ERYTHROPOIETIN ANALOG-IgG FUSION PROTEINS

• US 20090246194A1
• Filed: 03/18/2008
• Published: 10/01/2009
• Est. Priority Date: 06/15/1998
• Status: Abandoned Application

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Krane, Ian, Meade, Harry M.
• Application Number:
  US12/050,569
• Publication Number:
  US 20090246194A1
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/30 Bird
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

Erythropoietin analog-human IgG fusion protein (EPOa-IgG) fusion protein and methods of making and using the fusion protein.

Claims

1. An EPOa-IgG fusion protein, wherein at least one amino acid residue of the EPOa moiety of the fusion protein is altered such that a site which serves as a site for glycosylation in EPO does not serve as a site for glycosylation in EPOa.

2. The EPOa-IgG fusion protein of claim 1, wherein said fusion protein has the formula:

R1-L-R2;

R2-L-R1;

or R1-L-R2-L-R1,

wherein R1 is an erythropoietin analog amino acid sequence;

L is a peptide linker and R2 is a human IgG immunoglobulin amino acid sequence.

5. The EPOa-IgG fusion protein of claim 1, wherein the at least one amino acid residue of the EPOa moiety which serves as a site for glycosylation has been replaced with an amino acid residue which does not serve as a site for glycosylation.

6. The EPOa-IgG fusion protein of claim 1, wherein said at least one amino acid residue is selected from the group consisting of amino acid residues Asn24, Asn38, Asn83 and Ser126 of the EPO moiety.

8. The EPOa-IgG fusion protein of claim 1, wherein said glycosylation site is an N-linked glycosylation site and is altered by replacing an amino acid residue Asn of the EPO moiety with Gln.

9. The EPOa-IgG fusion protein of claim 1, wherein said glycosylation site is an O-linked glycosylation and is altered by replacing an amino acid residue Ser of the EPO moiety with Gln.

10. The EPOa-IgG fusion protein of claim 1, wherein the amino acid residues 24, 38, or 83 of the EPO moiety have been altered.

11. The EPOa-IgG fusion protein of claim 10, wherein the amino acid residues 24, 38, or 83 of the EPO moiety have been replaced with Gln.

12. The EPOa-IgG fusion protein of claim 1, wherein the amino acid residue 126 of the EPO moiety has been altered.

13. The EPOa-IgG fusion protein of claim 12, wherein said amino acid residue 126 of the EPO moiety has been replaced with Ala.

14. The EPOa-IgG fusion protein of claim 1, wherein the amino acid residues 24, 38, 83 and 126 of the EPO moiety have been altered such that none of them serves as a glycosylation site.

20. The EPOa-IgG fusion protein of claim 14, wherein the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.

3-4. -4. (canceled)

7. (canceled)

15-19. -19. (canceled)

21-26. -26. (canceled)

27. An isolated nucleic acid comprising a nucleotide sequence which encodes an EPOa-IgG fusion protein, wherein at least one amino acid residue of the encoded EPOa-IgG which can serve as a glycosylation site in EPO is altered such that it does not serve as a glycosylation site in EPOa.

28-32. -32. (canceled)

33. A method of making an EPOa-IgG fusion protein comprising:

providing a transgenic organism which includes a transgene which directs the expression of the EPOa-IgG fusion protein;

allowing the transgene to be expressed; and

,

recovering the EPOa-IgG fusion protein.

34-39. -39. (canceled)

40. A transgenic organism, which includes a transgene which encodes an EPOa-IgG fusion protein.

41-45. -45. (canceled)

46. A pharmaceutical composition having a therapeutically effective amount of an EPOa-IgG fusion protein.

47. A method of treating a subject in need of erythropoietin comprising administering a therapeutically effective amount of an EPOa-IgG fusion protein to the subject.

48-54. -54. (canceled)

55. A method for making an EPOa-IgG fusion protein in a cultured cell comprising supplying a cell which includes a nucleic acid which encodes an EPOa-IgG fusion protein, and expressing the EPOa-IgG fusion protein from the nucleic acid, thereby making the EPOa-IgG fusion protein.

61. The method of claim 55, wherein the EPOa-IgG fusion protein is made in a mammary gland of a transgenic mammal under the control of a milk specific promoter.

56. (canceled)

57. An EPOa-IgG fusion protein, wherein both the EPOa moiety and the human IgG moiety of the fusion protein are altered such that any site that serves as a site for glycosylation is altered such that it cannot serve as a site for glycosylation in the EPOa-IgG fusion protein, making the entire EPOa-IgG fusion protein non-glycosylated.

58-60. -60. (canceled)