PROJECT SUMMARY Despite increasingly aggressive standard of care treatments, patients with glioblastoma (GBM) have a poor prognosis that has remained unchanged for decades, and this represents a fundamental unmet medical need. Progress in immunotherapy across a broad range of tumor types provides hope that immunological approaches, including CAR T cell therapy, may improve outcomes for patients with GBM. This is supported by the ability of CD19-targeted CAR T cells to eliminate B cell malignancies in the central nervous system (CNS), as well as early clinical experiences showing safety and disease-modifying activity of CAR T cells in GBM. One major challenge for GBM immunotherapies is the phenotypic heterogeneity of GBM tumors coupled with the scarcity of targetable tumor-associated antigens. As an approach to address this challenge we have developed a novel CAR that exploits the selective and broad GBM-binding properties of chlorotoxin (CLTX). CLTX is a 36-amino acid peptide component of scorpion venom that binds specifically to GBM and other tumors with minimal cross- reactivity to non-malignant cells. Previous early phase clinical trials have established safety of CLTX when used as a GBM-targeting peptide for imaging and radiotherapy. Our preclinical studies demonstrate that CLTX-CAR T cells mediate potent antitumor activity in vitro and in vivo, with no observable effector activity against normal human cells or when adoptively transferred into cross-reactive mouse models. We now aim, in this proposal, to clinically test the hypothesis that CLTX-CAR T cells will be safe and mediate antitumor effects when locoregionally delivered to the CNS in patients with GBM. Aim 1 will evaluate the feasibility, safety and response rates of CLTX-CAR T cells in patients with recurrent GBM in a phase 1 clinical trial. CLTX-CAR T cells will be delivered locoregionally via intratumoral [ICT] and intracerebroventricular [ICV] catheters. Aim 2 will assess CAR-mediated immunological changes associated with response and resistance. Taking advantage of our catheter-based locoregional delivery strategy that allows sampling of the CSF throughout treatment, we will monitor surrogate markers of CAR T cell activity, including CAR T cell persistence and changes in inflammatory cytokines and endogenous immune subsets. We will also compare the intrinsic characteristics across different autologous CAR T cell products with clinical response. Aim 3 will investigate pathways of GBM tumor resistance and CAR-induced tumor evolution pre- and post-therapy, investigating antigen escape pathways and adaptive immunosuppressive mechanisms to CAR T treatment. The innovative use of scorpion-derived CLTX for tumor targeting would be the first example of a natural peptide−based CAR in the clinic, potentially expanding CAR designs beyond antibody- and ligand-based targeting domains. CLTX-CAR T cell targeting of a wide range of GBM cells within individual tumors is significant in that it will...