Glioblastoma (GBM) is a very aggressive brain tumor, associated with poor prognosis and survival. So far, the efficiency of available therapies is limited. After proving their effectiveness in targeting hematological malignancies, chimeric antigen receptor (CAR) T cells might provide a promising therapeutic approach for GBM. Here, we present our switchable Reverse CAR technology (RevCAR). Unlike conventional CAR T cells, RevCAR T cells contain an epitope in their extracellular receptor domain, and can only be activated via bispecific target modules (RevTM) which recognize the RevCAR T cells on one side and tumor cells on the other side. Once these RevTMs are eliminated, RevCARs are switched off. In addition, we have developed dual-targeting RevCARs, allowing the control of T cells according to the AND gate logic of Boolean algebra.
Novel RevTMs specific for GBM were developed. Subsequently, they were expressed in eukaryotic cells and purified with affinity chromatography. The ability of these RevTMs to bind GBM cells and RevCAR T cells was analyzed using flow cytometry. Moreover, the capability of the mono-specific and the dual-targeting RevCAR T cells to kill GBM cells was analyzed using luminescence-based cytotoxicity assay in the absence or presence of a range of RevTM concentrations. Secreted pro-inflammatory cytokines were also evaluated by ELISA. In addition to the in vitro assays, a proof of concept co-injection experiment was performed in vivo.
In this study, we show that GBM-specific RevTMs can bind both the RevCAR T cells and GBM cells. Importantly, the RevCAR T cells can be activated to secrete pro-inflammatory cytokines and to efficiently kill GBM cells via the RevTMs. Moreover, we were able to prove that dual-targeting RevCAR T cells can only be activated upon recognition of two different GBM targets, thereby allowing a highly specific and selective killing of GBM both in vitro and in vivo.
In conclusion, the switchable RevCAR platform is a novel therapeutic approach that provides improved safety, and allows combinatorial targeting of GBM.
Helmholtz-Zentrum Dresden-Rossendorf.
EU-Europa fördert Sachsen-Europäischer Fonds für regionale Entwicklung-der Sächsische Landtag.
All authors have declared no conflicts of interest.