Fusion polypeptides of human serum albumin and a therapeutically active polypeptide

• US 20030082747A1
• Filed: 09/10/2002
• Published: 05/01/2003
• Est. Priority Date: 01/31/1992
• Status: Active Grant

• Current Assignee: Novozymes Biopharma DK A/S (Novozymes)
• Original Assignee: Aventis Behring LLC (Sanofi )
• Inventors: Fournier, Alain, Jung, Gerard, Guitton, Jean-Dominique, Yeh, Patrice, Fleer, Reinhard
• Granted Patent: US 7,056,701 B2
• Time in Patent Office: Days
• US Class Current: 435/69.5
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/21 Interferons [IFN]
• CPC Class Codes: A61K 38/38 Albumins
• CPC Class Codes: A61K 47/642 the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: B65B 69/00 Unpacking of articles or materials, not otherwise provided for
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 14/76 Albumins
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 9/6456 Plasminogen activators

Abstract

Biologically active polypeptides comprising a therapeutically active polypeptide fused to human serum albumin or a variant thereof, methods for the preparation thereof, nucleotide sequences encoding such fusion polypeptides, expression cassettes comprising such nucleotide sequences, self-replicating plasmids containing such expression cassettes, and pharmaceutical compositions containing said fusion polypeptides.

Claims

1. Recombinant polypeptide containing an active part derived from a polypeptide having a therapeutic activity genetically coupled to an albumin or to a variant of albumin.

2. Polypeptide according to claim 1, characterized in that the polypeptide having a therapeutic activity is a polypeptide of human origin.

3. Polypeptide according to claim 2, characterized in that the polypeptide having a therapeutic activity is chosen from all or part of the enzymes, enzyme inhibitors, antigens, antibodies, hormones, coagulation factors, interferone, cytokines, growth and/or differentiation factors, factors involved in the genesis/resorption of bone tissues, chemotactic factors, cell motility or migration factors, cytostatic factors bactericidal or antifungal factors, or plasma or interstitial adhesive molecules or extracellular matrices.

4. Polypeptide according to one of claims 1 to 3, characterized in that the polypeptide having a therapeutic activity is chosen from any peptide sequence which is an agonist or antagonist of molecular and/or cellular interactions involved in the pathologies of the circulatory and interstitial compartments.

5. Polypeptide according to one of claims 1 to 4, characterized in that the active part has a structure chosen from:

(a) the entire peptide structure or

(b) a fragment of (a) or a structure derived from (a) by structural modification (mutation, substitution, addition and/or deletion of one or more residues) and preserving a therapeutic activity.

6. polypeptide according to one of claims 1 to 5, characterized in that the active part is coupled to the N-terminal end of albumin.

7. Polypeptide according to one of claims 1 to 5, characterized in that the active part is coupled to the C-terminal end of albumin.

8. Polypeptide according to one of claims 1 to 7, characterized in that the active part is represented therein several times.

9. Nucleotide sequence encoding a polypeptide according to any one of claims 1 to 8.

10. Nucleotide sequence according to claim 9, characterized in that it comprises a “

leader”

sequence permitting the secretion of the expressed polypeptide.

11. Expression cassette comprising a nucleotide sequence according to one of claims 9 or 10 under the control of a region for initiation of transcription and optionally a region for termination of transcription.

12. Self-replicating plasmid containing an expression cassette according to claim 11.

13. Eukaryotic or prokaryotic recombinant cell in which a nucleotide sequence according to one of claims 9 or 10 or an expression cassette according to claim 11 or a plasmid according to claim 12 has been inserted.

14. Recombinant cell according to claim 13, characterized in that it is a yeast, an animal cell, a fungus or a bacterium.

15. Recombinant cell according to claim 14, characterized in that it is a yeast.

16. Recombinant cell according to claim 15, characterized in that it is a yeast of the genus Saccharomyces or Kluyveromyces.

17. Process for preparing a polypeptide as defined in one of claims 1 to 8, characterized in that a recombinant cell according to one of claims 13 to 16 is cultured under conditions for expression, and the polypeptide produced is recovered.

18. Pharmaceutical composition comprising one or more polypeptides according to any one of claims 1 to 8.

19. Pharmaceutical composition comprising a nucleotide sequence according to any one of claims 9 to 11, which can be used in gene therapy.

20. A fusion protein comprising hormone and albumin or an albumin variant, wherein said albumin variant has a high plasma half-life.

