Adverse immune reactions such as cytokine release syndrome and neurotoxicity occur with CAR-T cell therapies. These are mostly treated only after symptoms appear, limiting the success of the treatment. Optimisation of CAR-T cell therapies can be achieved by designing rapid and reversible ON- and OFF- safety switch chimeric antigen receptors controlled by small molecules. As a part of our MSc thesis, we have developed a safety switch based on a proteolysis-targeting chimera (PROTAC) ARV-471, which specifically targets and degrades the estrogen receptor (ER). CAR expression was studied by flow cytometry and ELISA on Jurkat cell lines stably expressing modified CARs with added estrogen receptor (ER). The results showed that addition of the PROTAC ARV-471 induced degradation of CARs with bound estrogen receptor alpha or beta. We showed that increasing the amount of PROTAC ARV-471 decreases the percentage of maximal CAR expression and the IL-2 concentration in ER-positive CAR cell lines. We have also shown that stimulation of cells with PROTAC ARV-471 and 4-OHT with increasing amounts of 4-OHT results in an increase in the percentage of maximal CAR expression and IL-2 concentration in ER-positive CAR cell lines, making the system reversible.
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