Erythropoietin analog-human serum albumin fusion

• US 7,101,971 B2
• Filed: 02/20/2002
• Issued: 09/05/2006
• Est. Priority Date: 06/15/1998
• Status: Expired due to Fees

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Young, Michael, Meade, Harry M., Krane, Ian
• Primary Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US10/081,400
• Publication Number:
  US 20020155998A1
• Time in Patent Office: 1,658 Days
• Field of Search: 530/397, 530/395, 530/402
• US Class Current: 530/350
• 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 EPOa-hSA fusion protein, wherein the EPOa moiety is the full coding region of the human EPO sequence but wherein each amino acid residue of the EPOa moiety that serves as a site for glycosylation of the fusion protein is altered such that such a site does not serve as a site for glycosylation glycosylation in the EPOa;

and,

wherein both the albumin moiety and the EPOa moiety of the fusion protein is derived from a human sequence.

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

R1-L-R2;

R2-L-R1;

orR1-L-R2 L-R1, wherein R1 is an erythropoietin og amino acid sequence;

L is a peptide linker and R2 is a h serum albumin amino acid sequence.

3. The EPOa-hSA fusion protein of claim 2, wherein R1 and R2 are covalently linked via said pep de linker.

4. The EPOa-hSA fusion protein of claim 3, wherein said peptide linker is composed of a sequenc having the formula (Ser-Ser-Ser-Ser-Gly)y (SEQ. ID 5) wherein y is less than or equal to 8.

5. The EPOa.hSA fusion protein of claim 3, wherein said peptide linker is 10 to 30 amino acids in length.

6. The EPOa-hSA fusion protein of claim 5, wherein each of said amino acids in said peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and Ala.

7. The EPOa-hSA fusion protein of claim 5, wherein said peptide linker is composed of either 2 or tandem repeats of a sequence having the formula ((Ser-Ser-Ser-Ser-Gly)₃-Ser-Pro (SEQ. ID 4).

8. The EPOa-hSA fusion protein of claim 1, wherein each amino acid residue which serves as an ent point for glycosylation has been deleted.

9. The EPOa-hSA fusion protein of claim 1, wherein each amino acid residue of human EPO which serves as a site for glycosylation has been replaced with an amino acid residue which does not serve as a site for glycosylation.

10. The EPOa-hS fusion protein of claim 1, wherein said amino acid residue is selected from the up consisting of amino acid residues Asn24, Asn38, Asn83 and Ser126.

11. The EPOa-hSA fusion protein of claim 1, wherein said glycosylation sites altered include Serl2 Asn24, Asn38 and Asn83.

12. The BPOa-hSA fusion protein of claim 1, wherein saud glycosylation sites altered are either O-linked or N-linked glycosylation sites and are altered by replacing an amino acid residue Am or Ser with a Gln residue.

13. The EPOa-hSA fusion protein of claim 1, wherein each of the amino acid residues 24, 38, 83 and 126 have been replaced with Gin.

14. The EPOa-hSA fusion protein of claim 1, wherein each of the amino acid residues 24, 38, 83 and 126 have been deleted.

15. The EPOa-hSA fusion protein of claim 14, wherein each of the amino acid residues 24, 38 and 83 have been replaced with Gin and wherein said amino acid residue 126 has replaced with Ala.

16. The EPOa-hSA fusion protein of claim 1, wherein fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, GlnS3 and Ala126, a peptide linker, and hwnan serum albumin.

17. The EPOa-hSA fusion protein of claim 1, wherein the fusion protein is from left to right, GIn24,iGIn3S, GIn83, Alal26 EPO, apeptide linker having the formula ((Ser-Gly-Gly-Gly-(Gly)₃-Ser-Pro) (SEQ. ID 4) and human serum albwnin.

18. The EPOa-hSA fusion protein of claim 1, wherein the EPOa-hSA fusion protein includes, from teft to right, human serum albumin, a peptide linker, and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and A1a126.

19. The EPOa-hSA fusion protein of claim 18, wherein the EPOs is Gln24, Gln38, Gln83, Ala126 EPO.

20. The EPOa-hSA fusion protein of claim 1, wherein the fusion protein is from left to right, human serum albumin, a peptido linker having the formula ((Ser-Gly.Gly-Gly-Gly)₃-Ser-Pro) (SEQ. ID 4), and Gln24, Gln38, Gln83, Ala 126 EPO.