Methods and compositions for the prevention and treatment of anemia

• US 7,304,150 B1
• Filed: 11/27/2000
• Issued: 12/04/2007
• Est. Priority Date: 10/23/1998
• Status: Expired due to Fees

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Egrie, Joan C., Elliott, Steven G., Sitney, Karen C., Browne, Jeffrey K.
• Primary Examiner(s): Allen; Marianne P.
• Assistant Examiner(s): DeBerry; Regina M.
• Application Number:
  US09/723,955
• Time in Patent Office: 2,563 Days
• Field of Search: 530/350, 530/380, 530/397, 530/387.1, 530/388.22, 424/134.1
• US Class Current: 536/23.51
• CPC Class Codes: A61P 7/06 Antianaemics
• 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: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

Methods for increasing and maintaining hematocrit in a mammal comprising administering a hyperglycosylated analog of erythropoietin are disclosed. An analog may be administered less frequently than an equivalent molar amount of recombinant human erythropoietin to obtain a comparable target hematocrit and treat anemia. Alternatively, a lower molar amount of a hyperglycosylated analog may be administered to obtain a comparable target hematocrit and treat anemia. Also disclosed are new hyperglycosylated erythopoietin analogs, methods of production of the analogs, and compositions comprising the analogs.

Claims

1. A fusion protein having the in vivo biological activity of increasing hematocrit comprising a hyperglycosylated analog of erythropoietin and an immunoglobulin heavy chain constant region, wherein the analog comprises the amino acid sequence of human erythropoietin from residues 1-165 or 1-166 as shown in FIG. 1 (SEQ ID NO:

1) except for one or more amino acid changes which provide for one or more additional glycosylation site(s) as compared to human erythropoietin, wherein one additional site is introduced at any one of positions 52, 53, 55, 86 and 114 and an N-linked carbohydrate chain is attached at said one additional site.

2. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30 and 55 and N-linked carbohydrate chains are attached at the additional sites.

3. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30 and 114 and N-linked carbohydrate chains are attached at the additional sites.

4. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30, 55 and 88 and N-linked carbohydrate chains are attached at the additional sites.

5. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30, 55 and 114 and N-linked carbohydrate chains are attached at the additional sites.

6. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30, 88 and 114 and N-linked carbohydrate chains are attached at the additional sites.

7. The fusion protein of claim 1 wherein additional glycosylation sites are introduced at positions 30, 55, 88 and 114 and N-linked carbohydrate chains are attached at the additional sites.

8. The fusion protein of claim 1 wherein the analog comprises the amino acid sequence from residues 1-165 or 1-166 as shown in FIG. 1 (SEQ ID NO:

1) except for Asn at position 30, Thr at position 32, Asn at position 55, Thr at position 57, Val at position 87, Asn at position 88, Thr at position 90, Asn at position 114, Thr at position 116, and any one or more of Pro at position 124, Thr at position 125 and Thr at position 126.

9. The fusion protein of claim 1 wherein the analog comprises the amino acid sequence from residues 1-165 or 1-166 as shown in FIG. 1 (SEQ ID NO:

1) except for the amino acid changes selected from the group consisting of;

Asn⁵²Thr⁵⁴Epo;

Asn⁵³Thr⁵⁵Epo;

Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Asn⁵³Thr⁵⁵Val⁸⁷Asn⁸⁸Thr⁹⁰Epo;

Asn⁵⁵Thr⁵⁷Epo;

Asn⁶⁶Val⁸⁷Thr⁸⁸Epo;

Asn³⁰Thr³²Asn⁵³Thr⁵⁵Epo;

Asn³⁰Thr³²Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Asn⁵³Thr⁵⁵Val⁸⁷Asn⁸⁸Thr⁹⁰Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Val⁸⁷Asn⁸⁸Thr⁹⁰Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Val⁸⁷Asn⁸⁸Thr⁹⁰Asn¹¹⁴Thr¹¹⁶Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Val⁸⁷Asn⁸⁸Thr⁹⁰Pro¹²⁴Thr¹²⁵Thr¹²⁶Epo;

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Asn¹¹⁴Thr¹¹⁶Pro¹²⁴Thr¹²⁵Thr¹²⁶Epo; and

Asn³⁰Thr³²Asn⁵⁵Thr⁵⁷Val⁸⁷Asn⁸⁸Thr⁹⁰Asn¹¹⁴Thr¹¹⁶Pro¹²⁴Thr¹²⁵Thr¹²⁶Epo.

10. The fusion protein of claims 1-9 wherein the immunoglobulin constant region is an Fc region.

11. The fusion protein of claim 10 wherein the immunoglobulin constant region comprises the hinge, CH2 and CH3 regions.

12. The fusion protein of claim 10 wherein the Fc region is from human IgG or is derived from human IgG.

13. The fusion protein of claim 12 wherein the IgG is IgG1, IgG2, IgG3 or IgG4 or a combination thereof.

14. The fusion protein of claim 10 wherein the Fc region is fused to a hyperglycosylated analog of human erythropoietin by a linker molecule.

15. A composition comprising a therapeutically effective amount of the fusion protein of claims 1-9 together with a pharmaceutically acceptable diluent, adjuvant or carrier.

16. The composition of claim 15 further comprising a buffer solution of sodium phosphate.

17. The composition of claim 15 further comprising polysorbate.

18. The composition of claim 15 further comprising an amino acid.

19. A fusion protein comprising a hyperglycosylated analog of erythropoietin and an immunoglobulin heavy chain constant region, wherein the analog comprises an amino acid sequence which differs from the amino acid sequence of human erythropoietin from residues 1-165 or 1-166 as shown in FIG. 1 (SEQ ID NO:

1) by having one or more additional glycosylation site(s), wherein one additional site is introduced at about position 114 and an N-linked carbohydrate chain is attached at said one additional site.

20. The fusion protein of claim 19 comprising two additional glycosylation sites as compared to human erythropoietin.

21. The fusion protein of claim 19 comprising three additional glycosylation sites as compared to human erythropoietin.

22. The fusion protein of claim 19 comprising four additional glycosylation sites as compared to human erythropoietin.

23. The fusion protein of claim 19 comprising more than four additional glycosylation sites as compared to human erythropoietin.

24. The fusion protein of claim 19 further comprising conservative amino acid substitutions.

25. The fusion protein of claim 19 wherein the immunoglobulin constant region is an Fc region.

26. The fusion protein of claim 19 wherein the immunoglobulin constant region is a fragment, derivative or variant of an Fc region.

27. The fusion protein of claim 19 wherein the immunoglobulin constant region is an Fc region from human IgG or derived from human IgG.

28. The fusion protein of claim 19 wherein the immunoglobulin constant region is IgG1, IgG2, IgG3 or IgG4 or a combination thereof.