CD95L or trail fusion proteins

• US 8,147,843 B2
• Filed: 08/28/2007
• Issued: 04/03/2012
• Est. Priority Date: 08/28/2006
• Status: Active Grant

• Current Assignee: Apogenix AG
• Original Assignee: Apogenix Gmbh
• Inventors: Hill, Oliver, Gieffers, Christian, Thiemann, Meinolf
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US12/439,486
• Publication Number:
  US 20110027218A1
• Time in Patent Office: 1,680 Days
• Field of Search: None
• US Class Current: 424/192.1
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 2319/73 containing coiled-coiled motif (leucine zippers)

Abstract

The present invention refers to fusion proteins comprising a TNF superfamily (TNFSF) cytokine or a receptor binding domain thereof fused to a trimerization domain and a nucleic acid molecule encoding the fusion protein. The fusion protein is present as a trimeric complex or as an oligomer thereof and is suitable for therapeutic, diagnostic and/or research applications.

Claims

1. A fusion protein comprising:

(i) a TNF-superfamily cytokine selected from the group consisting of CD95L or TRAIL, or a receptor binding domain thereof;

(ii) a fibritin trimerization domain; and

(iii) a flexible linker between (i) and (ii),

wherein the fibritin trimerization domain is a bacteriophage RB69 trimerization domain, and the linker has 5-20 amino acid residues having the amino acid sequence (GSS)_z(SSG)_b(GS)_c(S)_d, a, b, c, d are independently 0, 1, 2, 3, 4, or 5.

2. The fusion protein according to claim 1, wherein the fibritin trimerization domain comprises amino acid residues 455-480 of SEQ ID NO:

24, amino acid residues or 456-480 of SEQ ID NO;

24.

3. The fusion protein according to claim 1, wherein the TNF-superfamily cytokine or receptor binding domain thereof is fused to the N-terminus of the linker and the linker is fused to the N-terminus of the fibritin trimerization domain.

4. The fusion protein according to claim 3, further comprising:

(iv) a N-terminal signal peptide.

5. The fusion protein according to claim 4, further comprising:

(v) a protease cleavage site C-terminal to the signal peptide.

6. The fusion protein according to claim 3, further comprising:

(iv) a C-terminal flexible element.

7. The fusion protein according to claim 6, further comprising:

(v) a recognition or purification domain fused in frame to the C-terminal flexible element.

8. The fusion protein according to claim 1, wherein the fibritin trimerization domain is fused to the N-terminus of the linker and the linker is fused to the N-terminus of the TNF-superfamily cytokine or receptor binding domain thereof.

9. The fusion protein according to claim 8, further comprising:

(iv) a N-terminal signal peptide.

10. The fusion protein according to claim 9, further comprising:

(v) a protease cleavage site C-terminal to the signal peptide.

11. The fusion protein according to claim 8, further comprising:

(iv) a C-terminal flexible element.

12. The fusion protein according to claim 11, further comprising:

(v) a recognition or purification domain fused in frame to the C-terminal flexible element.

13. The fusion protein according to claim 1, wherein the fusion protein forms a trimeric complex or an oligomer of a trimeric complex.

14. The fusion protein according to claim 13, wherein the complex consists of three identical fusion proteins.

15. The fusion protein according to claim 1, wherein said TNF-superfamily cytokine is CD95L.

16. The fusion protein according to claim 15, wherein said CD95L has the amino acid sequence of SEQ ID. NO:

30.

17. The fusion protein according to claim 15, wherein the CD95L comprises amino acid residues 142-281 or amino acid residues 144-281 of SEQ ID NO:

30.

18. The fusion protein according to claim 1, wherein the TNF-superfamily cytokine is TRAIL, which comprises amino acid residues 116-281, amino acid residues 118-281, or amino acid residues 120-281 of SEQ ID NO:

34.

19. The fusion protein according to claim 1, wherein the linker has a length of 6, 9, 12, 15 or 18 amino acid residues.

20. The fusion protein according to claim 1, wherein the linker has the amino acid sequence of GSS GSS GSS GS (SEQ ID NO:

46).

21. A pharmaceutical composition comprising:

the fusion protein according to claim 1, and

a pharmaceutically acceptable carrier, diluent or adjuvant.

22. A nucleic acid encoding the fusion protein according to claim 1.

23. An expression vector comprising the nucleic acid according to claim 22.

24. A cell transformed or transfected with the expression vector according to claim 23.