TNF superfamily collectin fusion proteins and encoding nucleic acids
The present invention refers to a fusion protein comprising a TNF-superfamily (TNFSF) cytokine or a receptor binding domain thereof fused to a collectin trimerization domain, to a nucleic acid molecule encoding the fusion protein, and to a cell comprising the nucleic acid molecule. The fusion protein is present as a trimeric complex or as an oligomer thereof. The fusion protein, the nucleic acid, and the cell is suitable as pharmaceutical composition or for therapeutic, diagnostic and/or research applications.
1. A fusion protein comprising:
(i) a TNF (tumor necrosis factor)-superfamily cytokine of CD95L, or a receptor binding domain thereof, and
(ii) a collectin trimerization domain comprising a neck domain or a neck and carbohydrate binding domain of surfactant protein-D, wherein (ii) is located C-terminally of (i).
2. The fusion protein of claim 1 , further comprising a flexible linker between (i) and (ii), wherein the flexible linker is a glycine/serine linker and has a length of 3-20 amino acids.
3. The fusion protein of claim 2 , wherein the flexible linker has the amino acid sequence of SEQ ID NO: 53, which is (GSS) a (SSG) b (GSG) c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6.
4. The fusion protein of claim 2 , wherein the flexible linker has a length of 9-15 amino acids.
5. The fusion protein of claim 2 , wherein the flexible linker has the amino acid sequence of SEQ ID NO: 54.
6. The fusion protein of claim 1 , wherein the CD95L has the amino acid sequence of SEQ ID NO: 6.
7. The fusion protein of claim 1 , wherein (i) comprises amino acids 142-281 or 144-281 of SEQ ID NO: 6.
8. The fusion protein of claim 1 , wherein (ii) comprises amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, or 225-375 of human surfactant protein-D of SEQ ID NO:21.
9. The fusion protein of claim 1 , wherein (ii) comprises amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:21.
10. The fusion protein of claim 1 , wherein (ii) comprises a mutant of SEQ ID NO:21 having only one amino acid substitution.
11. The fusion protein of claim 10 , wherein the amino acid substitution is at the amino acid position F355 of human surfactant protein-D of SEQ ID NO:21, and wherein the amino acid substitution is one of the following: F355A, F355S, F355T, F355E, F355D, F355K, or F355R.
12. The fusion protein of claim 1 , which comprises the sequence of SEQ ID NO: 40.
13. The fusion protein of claim 1 , which comprises amino acid residues 21-327 of SEQ ID NO: 40.
14. The fusion protein of claim 1 , which additionally comprises an N-terminal signal peptide domain.
15. The fusion protein of claim 14 , wherein the N-terminal signal peptide domain has the sequence SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.
16. The fusion protein of claim 1 , wherein the fusion protein further comprises a recognition/purification domain located at the N-terminus or at the C-terminus.
17. The fusion protein of claim 16 , wherein the recognition/purification domain is a Strep-tag or a poly His-domain.
18. A trimeric complex formed by covalent linkages between three of the fusion proteins of claim 1 .
19. A nucleic acid molecule encoding the fusion protein of claim 1 .
20. A vector comprising the nucleic acid molecule of claim 18 .