CD95L or trail fusion proteins
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.
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 .