• Est. Priority Date: 08/15/2007
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Schellenberger, Volker, Wang, Chia-wei, Stemmer, Willem P., Yin, Yong, Bogin, Oren, Geething, Nathan C.
• Granted Patent: US 8,933,197 B2
• Time in Patent Office: Days
• US Class Current: 424/85.5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 15/10 for impotence
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• 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 27/06 Antiglaucoma agents or miotics
• 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 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/24 : of the sex hormones
• 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 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07K 14/00 : Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/535 : Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/56 : IFN-alpha
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 16/40 : against enzymes
• CPC Class Codes: C07K 2317/22 : from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/569 : Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/626 : Diabody or triabody
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The present invention relates to biologically active polypeptides linked to one or more accessory polypeptides. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.

Claims

1. A method of producing a biologically active polypeptide, comprising:

a) providing a polynucleotide sequence coding for a modified polypeptide comprising the biologically active polypeptide linked with an accessory polypeptide such that expression of the modified polypeptide in a host cell yields a higher quantity of soluble form of biologically active polypeptide as compared to expression of the biologically active polypeptide by itself;

b) causing the modified polypeptide to be expressed in said host cell, thereby producing the biologically active polypeptide.

2. The method of claim 1, wherein expression of the modified polypeptide in a host cell yields at least about 2-fold more soluble form of biologically active polypeptide as compared to expression of the biologically active polypeptide by itself.

3. The method of claim 1, wherein expression of the modified polypeptide in a host cell yields at least about 5-fold more soluble form of biologically active polypeptide as compared to expression of the biologically active polypeptide by itself.

4. The method of claim 1, wherein expression of the modified polypeptide in a host cell yields at least about 10-fold more soluble form of biologically active polypeptide as compared to expression of the biologically active polypeptide by itself.

5. The method of claim 1, wherein expression of the modified polypeptide in a host cell yields between about 2-fold to about 10-fold more soluble form of biologically active polypeptide as compared to expression of the biologically active polypeptide by itself.

6. The method of claim 1, wherein the accessory polypeptide comprises more than about 50 aminoacids.

7. The method of claim 1, wherein the accessory polypeptide comprises more than about 100 aminoacids.

8. The method of claim 1, wherein the biologically active polypeptide is selected from the group consisting of human growth hormone (hGH), glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), interferon-alpha, interferon-beta, interferon-gamma, insulin, erythropoietin, tumor necrosis factor-alpha (TFN-alpha), IL-1RA, exenatide, uricase and pramlitide.

9. The method of claim 1, wherein the accessory polypeptide comprises at least 40 contiguous amino acids and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the accessory polypeptide, constitutes more than about 80% of the total amino acids of the accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

10. The method of claim 1, wherein the accessory polypeptide comprises at least 40 contiguous amino acids and wherein the accessory polypeptide has an in vitro serum half-life greater than about 4 hours in a subject, and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the accessory polypeptide, constitutes more than about 80% of the total amino acids of the accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

11. The method of claim 1, wherein at least 50% of all residues of the accessory polypeptide are glycine (G) residues.

12. The method of claim 1, wherein at least 80% of all residues of the accessory polypeptide are selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

13. The method of claim 1, wherein less than 50% of all residues of the accessory polypeptide are glycine (G) residues.

14. The method of claim 1, wherein at least 50% of all residues of the accessory polypeptide are serine (S) residues.

15. The method of claim 1, wherein at least 50% of all residues of the accessory polypeptide are either serine (S) and glycine (G) residues.

16. The method of claim 1, wherein the accessory polypeptide is characterized in that:

(a) it consists of three types of amino acids, and each type being selected from a group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), lysine (K), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and

(b) it comprises ten or more amino acids.

17. The method of claim 16, wherein the accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), proline (P), arginine (R), serine (S), and threonine (T).

18. The method of claim 16, wherein the accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

19. The method of claim 1, wherein the accessory polypeptide is characterized in that:

(a) it consists of two types of amino acids, one of which is glycine (G) and the other type is selected from the group consisting of aspartic acid (D), glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine (A), histidine (H), asparagine (N), tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I), and cysteine (C); and

(b) it comprises ten or more amino acid residues, of which 50% or less are glycine.

