Use of preTalpha or functional variant thereof for expanding TCRalpha deficient T cells
A method of expanding TCRalpha deficient T-cells by expressing pTalpha or functional variants thereof into said cells, thereby restoring a functional CD3 complex. This method is particularly useful to enhance the efficiency of immunotherapy using primary T-cells from donors. This method involves the use of pTalpha or functional variants thereof and polynucleotides encoding such polypeptides to expand TCRalpha deficient T-cells. Such engineered cells can be obtained by using specific rare-cutting endonuclease, preferably TALE-nucleases. The use of Chimeric Antigen Receptor (CAR), especially multi-chain CAR, in such engineered cells to target malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.
1. An isolated TCRalpha deficient primary human T cell,
wherein the TCRalpha gene in the T cell has been inactivated by introducing a rare-cutting endonuclease that induces a DNA cleavage within the TCRalpha gene,
comprising an exogenous nucleic acid encoding an exogenous pTalpha polypeptide that supports CD3 surface expression and supports expansion of the TCR-deficient primary T cell using stimulatory anti-CD3/CD28 antibodies.
2. The isolated TCRalpha deficient human T cell according to claim 1 , wherein said pTalpha is truncated from C-terminus.
3. The isolated TCRalpha deficient human T cell according to claim 2 , wherein said pTalpha is fused to a signal-transducing domain.
4. The isolated TCRalpha deficient human T cell according to claim 3 , wherein said signal-transducing domain is selected from the group consisting of: CD28, OX40, ICOS, CD137 and CD8.
5. The isolated TCRalpha deficient human T cell according to claim 4 , wherein said pTalpha comprises protein sequence selected from the group consisting of SEQ ID NO:107 to SEQ ID NO:124.
6. The isolated TCRalpha deficient human T cell according to claim 1 , wherein said pTalpha is fused to an extracellular ligand-binding domain.
7. The isolated TCRalpha deficient human T cell according to claim 6 , wherein said extracellular ligand-binding domain is a single chain antibody fragment (scFV).
8. The isolated TCRalpha deficient human T cell according to claim 1 , wherein said nucleic acid is an RNA.
9. The isolated TCRalpha deficient human T cell according to claim 1 , wherein said nucleic acid is a transgene.
10. The isolated TCRalpha deficient human T cell according to claim 1 , wherein rare-cutting endonuclease is TALE-nuclease.
11. The isolated TCRalpha deficient human T cell according to claim 10 , wherein TALE-nuclease is targeted against one of the gene target sequences of TCRalpha selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:57 to 60.
12. The isolated TCRalpha deficient human T cell according to claim 1 , further comprising a Chimeric Antigen Receptor.
13. The isolated TCRalpha deficient human T cell according to claim 1 , further comprising a TALE-nuclease able to selectively inactivate by DNA cleavage PDCD1 or CTLA-4 gene.
14. The isolated TCRalpha deficient human T cell according to claim 1 , wherein said T-cell is recovered from a patient diagnosed with cancer or infection.
15. A pharmaceutical composition comprising the isolated TCRalpha deficient human T cell according to claim 1 .
16. A population of at least 10 4 isolated TCRalpha deficient primary human T cells comprising an exogenous nucleic acid encoding an exogenous pTalpha polypeptide that supports CD3 surface expression.
17. The population of primary human T cells according to claim 16 , wherein the exogenous pTalpha polypeptide supports expansion of the TCR-deficient primary T cells using stimulatory anti-CD3/CD28 antibodies.