Method for in situ inhibition of regulatory T cells
The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are designed to express both a Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell, and a secreted inhibitor of regulatory T-cells (Treg). Preferably, such secreted inhibitor is a peptide inhibitor of forkhead/winged helix transcription factor 3 (FoxP3), a specific factor involved into the differentiation of T-cells into regulatory T-cells. The engineered T-cells of the invention direct their immune activity towards specific malignant or infected cells, while at the same time will prevent neighboring regulatory T-cells from modulating the immune response. The invention opens the way to standard and affordable adoptive immunotherapy strategies, especially for treating or preventing cancer, and bacterial or viral infections.
1. A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an engineered T-cell comprising:
a) a first exogenous nucleic acid molecule comprising a nucleotide sequence coding for a first chimeric antigen receptor (CAR) comprising an extracellular domain capable of binding to at least one antigen expressed at the surface of a malignant cell; and
b) at least a second exogenous nucleic acid molecule comprising a nucleotide sequence coding for a peptide-inhibitor of FoxP3 comprising the amino acid sequence of SEQ ID NO 1 or SEQ ID NO:3,
wherein the peptide-inhibitor of FoxP3 inhibits regulatory T-cell activity.
2. The method according to claim 1 , wherein the engineered T-cell further comprises an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a rare-cutting endonuclease that selectively inactivates a gene coding for at least one component of the T-Cell receptor (TCR).
3. The method according to claim 2 , wherein the at least one component of the TCR is TCR alpha or TCR beta.
4. The method according to claim 1 , wherein the inhibitor of FoxP3 is a cell penetrating peptide.
5. The method according to claim 1 , wherein the first CAR and the peptide inhibitor of FoxP3 are expressed by the T-cell.
6. The method according to claim 1 , further comprising a third exogenous nucleic acid molecule comprising nucleotide sequence coding for a second CAR comprising an extracellular domain capable of binding to at least one other antigen expressed at the surface of a regulatory T-cell.
7. The method according to claim 6 , wherein the second CAR is expressed by the T-cell.
8. The method according to claim 1 wherein the cell further comprises a deletion or mutation in at least one gene coding for one component of the TCR.
9. The method according to claim 1 , wherein the cell comprises an inactivated CD25.
10. The method according to claim 1 , wherein the first CAR is directed against B-lymphocyte antigen CD19.
11. The method according to claim 6 , wherein the first CAR is a single chain CAR.
12. The method according to claim 6 , wherein the first and second CARs are single chain CARs.
13. The method according to claim 6 , wherein the first and second CARs are multi-chain CARs.
14. The method according to claim 6 , wherein the second CAR is mono-specific or multi-specific.
15. The method according to claim 6 , wherein the second CAR is directed to CD25.
16. The method according to claim 1 , wherein the cell is derived from a cytotoxic T-lymphocyte.