Glioblastoma multiforme (GBM) is the most prevalent primary brain tumor among adults, with poor patient prognosis despite aggressive treatment regimens combining chemotherapy, surgery, and radiation. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells in the brain. Engineering T cells to express tumor-targeting chimeric antigen receptors (CARs) offers a tantalizing means to induce targets for CAR-T cell-based immunotherapies. Prior clinical trials have shown the safety of CAR-T cell therapy targeting interleukin-13 receptor subunit alpha-2 (IL-13Rα2), an antigen that is overexpressed by 58-78% of gliomas. However, the highly immunosuppressive tumor microenvironment (TME) is a major barrier hindering the effectiveness of these immunotherapies against GBM, with transforming growth factor-beta (TGF-β) playing a particularly prominent role. We have now developed a bispecific CAR that simultaneously targets TGF-β and IL-13Rα2, and observed superior in vivo anti-tumor efficacy against both patient-derived GBM xenografts and syngeneic gliomas in immunocompetent mice compared to single-input IL-13Rα2 CAR-T cells. Our central hypothesis is that IL-13Rα2/TGF-β bispecific CAR-T cells can directly attack GBM via IL-13Rα2 recognition and simultaneously disrupt the immunosuppressive feedback loop between TGF-β and tumor-infiltrating myeloid cells. In this new SPORE Project, we have planned a series of studies with the following specific aims: 1) to optimize clinicalgrade cell manufacturing of IL-13Rα2/TGF-β CAR-T cells for clinical testing; 2) to conduct a new first-in-human clinical trial of a novel IL-13Rα2/TGF-β bispecific CAR-T cell therapy for patients with recurrent glioblastoma; and 3) to determine correlates of response and potential resistance mechanisms to this novel therapeutic approach. At the conclusion of this grant, we hope to address some of the major hurdles of CAR T cell therapy for GBM by targeting both glioblastoma cells and the immunosuppressive tumor microenvironment. If successful, this project will expand our immunotherapeutic armamentarium and potentially help to enhance tumor eradication, thereby improving outcomes for patients with GBM.