Non‐Viral Manufacturing of Cellular Immunotherapy Using Simple Mechanical Transfection Device

Abstract In recent years, cellular immunotherapies, such as chimeric antigen receptor T (CAR‐T) therapy, have emerged as promising treatment options for cancer, demonstrating particularly strong efficacy against liquid tumors. By introducing tumor‐targeting CAR genes into patient immune cells ex vivo and reintroducing the modified cells into the patient, tumor cells expressing specific surface markers can be selectively killed. However, existing virus‐based methods for producing cellular products are plagued by high costs, which seriously limit their adoption. In this paper, the development of a mechanical transfection device is described, which passes cells through micron‐sized pores and is capable of delivering different molecules into cells. The effects of parameters such as pore size, flow rate, and payload concentration on transfection efficiency are studied and used to inform a standard transfection protocol. Finally, the delivery of CAR‐encoding mRNA into primary T‐cells is demonstrated to manufacture CAR‐T cells, which secrete IFN‐γ and TNF‐α in an antigen‐specific manner. As the method is developed from the outset to be easily deployable and scalable, it is envisioned that it will be able to impact cell manufacturing in the near future.