Multimeric TNF receptor fusion proteins and nucleic acids encoding same
The present invention refers to fusion proteins comprising a TNF receptor family extracellular domain fused to a trimerization domain, and a nucleic acid molecule encoding the fusion protein. The fusion protein may be present as a trimeric complex. It is suitable for therapeutic, diagnostic and/or research applications.
1. A fusion protein comprising
(i) a TNF-family receptor extracellular domain,
(ii) a flexible linker element comprising more than 2 amino acids between components (i) and (iii), and
(iii) a collectin trimerization domain comprising a neck and a carbohydrate binding domain.
2. The fusion protein of claim 1 wherein the TNF-family receptor is TNFRSF6 (SEQ ID NO: 6).
3. The fusion protein of claim 2 wherein component (i) comprises amino acids 1 to 169 of TNFRSF6 (SEQ ID NO: 6).
4. The fusion protein of claim 1 wherein component (ii) has a length of from 2-20 amino acids.
5. The fusion protein of claim 4 wherein component (ii) is a glycine/serine linker.
6. The fusion protein of claim 5 wherein component (ii) has the amino acid sequence (GSS) a (GSG) b (SEQ ID NO: 41) or (GTT) a (GTG) b (SEQ ID NO: 42) wherein a or b is 0, 1, 2, 3, 4 or 5, wherein when a=0 then b is ≧1 and when b=0 then a is ≧1.
7. The fusion protein of claim 1 wherein the collectin is surfactant protein-D, surfactant protein-A, mannan-binding protein-A, mannan-binding-protein-C, collectin liver 1, collectin placenta 1, or collectin-11.
8. The fusion protein of claim 7 wherein component (iii) comprises amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, 225-375 of human surfactant protein-D of SEQ ID NO: 59.
9. The fusion protein of claim 1 wherein the collectin comprises only one amino acid substitution of SEQ ID NO: 59 or 60.
10. The fusion protein of claim 9 wherein the amino acid substitution affects amino acid position F355 of human surfactant protein-D of SEQ ID NO: 59.
11. The fusion protein of claim 10 wherein the amino acid substitution is one of the following: F355A, F355S, F355T, F355E, F355D, F355K, or F355R.
12. The fusion protein of claim 9 wherein the collectin is a mutant which does not bind to mannose.
13. The fusion protein of claim 1 wherein component (iii) comprises amino acids 110-271, 116-271, or 121-271 of human collectin-11 of SEQ ID NO: 60.
14. The fusion protein of claim 1 wherein component (i) is located N-terminally and component (iii) is located C-terminally.
15. The fusion protein of claim 1 wherein component (iii) is located N-terminally and component (i) is located C-terminally.
16. The fusion protein of claim 1 which additionally comprises an N-terminal signal peptide domain, which optionally comprises a protease cleavage site.
17. The fusion protein of claim 1 which additionally comprises a C-terminal flexible element which optionally comprises and/or connects to a recognition/purification domain.
18. A trimeric complex consisting of three identical fusion proteins of claim 1 .
19. The fusion protein of claim 2 wherein component (i) comprises Arg17-Glu168 of TNFRSF6 (SEQ ID NO: 6).
20. The fusion protein of claim 7 wherein the collectin is collectin-11.
21. An isolated nucleic acid molecule encoding the fusion protein of claim 1 , optionally operatively linked to an expression control sequence.
22. An isolated cell transformed or transfected with the nucleic acid molecule of claim 21 .