Combinatorial tumor targeting using a novel switchable RevCAR system

Background
Although T-cells genetically modified to express chimeric antigen receptors (CARs) are successfully used to treat hematological malignancies, patients still suffer from several drawbacks of conventional CAR (cCAR) therapy. CAR-T-cells can cause severe to life-threatening adverse reactions like on-target, off-tumor toxicities which cannot be controlled in patients. Moreover, cCAR therapy often fails to successfully affect solid tumors and bears the risk to encourage tumor escape variants upon targeting of only one single tumor-associated antigen (TAA). In order to overcome these problems, we have established a novel on/off-switchable RevCAR system facilitating combinatorial targeting strategies.
Methods
For combinatorial targeting one T-cell has to be modified with two separate CARs recognizing different TAAs. The first CAR mediates the activation and the second CAR the costimulatory signal. In case of ‘AND’ gate targeting, dual-CAR-T-cells have to recognize both TAAs on the surface of the target cells to get activated. However, such combinatorial targeting strategies are struggling with several challenges including the adjustment of signal strength and affinity of both split CARs as well as the CAR size limiting the number of transduced specificities. In order to overcome these obstacles, our idea was to construct small RevCARs comprising only a small peptide epitope as extracellular domain. By removing the extracellular single-chain variable fragment (scFv) of cCARs, RevCARs avoid tonic signaling induced by scFv dimerization. As RevCARs do not have an extracellular antigen binding moiety, they cannot bind to any antigen per se. Thus, actually they are switched off. Only in the presence of a bispecific target module (RevTM), RevCAR-T-cells can be redirected to tumor cells and switched on. Finally, short-living RevTMs allow a repeatedly on/off-switch and controllability of RevCAR-T-cells and furthermore a flexible redirection of RevCAR-T-cells to any target.
Results
For proof of concept two small peptide epitopes were selected to construct the respective RevCARs. Additionally, a series of different RevTMs was generated recognizing one of the two peptide epitopes and simultaneously any potential TAA. RevTMs were able to efficiently redirect RevCAR-T-cells specifically against different tumor targets. Moreover, we show that combinatorial targeting can be achieved using our RevCAR system. Here, dual-RevCAR-T-cells were efficiently activated only after engagement by two RevTMs targeting the activating or costimulatory RevCAR and different TAAs.
Conclusions
Taken together, we developed a switchable RevCAR platform showing high effectiveness, increased specificity, improved safety, easy controllability, and small size facilitating combinatorial tumor targeting.