PROJECT SUMMARY This proposal centers on the use of T cell receptor (TCR) directed therapy in preclinical glioblastoma (GBM) models. GBM remains a difficult cancer to treat, and clinical outcomes remain poor. However, despite the seismic influence of immunotherapy in cancer, there remain no FDA approved immunotherapies for GBM. There are several reasons that underlie the difficulty in extending immune-based treatments to the central nervous system (CNS). GBM harbors few T cells and is considered “non-inflamed”, there is a paucity of dendritic cells in the brain parenchyma, and a myriad of immunosuppressive features has been identified in patients. The CNS is also immunologically specialized due to the presence of site-specific elements not seen elsewhere—e.g., lack of lymph nodes, presence of dural lymphatics, and the blood-brain barrier, among others. Moreover, the genomic landscape of GBM introduces additional obstacles in that many antigenic and neoantigenic targets are heterogeneously distributed. Importantly, although heterogeneity is a formidable challenge to immunotherapy, it is poorly modeled in preclinical settings. Despite these barriers, however, the final goal of CNS immunotherapy—T cell clonal expansion—remains the same. Here, we will test the use of adoptively transferred TCR directed T cells to treat preclinical GBM as a gateway to understand key mechanistic principles for ultimate clinical translation. We have assembled a team with Dr. Charest and Dr. Petti that brings diverse expertise to this work. The proposed work focuses on a novel transgenic mouse that targets an endogenous neoantigen, mutant Imp3 (mImp3), in the GL261 mouse model. This mouse, the Mutant Imp3 Specific TransgenIC (MISTIC) mouse, expresses a TCR that recognizes the H2-Db restricted mImp3 neoantigen and thus represents an exciting model for TCR-directed cell therapy. In Aim 1, we will dissect the mechanisms underlying MISTIC therapy and also understand the requirement for endogenous RAG-dependent lymphocyte populations in effective treatment. Additionally, we will develop and study a new, autochthonous model of spontaneous, EGFR-driven and neoantigenically-defined GBM model to allow us to study MISTIC cell therapy in physiologic settings. In Aim 2, we will study the observation that a small subset of mice escape MISTIC therapy and progress after prolonged survival by interrogating the molecular and cellular basis of resistance by characterizing changes at both the level of the tumor and microenvironment. In Aim 3, we will use our isogenic, CRISPR-edited GL261 clones with wild type Imp3 to model heterogeneity and explore the engineering of MISTIC cells with cytokines and chemokines designed to remodel the GBM microenvironment as a platform to target heterogeneous tumors. We envision this approach as a proof-of - concept to use single-antigen systems to unleash epitope spreading. Together, these Aims will reveal new insights from a TCR cell therapy model that wil...