Here, we developed a straightforward methodology to generate TCRαβ negative (allogeneic) cells for CAR-T cell therapies. With an early and transient expression of an anti-CD3 CAR in the engineered donor T cells, we programmed these cells to self-eliminate the TCR+ cell population and obtained an ultrapure TCRαβ– population (99–99.9%) at the end of the CAR-T production. This novel and easy-to-implement procedure preserves the production yield and cell fitness and has the potential to streamline the manufacturing of “off-the-shelf” CAR T-cell therapies.
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