Latency associated protein construct with aggrecanase sensitive cleavage site
The present provides a fusion protein comprising a latency associated peptide (LAP) and a pharmaceutically active agent in which the LAP and the pharmaceutically active agent are connected by an amino acid sequence comprising an aggrecanase proteolytic cleavage site.
1. A fusion protein comprising a latency associated peptide (LAP) which is the precursor domain of TGFβ-1, -2, -3, -4 or -5 and a cytokine in which the LAP and cytokine are connected by an amino acid sequence comprising an aggrecanase proteolytic cleavage site which is DVQEFRGVTAVIR (SEQ ID NO:24).
2. A fusion protein as claimed in claim 1 , in which a peptide linker sequence is present adjacent to the aggrecanase cleavage site.
3. A fusion protein as claimed in claim 2 , in which the peptide linker sequence is GGGGS (SEQ ID NO:57), or a multimer thereof.
4. A pharmaceutical composition comprising a fusion protein as claimed in claim 1 .
5. A nucleic acid construct comprising a first nucleic acid sequence encoding a cytokine, a second nucleic acid sequence encoding a LAP which is the latency associated peptide (LAP) of TGFβ-1, -2, -3, -4 or -5, and a nucleic acid sequence encoding an aggrecanase proteolytic cleavage site which is DVQEFRGVTAVIR (SEQ ID NO: 24) and wherein said site is provided between the first and second nucleic acid sequences.
6. A vector comprising a nucleic acid construct as claimed in claim 5 .
7. A fusion protein encoded by a nucleic acid construct comprising a first nucleic acid sequence encoding a cytokine, a second nucleic acid sequence encoding a LAP which is the latency associated peptide (LAP) of TGFβ-1, -2, -3, -4 or -5 wherein a nucleic acid sequence encoding an aggrecanase proteolytic cleavage site which is DVQEFRGVTAVIR (SEQ ID NO: 24) is provided between the first and second nucleic acid sequences.
8. A cell comprising a vector of claim 6 .
9. A composition comprising a nucleic acid construct as claimed in claim 5 .
10. A kit of parts comprising a fusion protein as claimed in claim 1 and an administration vehicle.
11. A process for preparing the fusion protein as claimed in claim 1 , comprising production of the fusion protein recombinantly by expression in a host cell and purification of the expressed fusion protein.
12. A process for preparing a nucleic acid construct as claimed in claim 5 comprising ligating together nucleic acid sequences encoding the LAP, the aggrecanase cleavage site, and the cytokine, optionally including a linker sequence on either side of the aggrecanase cleavage site.
13. A cell comprising a nucleic acid construct as claimed in claim 5 .
14. A composition comprising a cell as claimed in claim 8 .
15. A kit of parts comprising a nucleic acid construct as claimed in claim 5 and an administration vehicle.
16. A kit of parts comprising a cell as claimed in claim 8 and an administration vehicle.