20. The method of claim 1, wherein the accessory polypeptide is characterized in that:

(a) it comprises 50 or more amino acids;

(b) it consists of two types of amino acids, and

(c) 50% or less of the total amino acids are selected from the group consisting of alanine (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), and histidines (H).

21. The method of claim 1, wherein the accessory polypeptide comprises ten or more repeating motifs.

22. The method of claim 1, comprising multiple accessory polypeptides.

23. A host cell expressing the modified polypeptide of claim 1.

24. A genetic vehicle comprising the polynucleotide sequence of claim 1.

25. The method of claim 1, wherein the accessory polypeptide consists of at most 10 copies of a repeat sequence, wherein each of the repeat sequence has about 8 to about 12 amino acids.

26. The method of claim 1, wherein the modified polypeptide is linked via a protease cleavage site to the accessory protein.

27. The method of claim 25, wherein the protease cleavage site is not part of the sequence coding for the biologically active polypeptide.

28. The method of claim 26, wherein the protease cleavage site is chosen from the group consisting of TEV protease, enterokinase, Factor Xa, thrombin, PreScission™

protease, 3C protease, sortase A, and granzyme B.

29. The method of claim 1, wherein the accessory polypeptide comprises an affinity tag.

30. The method of claim 28, wherein the affinity tag is chosen from the group consisting of FLAG, His-tag, CBD, GST, MBP and TRX.

31. The method of claim 1, wherein the host cell is a prokaryotic cell.

32. The method of claim 1, wherein the host cell is E. Coli.

33. The method of claim 1, wherein host cell is a eukaryotic cell.

34. The method of claim 1, wherein the host cell is selected from the group consisting of a mammalian cell and a yeast cell.

53. The method of claim 1, wherein the accessory polypeptide provides a net positive charge of the modified polypeptide about +0.1 or above.

54. The method of claim 1, wherein the accessory polypeptide provides a net negative charge of the modified polypeptide about −

0.1 or lower.

35. A composition comprising soluble form of a biologically active polypeptide linked with an accessory polypeptide, wherein said accessory polypeptide when linked with the biologically active polypeptide increases solubility of the biologically active polypeptide in a cytosolic fraction of a host cell in which the linked biologically active polypeptide is expressed.

36. The composition of claim 35, wherein the biologically active polypeptide is linked via a protease cleavage site to the accessory polypeptide.

37. The composition of claim 35, wherein the biologically active polypeptide is selected from the group consisting of human growth hormone (hGH), glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), interferon-alpha, interferon-beta, interferon-gamma, insulin, erythropoietin, tumor necrosis factor-alpha (TFN-alpha), IL-1RA, exenatide, uricase and pramlitide.

38. The composition of claim 35, wherein the accessory polypeptide comprises at least 40 contiguous amino acids and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the accessory polypeptide, constitutes more than about 80% of the total amino acids of the accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

39. The composition of claim 35, wherein the accessory polypeptide comprises at least 40 contiguous amino acids and wherein the accessory polypeptide has an in vitro serum half-life greater than about 4 hours in a subject, and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the accessory polypeptide, constitutes more than about 80% of the total amino acids of the accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

40. The composition of claim 35, wherein at least 50% of all residues of the accessory polypeptide are glycine (G) residues.

41. The composition of claim 35, wherein at least 80% of all residues of the accessory polypeptide are selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

42. The composition of claim 35, wherein less than 50% of all residues of the accessory polypeptide are glycine (G) residues.

43. The composition of claim 35, wherein at least 50% of all residues of the accessory polypeptide are serine (S) residues.

44. The composition of claim 35, wherein at least 50% of all residues of the accessory polypeptide are either serine (S) and glycine (G) residues.

45. The composition of claim 35, wherein the accessory polypeptide is characterized in that:

(a) it consists of three types of amino acids, and each type being selected from a group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), lysine (K), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and

(b) it comprises ten or more amino acids.

46. The composition of claim 45, wherein the accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), proline (P), arginine (R), serine (S), and threonine (T).

