Background: Glioblastoma (GBM) is the most common and most aggressive primary brain tumor. Around 294,900 new cases are diagnosed globally with 241,000 deaths each year. The 5-year survival is only 5%. Median overall survival from first recurrence is only 5-8 months. There is no established standard of care for recurrent GBM. City of Hope (COH) has developed and optimized a CAR T cell therapy utilizing the chlorotoxin peptide (CLTX) as the CAR’s tumor recognition domain against GBM. CLTX-CAR T cells specifically and broadly target GBM through recognition of a receptor complex including membrane-bound matrix metalloprotease 2 (MMP-2). CLTX-CAR T cells do not exhibit off-tumor recognition of normal human or murine cells and tissues in preclinical models. In in vitro studies, COH evaluated patient-derived brain tumor (PBT) cell lines for CLTX binding and expression of IL13Rα2, HER2 and EGFR, three targets of CAR T cell trials for GBMs. Strong CLTX binding to tumor cells was observed in of the majority of primary GBM lines, independent of these other antigens. In preclinical studies using in vivo mouse models, a single intratumoral (ICT) injection of CLTX-CAR T cells (1×10⁶ CAR+ T cells) exhibited robust anti-tumor activity against ffLuc+ PBT106 tumors orthotopically-engrafted in NSG mice. Overall, when compared to mice treated with mock-transduced Tn/mem (no CAR) T cells, the CLTX(EQ)28ζ/CD19t+ T cells reduced tumor burden and significantly increased survival. Taken together, these preclinical findings support the potential safety and efficacy of CLTX-CAR T cells, and provide the rationale for clinical testing of this therapy. As cellular heterogeneity intrinsic to GBM likely contributes to resistance to therapy and limited response rates, CLTX-CAR T cells may provide greater tumor eradication in a higher proportion of patients with GBM. Methods: This study is a phase 1, single center, safety and maximum tolerated dose (MTD) finding study of CLTX-CAR T cells for subjects with MMP2+ recurrent or progressive GBM. A safety lead-in of 3−6 participants receiving CLTX-CAR T cells by ICT delivery will be completed first. Subsequently, subjects would receive cells administered through both ICT and intraventricular (ICV catheters) (i.e. dual delivery) in two dose schedules. Subjects will be evaluated for safety and tolerability, and may continue to receive treatment until disease progression. Time to progression, overall survival, and disease response by Response Assessment in Neuro-Oncology (RANO) criteria, will be evaluated and descriptively compared to historical data. The study is actively enrolling patients. Clinical trial information: NCT04214392