Recombinant polypeptides of the members of the TNF ligand family and use thereof
The invention relates to polypeptides comprising, as constituent A, at least three monomers of a member of the TNF ligand family and, as constituent B, at least two peptide linkers that link the monomers of the member of the TNF ligand family to one another. The invention also relates to the use of these polypeptides for treating diseases and for producing a medicament or a vaccine. The invention also relates to methods for producing and isolating these polypeptides, to nucleic acids that code for these polypeptides, to vectors containing these nucleic acids, to host cells transfected with these vectors, and to pharmaceutical compositions containing these inventive objects. Finally, the invention relates to methods for the extracorporeal manipulation, depletion and/or removal of components contained in body fluids, e.g. by means of apheresis.
1. A method for extracorporeal depletion or removal of tumor necrosis factor receptor (TNFR) from blood or blood fractions comprising the following steps:
a) Optional separation of the blood into one or more blood fractions with solid or liquid components;
b) Binding of the blood or the blood fractions expressing a TNFR to a surface or particle coupled to a polypeptide wherein the polypeptide comprises at least three components A and at least two components B, wherein each component A comprises a tumor necrosis factor (TNF) monomer or a functional fragment or a functional variant thereof, and each component B is a peptide linker under conditions allowing binding of TNFR in the blood or the blood fractions to the surface or the particle; and
c) Separating the bound TNFR from the blood or the blood fractions, thereby depleting or removing TNFR from the blood or the blood fractions.
2. The method according to claim 1 , wherein before step a) or b) blood is taken from a patient.
3. The method according to claim 1 , wherein after a step b) or c), the blood or blood fraction is injected or reinjected into a patient.
4. The method according to claim 1 , wherein components A are identical or different.
5. The method according to claim 1 , wherein the blood or the blood fraction is from the same organism or different organisms.
6. The method according to claim 1 , wherein components B each link together two of the at least three components A.
7. The method according to claim 1 , wherein at least one of components B has the amino acid sequence (GGGS) 3 (SEQ ID NO:2) or (GGGS) 4 (SEQ ID NO:3).
8. The method according to claim 1 , wherein components A and components B form a trimeric protein structure.
9. The method according to claim 8 , wherein components A and components B form a homotrimeric protein structure.
10. The method according to claim 8 , wherein components A and components B form a heterotrimeric protein structure.
11. The method according to claim 1 , wherein components B are identical or different.
12. The method according to claim 1 , wherein components B are from the same organism or different organisms.
13. The method according to claim 1 , wherein the polypeptide has an N-terminal tag sequence.
14. The method according to claim 13 , wherein the N-terminal tag sequence is a His tag sequence or a Flag tag sequence.
15. The method according to claim 1 , wherein the polypeptide has an N-terminal leader peptide sequence.
16. The method according to claim 1 , wherein the polypeptide further comprises component C, wherein component C comprises an antibody fragment or a different protein or peptide, which selectively recognizes a specific target molecule on a cell surface.
17. The method according to claim 16 , wherein component C is an antibody fragment from a mammal.
18. The method according to claim 17 , wherein the mammal is human.
19. The method according to claim 17 , wherein the antibody fragment is a humanized antibody fragment.
20. The method according to claim 16 , wherein the antibody fragment comprises scFv.
21. The method according to claim 1 , wherein at least one component A comprises a soluble tumor necrosis factor (TNF) monomer.
22. The method according to claim 1 , wherein each component A comprises a soluble tumor necrosis factor (TNF) monomer.
23. The method according to claim 1 , wherein the polypeptide is coupled to the surface or particle by a cysteine residue in the first 1-15 N-terminal amino acids of the polypeptide.