Project Abstract: Pediatric HGGs comprise glioblastoma [glioblastoma multiforme (GBM)], anaplastic astrocytoma, and diffuse intrinsic pontine glioma (DIPG) and collectively are the most common malignant brain tumors in children. Patients with HGGs have a very dismal prognosis, with <5% 5-year survival for GBM patients and a median survival of <1 year for patients with DIPG, pointing to an urgent need for alternative therapeutic approaches. Chimeric antigen receptors (CARs) are engineered cell surface molecules that combine the specificity of antibodies with the downstream signaling of effector cells such as T cells. The expression of CARs on T cells has shown enormous promise by effectively treating malignancies by directly killing them. However, CAR-T cell therapy is often hampered by the inability of T cells to penetrate solid tumors and the inhibitory tumor microenvironment (TME). Recently there has been great interest in developing CAR-macrophages against solid tumors to improve phagocytic activity and antigen presentation of macrophages against tumors. Here we hypothesize that this distinctive proficiency of macrophages and microglia to infiltrate malignant brain tumors provides a unique opportunity to exploit their phagocytic properties by engineering the activation of these professional phagocytes using exogenous expression of Chimeric Antigen Receptor (CARs) targeted towards a brain tumor-specific antigen. In Aim 1 we will engineer the most effective CAR-expressing phagocyte for efficient targeting of pHGG and demonstrate the superior efficacy of B7H3 CAR phagocyte therapy in combination with blockage of phagocytosis checkpoint (anti-CD47) immunotherapy in humanized xenograft models of pHGG. In Aim 2 we will Identify the most efficient cell type that can function as a CAR-expressing phagocyte that can most efficiently traffic to the brain and target pHGG tumors in combination with blockage of phagocytosis checkpoint (anti-CD47) immunotherapy and finally in Aim 3 we will study the in vivo biology of CAR-macrophages and its effect on the TME in syngeneic mouse models of high-grade pediatric glioma.