Engineering a Dual Small Molecule Gated ZAP70 Switch in T Cells

[Image: see text] T cells expressing tumor-specific T cell receptors are promising cancer therapeutic agents, but safety control switches are needed to manage potential side effects arising from overactivity. Here, we present the first dual small molecule-gated ZAP70 signaling switch for the regulation of T cell activity. We show that when an analogue-sensitive ZAP70 allele is fused to the engineered ligand binding domain of the estrogen receptor, ER(T2), its activity can be upregulated to an extent by a metabolite of an FDA-approved tamoxifen, 4-hydroxy-tamoxifen, and downregulated by an ATP analogue, 3-MB-PP1. The strength of early T cell signaling can also be modulated by varying the concentrations of activator and inhibitor, and the switch exhibits temporal control on the time scale of minutes. Interestingly, the switch has the ability to control CD69 and calcium levels in T cells but has limited capabilities in the regulation of downstream cytokine release, suggesting further investigation is needed before it can be implemented in adoptive T cell therapy.