The control of immune T cells expressing synthetic chimeric antigen receptors (CAR-T cells) using destabilization domains is an important tool for precise and adaptive regulation of CAR-T cell activity. Destabilization domains serve as safety switches and allow the regulation of CAR-T cell activity by modulating the stability of the CAR receptor on the cell surface using small molecules. Their use could limit the side effects and serious complications associated with CAR-T cell therapy, which are mainly due to their over-activation. To date, several control systems based on destabilization domains are known (e.g. ecDHFR or FKBP destabilization domains), but their number is limited. These systems have limited therapeutic utility because their ligands are molecules that are toxic to human cells, or the protein components of these systems are derived from other organisms and can elicit an immune response in humans. The focus of the MSc thesis was therefore to extend the current set of destabilization domains. We used destabilization domains based on the ligand binding domain of the estrogen receptor α (ERα), which are activated by the FDA approved drug, molecule 4-OHT. The prepared constructs were inserted into mammalian cell lines and the expression of the CAR receptor was verified after stimulation of the cells with the small target molecule 4-OHT. For the destabilization domains to which CD19 CAR was added, the presence of the ligand ensured stable expression of the receptor on the cell surface. We expect that the designed destabilization domains will help to extend the range of rapid, reversible and scalable methods to regulate protein stability in living cells using small molecules.
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