Erythropoietin analog-human serum albumin fusion

• US 6,548,653 B1
• Filed: 06/15/1999
• Issued: 04/15/2003
• Est. Priority Date: 06/15/1998
• Status: Expired due to Fees

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: Genzyme Transgenics Corporation
• Inventors: Young, Michael, Krane, Ian, Meade, Harry
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/333,213
• Time in Patent Office: 1,400 Days
• Field of Search: 800/7, 800/295, 435/325, 435/419, 435/254.2, 536/23.1, 536/23.4, 514/44
• US Class Current: 536/23.4
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 2319/02 containing a signal sequence

Abstract

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

Claims

1. An isolated nucleic acid comprising a nucleotide sequence which encodes an erythropoietin analog-human serum albumin (EPOa-hSA) fusion protein, wherein each of amino acid resdues 24, 38, 83 and 126 of human erythropoietin (EPO) has been altered such that it does not serve as a glycosylation site in the EPOa.

2. An expression vector or construct which comprises the nucleic acid of claim 1.

3. A cell which comprises the vector or construct of claim 2.

4. The vector of claim 2, further comprising a promoter.

5. The vector of claim 4, wherein the promoter is a promoter which directs expression in a tissue specific manner.

6. The vector of claim 4, wherein the promoter is a promoter which directs expression in mammary epithelial cells.

7. The nucleic acid of claim 1, wherein the encoded EPOa-hSA 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 human serum albumin amino acid sequence.

8. The nucleic acid of claim 1, wherein at least one amino acid residue of the encoded EPOa-hSA which can serve as a glycosylation site in EPO has been deleted.

9. The nucleic acid of claim 1, wherein at least one amino acid residue of the encoded EPOa-hSA which can serve as a glycosylation site in EPO has been replaced with an amino acid which does not serve as a site for glycosylation.

10. The nucleic acid of claim 9, wherein the amino acid residue which is replaced is an N-linked glycosylation site of EPO.

11. The nucleic acid of claim 10, wherein the N-linked glycosylation site is altered by replacing an amino acid residue Asn with Gln.

12. The nucleic acid of claim 10, wherein amino acid residue 24 of human EPO has been replaced with Gln.

13. The nucleic acid of claim 10, wherein amino acid residue 38 of human EPO has been replaced with Gln.

14. The nucleic acid of claim 10, wherein amino acid residue 83 of human EPO has been replaced with Gln.

15. The nucleic acid of claim 9, wherein the amino acid residue which is replaced is an O-linked glycosylation site of EPO.

16. The nucleic acid of claim 15, wherein the O-linked glycosylation site is altered by replacing an amino acid residue Ser with Ala.

17. The nucleic acid of claim 15, wherein amino acid residue 126 of human EPO has been replaced with Ala.

18. The nucleic acid of claim 9, wherein amino acid residues 24 of human EPO is replaced with a Gln, amino acid residues 38 of human EPO is replaced with a Gln, amino acid residue 83 of human EPO is replaced with a Gln and amino acid residue 126 of human EPO have been replaced with an Ala.

21. A method for making an EPOa-hSA fusion protein comprising:

supplying a cell of claim 3 which comprises a nucleic acid which encodes an EPOa-hSA fusion protein and

expressing said EPOa-hSA fusion protein from said nucleic acid, thereby making said EPOa-hSA fusion protein.

22. A method for making an EPOa-hSA fusion protein in a cultured cell comprising supplying a cell which includes a nucleic acid of claim 1, and expressing the EPOa-hSA fusion protein from the nucleic acid, thereby making the EPOa-hSA fusion protein.

19. An isolated nucleic acid comprising a nucleotide sequence which encodes an erythropoietin analog-human serum albumin (EPOa-hSA) fusion protein, wherein sequence in which a nucleic acid which comprises a nucleotide sequence encoding an EPOa is operably linked to a nucleic acid which comprises a nucleotide sequence encoding human serum albumin, wherein each of amino acid residues 24, 38, 83 and 126 of human erythropoietin (EPO) has been altered such that it does not serve as a glycosylation site in the EPOa.

20. The method of claim 19, wherein said cell is selected from a group consisting of a mammalian, yeast, plant, insect or a bacterial cell.