PROJECT SUMMARY Glioblastoma (GBM) is a deadly brain tumor with limited treatment options. We have developed a promising new treatment for GBM using chimeric antigen receptor (CAR)-T cells. CAR-T cells are derived from the patient’s own T cells and genetically modified to express a receptor that will target the cancer cells through a specific antigen. We first developed CAR-T cells targeting epidermal growth factor receptor variant III (EGFRvIII), a mutated form of EGFR that is uniquely expressed in GBM. Treating patients with EGFRvIII-CAR-T cells resulted in decreased EGFRvIII-expressing cells, while EGFR expression remained the same or increased. With EGFRvIII-CAR-T cell treatment, there was also infiltration of T regulatory cells (Tregs), which are known to suppress T cell function and may have limited CAR-T cell killing of tumor cells. To improve this therapy, we engineered the EGFRvIII-CAR-T cell to secrete a T cell-engaging antibody molecule (TEAM) that binds to EGFR and CD3 (CARv3.TEAM-E cells). The TEAM allows the CAR-T cell to target more than one antigen while also activating other (non-CAR) T cells within the tumor, including converting Tregs to killer cells. We tested CARv3.TEAM-E cells in vitro and in mouse xenograft models and found they safely and effectively target EGFRvIII+EGFR+ and EGFRvIII–EGFR+ GBM. When these cells were injected into the mouse brain for direct access to the tumor, very few escaped into the peripheral circulation, which protected normal tissues with EGFR expression from being targeted by the TEAM. Based on these preclinical results, we are initiating a phase I clinical trial of the CARv3.TEAM-E cells for the treatment of primary or recurrent GBM. The CARv3.TEAM-E cells will be injected into the cerebrospinal fluid (CSF) via an Ommaya reservoir. We will measure the safety of this treatment by monitoring patients for adverse events related to CAR-T treatment and EGFR targeting. Additionally, we will perform correlative studies to monitor CAR-T cell expansion, persistence, and phenotype in the CSF and peripheral blood as well as infiltration into the tumor. We will examine the tumor for changes in antigen expression (EGFRvIII, EGFR, etc.) with CARv3.TEAM-E cell infiltration and changes in other immune cells that could be influencing CARv3.TEAM-E cell function, including Tregs and myeloid cells. We will correlate these parameters with responses to treatment or toxicities that we observe in each patient. Overall, these studies will inform us about the safety and feasibility of CARv3.TEAM-E cells for treating GBM, as well as provide insights into future modifications that could improve the efficacy of these CAR-T cells or lessen their side effects. CARv3.TEAM-E cells hold promise for improving the outcome of this dismal disease.