47. The composition of claim 45, wherein the accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

48. The composition of claim 35, wherein the accessory polypeptide is characterized in that:

(a) it consists of two types of amino acids, one of which is glycine (G) and the other type is selected from the group consisting of aspartic acid (D), glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine (A), histidine (H), asparagine (N), tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I), and cysteine (C); and

(b) it comprises ten or more amino acid residues, of which 50% or less are glycine.

49. The composition of claim 35, wherein the accessory polypeptide is characterized in that:

(a) it comprises 50 or more amino acids;

(b) it consists of two types of amino acids, and

(c) 50% or less of the total amino acids are selected from the group consisting of alanine (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), and histidine (H).

50. The composition of claim 35, wherein the accessory polypeptide comprises ten or more repeating motifs.

51. The composition of claim 35, wherein the accessory polypeptide provides a net positive charge of the linked biologically active polypeptide of about +0.1 or above.

52. The composition of claim 35, wherein the accessory polypeptide provides a net negative charge of the linked biologically active polypeptide of about −

0.1 or lower.

55. A pharmaceutical composition comprising

a) a slow release agent,

b) a modified polypeptide comprising a biologically active polypeptide linked to an accessory polypeptide, wherein said accessory polypeptide increases the protease resistance of said biologically active polypeptide.

56. The pharmaceutical composition of claim 55, wherein said modified polypeptide yields an apparent molecular weight factor of greater than 3, and further wherein said apparent molecular weight factor is determined as a ratio of an apparent molecular weight of the modified polypeptide as measured by size exclusion chromatography relative to a predicted molecular weight of the modified polypeptide.

57. The composition of claim 55, wherein the apparent molecular weight factor of said modified polypeptide is greater than 5.

58. The composition of claim 55, wherein the apparent molecular weight factor of said modified polypeptide is greater than 7.

59. The composition of claim 55, wherein the apparent molecular weight factor of said modified polypeptide is greater than 9.

60. The composition of claim 55, wherein said accessory polypeptide comprises more than about 50 aminoacids.

61. The composition of claim 55, wherein said accessory polypeptide comprises more than about 100 aminoacids.

62. The composition of claim 55, wherein the biologically active polypeptide is selected from the group consisting of human growth hormone (hGH), glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), interferon-alpha, interferon-beta, interferon-gamma, insulin, erythropoietin, tumor necrosis factor-alpha (TFN-alpha), IL-1RA, exenatide, uricase and pramlitide.

63. The composition of claim 55, wherein said accessory polypeptide comprises at least 40 contiguous amino acids and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in said accessory polypeptide, constitutes more than about 80% of the total amino acids of said accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

64. The composition of claim 55, wherein said accessory polypeptide comprises at least 40 contiguous amino acids and wherein said accessory polypeptide has an in vitro serum half-life greater than about 24 hours in a subject, and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in said accessory polypeptide, constitutes more than about 80% of the total amino acids of said accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

65. The composition of claim 55, wherein at least 50% of all residues of said accessory polypeptide are glycine (G) residues.

66. The composition of claim 55, wherein at least 80% of all residues of said accessory polypeptide are selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

67. The composition of claim 55, wherein less than 50% of all residues of said accessory polypeptide are glycine (G) residues.

68. The composition of claim 55, wherein at least 50% of all residues of said accessory polypeptide are serine (S) residues.

69. The composition of claim 55, wherein at least 50% of all residues of said accessory polypeptide are either serine (S) and glycine (G) residues.

70. The composition of claim 55, wherein said accessory polypeptide is characterized in that:

(a) it consists of three types of amino acids, and each type being selected from a group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), lysine (K), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and

(b) it comprises ten or more amino acids.

71. The composition of claim 70, wherein said accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), proline (P), arginine (R), serine (S), and threonine (T).

72. The composition of claim 70, wherein said accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

73. The composition of claim 55, wherein the accessory polypeptide is characterized in that:

(a) it consists of two types of amino acids, one of which is glycine (G) and the other type is selected from the group consisting of aspartic acid (D), glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine (A), histidine (H), asparagine (N), tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I), and cysteine (C); and

(b) it comprises ten or more amino acid residues, of which 50% or less are glycine.

