Erythropoietin and albumin fusion protein, nucleic acids, and methods thereof
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.
1. A nucleic acid molecule comprising a polynucleotide encoding a fusion protein wherein said fusion protein comprises erythropoietin and albumin or an albumin variant, wherein (i) said fusion protein has a higher plasma stability than unfused erythropoietin, (ii) said fusion protein retains the therapeutic activity of unfused erythropoietin, and (iii) said albumin or albumin variant is located either at the N-terminus or C-terminus of said fusion protein.
2. The nucleic acid molecule of claim 1 , wherein said fusion protein comprises albumin.
3. The nucleic acid molecule of claim 1 , wherein said fusion protein comprises an albumin variant.
4. The nucleic acid molecule of claim 3 , wherein said albumin variant is a fragment of albumin.
5. The nucleic acid molecule of claim 3 , wherein said albumin variant has a mutation of one or more residues.
6. The nucleic acid molecule of claim 3 , wherein said albumin variant has a deletion of one of more residues.
7. The nucleic acid molecule of claim 3 , wherein said albumin variant has a mutation and a deletion of one or more residues.
8. The nucleic acid molecule of claim 3 , wherein said albumin variant has an addition of one or more residues.
9. The nucleic acid molecule of claim 1 , wherein said fusion protein comprises an N-terminal methionine.
10. The nucleic acid molecule of claim 1 , wherein said fusion protein comprises a peptide linker.
11. The nucleic acid molecule of claim 1 , wherein said fusion protein comprises a secretion signal sequence.
12. The nucleic acid molecule of claim 11 , wherein said secretion signal sequence is the natural leader sequence of erythropoietin.
13. The nucleic acid molecule of claim 1 , wherein said erythropoietin is fused to the N-terminal end of said albumin or albumin variant.
14. The nucleic acid molecule of claim 1 , wherein said erythropoietin is fused to the C-terminal end of said albumin or albumin variant.
15. The nucleic acid molecule of claim 1 , which further comprises a heterologous polynucleotide.
16. The nucleic acid molecule of claim 15 , wherein said heterologous polynucleotide is a vector sequence.
17. The nucleic acid molecule of claim 15 , wherein said heterologous polynucleotide is a promoter sequence.
18. The nucleic acid molecule of claim 17 , wherein said promoter sequence is a promoter selected from:
(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 phosphatase (PHO5) 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.
19. The nucleic acid molecule of claim 15 , wherein said heterologous polynucleotide is a selectable marker.
20. The nucleic acid molecule of claim 19 , wherein said selectable marker is a selectable marker selected from:
(a) the URA3 gene;
(b) geneticin resistance;
(c) metal iron resistance; and
(d) ampicillin resistance.
21. The nucleic acid molecule of claim 15 , wherein said heterologous polynucleotide is a region for termination of transcription.
22. A vector comprising the nucleic acid molecule of claim 1 .
23. An isolated host cell comprising the vector of claim 22 .
24. An isolated host cell comprising the nucleic acid molecule of claim 1 .
25. The isolated host cell of either claim 23 or 24 , wherein said isolated host cell is prokaryotic or eukaryotic.
26. The isolated host cell of claim 25 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
27. The isolated host cell of either claim 23 or 24 , wherein said isolated host cell is an animal cell.
28. The isolated host cell of claim 27 , wherein said animal cell is a CHO cell.
29. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of either claim 23 or 24 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
30. An isolated host cell comprising the nucleic acid molecule of claim 13 .
31. The isolated host cell of claim 30 , wherein said isolated host cell is prokaryotic or eukaryotic.
32. The isolated host cell of claim 31 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
33. The isolated host cell of claim 30 , wherein said isolated host cell is an animal cell.
34. The isolated host cell of claim 33 , wherein said animal cell is a CHO cell.
35. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of claim 30 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
