Use of pre T alpha or functional variant thereof for expanding TCR alpha 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. A method to obtain modified TCR alpha deficient human T-cells, wherein said TCR alpha deficient human T-cells can proliferate independently of the Major Histocompatibility Complex signaling pathway, said method comprising the following steps:
(a) recovering cells from a human;
(b) genetically modifying said recovered cells ex-vivo by inactivating TCR alpha;
(c) introducing into said genetically modified cells an exogenous pTalpha polypeptide that supports CD3 surface expression; and
(d) expanding TCR alpha deficient human T-cells.
2. The method of claim 1 , wherein said recovered cells are stem cells.
3. The method of claim 1 , wherein said recovered cells are T-cells.
4. The method of claim 1 , wherein said TCR alpha deficient primary T-cells are expanded through stimulation of the CD3 complex.
5. The method of claim 1 , wherein said pTalpha polypeptide is truncated from C-terminus.
6. The method of claim 1 , wherein said pTalpha polypeptide is fused to a signal-transducing domain.
7. The method of claim 6 , wherein said signal-transducing domain is selected from the group consisting of: CD28, OX40, ICOS, CD137 and CD8.
8. The method of claim 1 , wherein said pTalpha polypeptide comprises a protein sequence selected from the group consisting of SEQ ID NO: 107 to SEQ ID NO: 124.
9. The method of claim 1 , wherein said pTalpha is fused to an extracellular ligand-binding domain.
10. The method of claim 9 , wherein said extracellular ligand-binding domain is a single chain antibody fragment (scFV).
11. The method of claim 1 , wherein said pTalpha polypeptide is introduced into the TCR alpha deficient cell by RNA electroporation.
12. The method of claim 1 , wherein deficient TCR alpha primary T cells are obtained by a step of inactivating the TCRalpha gene.
13. The method of claim 12 , wherein said TCRalpha gene is inactivated by introducing a rare-cutting endonuclease capable of inducing a DNA cleavage within TCRalpha gene.
14. The method of claim 13 , wherein rare-cutting endonuclease is TALE-nuclease.
15. The method of claim 14 , wherein TALE-nuclease is targeted against one of the gene target sequence of TCRalpha selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 57 to 60.
16. The method of claim 1 , wherein said TCR alpha deficient T-cells express a Chimeric Antigen Receptor.
17. The method of claim 1 , further comprising introducing into said TCR alpha deficient T-cells a TALE-nuclease able to selectively inactivate by DNA cleavage PDCD1 or CTLA-4 gene.
18. The method of claim 1 , wherein the recovered cell is from a patient diagnosed with cancer or infection.