2008 Striking Twice With One Hit: Enhancing Neurosurgery Through Patient-Derived CAR T-Cell Therapy Against Human Glioblastoma and Tumor Microenvironment via Locoregional and Systemic Infusion, Bolstered by Anti PD-L1 Strategy

INTRODUCTION: Glioblastoma Multiforme (GBM), the most common and devastating primary brain cancer, resists standard of care. Gene therapy, anti-angiogenics, and immune checkpoint inhibitors (ICI) struggle to gain FDA approval and have failed against GBM. Adoptive T-cell immunotherapy; Chimeric Antigen Receptor (CAR) T-cell therapy has shown safety and efficacy in recent clinical trials. Success is still impeded by tumor heterogeneity, tumor antigen loss and the immunosuppressive tumor microenvironment (TME). METHODS: Our CAR T-cells were tested in vitro and in vivo against GBM patient-derived primary lines and TME. We performed xCELLigence impedance, degranulation, and ELISA assays to measure granule secretion (e.g. granzyme B). We tested efficacy and safety of our MC9999 CAR-T cells in vivo in mice bearing patient-derived GBM tumors via both intravenous/systemic and locoregional delivery, compared to non-armored T-cells and controls (n=15/group). For antigen analysis after treatment, we used Akoya 6-plex staining. To elucidate molecular mechanisms, we performed Sc-RNA sequencing in mice infused with CAR-T cells vs. controls. RESULTS: Our novel MC9999 CAR construct is highly specific and can effectively target primary GBM cell lines and tumor associated macrophages in vitro. (p <0.001). Our MC9999 CAR T-cells achieved safe complete tumor erradication in vivo after systemic/intravenous and locoregional delivery, without tumor recurrence in over 150 days in patient derived GBM and commercial cell lines in repeated experiments. (CAR T vs Control p=0.0043) (CAR-T vs non-CAR T-cells: p=0.0031). CONCLUSIONS: Our novel MC9999 CAR T-cell construct emerges as a beacon of hope against GBM and its intricate microenvironment. Proven effective and safe directed at PD-L1, it stands as a formidable contender for imminent clinical trials.