Methods for engineering highly active T cell for immunotherapy
The present invention relates to methods for developing engineered T-cells for immunotherapy and more specifically to methods for modifying T-cells by inactivating at immune checkpoint genes, preferably at least two selected from different pathways, to increase T-cell immune activity. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to highly efficient adoptive immunotherapy strategies for treating cancer and viral infections.
1. A population of isolated primary human T cells comprising at least 10 5 isolated primary human T cells that:
a) express a chimeric antigen receptor (CAR),
wherein the CAR comprises the amino acid sequence of SEQ ID NO:73;
b) have the TCRα or TCRβ alleles in the cells inactivated; and
c) have the PD-1 gene inactivated,
wherein inactivation of the PD1 gene enhances the anti-tumor activity of the population of isolated primary human T cells.
2. The population of isolated primary human T cells of claim 1 , having the TCRa alleles in the cells inactivated.
3. The population of isolated primary human T cells of claim 1 , having the TCRβ alleles in the cells inactivated.
4. The population of isolated primary human T cells of claim 1 , having the TCRα alleles and the TCRβ alleles in the cells inactivated.
5. The population of isolated primary human T cells of claim 1 , further having the CD52 alleles in the cells inactivated.
6. The population of isolated primary human T cells of claim 1 , further having the glucocorticoid receptor alleles in the cells inactivated.
7. The population of isolated primary human T cells of claim 2 , further expressing a fragment of preTalpha sufficient to support CD3 surface expression.
8. The population of isolated primary human T cells of claim 1 , having the PD-1 gene and CTLA-4 alleles in the cells inactivated.
9. A population of isolated primary human T cells comprising at least 10 5 isolated primary human T cells that:
a) express a chimeric antigen receptor (CAR) ,
wherein the CAR comprises the amino acid sequence of SEQ ID NO:73;
b) have the TCRa or TCRβ alleles in the cells inactivated; and
c) have the PD-1 or CTLA-4 alleles in the cells inactivated,
wherein inactivation of the PD1 alleles enhances the anti-tumor activity of the population of isolated primary human T cells.
10. The population of isolated primary human T cells of claim 9 , having the TCRα alleles in the cells inactivated.
11. The population of isolated primary human T cells of claim 9 , having the TCRβ alleles in the cells inactivated.
12. The population of isolated primary human T cells of claim 9 , having the TCRα alleles and the TCRβ alleles in the cells inactivated.
13. The population of isolated primary human T cells of claim 9 , further having the CD52 alleles in the cells inactivated.
14. The population of isolated primary human T cells of claim 9 , further having the glucocorticoid receptor alleles in the cells inactivated.
15. The population of isolated primary human T cells of claim 9 , further expressing a fragment of preTalpha sufficient to support CD3 surface expression.