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Fusing Drug- and Light-Responsive Proteins to Reprogram the Activation of CAR T Cells with AND Logic


Despite its incredible potential in treating cancer, chimeric antigen receptor (CAR) T cell therapy carries the deadly risk of cytokine release syndrome. For CAR T cell therapy to be an effective therapeutic, it must be controlled in a spatiotemporal manner to prevent unwanted CAR activation. To address this, a cre-lox recombination system called the “TamPA-Cre” system was developed that requires both the presence of 4OHT, and blue light for recombination. Genetic cassettes containing CARs, which only get transcribed after recombination, were then inserted into T cells, so both blue light and 4OHT could induce the activation of CARs. First, plasmids for the TamPA-Cre system were developed by molecular cloning, and then transfected into mCherry-EGFP reporter HEK293T cells. Initial testing demonstrated that both blue light and 4OHT were required for recombination, confirming the AND logic. Second, the TamPA-Cre system was optimized by manipulating: the light stimulation pattern, the time to begin light stimulation after 4OHT addition, the molar ratio between the CreN and CreC halves, and the affinity of nMag. Next, CD19CAR genetic constructs were validated through coculture with CD19+ cells. Lastly, a Jurkat cell line containing the TamPA-Cre and CD19CAR construct showed the expression of CD69 only when these cells were: cocultured with CD19+ cells, given 4OHT, and stimulated with blue light. Overall, the TamPA-Cre system provides a platform for the spatiotemporal control of CAR T cells using AND logic.

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