DNA encoding chimeric toxin peptide fusion proteins and vectors and mammalian cells for recombinant expression

• US 8,043,829 B2
• Filed: 10/25/2007
• Issued: 10/25/2011
• Est. Priority Date: 10/25/2006
• Status: Expired due to Fees

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Arora Khare, Taruna, Nguyen, Hung Q., Adler, Beverly S., Miranda, Leslie P., McGivern, Joseph G., Sullivan, John K., Martin, Francis H., Walker, Kenneth W., Gegg, Colin V. Jr., Hu, Shaw-Fen Sylvia
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US11/978,104
• Publication Number:
  US 20090299044A1
• Time in Patent Office: 1,461 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/43522 from scorpions
• 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: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

Disclosed is a composition of matter comprising a ShK toxin peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

Claims

1. A DNA encoding a recombinant fusion protein, wherein the fusion protein comprises a ShK toxin peptide, wherein said fusion protein comprises, in the N-terminal to C-terminal direction:

(a) an immunoglobulin or a portion thereof comprising;

(i) an immunoglobulin light chain and an immunoglobulin heavy chain;

or

(ii) a first immunoglobulin Fc domain or a CH2 domain thereof;

(b) a peptidyl linker 4 to 40 amino acid residues long and comprising at least one protease cleavage site; and

(c) an immunoglobulin Fc domain or a CH2 domain thereof, wherein if (a) comprises (ii), (c) is a second immunoglobulin Fc domain or a CH2 domain thereof;

and wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO;

914, SEQ ID NO;

915, SEQ ID NO;

916, or SEQ ID NO;

917, covalently linked to the N-terminal end of (a) or C-terminal end of (c).

2. The DNA of claim 1, wherein the immunoglobulin in (a) comprises the immunoglobulin light chain and the immunoglobulin heavy chain.

3. The DNA of claim 1, wherein the immunoglobulin or the portion thereof in (a) is the first immunoglobulin Fc domain or the CH2 domain thereof.

4. The DNA of claim 1, wherein the at least one protease cleavage site is a furin cleavage site.

5. The DNA of claim 1, wherein the immunoglobulin of (a) and (c) is in each instance independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

6. The DNA of claim 1, wherein the DNA, 5′

to the coding sequence of (a), further encodes a signal peptide sequence operably linked to the expressed fusion protein.

7. An expression vector comprising the DNA of claim 1.

8. An isolated mammalian host cell comprising the expression vector of claim 7.

9. The isolated mammalian host cell of claim 8, wherein the cell is a CHO cell or HEK 293 cell.

16. The DNA of any of claims 1, 10, or 12, wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO:

914.

17. The DNA of any of claims 1, 10, or 12, wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO:

915.

18. The DNA of any of claims 1, 10, or 12, wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO:

916.

19. The DNA of any of claims 1, 10, or 12, wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO:

917.

10. A DNA encoding a recombinant fusion protein, wherein the fusion protein comprises a ShK toxin peptide, wherein said fusion protein comprises, in the N-terminal to C-terminal direction:

(a) an immunoglobulin light chain;

(b) a first peptidyl linker 4 to 40 amino acid residues long comprising at least one protease cleavage site;

(c) an immunoglobulin heavy chain;

(d) a second peptidyl linker 4 to 40 amino acid residues long comprising at least one protease cleavage site; and

(e) an immunoglobulin Fc domain or a CH2 domain thereof;

and wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO;

914, SEQ ID NO;

915, SEQ ID NO;

916, or SEQ ID NO;

917, covalently linked to the N-terminal end of (a) or C-terminal end of (e) in the expressed fusion protein.

11. The DNA of claim 10, wherein the ShK toxin peptide is covalently linked to the C-terminal end of (e).

14. The DNA of claim 10 or claim 12, wherein the at least one protease cleavage site is a furin cleavage site.

15. The DNA of claim 10 or claim 12, wherein the DNA, 5′

to the coding sequence of (a), further encodes a signal peptide sequence operably linked to the expressed fusion protein.

12. A DNA encoding a recombinant fusion protein, wherein the fusion protein comprises a ShK toxin peptide, wherein said fusion protein comprises, in the N-terminal to C-terminal direction:

(a) a first immunoglobulin Fc domain or a CH2 domain thereof;

(b) a peptidyl linker 4 to 40 amino acid residues long comprising at least one protease cleavage site; and

(c) a second immunoglobulin Fc domain or a CH2 domain thereof;

and wherein the amino acid sequence of the ShK toxin peptide is SEQ ID NO;

914, SEQ ID NO;

915, SEQ ID NO;

916, or SEQ ID NO;

917, covalently linked to the N-terminal end of (a) or C-terminal end of (c) in the expressed fusion protein.

13. The DNA of claim 12, wherein the ShK toxin peptide is covalently linked to the C-terminal end of (c).