Harnessing Neurosurgery to Empower GBM-Targeted CAR-T Immunotherapies Uniquely Designed to Combat the Tumor Microenvironment”

T-cell immunotherapies, including chimeric antigen receptor (CAR) T cells and tumor-infiltrating lymphocytes (TILs), have shown success in treating certain cancers; however, their efficacy against glioblastoma (GBM) remains limited. GBM presents challenges, such as the blood-brain-barrier (BBB), tumor heterogeneity, and the immunosuppressive tumor microenvironment (TME). TME impairs the fitness of TILs. New strategies are required to tackle these challenges. Our long-term goal is to develop an immunotherapeutic treatment regimen to combat GBM. The aim of this grant is to apply a “Double-Hit” strategy that applies an adoptive T cell therapy for GBM. In the First Hit (Aim 1), we propose a bicistronic CAR T-cell therapy that targets EGFRvIII, a GBM-specific antigen, and programmed death-ligand 1 (PD-L1). PD-L1 is an ideal target, expressed on tumor and immunosuppressive cells. With an intratumoral administration (Ommaya reservoir) of PD-L1/EGFRvIII bicistronic CAR T-cells we will i) bypass the BBB to facilitate localized T cell infiltration that can eliminate TAMs, ii) kill residual tumor cells, and iii) seize the window of opportunity between surgery and postoperative treatment to apply treatment to prevent tumor progression. In the Second Hit (Aim 2), TILs will be isolated from our patients at time of surgery and will be used to create a malic enzyme 1 (ME1)- armored PD-L1 CAR-TILs, which will be administrated postoperatively using the same Ommaya reservoir as in the first hit, which allows administration of our CAR-TILs directly into the brain. This approach leverages the TILs’ ability to specifically recognize GBM antigens via T cell receptor (TCR) to overcome antigen heterogeneity. Armoring TILs with ME1, a critical enzyme in intracellular energy production and metabolism, aims to rescue TIL metabolic fitness and enhance functionality. In our human translational study, we will implant a reservoir intracavitary at the time of resection to enable local delivery and potentially repeated doses of both CAR T-cells (First-Hit) and TILs (Second-Hit).Our preliminary data strongly support 1) PD-L1 as a viable target for eliminating immunosuppressive cells and tumor eradication; 2) the role of ME1 in rescuing TIL cytotoxicity; and 3) the safety of intracranial delivery of PD-L1 CAR T-cells. Our unique "Double-Hit" strategy introduces key innovations: 1) attacking the challenge of tumor heterogeneity using TIL-mediated diverse antigen recognition combined with a bicistronic CAR to prevent antigen escape; 2) targeting the diverse population of PD-L1-expressing cells in solid tumors with CAR T-cells to eliminate immunosuppressive cells in the TME, thereby enhancing T-cell antitumor activity; (3) restoring T-cell responsiveness and cytotoxicity by supplementing TILs with ME1; and (4) maximizing therapeutic efficacy through sequential intracavitary therapy—first clearing the TME of immunosuppressive cells and reducing tumor burden, followed by armored TIL therapy to target residual disease. Our study aims will advance toward an IND application, offering new hope for GBM patients.