CAR-T cells are currently manufactured for clinical use by infection of human T cells with viral vectors containing the CAR gene. T lymphocytes have to be stimulated and expanded ex vivo because the viral vectors infect fresh natural lymphocytes very poorly. NK cells are also commonly expanded ex vivo before they are subject to electroporation. Possibly influenced by virus infection protocols, T cell and NK cell electroporation is often done after they are ex vivo expanded. We developed a novel electroporation technology that corrected many physics mistakes in the field. We can now achieve very high transfection efficiency for fresh T cells while maintaining cell survival. For Sleeping Beauty transposon-based CAR expression, we found that over a period of two to three weeks the efficiency can get to 60% to 90% with fast cell proliferation. The protein expression time after electroporation is very short. For simple GFP plasmids we can observe GFP expression after only 30 minutes. Similarly, for NK cells, we were able to achieve 42% transfection efficiency with fresh nonexpanded NK cells. We further compared post-electroporation cell proliferation potential between nonexpanded fresh T cells and stimulated/expanded T cells. It turned out that although fresh T cells are smaller in volume, they are much better than expanded T cells in survival after electroporation. Expanded NK cells also showed much higher cytotoxicity than fresh NK cells after electroporation. With our new electroporation technology, CAR-T and CAR-NK can now be manufactured quicker and better. Our new protocols focusing on fresh T cells and fresh NK cells would be a great help for the field.

Citation Format: Jian Chen, Xiaofeng Xia. High-efficiency electroporation of nonexpanded T cells and NK cells for CAR-T and CAR-NK [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A44.