36. An isolated host cell comprising the nucleic acid molecule of claim 14 .
37. The isolated host cell of claim 36 , wherein said isolated host cell is prokaryotic or eukaryotic.
38. The isolated host cell of claim 37 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
39. The isolated host cell of claim 36 , wherein said isolated host cell is an animal cell.
40. The isolated host cell of claim 39 , wherein said animal cell is a CHO cell.
41. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of claim 36 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
42. A fusion protein produced by the method of claim 29 .
43. A fusion protein produced by the method of claim 35 .
44. A fusion protein produced by the method of claim 41 .
45. A nucleic acid molecule comprising a polynucleotide encoding a fusion protein wherein said fusion protein comprises erythropoietin and a mature form of albumin, wherein (i) said fusion protein has a higher plasma stability than unfused erythropoietin, (ii) said fusion protein retains the therapeutic activity of unfused erythropoietin, (iii) and said mature form of albumin is located either at the N-terminus or C-terminus of said fusion protein.
46. The nucleic acid molecule of claim 45 , wherein said fusion protein comprises an N-terminal methionine.
47. The nucleic acid molecule of claim 45 , wherein said fusion protein comprises a peptide linker.
48. The nucleic acid molecule of claim 45 , wherein said fusion protein comprises a secretion signal sequence.
49. The nucleic acid molecule of claim 48 , wherein said secretion signal sequence is the natural leader sequence of erythropoietin.
50. The nucleic acid molecule of claim 45 , wherein said erythropoietin is fused to the N-terminal end of said mature form of albumin.
51. The nucleic acid molecule of claim 45 , wherein said erythropoietin is fused to the C-terminal end of said mature form of albumin.
52. The nucleic acid molecule of claim 45 , which further comprises a heterologous polynucleotide.
53. The nucleic acid molecule of claim 52 , wherein said heterologous polynucleotide is a vector sequence.
54. The nucleic acid molecule of claim 52 , wherein said heterologous polynucleotide is a promoter sequence.
55. The nucleic acid molecule of claim 54 , wherein said promoter sequence is a promoter selected from:
(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 phosphatase (PHO5) 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.
56. The nucleic acid molecule of claim 52 , wherein said heterologous polynucleotide is a selectable marker.
57. The nucleic acid molecule of claim 56 , wherein said selectable marker is a selectable marker selected from:
(a) the URA3 gene;
(b) geneticin resistance;
(c) metal iron resistance; and
(d) ampicillin resistance.
58. The nucleic acid molecule of claim 52 , wherein said heterologous polynucleotide is a region for termination of transcription.
59. A vector comprising the nucleic acid molecule of claim 45 .
60. An isolated host cell comprising the nucleic acid molecule of claim 45 .
61. An isolated host cell comprising the vector of claim 59 .
62. The isolated host cell of either claim 60 or 61 , wherein said isolated host cell is prokaryotic or eukaryotic.
63. The isolated host cell of claim 62 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
64. The isolated host cell of either claim 60 or 61 , wherein said isolated host cell is an animal cell.
65. The isolated host cell of claim 64 , wherein said animal cell is a CHO cell.
66. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of either claim 60 or 61 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
67. An isolated host cell comprising the nucleic acid molecule of claim 50 .
68. The isolated host cell of claim 67 wherein said isolated host cell is prokaryotic or eukaryotic.
69. The isolated host cell of claim 68 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
70. The isolated host cell of claim 67 , wherein said isolated host cell is an animal cell.
71. The isolated host cell of claim 70 , wherein said animal cell is a CHO cell.
72. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of claim 67 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
73. An isolated host cell comprising the nucleic acid molecule of claim 51 .
74. The isolated host cell of claim 73 , wherein said isolated host cell is prokaryotic or eukaryotic.
75. The isolated host cell of claim 74 , wherein said isolated host cell is selected from:
(a) a bacteria cell;
(b) a yeast cell;
(c) a SACCHAROMYCES cell;
(d) a COS cell; and
(e) a fungi cell.
76. The isolated host cell of claim 73 , wherein said isolated host cell is an animal cell.
77. The isolated host cell of claim 76 , wherein said animal cell is a CHO cell.
78. A method for producing a fusion protein comprising:
(a) culturing the isolated host cell of claim 73 under conditions suitable to produce the fusion protein encoded by said polynucleotide; and
(b) recovering said fusion protein.
79. A fusion protein produced by the method of claim 66 .
80. A fusion protein produced by the method of claim 72 .
81. A fusion protein produced by the method of claim 78 .