74. The composition of claim 55, wherein the accessory polypeptide is characterized in that:

(a) it comprises 50 or more amino acids;

(b) it consists of two types of amino acids, and

(c) 50% or less of the total amino acids are selected from the group consisting of alanine (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), and histidine (H).

75. The composition of claim 55, wherein said accessory polypeptide comprises ten or more repeating motifs.

76. The composition of claim 55, comprising multiple accessory polypeptides.

77. The composition of claim 55, wherein said slow release agent is a polymeric matrix.

78. The composition of claim 77, wherein said polymeric matrix is selected from the group consisting of poly-d,l-lactide (PLA), poly-(d,l-lactide-co-glycolide) (PLGA), PLGA-PEG copolymers, alginate, dextran and chitosan.

79. The composition of claim 55, wherein said slow release agent is a transdermal patch.

80. The composition of claim 55, wherein said accessory polypeptide comprises charged aminoacids.

81. The composition of claim 55, wherein said accessory polypeptide comprises charged aminoacids, and further wherein the slow release agent comprises a charged polymeric matrix.

82. A method of producing modified polypeptide, comprising:

(a) providing a polynucleotide sequence encoding the modified polypeptide of claim 55;

(b) causing said modified polypeptide to be expressed in a host cell, thereby producing said modified polypeptide.

83. A genetic vehicle comprising a nucleic acid sequence encoding the modified polypeptide of claim 55.

84. A host cell expressing the modified polypeptide of claim 55.

85. A method of making a controlled release composition, said method comprising:

(a) providing the modified polypeptide of claim 55;

(b) mixing said modified polypeptide with a polymer matrix.

86. A pharmaceutical composition comprising

a) a slow release agent,

b) a modified polypeptide comprising a biologically active polypeptide linked to an accessory polypeptide, wherein said accessory polypeptide increases the solubility of said biologically active polypeptide.

87. The pharmaceutical composition of claim 86, wherein said modified polypeptide yields an apparent molecular weight factor of greater than 3, and further wherein said apparent molecular weight factor is determined as a ratio of an apparent molecular weight of the modified polypeptide as measured by size exclusion chromatography relative to a predicted molecular weight of the modified polypeptide.

88. The composition of claim 86, wherein the apparent molecular weight factor of said modified polypeptide is greater than 5.

89. The composition of claim 86, wherein the apparent molecular weight factor of said modified polypeptide is greater than 7.

90. The composition of claim 86, wherein the apparent molecular weight factor of said modified polypeptide is greater than 9.

91. The composition of claim 86, wherein said accessory polypeptide comprises more than about 50 aminoacids.

92. The composition of claim 86, wherein said accessory polypeptide comprises more than about 100 aminoacids.

93. The composition of claim 86, wherein the biologically active polypeptide is selected from the group consisting of human growth hormone (hGH), glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), interferon-alpha, interferon-beta, interferon-gamma, insulin, erythropoietin, tumor necrosis factor-alpha (TFN-alpha), IL-1RA, exenatide, uricase and pramlitide.

94. The composition of claim 86, wherein said accessory polypeptide comprises at least 40 contiguous amino acids and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in said accessory polypeptide, constitutes more than about 80% of the total amino acids of said accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

95. The composition of claim 86, wherein said accessory polypeptide comprises at least 40 contiguous amino acids and wherein said accessory polypeptide has an in vitro serum half-life greater than about 24 hours in a subject, and further wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in said accessory polypeptide, constitutes more than about 80% of the total amino acids of said accessory polypeptide;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

96. The composition of claim 86, wherein at least 50% of all residues of said accessory polypeptide are glycine (G) residues.

97. The composition of claim 86, wherein at least 80% of all residues of said accessory polypeptide are selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

98. The composition of claim 86, wherein less than 50% of all residues of said accessory polypeptide are glycine (G) residues.

99. The composition of claim 86, wherein at least 50% of all residues of said accessory polypeptide are serine (S) residues.

