Collectin fusion proteins comprising TNF or trail
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 selected from the group consisting of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and LIGHT, 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 element 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 element has a length of 9-15 amino acids.
5. The fusion protein of claim 1 wherein (i) is TRAIL (SEQ ID NO:10), or LIGHT (SEQ ID NO:16), or a receptor binding domain thereof.
6. The fusion protein of claim 1 wherein (i) comprises amino acids 91-240 of LIGHT (SEQ ID NO:16).
7. The fusion protein of claim 1 wherein (i) is TRAIL or a receptor binding domain thereof, which comprises amino acids 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO:10).
8. The fusion protein of claim 1 wherein (i) comprises a mutant of SEQ ID NO:10 or a receptor binding domain thereof, which binds and/or activates TRAILR1 and/or TRAILR2, wherein the mutant comprises amino acid substitutions only in one or more of the following amino acid positions of SEQ ID NO:10: R130, G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, I266, D267, and D269.
9. 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.
10. 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.
11. The fusion protein of claim 1 wherein (ii) comprises a mutant of SEQ ID NO:21 having only one amino acid substitution.
12. The fusion protein of claim 11 wherein the amino acid substitution affects 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.
13. The fusion protein of claim 11 wherein (ii) comprises a mutant which does not bind to mannose.
14. The fusion protein of claim 1 which additionally comprises an N-terminal signal peptide domain.
15. The fusion protein of claim 1 which comprises the sequence of SEQ ID NO: 41, 43, or 44.
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. The fusion protein of claim 1 which additionally comprises a terminal flexible element.
19. The fusion protein of claim 1 which is present as a trimeric complex.
20. The fusion protein of claim 19 wherein the complex is formed by covalent linkage between three fusion proteins.
21. The fusion protein of claim 1 wherein the complex consists of three identical fusion proteins.
22. A nucleic acid molecule encoding the fusion protein of claim 1 .
23. The nucleic acid molecule of claim 22 which is operatively linked to an expression control sequence.
24. A vector comprising the nucleic acid molecule of claim 22 .
25. An isolated cell transformed or transfected with the nucleic acid molecule of claim 22 .
26. The fusion protein of claim 8 , wherein the amino acid substitutions are one or more of the following: R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H, D269R, and D269K.
27. The fusion protein of claim 20 , wherein the covalent linkage consists of disulfide bridges between cysteines of (ii).
28. The fusion protein of claim 2 , wherein the flexible linker has the amino acid sequence of SEQ ID NO: 54.
29. The fusion protein of claim 14 wherein the N-terminal signal peptide domain comprises the sequence SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.