Targeted protein degradation
The invention relates to a method of controlling the level of a polypeptide sequence comprising administering a polypeptide sequence fused to a ubiquitin targeting protein which comprises a minimal degron structural motif. In particular, the polypeptide sequence comprises a chimeric antigen receptor therefore the present invention is useful in methods of cell and gene therapy where the activity of the chimeric antigen receptor needs to be controlled.
1. A chimeric antigen receptor (CAR) comprising: an extracellular ligand binding domain; a transmembrane domain; an intracellular signalling domain; and, a ubiquitin targeting protein consisting of less than 135 amino acids in length which comprises the hairpin motif of a cereblon binding site, which is capable of being bound by ubiquitin ligase in the presence of a compound which mediates binding of the ubiquitin targeting protein and cereblon.
2. An isolated polynucleotide encoding the chimeric antigen receptor (CAR) of claim 1 .
3. An expression vector comprising a polynucleotide of claim 2 .
4. A cell comprising a polynucleotide of claim 2 .
5. A cell of claim 4 , which is an immunomodulatory cell.
6. A veil according to claim 5 , which is a T-cell.
7. A pharmaceutical composition comprising a plurality of cells of claim 4 .
8. The pharmaceutical composition of claim 7 , which additionally comprises a pharmaceutically acceptable excipient, carrier, or diluent.
9. A method of engineering an immunomodulatory cell, comprising: (a) providing an immunomodulatory cell; (b) transducing or transfecting the polynucleotide of claim 2 into said immunomodulatory cell; and (c) expressing said polynucleotide in the immunomodulatory cell.
10. A method of affecting the amount of a polypeptide in a system comprising:
a) contacting a fusion protein comprising said polypeptide and a ubiquitin targeting protein consisting of less than 135 amino acids in length which comprises the hairpin motif of a cereblon binding site, wherein said polypeptide is a transmembrane protein, and
b) contacting a compound which mediates binding of the ubiquitin targeting protein and cereblon;
wherein the binding of the ubiquitin targeting protein to cereblon affects the amount of polypeptide in a system.
11. A method of affecting the amount of a polypeptide in a system comprising:
a) contacting a fusion protein comprising said polypeptide and a ubiquitin targeting protein consisting of less than 135 amino acids in length which comprises the hairpin motif of a cereblon binding site, wherein said polypeptide is a chimeric antigen receptor (CAR), and
b) contacting a compound which mediates binding of the ubiquitin targeting protein and cereblon;
wherein the binding of the ubiquitin targeting protein to cereblon affects the amount of polypeptide in a system.