21. The fusion protein of claim 20, comprising albumin.

22. The fusion protein of claim 20, comprising an albumin variant.

23. The fusion protein of claim 22, wherein said albumin variant is a fragment of albumin.

24. The fusion protein of claim 22, wherein said albumin variant is a mature form of albumin.

25. The fusion protein of claim 22, wherein said albumin variant has a mutation of one or more residues.

26. The fusion protein of claim 22, wherein said albumin variant has a deletion of one or more residues.

27. The fusion protein of claim 22, wherein said albumin variant has a mutation and deletion of one or more residues.

28. The fusion protein of claim 22, wherein said albumin variant has an addition of one or more residues.

29. The fusion protein of claim 20, wherein said fusion protein comprises an N-terminal Methionine.

30. The fusion protein of claim 20, wherein said fusion protein comprises a peptide linker.

31. The fusion protein of claim 20, wherein said fusion protein comprises a secretion signal sequence.

32. The fusion protein of claim 31, wherein said secretion signal sequence is the natural leader sequence of hormone.

33. The fusion protein of claim 20, wherein said hormone is fused to the N-terminal end of said albumin or albumin variant.

34. The fusion protein of claim 33, which comprises a polypeptide having therapeutic activity fused to the C-terminal end of said albumin or albumin variant.

35. The fusion protein of claim 34, wherein said polypeptide having therapeutic activity is hormone.

36. The fusion protein of claim 20, wherein said hormone is fused to the C-terminal end of said albumin or albumin variant.

37. The fusion protein of claim 36, which comprises a polypeptide having therapeutic activity fused to the N-terminal end of said albumin or albumin variant.

38. The fusion protein of claim 20, wherein said fusion protein is expressed by a prokaryotic cell.

39. The fusion protein of claim 38, wherein said fusion protein is expressed by a bacteria.

40. The fusion protein of claim 20, wherein said fusion protein is expressed by a eukaryotic cell.

41. The fusion protein of claim 40, wherein said fusion protein is expressed by an animal cell.

42. The fusion protein of claim 41, wherein said animal cell is a CHO cell.

43. The fusion protein of claim 41, wherein said animal cell is a COS cell.

44. The fusion protein of claim 20, wherein said fusion protein is expressed by a yeast.

45. The fusion protein of claim 44, wherein said yeast is Saccharomyces.

46. The fusion protein of claim 40, wherein said fusion protein is expressed by a fungi.

47. A nucleic acid molecule comprising a polynucleotide encoding the fusion protein of claim 20.

48. A nucleic acid molecule of claim 47, which comprises a heterologous polynucleotide.

49. The nucleic acid molecule of claim 48, wherein said heterologous polynucleotide is a vector sequence.

50. The nucleic acid molecule of claim 49, wherein said heterologous polynucleotide is a promoter sequence.

51. The nucleic acid molecule of claim 50, wherein said promoter sequence is any one selected from the group:

a. a hybrid promoter;

b. a constitutive promoter;

c. a regulatable promoter;

d. a yeast phosphoglycerate kinase (PGK) promoter;

e. a yeast glyceraldehyde-3-phosphate dehydrogenase (GDP) promoter;

f. a yeast lactase (LAC4) promoter;

g. a yeast enolase (ENO) promoter;

h. a yeast alcohol dehydrogenase (ADH) promoter;

i. a yeast acid phosphatase (PHOS) promoter;

j. a lambda bacteriophage P_L promoter;

k. a lambda bacteriophage P_R promoter;

l. a tryptophan P_trp promoter; and

m. a lactose P_lac promoter.

52. The nucleic acid molecule of claim 48, wherein said heterologous polynucleotide is a selectable marker.

53. The nucleic acid molecule of claim 52, wherein said selectable marker is any one selected from the group:

a. the URA3 gene;

b. geneticin resistance;

c. metal ion resistance; and

d. ampicillin resistance.

54. The nucleic acid molecule of claim 48, wherein said heterologous polynucleotide is a region for termination of transcription.

55. A host cell comprising the nucleic acid molecule of claim 47.

56. A host cell comprising the nucleic acid molecule of claim 48.

57. A method for producing a fusion protein, comprising:

a. culturing the host cell of claim 55 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and

b. recovering said fusion protein.

58. The method of claim 57, wherein the host cell is a CHO cell.

59. A method for producing a fusion protein, comprising:

a. culturing the host cell of claim 56 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and

b. recovering said fusion protein.

60. The method of claim 59, wherein the host cell is a CHO cell.

61. A fusion protein produced by the method of claim 57.

62. A fusion protein produced by the method of claim 58.

63. A fusion protein produced by the method of claim 59.

64. A fusion protein produced by the method of claim 60.

65. A method of treating a disease or disorder in a patient, comprising the step of administering the fusion protein of claim 20.

66. A composition comprising one or more fusion proteins of claim 20.