Abstract IA004: Interplay between infused and endogenous immune system: Implications for next-generation CAR-T engineering for GBM

Abstract We conducted a completed clinical trial of locoregionally delivered bivalent CAR-T cells targeting EGFR and IL13Rα2 in eighteen patients with recurrent EGFR-amplified glioblastoma (rGBM) using a 3+3 dose-escalation design (5.0 × 106, 1.0 × 107, and 2.5 × 107 cells). Patients received a single intracerebroventricular infusion without lymphodepletion, as previously reported. Pharmacokinetic analyses demonstrated superior CAR-T expansion and cerebrospinal fluid (CSF) exposure at the highest dose, which was selected for subsequent trials. Despite heavy pretreatment, including multifocal disease in over half of patients, 12-month overall survival was 56%. Longitudinal immune profiling of CSF revealed a strong association between endogenous immune remodeling and clinical outcome. Responders exhibited expansion of cytotoxic NK cells, whereas non-responders showed early regulatory T-cell enrichment and increased T-cell clonality. CAR-T therapy also reshaped the myeloid compartment, and a baseline scavenger-like immunosuppressive phenotype was associated with inferior survival, underscoring the importance of pre-existing innate immune states. Patients retreated at progression in the original study showed reduced CAR-T engraftment compared with their initial infusion, prompting prospective evaluation of immune conditioning and dosing strategies. We have since opened additional trials using the same bivalent product in newly diagnosed GBM following radiation without lymphodepletion and in rGBM after lymphodepleting chemotherapy. In parallel, a separate rGBM cohort received two maximal-dose infusions 14 days apart; repeat dosing again resulted in diminished CSF CAR-T expansion, consistent with adaptive resistance previously observed with systemic monovalent CAR-T therapy. Together, these data highlight a dynamic interplay between infused CAR-T cells and endogenous immune populations in the central nervous system, with NK cells, regulatory T cells, and myeloid states emerging as critical determinants of response and resistance. These insights are now guiding development of next-generation multivalent CAR-T platforms for GBM designed to enhance persistence, enable repeat dosing, and remodel suppressive innate immune niches. Citation Format: Donald M. O'Rourke. Interplay between infused and endogenous immune system: Implications for next-generation CAR-T engineering for GBM [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr IA004.