Engineering human T cells with a novel switchable CAR technology for tumor immunotherapy

With the first approvals of chimeric antigen receptor (CAR) T cell therapies by the FDA the use of genetically modified T cells in the immunotherapy of tumors has recently become a very promising approach. CAR T cells are able to recognize tumor-associated antigens (TAAs) via specific single-chain variable fragments (scFvs) in a major histocompatibility-complex (MHC)-independent manner. Although highly efficient, the inability to regulate the activity of CAR T cells can cause severe even
life-threatening side effects such as cytokine-release syndrome (CRS) and on-target, off-tumor toxicities. Modular CAR systems may overcome these limitations allowing to switch the activity of CAR T cells repeatedly “ON” and “OFF”. Alternatively or in addition, the safety of CAR T cells could also be improved by “gated” targeting strategies e.g. by splitting the signaling and costimulatory motifs to independent CARs of different specificities. Theoretically, the idea of gated targeting could be extended to include further e.g. inhibitory signals. However, the size of current CARs limit the number of specificities that can be simultaneously transduced into a T cell. We therefore developed a novel switchable modular universal artificial receptor having a minimal size. The platform was termed RevCAR system.
In order to reduce the size of the artificial receptor the original idea was to replace the extracellular scFv domain of a conventional CAR with a small peptide epitope and to engage the resulting RevCAR T cell via a bispecific target module which we termed RevTM. For proof of concept two small peptide epitopes were selected and the respective RevCARs constructed. In addition, a series of different RevTMs were constructed. On the one hand the RevTM recognized one of the two peptide epitopes on the other hand the RevTM was directed to a potential tumor associated antigen (TAA). Until now a series of such pairs of RevTMs were constructed and functionally analyzed. RevCAR T cells armed via the respective RevTM were able to specifically lyse their respective target cell in a peptide epitope specific and target specific as well as target dependent manner. These data are supported by analysis of cytokine secretion. We only observed a specific cytokine release from RevCAR T cells in the presence of both target cells and the respective RevTM. Released cytokines detected were IFN-gamma, GM-CSF, TNF, and IL-2.
Taken together these results demonstrate the high anti-tumor efficiency of the novel RevCAR platform which is characterized by a small size, an improved safety, easy controllability as well as high flexibility.