100. The composition of claim 86, wherein at least 50% of all residues of said accessory polypeptide are either serine (S) and glycine (G) residues.

101. The composition of claim 86, wherein said accessory polypeptide is characterized in that:

(a) it consists of three types of amino acids, and each type being selected from a group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), lysine (K), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and

(b) it comprises ten or more amino acids.

102. The composition of claim 101, wherein said accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), proline (P), arginine (R), serine (S), and threonine (T).

103. The composition of claim 101, wherein said accessory polypeptide consists of three types of amino acids, each type being selected from the group consisting of glutamic acid (E), glycine (G) and serine (S).

104. The composition of claim 86, wherein the accessory polypeptide is characterized in that:

(a) it consists of two types of amino acids, one of which is glycine (G) and the other type is selected from the group consisting of aspartic acid (D), glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine (A), histidine (H), asparagine (N), tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I), and cysteine (C); and

(b) it comprises ten or more amino acid residues, of which 50% or less are glycine.

105. The composition of claim 86, wherein the accessory polypeptide is characterized in that:

(a) it comprises 50 or more amino acids;

(b) it consists of two types of amino acids, and

(c) 50% or less of the total amino acids are selected from the group consisting of alanine (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), and histidine (H).

106. The composition of claim 86, wherein said accessory polypeptide comprises ten or more repeating motifs.

107. The composition of claim 86, comprising multiple accessory polypeptides.

108. The composition of claim 86, wherein said slow release agent is a polymeric matrix.

109. The composition of claim 108, wherein said polymeric matrix is selected from the group consisting of poly-d,l-lactide (PLA), poly-(d,l-lactide-co-glycolide) (PLGA), PLGA-PEG copolymers, alginate, dextran and chitosan.

110. The composition of claim 86, wherein said slow release agent is a transdermal patch.

111. The composition of claim 86, wherein said accessory polypeptide comprises charged aminoacids.

112. The composition of claim 86, wherein said accessory polypeptide comprises charged aminoacids, and further wherein the slow release agent comprises a charged polymeric matrix.

113. A method of producing modified polypeptide, comprising:

(a) providing a polynucleotide sequence encoding the modified polypeptide of claim 86;

(b) causing said modified polypeptide to be expressed in a host cell, thereby producing said modified polypeptide.

114. A genetic vehicle comprising a nucleic acid sequence encoding the modified polypeptide of claim 86.

115. A host cell expressing the modified polypeptide of claim 86.

116. A method of making a controlled release composition, said method comprising:

(a) providing the modified polypeptide of claim 86;

(b) mixing said modified polypeptide with a polymer matrix.

117. An isolated polypeptide comprising a biologically active polypeptide and an accessory polypeptide, wherein the accessory polypeptide is characterized in that:

(i) it consists of three types of amino acids, and each type being selected from a group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), histidine (H), lysine (K), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and

(ii) it comprises ten or more amino acids.

118. The isolated polypeptide of claim 117, wherein the accessory polypeptide consists of three types of amino acids, and each type being selected from a group consisting of D, E, G, K, P, R, S, and T.

119. The isolated polypeptide of claim 117, wherein the accessory polypeptide consists of three types of amino acids, and each type being selected from a group consisting of E, S, G, R, and A.

120. The isolated polypeptide of claim 117, wherein the biologically active polypeptide is a therapeutic polypeptide.

121. The isolated polypeptide of claim 117, wherein the accessory polypeptide consists of three types of amino acids, and each type being selected from a group consisting of E, G, and S.

126. The isolated polypeptide of any one of claims 121, 122, and 123, wherein the two different types of amino acids are represented in 1:

2 ratio.

127. The isolated polypeptide of any one of claims 121, 122, and 123, wherein the two different types of amino acids are represented in 2:

3 ratio.

128. The isolated polypeptide of any one of claims 121, 122, and 123, wherein the two different types of amino acids are represented in 3:

4 ratio.

129. The isolated polypeptide of claim 128, wherein the accessory polypeptide comprises four or more repeating motifs, each of which comprises two to five hundred amino acids and is made of two different types of amino acids.

