Natural killer cell-based signaling in EGFR-targeted KIR-CAR T overcomes CD3-based CAR T functional deficits to eliminate resistant glioblastomas in vivo

Glioblastoma (GBM) is the most common primary brain cancer in adults, with a prognosis of 15-18 months' survival despite standard treatment of resection, chemotherapy, and radiotherapy. Recent clinical trials investigating autologous chimeric antigen-receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) variants in GBM through single-chain variable fragments (scFv) recombined with 41BB-co-stimulation and CD3ζ activation showed encouraging radiographic evidence of early tumor reductions, within days of CAR T treatment. Unfortunately, this anti-tumor function was short-lived, with tumor outgrowth generally occurring within days to weeks, potentially attributed to rapid loss of T cell function due to exhaustion, or target antigen loss. In efforts to bypass CD3-based CAR signaling exhaustion and prolong CAR T anti-tumor function, the recombinant single-chain CD3ζ-based CAR was replaced with a more physiologic split-signal system derived from natural killer (NK) cells, utilizing killer immunoglobulin-like receptor (KIR) and DNAX-activation protein of 12 kDa (DAP12) activation to allow a more natural cellular activation/rest cycle upon target cell engagement and disengagement. Second-generation EGFR/EGFRvIII-targeted single-chain 41BB-CD3ζ CAR was replaced with multi-chain KIR/DAP12 CAR signaling, and CAR and KIR T cells were compared in vitro for recognition specificity, cytolytic function, cytokine secretion, and T cell differentiation phenotype. In vivo efficacy was evaluated in established CAR-resistant immunocompromised NOD/SCID/gamma-chain -/- (NSG) murine GBM xenograft models, where tumor size was evaluated by in vivo imaging and caliper measurements and survival per Kaplan-Meier graph. Following lentiviral transduction and ex vivo expansion, KIR-CAR T displayed an increased naïve-like phenotype, with reduced effector-memory cells compared with CD3-based CAR T. In vitro, both CAR and KIR-signaling formats conferred comparable target specificity, cytotoxicity, T cell activation and cytokine release. In contrast, in vivo KIR-CAR T cells repeatedly outperformed 41BB-CD3ζ CARs, with the most effective treatment delivered by an scFv targeting multiple EGFR tumor-associated variants, demonstrating superior tumor regressions, including complete anti-tumor responses and increased survival. With the same targeting specificity, similar cytokine production in vitro, and significantly increased anti-tumor functional impact and prolonged survival in vivo, EGFR-KIR-CAR has the potential for rapid translation to the clinic to overcome the limitations of CD3-based single-chain CAR T in treating patients with GBM. Funded by NIH grant DP2CA174502 (LAJ).