Project Summary/Abstract The development of safe and effective chimeric antigen receptor (CAR)-transduced T cell (CART) therapy for glioblastoma (GBM) needs to overcome multiple challenges, including heterogeneity of antigen expression, on- target off-tumor toxicity, and exhaustion of CART cells. To date, there are no GBM-specific antigens that are uniformly present on all GBM cells. On the other hand, while non-mutant GBM-associated antigens (GAAs), including Ephrin type A receptor 2 (EphA2) and interleukin-13 receptor α2 (IL-13Rα2), are expressed in the majority of GBM cells, they are also expressed in some non-central nervous system (CNS) organs, raising the concern of off-tumor toxicity. As a way to safely and effectively target GAAs, we have adopted a novel synthetic Notch “synNotch” receptor system and developed innovative “prime-and-kill” T cell circuits. In this system, the first antigen, which is expressed exclusively on GBM or CNS cells, primes the T cells to induce the expression of a CAR that recognizes IL-13Rα2 and EphA2, thereby eradicating GBM cells expressing either EphA2 or IL- 13Rα2. The first priming antigen should be restrictedly expressed on CNS cells but not on cells of any (non- CNS) systemic organs. Then the system should be safe because, within the CNS, EphA2 and IL-13Rα2 are expressed only on tumor cells and not on normal CNS cells. We found Brevican (BCAN), a proteoglycan localized to the neuronal and glial cell surface, as the most promising priming antigen. When mice bearing well-established day 9 intracerebral GBM6 PDX tumors received a single IV infusion of T cells engineered with the α-BCAN synNotchα-EphA2/IL-13Rα2 CAR (B-SYNC) circuit, all mice demonstrated complete regression of tumor without attacking EphA2/IL-13Rα2-positive cells outside of CNS. Furthermore, these B-SYNC T cells were significantly more efficacious than conventional, constitutively expressed EphA2/IL-13Rα2 CART cells, with superior CNS tumor-homing and less exhausted phenotype compared to control T cells. We will develop a phase I study to evaluate our hypothesis that B-SYNC T cells can be safely administered as a single IV infusion in patients with GBM and that B-SYNC T cells are able to infiltrate the GBM tissue, wherein they will be primed to express the CAR against EphA2 and IL-13Rα2. Concurrently, using syngeneic mouse models, we will evaluate our 2nd hypothesis that modulation of immunoregulatory mechanisms may further improve the function and efficacy of B-SYNC T cells. We will evaluate the following two specific aims. Aim 1. Determine the safety as well as the homing and priming status (i.e., expression of CAR) of the IV-infused B-SYNC T cells in patients with GBM. Aim 2. Assess potential resistance mechanisms and integrate mitigation strategies into the B-SYNC regimen in preclinical syngeneic models. In a prior trial with anti-EGFRvIII CART cells, the CART cells infiltrated the GBM and induced immunoregulatory molecules, such as progra...