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. The viral vector approach is extremely expensive due to the high cost of virus production and the high cost of long-term cell expansion that could take 10-14 days in a GMP facility. The viral vector approach also has a huge biologic downside: ex vivo expanded T-cells become bulky and lose efficacy against tumor cells. The use of electroporation technology in CAR-T-cell manufacturing has attracted increasing interests for its low cost and the wide range of applications, including transposon based stable CAR expression, transient expression and genome editing. However, actual clinical use of electroporation technology in CAR-T has been difficult and several clinical trials have met significant problems due to the poor transfection efficiency and decreased T-cell survival with traditional electroporation methods. Through theoretical analysis of the other existing electroporation technologies, we found that they all have problems in electrophysical design. The problems include physical design of devices, electrical pulse selection, and buffer composition. With a redesigned electroporation system, we can now achieve very high transfection efficiency for 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. Unlike viral vectors, electroporation works well on fresh natural T-cells, thereby eliminating the need for expensive cell expansion and virus production altogether and cutting the huge economic burden of CAR-T therapy. By reinfusion of more natural T-cells, the antitumor efficacy of CAR-T-cells could be improved while the side effects of cytokine release syndrome could be minimized.
Citation Format: Jian Chen, Xiaofeng Xia. Novel electroporation method for quick CAR-T-cell manufacture [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A026.