Methods and compositions for the prevention and treatment of anemia

• US 8,268,588 B2
• Filed: 06/24/2011
• Issued: 09/18/2012
• 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., Browne, Jeffrey K., Sitney, Karen C.
• Primary Examiner(s): Kemmerer, Elizabeth C
• Assistant Examiner(s): Deberry, Regina M
• Application Number:
  US13/168,913
• Publication Number:
  US 20110256586A1
• Time in Patent Office: 452 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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 nucleic acid molecule encoding an analog of human erythropoietin comprising the amino acid sequence of human erythropoietin from residues 1-165 as shown in 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 position 52, 53, 55, 86 or 114 and an N-linked carbohydrate chain is attached at said one additional site.

2. The nucleic acid molecule of claim 1 wherein the analog comprises three, four, or more than four additional glycosylation sites wherein a carbohydrate chain is attached at each said additional site.

3. The nucleic acid molecule 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.

4. The nucleic acid molecule 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.

5. The nucleic acid molecule 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.

6. The nucleic acid molecule 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.

7. The nucleic acid molecule 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.

8. The nucleic acid molecule of claim 1 wherein additional glycosylation sites are introduced at positions 30, 55, 88 and 114 and an N-linked carbohydrate chain is attached at the additional sites.

9. The nucleic acid molecule of claim 1 further comprising an additional glycosylation site at position 125 and wherein an O-linked carbohydrate chain is attached at the additional site.

12. An expression vector comprising the nucleic acid molecule of claim 1, 10 or 11.

13. A eucaryotic host cell in culture comprising the nucleic acid molecule of claim 1, 10 or 11.

14. A eucaryotic host cell in culture comprising the expression vector of claim 12.

15. The host cell of claim 13 or 14 which is a mammalian host cell.

16. The host cell of claim 13 or 14 which is a human host cell.

17. The host cell of claim 14 which is selected from the group consisting of COS-7, BHK, CHO, 293, HeLa, L-929 and 3T3 cells.

18. A method of producing an analog of human erythropoietin comprising culturing a host cell of claim 13 or 14 under conditions that permit expression of the analog and recovering the analog.

10. A nucleic acid molecule encoding an analog of human erythropoietin comprising the amino acid sequence from residues 1-165 as shown in SEQ ID N0:

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.

11. A nucleic acid molecule encoding an analog of human erythropoietin comprising the amino acid sequence from residues 1-165 as shown in SEQ ID NO:

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

Asn⁵² Thr⁵⁴ Epo;

Asn⁵³ Thr⁵⁵ Epo;

Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Asn⁵³ Thr⁵⁵ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Epo;

Asn⁵⁵ Thr⁵⁷ Epo;

Asn⁸⁶ Val⁸⁷ Thr⁸⁸ Epo;

Asn³⁰ Thr³² Asn⁵³ Thr⁵⁵ Epo;

Asn³⁰ Thr³² Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Asn⁵³ Thr⁵⁵ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Asn¹¹⁴ Thr¹¹⁶ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Pro¹²⁴ Thr¹²⁵ Thr¹²⁶ Epo;

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Asn¹¹⁴ Thr¹¹⁶ Pro¹²⁴ Thr¹²⁵ Thr¹²⁶ Epo; and

Asn³⁰ Thr³² Asn⁵⁵ Thr⁵⁷ Val⁸⁷ Asn⁸⁸ Thr⁹⁰ Asn¹¹⁴ Thr¹¹⁶ Pro¹²⁴ Thr¹²⁵ Thr¹²⁶ Epo.