Methods for engineering allogeneic and immunosuppressive resistant T cell for immunotherapy
Methods for developing engineered T-cells for immunotherapy that are both non-alloreactive and resistant to immunosuppressive drugs. The present invention relates to methods for modifying T-cells by inactivating both genes encoding target for an immunosuppressive agent and T-cell receptor, in particular genes encoding CD52 and TCR. 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 method for generating a population of doubly-inactivated primary human T-cells for immunotherapy comprising:
providing at least 5×10 6 primary human T cells from a single donor;
co-electroporating into said primary human T-cells:
(a) two different RNAs each encoding one half TALE-nuclease, wherein the two half TALE-nucleases dimerize to form a first TALE-nuclease that binds and cleaves the T-cell receptor alpha (TCRα) sequence consisting of SEQ ID NO:37 in a TCRα gene, and
(b) two different RNAs each encoding one half TALE-nuclease, wherein the two half TALE-nucleases dimerize to form a second TALE-nuclease that binds and cleaves the CD52 sequence consisting of SEQ ID NO:40 in a CD52 gene,
to generate a population of transfected human T cells comprising doubly-inactivated human T-cells having both TCRα and CD52 genes inactivated; and
expanding the population of doubly inactivated human T-cells; whereby a population of doubly-inactivated human T-cells sufficient for infusing into an individual for immunotherapy treatment is generated.
2. The method of claim 1 , wherein the population of doubly inactivated human T-cells is further modified to express a chimeric antigen receptor.
3. The method of claim 1 , wherein the first TALE-nuclease is encoded by the nucleotide sequences of SEQ ID NO:49 and SEQ ID NO:50.
4. The method of claim 3 , wherein the population of doubly inactivated human T-cells is further modified to express a chimeric antigen receptor.
5. The method of claim 1 , wherein the second TALE-nuclease is encoded by the nucleotide sequences of SEQ ID NO:55 and SEQ ID NO:56.
6. The method of claim 5 , wherein the population of doubly inactivated human T-cells is further modified to express a chimeric antigen receptor.
7. The method of claim 2 , further comprising purifying the doubly-inactivated cells.
8. The method of claim 4 , further comprising purifying the doubly-inactivated cells.
9. The method of claim 6 , further comprising purifying the doubly-inactivated cells.