CD40L collectin fusion proteins and encoding nucleic acid molecules
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 CD40 ligand (CD40L) 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 , additionally 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 1 , wherein the CD40L has the sequence of SEQ ID NO:5.
6. The fusion protein of claim 1 , wherein (i) comprises amino acids 112-261 or 117-261 of a human CD40L of SEQ ID NO:5.
7. 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 a human surfactant protein-D of SEQ ID NO:21.
8. 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 a human surfactant protein-D of SEQ ID NO:21.
9. The fusion protein of claim 1 , wherein (ii) comprises a mutant of human surfactant protein-D of SEQ ID NO:21 having only one amino acid substitution.
10. The fusion protein of claim 9 , wherein the amino acid substitution affects amino acid position F355 of SEQ ID NO:21, and the amino acid substitution is one of the following: F355A, F355S, F355T, F355E, F355D, F355K, or F355R.
11. The fusion protein of claim 9 , wherein (ii) comprises a mutant which does not bind to mannose.
12. The fusion protein of claim 1 , which additionally comprises an N-terminal signal peptide domain.
13. 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.
14. The fusion protein of claim 13 , wherein the recognition/purification domain is a Strep-tag or a poly His-domain.
15. The fusion protein of claim 1 , which additionally comprises a C-terminal flexible element.
16. A trimeric complex comprising three fusion proteins of claim 1 .
17. The trimeric complex of claim 16 , wherein the complex is formed by covalent linkage between the three fusion proteins.
18. The trimeric complex of claim 17 , wherein the complex consists of three identical fusion proteins.
19. A nucleic acid molecule encoding the fusion protein of claim 1 .
20. The nucleic acid molecule of claim 19 , which is operatively linked to an expression control sequence.
21. A vector comprising the nucleic acid molecule of claim 20 .
22. An isolated cell transformed or transfected with the nucleic acid molecule of claim 20 .