DNA sequences encoding erythropoietin

• US 4,703,008 A
• Filed: 11/30/1984
• Issued: 10/27/1987
• Est. Priority Date: 12/13/1983
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Kiren-Amgen Incorporated
• Inventors: Lin, Fu-Kuen
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US06/675,298
• Time in Patent Office: 1,061 Days
• Field of Search: 435/68, 435/317, 435/172.3, 435/253, 435/240, 935/6, 935/10, 935/11, 935/27, 935/69, 935/73, 935/13
• US Class Current: 435/360
• 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 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells

Abstract

Disclosed are novel polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties of mammalian erythropoietin ("EPO") which are characterized in preferred forms by being the product of procaryotic or eucaryotic host expression of an exogenous DNA sequence. Illustratively, genomic DNA, cDNA and manufactured DNA sequences coding for part or all of the sequence of amino acid residues of EPO or for analogs thereof are incorporated into autonomously replicating plasmid or viral vectors employed to transform or transfect suitable procaryotic or eucaryotic host cells such as bacteria, yeast or vertebrate cells in culture. Upon isolation from culture media or cellular lysates or fragments, products of expression of the DNA sequences display, e.g., the immunological properties and in vitro and in vivo biological activities of EPO of human or monkey species origins. Disclosed also are chemically synthesized polypeptides sharing the biochemical and immunological properties of EPO. Also disclosed are improved methods for the detection of specific single stranded polynucleotides in a heterologus cellular or viral sample prepared from, e.g., DNA present in a plasmid or viral-borne cDNA or genomic DNA "library".

Claims

1. A purified and isolated DNA sequence encoding erythropoietin, said DNA sequence selected from the group consisting of:

(a) the DNA sequences set out in FIGS. 5 and 6 or their complementary strands; and

(b) DNA sequences which hybridize under stringent conditions to the DNA sequences defined in (a).

4. A procaryotic or eucaryotic host cell transformed or transfected with a DNA sequence according to claim 1, 2 or 3 in a manner allowing the host cell to express erythropoietin.

5. A biologically functional circular plasmid or viral DNA vector including a DNA sequence according to claim 1, 2, or 3.

6. A procaryotic or eucaryotic host cell stably transformed or transfected with a DNA vector according to claim 5.

2. A purified and isolated DNA sequence consisting essentially of a DNA sequence encoding human erythropoietin.

3. A purified and isolated DNA sequence consisting essentially of a DNA sequence encoding monkey erythropoietin.

7. A purified and isolated DNA sequence consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

8. A cDNA sequence according to claim 7.

9. A monkey species erythropoietin coding DNA sequence according to claim 8.

10. A DNA sequence according to claim 9 and including the protein coding region set forth in FIG. 5.

11. A genomic DNA sequence according to claim 7.

12. A human species erythropoietin coding DNA sequence according to claim 11.

13. A DNA sequence according to claim 12 and including the protein coding region set forth in FIG. 6.

14. A DNA sequence according to claim 7 and including one or more codons preferred for expression in E.coli cells.

15. A DNA sequence according to claim 14, coding for expression of human species erythropoietin.

16. A DNA sequence according to claim 15 including the protein coding region set forth in FIG. 7.

17. A DNA sequence according to claim 7 and including one or more codons preferred for expression in yeast cells.

18. A DNA sequence according to claim 17, coding for expression of human species erythropoietin.

19. A DNA sequence according to claim 18 including the protein coding region set forth in FIG. 8.

20. A DNA sequence according to claim 7 covalently associated with a detectable label substance.

21. A DNA sequence according to claim 20 wherein the detectable label is a radiolabel.

22. A single-strand DNA sequence according to claim 20.

23. A procaryotic or eucaryotic host cell transformed or transfected with a DNA sequence according to claim 7, 8, or 11 in a manner allowing the host cell to express said polypeptide.

24. A transformed or transfected host cell according to claim 23 which host cell is capable of glycosylating said polypeptide.

25. A transformed or transfected mammalian host cell according to claim 24.

26. A transformed or transfected COS cell according to claim 25.

27. A transformed or transfected CHO cell according to claim 25.

28. A biologically functional circular plasmid or viral DNA vector including a DNA sequence according to claim 7.

29. A procaryotic or eucaryotic host cell stably transformed or transfected with a DNA vector according to claim 28.

30. A DNA sequence according to claim 7 coding for [Phe¹⁵ ]hEPO, [Phe⁴⁹ ]hEPO, [Phe¹⁴⁵ ]hEPO, [His⁷ ]hEPO, [Asn² des-Pro² through Ile⁶ ]hEPO, [des-Thr¹⁶³ through Arg¹⁶⁶ hEPO, or [Δ

27-55]hEPO.

31. A purified and isolated DNA sequence as set out in FIGS. 5 or 6 or the complementary strand of such a sequence.