Erythropoietin forms with improved properties

• US 7,211,253 B1
• Filed: 11/09/2000
• Issued: 05/01/2007
• Est. Priority Date: 11/12/1999
• Status: Expired due to Fees

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Way, Jeffrey
• Primary Examiner(s): Allen; Marianne P.
• Assistant Examiner(s): DeBerry; Regina M.
• Application Number:
  US09/708,506
• Time in Patent Office: 2,364 Days
• Field of Search: 530/300, 530/356, 530/387.1, 530/387.3, 530/397, 530/399, 424/130.1, 424/134.1
• US Class Current: 424/134.1
• 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: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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

Abstract

The invention relates to novel modified erythropoietin (EPO) forms such as fusion proteins comprising a Fc portion of an Ig molecule and a target molecule having the biological activity of EPO. By selective altering of the amino acid sequences of the erythropoietin moiety as well as of the immunoglobulin moiety and the glycosylation pattern of erythropoietin fusion proteins with enhanced biological activity can be obtained. The invention relates also to novel nun-fused EPO molecules which have a pattern of cysteines or disulfide bonding which is distinct from human or animal EPO.

Claims

1. A fusion protein comprising an Fc portion of an Ig molecule and an erythropoietin (EPO) portion, wherein (i) the Fc portion is fused covalently via its C-terminus directly or indirectly to the EPO portion, (ii) the EPO portion comprises a Cys at a position corresponding to Trp₈₈ of human erythropoietin and an amino acid other than Cys at a position corresponding to position 33 of human erythropoietin such that the EPO portion comprises a Cys₂₉–

Cys₈₈ disulfide bond, and (iii) the EPO portion retains erythropoietin activity.

2. The fusion protein of claim 1, wherein the EPO portion is derived from human erythropoietin.

3. The fusion protein of claim 1, wherein the Fc portion is mutated or truncated in its amino acid sequence.

4. The fusion protein of claim 1, wherein the Fc portion is modified in its glycosylation pattern.

5. The fusion protein of claim 1, wherein the Fc portion is derived from an IgG chain and comprises a mutation of the glycosylation site within the Fc portion of the IgG chain.

6. The fusion protein of claim 5, wherein the mutation is of an asparagine at an amino acid position corresponding to position 297 of IgG1.

7. The fusion protein of claim 1 further comprising a linker between the Fc portion and the EPO portion.

8. The fusion protein of claim 7, wherein the linker has no protease cleavage site.

9. The fusion protein of claim 1 having improved biological activity compared to naturally-occurring human erythropoietin.

10. The fusion protein of claim 1 having an extended serum half-life compared to naturally-occurring human erythropoietin.

11. The fusion protein of claim 10, wherein said extended serum half-life is greater than 20 hours.

12. The fusion protein of claim 1, comprising a whole Ig molecule.

13. The fusion protein of claim 1, wherein the Fc portion and the EPO portion are of mammalian origin.

14. The fusion protein of claim 13, wherein the Fc portion is derived from human IgG.

15. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

16. The pharmaceutical composition of claim 15 containing at least one additional pharmaceutically effective drug and/or adjuvants.

17. A fusion protein comprising an Fc portion of an Ig molecule and an erythropoietin (EPO) portion, wherein (i) the Fc portion is fused covalently via its C-terminus directly or indirectly to the EPO portion, (ii) the EPO portion comprises a Cys at a position corresponding to Trp₈₈ of human erythropoietin and an amino acid other than Cys at a position corresponding to position 33 of human erythropoietin such that the EPO portion comprises a Cys₂₉–

Cys₈₈ disulfide bond, (iii) the EPO portion retains erythropoietin activity and is derived from human erythropoietin; and

(iv) the EPO portion comprises at least one of the following mutations;

His₃₂→

Gly, Ser₃₄→

Arg, and Pro₉₀→

Ala.

18. A fusion protein comprising an Fc portion of an Ig molecule and an erythropoietin (EPO) portion, wherein (i) the Fc portion is fused covalently via its C-terminus directly or indirectly to the EPO portion, (ii) the EPO portion comprises a Cys at a position corresponding to Trp₈₈ of human erythropoietin and an amino acid other than Cys at a position corresponding to position 33 of human erythropoietin such that the EPO portion comprises a Cys₂₉–

Cys₈₈ disulfide bond, (iii) the EPO portion retains erythropoietin activity and is derived from human erythropoietin; and

(iv) the EPO portion comprises the following substitutions;

His₃₂→

Gly, Cys₃₃→

Pro, Trp₈₈→

Cys, and Pro₉₀→

Ala.