Methods for engineering allogeneic and immunosuppressive resistant T cell for immunotherapy

A method of preparing engineered T-cells for immunotherapy comprising

• (a) inactivating PDCD1 in the T-cells comprising introducing into the T-cells by electroporation of RNAs encoding at least two half Transcription activator-like effector nucleases (TALE-nucleases) able to selectively inactivate by DNA cleavage a gene encoding PDCD1, to produce engineered T-cells;
• and
• (b) expanding said engineered T-cells,
• wherein said at least two half TALE-nucleases recognize and cleave a target sequence selected from SEQ ID NO:77 and SEQ ID NO:78;
• wherein introducing said at least two half Transcription activator-like effector nucleases (TALE-nucleases) into said T-cells comprises contacting said engineered T-cells with RNAs encoding said at least two half TALE-nucleases;
• and
• i. applying an agile pulse sequence consisting of:
• one electrical pulse with a voltage range from 2250 to 3000 V per centimeter, a pulse width of 0.1 ms and a pulse interval of 0.2 to 10 ms between the electrical pulses of steps (i) and (ii);
• ii. one electrical pulse with a voltage range from 2250 to 3000 V with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (ii) and the first electrical pulse of step (iii);
• and
• iii. 4 electrical pulses with a voltage of 325 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses.