Preclinical efficacy of multi-targeting mRNA-based CAR T cell therapy in resection models of glioblastoma

Traditional viral-based chimeric antigen receptor (CAR) T cell therapies have vanquished multiple blood malignancies with decade-long remissions yet struggle against solid tumors. Nonviral engineering of CAR T cells via electroporation or lipid nanoparticle (LNP) delivery of CAR-encoding mRNA results in highly efficient yet transient CAR expression, challenging the adequacy of available preclinical models for mRNA-based CAR T cell evaluation. This study presents a unique three-pronged approach that combines mRNA-based CAR T cells, multi-targeting of glioblastoma (GBM)-associated receptors, and maximal surgical resection as a novel and readily translatable platform for preclinical evaluation of mRNA-based CAR T cells against solid tumors. We performed head-to-head <i>in vitro</i> and <i>in vivo</i> analyses of mRNA-based CAR T cells generated using different expansion conditions, mRNA delivery methods, or combination approaches. Besides potent <i>in vitro</i> cytotoxicity, our findings unveil a therapeutic window of anti-tumor efficacy, as well as robust and durable complete remissions in xenograft mouse models of GBM receiving maximal surgical resection and locoregional injections of multivalent CAR T cells (MVCAR). Such efficacies were significantly better in 5-day expanded versus quiescent T cells. Interestingly, MVCAR T cells were superior to pooled CAR T cells (CARPool) expressing the same CAR scFv combinations in an orthotopic resection model of GBM.