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

• US 20090299044A1
• Filed: 10/25/2007
• Published: 12/03/2009
• Est. Priority Date: 10/25/2006
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Nguyen, Hung Q., Adler, Beverly S., Miranda, Leslie P., McGivern, Joseph G., Khare, Taruna Arora, Sullivan, John K., Martin, Francis H., Walker, Kenneth W., Hu, Shaw-Fen Sylvia, Gegg, Colin V. JR.
• Granted Patent: US 8,043,829 B2
• Time in Patent Office: Days
• US Class Current: 536/23.53
• 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 an OSK1 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 for expression in a mammalian cell, said fusion protein comprising, in the N-terminal to C-terminal direction:

(a) an immunoglobulin or a portion thereof;

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

(c) an immunoglobulin Fc domain or a portion thereof;

and wherein the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c).

2. The DNA of claim 1, wherein the portion of the immunoglobulin in (a) comprises:

(i) an immunoglobulin light chain or the variable region thereof, and

(ii) an immunoglobulin heavy chain or the variable region thereof.

3. The DNA of claim 1, wherein the portion of the immunoglobulin in (a) is a first immunoglobulin Fc domain or a portion 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 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. The DNA of claim 1, wherein the toxin peptide is selected from ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, and Pi3 toxin peptide, or is a peptide analog of any of these.

8. The DNA of claim 1, wherein the toxin peptide is OSK1 or an OSK1 peptide analog.

9. The DNA of claim 1, wherein the toxin peptide is ShK or a ShK peptide analog.

10. The DNA of claim 8, wherein the OSK1 peptide analog comprises an amino acid sequence selected from SEQ ID NOS:

25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Tables 7 and Tables 7A-J.

11. The DNA of claim 9, wherein the ShK peptide analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:

5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2.

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

13. A mammalian host cell comprising the expression vector of claim 12.

14. The mammalian host cell of claim 13, wherein the cell is a CHO cell or HEK 293 cell.

15. A DNA encoding a recombinant fusion protein for expression in a mammalian cell, said fusion protein comprising, in the N-terminal to C-terminal direction:

(a) an immunoglobulin light chain;

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

(c) an immunoglobulin heavy chain;

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

(e) an immunoglobulin Fc domain or a portion thereof; and

wherein the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a), (c), or (e) in the expressed fusion protein.

16. The DNA of claim 15, wherein the toxin peptide is covalently linked to the C-terminal end of (e).

19. The DNA of claim 15 or claim 17, wherein the at least one protease cleavage site is a furin cleavage site.

20. The DNA of claim 15 or claim 17, wherein the DNA, 5′

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

17. A DNA encoding a recombinant fusion protein for expression in a mammalian cell, said fusion protein comprising, in the N-terminal to C-terminal direction:

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

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

(c) a second immunoglobulin Fc domain or a portion thereof; and

wherein the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) in the expressed fusion protein.

18. The DNA of claim 17, wherein the toxin peptide is covalently linked to the C-terminal end of (c).