130. The isolated polypeptide of claim 128, wherein the repeating motif comprises more than 8 amino acids.

131. The isolated polypeptide of any one of claim 128, wherein the four or more repeating motifs are identical.

132. The isolated polypeptide of any one of claim 128, wherein the four or more repeating motifs comprise different amino acid sequences.

133. The isolated polypeptide of claim 128, wherein the accessory polypeptide comprises at least ten repeating motifs.

134. The isolated polypeptide of claim 128, wherein the accessory polypeptide substantially lacks secondary structure.

135. The isolated polypeptide of claim 128, wherein the apparent molecular weight of the isolated polypeptide is greater than that of a corresponding polypeptide lacking the accessory polypeptide.

136. The isolated polypeptide of claim 128, wherein the apparent molecular weight of the accessory polypeptide is at least 3 times greater than its actual molecular weight.

137. The isolated polypeptide of claim 128, exhibiting a two-fold longer serum half-life as compared to a corresponding polypeptide lacking the accessory polypeptide.

138. The isolated polypeptide of claim 128, wherein the biologically active polypeptide and the accessory polypeptide are linked via a peptide bond.

139. The isolated polypeptide of any one of claim 117, wherein the accessory polypeptide comprises an amino acid sequence (GGEGGS)n (SEQ ID NO:

5), wherein n is an integer of 3 or greater.

140. The isolated polypeptide of any one of claim 117, wherein the accessory polypeptide comprises an amino acid sequence (GES)n, wherein G, E, and S can be in any order and n is an integer of 3 or greater.

141. The isolated polypeptide of any one of claim 117, wherein the accessory polypeptide comprises an amino acid sequence (GGSGGE)n, wherein G, E, and S can be in any order and n is an integer of 3 or greater.

142. The isolated polypeptide of any one of claim 117, wherein the accessory polypeptide comprises an amino acid sequence (GGEGGEGGES)n (SEQ ID NO:

6), wherein n is an integer of 1 or greater.

143. A method of producing an isolated polypeptide, comprising:

a) providing a polynucleotide sequence encoding the isolated polypeptide of any one of claims 117, 121, 122, 123, or 124;

b) causing said polypeptide to be expressed in a host cell, thereby producing said polypeptide.

144. A genetic vehicle comprising a nucleic acid sequence encoding the isolated polypeptide of any one of claims 117, 121, 122, 123, or 124.

145. A host cell expressing the isolated polypeptide of any one of claims 117, 121, 122, 123, or 124.

146. A library of the isolated polypeptide of any one of claims 117, 121, 122, 123, or 124.

147. The library of claim 145 displayed on phage particles.

122. An isolated polypeptide comprising a biologically active polypeptide and an accessory polypeptide, wherein the accessory polypeptide is characterized in that:

(i) it consists of two types of amino acids, one of which is glycine (G) and the other type is selected from the group consisting of aspartic acid (D), glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine (A), histidine (H), asparagine (N), tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I), and cysteine (C); and

(ii) it comprises ten or more amino acid residues, of which 50% or less are glycine.

123. An isolated polypeptide comprising a biologically active polypeptide and an accessory polypeptide, wherein the accessory polypeptide is characterized in that:

(i) it comprises ten or more amino acids;

(ii) it consists of two types of amino acids, 50% or less of the total amino acids are selected from the group consisting of A;

S, T, D, E, and H.

124. An isolated polypeptide comprising a biologically active polypeptide and an accessory polypeptide, wherein the accessory polypeptide is characterized in that:

(i) it consists of two types of amino acids, one of which is selected from the group consisting of P, R, L, V, Y, W, M, F, I, K, and C; and

(ii) it comprises ten or more amino acids.

125. An isolated polypeptide comprising a biologically active polypeptide and an accessory polypeptide, wherein the accessory polypeptide comprises at least 10 amino acids in length and consists of two different types of amino acids represented in equal numbers.

148. A pharmaceutical composition comprising

a) a slow release agent,

b) a modified polypeptide comprising a modified polypeptide comprising a biologically active polypeptide linked to a PEG group of greater than 5 kD in size.