Glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) single-chain fusion polypeptides
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 glucocorticoid-induced tumor necrosis factor receptor ligand ligand (GITRL) cytokine domain,
(ii) a first peptide linker,
(iii) a second soluble GITRL cytokine domain,
(iv) a second peptide linker, and
(v) a third soluble GITRL cytokine domain,
wherein each of the soluble GITRL cytokine 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 first, the second, and the third soluble GITRL cytokine domains are independently an N-terminally shortened domain of SEQ ID NO:18, and optionally consists of an amino acid mutation.
3. The polypeptide of claim 1 , wherein the first, the second, and the third soluble GITRL cytokine domains independently have the amino sequence of 52-177 of SEQ ID NO: 18.
4. The polypeptide of claim 1 , wherein the first and second peptide linkers are, independently, glycine/serine linkers.
5. The polypeptide of claim 4 , wherein the glycine/serine linkers comprise substituted asparagine residues.
6. The polypeptide of claim 1 , wherein the first and second peptide linkers are independently selected from the group consisting of SEQ ID NO: 52-53 and SEQ ID NO: 1-26.
7. The polypeptide of claim 1 , which additionally comprises an N-terminal signal peptide domain.
8. The polypeptide of claim 7 , wherein the N-terminal signal peptide domain comprises a protease cleavage site.
9. The polypeptide of claim 1 , which additionally comprises a further domain at the N-terminal and/or C-terminal end.
10. The polypeptide of claim 1 , which additionally comprises a further domain and a third peptide linker at the C-terminal end.
11. A dimer comprising two polypeptides of claim 10 , wherein the two polypeptides are dimerized via disulfide bridges of the third peptide linkers.
12. An isolated nucleic acid molecule encoding the fusion polypeptide of claim 1 .
13. An isolated host cell or a non-human organism transformed or transfected with the nucleic acid molecule of claim 12 .