Method for the production of fusion proteins in transgenic mammal milk

• US 20060105347A1
• Filed: 09/03/2004
• Published: 05/18/2006
• Est. Priority Date: 09/05/2003
• Status: Active Grant

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Cox, Geoffrey F., Meade, Harry
• Granted Patent: US 7,550,263 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• 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 19/10 for osteoporosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/40 : being an insulator
• CPC Class Codes: C12N 2830/85 : mammalian

Abstract

Desirable fusion proteins can be produced in and purified from the milk of transgenic animals. The peptides are made as fusion proteins with a suitable fusion partner such as human alpha-fetoprotein. The fusion partner protein acts to promote and increase the half-life of the overall molecule as well as having therapeutic effects on its own. The fusion protein is typically produced through the use of transgenic animals and can be purified away from the now the milk or other bodily fluid of such an animal by an affinity purification method. A particular advantage of producing peptides via this route, in addition to the obvious advantages of high yield and biocompatibility, is that specific post-translational modifications, such as carboxy terminal amidation, can be performed in the mammary gland. Biologically active polypeptides comprising a therapeutically active polypeptide fused to human alpha-fetoprotein fragment 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. A bifunctional fusion protein, encoded by a transgene DNA construct comprising a first polypeptide domain which has a desired bioactivity, and a second polypeptide domain which has a desired bioactivity, together comprising said fusion protein wherein each said first and said second polypeptide domains retain their desired bioactivity wherein the encoded polypeptide of said first polypeptide domain is human alpha-fetoprotein or a fragment thereof having the biological activity of human alpha-fetoprotein.

2. The fusion protein of claim 1, wherein said fusion protein is the product of a contiguous coding sequence of DNA.

3. The fusion protein of claim 1, wherein the encoded mammalian polypeptide of said second polypeptide domain is human IFN-β

or a fragment thereof having the biological activity of IFN-β

.

4. The fusion protein of claim 2, wherein said second polypeptide domain is selected from a group consisting of:

antithrombin III, lactoferrin, ferritin, calcitonin, urokinase, Platelet Factor 4, alpha-fetoprotein, alpha-1-antitrypsin, C-1 esterase inhibitor, decorin, prolactin, tau interferon, alpha interferon, ferritin, prolactin, CFTR, Muellerian Inhibitory Substance, blood Factor X, blood Factor VIII, truncated ATIII, and erthyropoietin.

5. The fusion protein of claim 1 further comprising a glutathione S-transferase polypeptide sequence.

22. A recombinant DNA vector comprising the nucleic acid sequence of the fusion protein of claim 1.

23. A host cell transformed with said recombinant DNA vector of claim 22.

24. A recombinant DNA vector comprising the nucleic acid sequence of the fusion protein of claim 1 wherein said vector is an expression vector comprising a promoter operably linked to a fusion protein of claim 1.

25. The method of claim 1 wherein said DNA construct encoding a desired fusion protein is actuated by at least one beta-casein promoter.

26. A fusion protein produced by a method comprising:

(a) expressing the fusion protein of claim 1 by a cell; and

(b) recovering the protein.

27. The fusion protein of claim 26, wherein said fusion protein further comprises an N-terminal methionine.

39. The fusion protein of claim 26, wherein said fusion protein is expressed by a prokaryotic cell.

40. The fusion protein of claim 26, wherein said fusion protein is expressed by a bacteria.

41. The fusion protein of claim 26, wherein said fusion protein is expressed by a eukaryotic cell.

42. The fusion protein of claim 26, wherein said fusion protein is expressed by an animal cell.

43. The fusion protein of claim 26, wherein said animal cell is a CHO cell.

44. The fusion protein of claim 26, wherein said animal cell is a COS cell.

45. The fusion protein of claim 26, wherein said fusion protein is expressed by a yeast.

46. The fusion protein of claim 26, wherein said yeast is Saccharomyces.

47. A fusion protein produced by a method comprising:

(a) expressing the fusion protein of claim 1 by a transgenic animal; and

(b) recovering the protein.

69. The fusion protein of claim 3 wherein said human alpha-fetoprotein or an human alpha-fetoprotein variant has a high plasma half-life

70. The fusion protein of claim 69, wherein said human alpha-fetoprotein variant has a mutation of one or more residues.

71. The fusion protein of claim 69, wherein said human alpha-fetoprotein variant has a deletion of one or more residues.

73. The fusion protein of claim 69, wherein said human alpha-fetoprotein variant has an addition of one or more residues.

74. The fusion protein of claim 69, wherein said fusion protein further comprises a peptide linker.

75. The fusion protein of claim 69, wherein said fusion protein comprises a secretion signal sequence.

76. The fusion protein of claim 69, wherein said human IFN-β

is fused to the N-terminal end of said human alpha-fetoprotein or human alpha-fetoprotein variant.

77. The fusion protein of claim 69, wherein said human IFN-β

is fused to the C-terminal end of said human alpha-fetoprotein or human alpha-fetoprotein variant.

6. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

10.

7. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

11.

8. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

12.

9. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

13.

10. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

14.

79. The isolated nucleic acid sequences as shown in claims 6-21 further comprising a amino acid linking sequence.

11. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

15.

12. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

16.

13. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

17.

14. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

18.

15. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

19.

16. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

20.

17. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

21.

18. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

22.

19. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

23.

20. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

24.

21. An isolated polynucleotide encoding the amino acid sequence shown in SEQ. ID NO:

4 linked to SEQ. ID NO;

25.

