CAR-T cell therapy has resulted in extremely effective and sustained clinical results in the treatment of cancer. However, there are limitations to CAR-T therapy, including life-threatening cytokine release syndrome and neurotoxicity. To alleviate the symptoms of side effects active substances are mostly used, but for effective and safe therapy it would be prefereable to introduce mechanisms that allow the regulation of side effects at the CAR-T cell signaling level. In our master's thesis, we designed six different inhibitory domains based on native T cell signaling mechanisms to regulate CAR-T cell signaling. Inhibitory domains and CAR were fused to chemically inducible dimerization domains, so that only with the addition of small exogenous molecule CAR-T signaling is inhibited. A small molecule danoprevir was used to induce heterodimerization. The expression of constructs and the functionality of inhibitory domains were studied on Jurkat cells by flow cytometry and ELISA. Jurkat cells were successfully electroporated with prepared constructs and their expression on the cell surface was confirmed. All constructs induced cell activation in the absence of danoprevir. With the addition of danoprevir, which is not cytotoxic at low concentrations, only construct with the CD45-based inhibitory domain achieved complete inhibition. Partial inhibition occurred withZAP-70 inhibitory domains and no inhibition with PD1 inhibitory domain.
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