IL-12-secreting CAR-T cells reprogram the tumor microenvironment and improve efficacy against heterogeneous models of glioblastoma

Background Glioblastoma (GBM) remains uniformly lethal due to pronounced intratumoral heterogeneity and a highly immunosuppressive microenvironment that limits the efficacy of targeted therapies. Methods We engineered chimeric antigen receptor (CAR) T cells targeting Epidermal Growth Factor Receptor variant III (EGFRvIII) and armored them with a single-chain interleukin-12 (scIL12) payload. These cells were tested in syngeneic, orthotopic GBM mouse models exhibiting heterogeneous EGFRvIII expression. CAR T cells were delivered intracranially without lymphodepletion. Results Intracranial administration of scIL12-secreting CAR-T cells eradicated tumors without requiring lymphodepletion, achieving 50% long-term survival. Survival benefits depended entirely on endogenous CD8 + T cells, as efficacy was abolished in CD8-deficient hosts and unaffected by natural killer cell depletion. Notably, therapeutic efficacy was abrogated by lymphodepletion, underscoring the necessity of an intact endogenous immune response. Mechanistically, scIL12 enhanced CAR-T cell persistence and reprogrammed tumor-associated microglia, indicating potential antigen spreading through polyclonal endogenous CD8+T cell responses, which facilitate the elimination of EGFRvIII-negative tumor cells. Conclusions This study demonstrates the pleiotropic benefits of IL-12 armored CAR-T cells with improved targeting of antigen-positive tumor cells and simultaneous remodeling of the microenvironment to engage adaptive immunity against antigen-negative clones. This strategy offers a potential clinically actionable approach to improve outcomes in GBM by circumventing the need for toxic lymphodepletion and addressing tumor heterogeneity.