Engineering a Controllable and Reversible Switch for CAR-T Cell Immunotherapy Via a Genetic Code Expansion System

Background

As one of the most promising adoptive cell therapies, CAR-T therapy has achieved notable clinical effects in patients with hematological tumors. However, several obstacles remain in CAR-T therapy, such as low killing effects for some tumor targets, high-frequency recurrence, and cytokine release syndrome. These obstacles severely limit the long-term effects and the further application of CAR-T therapy. Therefore, the optimization of CAR-T cells for increased controllability and safety is urgently needed.

Methods

In this study, we engineered a genetic code expansion-based therapeutic system to achieve rapid CAR protein regulation in response to cognate unnatural amino acid at the translational level, in which the termination codon TAG was inserted into the CAR coding region. When the unnatural amino acid, N-ε-((tert-butoxy) carbonyl)-l-lysine (BOCK) is absent, the CAR protein cannot be completely translated, and CAR-T is “closed”. When BOCK is present, it recognizes and combines with the TAG, the whole CAR protein is translated, and CAR-T is “open”. Based on these findings, we subsequently investigated whether BOCK can control CAR protein expression and regulate CAR-T cell function via a series of in vitro experiments and a xenograft mouse tumor model.

Results

First, we verified that the BOCK-induced genetic code expansion system enables regulate protein expression as a controllable switch. Subsequently, we demonstrated that BOCK effectively and precisely controlled CAR protein expression and induced the activation of CAR signaling, the expression of activation markers, CD69 and CD25, and cytokine secretion. When incubated with tumor cells, BOCK regulated CAR-T cell cytotoxicity in a dose-dependent manner. Finally, a NOG mouse model analysis showed that BOCK can control CAR-T cell function and anti-tumor effect in vivo.

Conclusions

We systematically demonstrated that the BOCK-induced genetic code expansion system can effectively and precisely regulate CAR protein expression and control CAR-T cell anti-tumor effects in vitro and in vivo. We conclude that this controllable and reversible switch has a potential for more effective, secure, and clinically available CAR-based cellular immunotherapies.