28. A method of treating a disease condition comprising the administering of an effective amount of a fusion protein, said fusion protein being encoded by a transgene DNA construct comprising a first polypeptide domain which has a desired bioactivity, and a second polypeptide domain which has a desired bioactivity, together comprising said fusion protein wherein each said first and said second polypeptide domains retain their desired bioactivity wherein the encoded polypeptide of said first polypeptide domain is human alpha-fetoprotein or a fragment thereof having the biological activity of human alpha-fetoprotein

29. The method of claim 28 wherein said disease condition is a viral infection.

30. The method of claim 29 wherein said viral infection is caused by the Human Immunodeficiency Virus.

31. The method of claim 28 wherein the encoded polypeptide of said second polypeptide domain is human IFN-β

or a fragment thereof having the biological activity of human IFN-β

.

32. The method of claim 28 wherein said disease condition is Cancer.

33. The method of claim 28 wherein said disease condition is Rheumatoid Arthritis.

34. The method of claim 28 wherein said disease condition is Multiple Sclerosis.

35. The method of claim 28 wherein said disease condition is Osteoporsis.

36. The method of claim 28 wherein said disease condition is psoriasis.

37. The method of claim 28 wherein said disease condition is Myasthenia Gravis.

38. The method of claim 32 wherein said disease condition is Cancer and treatment of said disease condition further comprises treatment with an effective amount of a second composition said second composition being selected from the group consisting of:

doxorubicin;

methotrexate;

tamoxifen;

cisplatin;

vinblastine;

or vincristine

48. A method for the production of transgenic animals capable of producing a fusion protein of interest comprising:

transfecting a non-human mammalian cell-line with a transgene DNA construct encoding a first protein polypeptide domain and a second protein polypeptide domain;

selecting a cell line(s) in which said transgene DNA construct has been inserted into the genome of that cell or cell-line; and

performing a first nuclear transfer procedure to generate a first transgenic animal heterzygous for the desired gene.

49. The method of claim 48 further comprising:

characterizing the genetic composition of said first heterzygous transgenic animal;

selecting cells homozygous for said desired transgene DNA construct through the use of a selective agent;

characterizing surviving cells using known molecular biolology methods; and

picking surviving cells or cell colonies cells for use in a second round of nuclear transfer or embryo transfer; and

producing a second transgenic animal homozygous for said desired transgene DNA construct.

50. The method of claim 49, wherein said surviving cell are characterized by one of several known molecular biology methods including without limitation FISH, Southern Blot, PCR.

51. The method of claim 48, wherein said non-human mammalian cell-line to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

52. The method of either claims 48 or 51, wherein said non-human mammalian cell-line to be used as a donor cell or donor cell nucleus is from an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo.

53. The method of claim 48, wherein said non-human mammalian cell-line to be used as a source of donor nuclei or donor cell nucleus is a differentiated mammalian cell from an adult non-human mammalian somatic cell.

54. The method of claim 48, wherein said first transgenic animal is a rodent.

55. The method of claim 48, wherein said first transgenic animal is an ungulate.

56. The method of claim 48, wherein said first transgenic animal is from an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo

57. The resultant offspring of the methods of claim 48 or 49.

58. The method of claim 48, wherein cytocholasin-B is used in said nuclear transfer protocol.

59. The method of claim 48, wherein cytocholasin-B is not used in said nuclear transfer protocol.

60. The method of claims 48 or 48, wherein the techniques used generate a homozygous cell line.

61. The method of claims 48 or 49 wherein said first and second protein polypeptide domains nucleic acid sequences are linked and are translated as a single amino acid sequence.

62. The method of claim 48 wherein said transgene DNA construct is operatively linked to a mammary tissue-specific promoter which enables the bi-functional fusion protein product of said transgene DNA construct to be produced in the milk of a transgenic non-human placental mammal.

63. The method of claim 49 wherein said transgene DNA construct is operatively linked to a mammary tissue-specific promoter which enables the bi-functional fusion protein product of said transgene DNA construct to be produced in the milk of a transgenic non-human placental mammal.

64. The transgenic mammal of claims 62 or 63, wherein said promoter is the beta-lactoglobulin promoter.

65. The transgenic mammal of claims 62 or 63, wherein said promoter is the beta-casein promoter.

66. The method of claim 48 wherein said first protein polypeptide domain encodes human alpha-fetoprotein and wherein said second protein polypeptide domain encodes the desired gene codes for a biopharmaceutical protein product selected from the group consisting of:

antithrombin III, lactoferrin, urokinase, Platelet Factor 4, alpha-fetoprotein, alpha-1-antitrypsin, C-1 esterase inhibitor, decorin, beta interferon, alpha interferon, ferritin, prolactin, CFTR, Muellerian Inhibitory Substance, Prolactin, blood Factor X, blood Factor VIII, truncated ATIII, and erthyropoietin.

67. The resultant milk derived from the offspring of the methods of claim 48 or 49.

68. A process for treating a human cancer or tumor cell with a composition of matter containing a fusion protein, said fusion protein being encoded by a transgene DNA construct comprising a first polypeptide domain which has a desired bioactivity, and a second polypeptide domain which has a desired bioactivity, together comprising said fusion protein wherein each said first and said second polypeptide domains retain their desired bioactivity and wherein said first polypeptide domain has the activity of human alpha-fetoprotein, an improvement comprising treating a patient with a substantially pharmaceutically pure composition of said fusion protein with an effective amount of 5-fluorouracil.

72. The fusion protein of claim 68, wherein said human alpha-fetoprotein variant has a mutation and deletion of one or more residues.

78. An isolated nucleic acid sequence as shown in SEQ. ID NO:

1.