Abstract LB138: Natural killer cell-based signaling in EGFR-targeted KIR-CAR T overcomes CD3-based CAR T functional deficits to eliminate resistant glioblastomas in vivo

Abstract 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). Citation Format: Jun Xu, Radhika Thokala, Yibo Yin, Chong Xu, Alina C. Boesteanu, Alexandria P. Cogdill, Zev A. Binder, Logan Zhang, Jiasi Vicky Zhang, Enxiu Wang, Carl H. June, Donald M. O'Rourke, Michael C. Milone, Laura A. Johnson. Natural killer cell-based signaling in EGFR-targeted KIR-CAR T overcomes CD3-based CAR T functional deficits to eliminate resistant glioblastomas in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB138.