Mechanistically Guided Immunometabolic CAR-TIL Therapy for Glioblastoma Heterogeneity and Immune Suppression - PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM), the most common malignant primary brain tumor, has a poor prognosis despite standard treatments such as surgery, chemotherapy (temozolomide), and radiation. With a median survival of ~14.6 months and a five-year survival rate of ~15%, GBM remains a major therapeutic challenge due to its cellular heterogeneity and highly immunosuppressive tumor microenvironment (TME). Intracranial chimeric antigen receptor (CAR) T cell therapies have shown promise, but their efficacy is often limited by tumor antigen loss and TME-mediated immune suppression, allowing cancer cells to evade immune detection. Pharmacologic modulation of tumor metabolism represents an additional therapeutic opportunity, as GBM cells are highly dependent on cholesterol and lipid metabolism. In my current research (K99 phase), I am developing dual-engineered ME1-armored anti–PD-L1 CAR tumor- infiltrating lymphocytes (CAR-TILs) in combination with intracranial administration of clemastine and bexarotene to exploit tumor metabolic vulnerabilities. These engineered TILs leverage their endogenous TCR diversity to target multiple tumor antigens while ME1 enhances metabolic fitness, cytotoxicity, and persistence in the nutrient- and lipid-deprived TME. The MC9999 CAR construct targets PD-L1, disrupting both tumor cells and tumor- associated macrophages (TAMs), key drivers of immune suppression in GBM. Preliminary data demonstrate that ME1-armored CAR-TILs exhibit superior cytotoxicity and persistence compared to non-armored counterparts, and that the combination of clemastine and bexarotene sensitizes GBM cells by disrupting lipid metabolism and upregulating PD-L1. To further investigate these effects, I will assess metabolic changes in CAR- TILs versus naïve and non-armored counterparts, evaluate functional efficacy using patient-derived GBM cells and TAMs, and identify mechanistic pathways through RNA sequencing and protein analyses in vivo. In my independent phase (R00 phase), I will expand this work to explore the broader effects of CAR-TILs and combination therapy on the central nervous system (CNS), including neurons, astrocytes, and oligodendrocytes. Using humanized GBM models, I will perform single-cell RNA sequencing at multiple time points to assess T cell persistence, TME remodeling, and CNS cellular responses,. For translational relevance, I will collaborate with clinical partners to analyze postmortem tissue from patients who received intracranial CAR T therapy, allowing direct comparison between human and animal models. By integrating tumor metabolic sensitization with dual-engineered CAR-TIL therapy, this project aims to overcome GBM heterogeneity, remodel the immunosuppressive microenvironment, and investigate the impact of these therapies on CNS cellular populations. These studies will pave the way for a future R01-supported clinical trial of ME1-armored CAR-TILs in glioblastoma patients.
