Single chain trail fusion polypeptides and encoding nucleic acids
The present invention refers to single-chain fusion proteins comprising three soluble TNF superfamily (TNFSF) cytokine domains and nucleic acid molecules encoding these fusion proteins. The fusion proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.
1. A single-chain fusion polypeptide comprising:
(i) a first soluble TNF-related apoptosis-inducing ligand (TRAIL) cytokine domain,
(ii) a first peptide linker,
(iii) a second soluble TRAIL cytokine domain,
(iv) a second peptide linker, and
(v) a third soluble TRAIL cytokine domain,
wherein each of the soluble TRAIL domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids.
2. The polypeptide of claim 1 , wherein the second and/or third soluble TRAIL domain is an N-terminally shortened domain which optionally comprises amino acid sequence mutations.
3. The polypeptide of claim 1 , wherein at least one of the soluble TRAIL domains has an N-terminal sequence which starts at amino acid Gln120, Arg121, or Val122 of human TRAIL (SEQ ID NO: 10), and the Arg121 is optionally replaced by a neutral amino acid of serine or glycine.
4. The polypeptide of claim 3 , wherein at least one of the soluble TRAIL domains has an N-terminal sequence selected from
(a) Arg121-Val122-Ala123 and
(b) (Gly/Ser)121-Val122-Ala123.
5. The polypeptide of claim 3 , wherein at least one of the soluble TRAIL domains ends with amino acid Gly281 of human TRAIL and optionally comprises one or more mutations at positions R130, G160, H168, R170, H177, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, I266, D267 or D269.
6. The polypeptide of claim 1 , which additionally comprises an N-terminal signal peptide domain.
7. The polypeptide of claim 6 , wherein the N-terminal signal peptide domain comprises a protease cleavage site.
8. The polypeptide of claim 1 , which additionally comprises a further domain at the N-terminal and/or C-terminal end.
9. The polypeptide of claim 8 , wherein the further domain is a Fab or Fc fragment domain.
10. A pharmaceutical composition comprising the fusion polypeptide of claim 1 and a pharmaceutically acceptable carrier, diluent and/or adjuvant.
11. The polypeptide of claim 1 , wherein the first and second peptide linkers are independently glycine/serine linkers.
12. The polypeptide of claim 11 , wherein the glycine/serine linkers comprises substituted asparagine residues.
13. The polypeptide of claim 9 , wherein the further domain is a Fc fragment domain at the C-terminal end.
14. The polypeptide of claim 9 , wherein the further domain is an Fc fragment domain at the N-terminal end.
15. A dimer comprising two polypeptides of claim 13 , fused via disulfide bridges between the Fc fragment domains.
16. A dimer comprising two polypeptides of claim 14 , fused via disulfide bridges between the Fc fragment domains.
17. An isolated nucleic acid molecule encoding the fusion polypeptide of claim 1 .
18. An isolated host cell or a non-human organism transformed or transfected with the nucleic acid molecule of claim 17 .