Single chain CD40L 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 CD40 ligand (CD40L) cytokine domain,
(ii) a first peptide linker,
(iii) a second soluble CD40L cytokine domain,
(iv) a second peptide linker, and
(v) a third soluble CD40L cytokine domain,
wherein each of the soluble CD40L 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 CD40L cytokine domains are independently an N-terminally shortened domain of SEQ ID NO: 5, and optionally comprises an amino acid mutation.
3. The polypeptide of claim 1 , wherein the first, the second, and the third soluble CD40L cytokine domains independently have the amino sequence of 112-261, 117-261, or 121-261 of SEQ ID NO: 5.
4. The polypeptide of claim 1 , wherein the first, the second, and the third soluble CD40L cytokine domains have the amino sequence of 121-261 of SEQ ID NO: 5.
5. The polypeptide of claim 1 , wherein the first and second peptide linkers are independently glycine/serine linkers.
6. The polypeptide of claim 5 , wherein the glycine/serine linkers comprise substituted asparagine residues.
7. The polypeptide of claim 1 , wherein the first and second peptide linkers are independently selected from the group consisting of SEQ ID NOs:52-53 and SEQ ID NO:21-26.
8. The polypeptide of claim 1 , which additionally comprises an N-terminal signal peptide domain.
9. The polypeptide of claim 8 , wherein the N-terminal signal peptide domain comprises a protease cleavage site.
10. The polypeptide of claim 1 , which additionally comprises a further domain at the N-terminal and/or C-terminal end.
11. The polypeptide of claim 10 , wherein the further domain is a Fab or Fc fragment domain.
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 .
14. A pharmaceutical composition comprising the fusion polypeptide of claim 1 and a pharmaceutically acceptable carrier, diluent and/or adjuvant.
15. The polypeptide of claim 1 , which additionally comprises a further domain of Fc fragment and a third peptide linker at the C-terminal end.
16. A dimer comprises two polypeptides of claim 15 , wherein the two polypeptides are dimerized via disulfide bridges of the third peptide linkers.