Methods for engineering allogeneic and highly active T cell for immunotheraphy
The present invention relates to methods for developing engineered T-cells for immunotherapy that are non-alloreactive. The present invention relates to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and an immune checkpoint gene to unleash the potential of the immune response. 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 standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.
1 . A T-cell comprising a first gene selectively inactivated by DNA cleavage by a first rare-cutting nuclease, wherein said first rare-cutting nuclease is a first TALE-nuclease directed against a PDCD1 (PD1) gene, wherein said first TALE-nuclease is directed against one of the gene target sequences of the PD-1 gene selected from the group consisting of SEQ ID NO:77 and SEQ ID NO:78, and wherein
(a) said first TALE-nuclease comprises amino acid sequences selected from the group consisting of (i) SEQ ID NOs:85 and 86, and (ii) SEQ ID NOs:87 and 88; and/or
(b) said first TALE-nuclease comprises amino acid sequences encoded by nucleic acid sequences selected from the group consisting of (i) SEQ ID NOs:95 and 96, and (ii) SEQ ID NOs:97 and 98.
2 . The T-cell of claim 1 , further comprising a second gene selectively inactivated by DNA cleavage by a second rare-cutting nuclease, wherein said second rare-cutting nuclease is a second TALE-nuclease directed against a CTLA-4, LAG3, or TIGIT gene.
3 . The T-cell according to claim 1 , further comprising a second gene selectively inactivated by DNA cleavage by a second rare-cutting nuclease, wherein said second rare-cutting nuclease is a second TALE-nuclease directed against a Tim3, BTLA, BY55 (CD160), TIGIT, B7H5, LAIR1, SIGLEC10, or 2B4 gene.
4 . The T-cell according to claim 2 , wherein each of the first and the second rare-cutting nucleases are encoded by RNA.
5 . The T-cell according to claim 4 , wherein each of the first and the second rare-cutting nucleases are introduced into said T-cell by way of RNA electroporation.
6 . The T-cell according to claim 2 , wherein said second TALE-nuclease is directed against one of the gene target sequences of CTLA-4 selected from the group consisting of SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.
7 . The T-cell according to claim 6 , wherein:
(a) said second TALE-nuclease comprises amino acid sequences selected from the group consisting of (i) SEQ ID NOs: 79 and 80, (ii) SEQ ID NOs: 81 and 82, and (iii) SEQ ID NOs:83 and 84; and/or
(b) said second TALE-nuclease comprises amino acids encoded by nucleic acid sequences selected from the group consisting of (i) SEQ ID NOs: 89 and 90, (ii) SEQ ID NOs: 91 and 92, and (iii) SEQ ID NOs: 93 and 94.