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Abstract BACKGROUND Immunotherapy is a developing but challenging field of research for the treatment of central nervous system malignancies. Chimeric antigen receptor (CAR)-T cell therapy has been effective in treating hematologic malignancies, but that success has not been clinically translated to solid tumors in children. B7-H3 has been identified as a promising immunotherapy target as it is highly expressed in pediatric glioblastoma and correlates with tumor progression and poor prognosis. We aim to evaluate a nanobody-based CAR-T cell therapy that binds B7-H3 with high affinity and hypothesize that anti-B7-H3 nanobody CAR-T cells can effectively kill B7-H3-expressing tumor cells. METHODS B7-H3 nanobodies were isolated from camel phage libraries and cloned into lentiviral CAR-T cell constructs. Human T cells were transduced by lentivirus to express CAR on the surface. Nanobody CAR-T cell functionality was evaluated by an impedance-based cytotoxicity assay and cytokine release was measured by ELISA. CAR-T avidity was quantified by the Lumicks z-Movi assay. In vivo studies are evaluating the efficacy of B7-H3 CAR-T cells in an orthotopic glioblastoma model in immunodeficient mice. Mice were treated with B7-H3 CAR-T cells by intracerebroventricular or intravenous delivery. RESULTS We confirmed high expression of B7-H3 on the surface of three pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. We confirmed effective killing of B7-H3-expressing tumor cells and the release of high levels of cytokines. B7-H3 nanobody CAR-T cells were observed to have high avidity compared to control CAR-T cells. In vivo studies are currently ongoing and treatment response will be evaluated by luminescence imaging and endpoint histologic analysis. CONCLUSION We have shown that B7-H3 nanobody CAR-T cells demonstrate significant preclinical efficacy. It is critical to study CAR-T cell therapy in immunocompetent in vivo models for further development and clinical application.