Mentor Name: Timothy Spear

High-risk neuroblastoma (HR NB) is a diverse and enigmatic malignancy arising from the developing sympathetic nervous system that remains lethal in 50% of patients despite intensive multi-modal therapy. There remains an urgent unmet need for developing novel therapeutic interventions to decrease the incidence of relapse, increase overall survival, and reduce devastating toxicities associated with standard therapy. Chimeric antigen receptor (CAR) T cells have revolutionized treatment for a subset of pediatric leukemias, but their success in solid tumors, such as HR NB, has traditionally been limited by ineffective penetrance into solid lesions and poor longitudinal persistence in patients leading to CAR-refractory or relapsed disease. Thus, developing strategies to enhance CAR T cell efficacy and persistence is critical. The primary goal of this project is to develop a vaccination strategy to enhance the anti-tumor activity of CAR T cells directed against HR NB-specific antigens. The central hypothesis is that vaccination with CAR scFv-targeted antigen will facilitate T cell activation by professional antigen presenting cells (APCs) outside of the immunosuppressive tumor microenvironment (TME) and subsequently enhance the ability for CAR T cells to eradicate tumors and prevent relapse. We have generated an innovative syngeneic murine model for a novel class of peptide-centric (PC)-CAR T cells targeting the intracellular oncogenic driver PHOX2B (9mer peptide on HLA-A*24:02 in order to test our hypothesis). We will characterize human dendritic cell (DC) line THP-1 genetically engineered to express HLA-A*24:02 as well as monocyte-derived DCs (moDCs) isolated from HLA-A24+ healthy donor peripheral blood mononuclear cells. We will simultaneously manufacture PC-CAR T cells targeting PHOX2B/A24 from autologously matched donor T cells. We will treat these DC sources with lipid nanoparticles (LNPs) encapsulating mRNA encoding PHOX2B, testing their ability to stimulate PC-CARs in vitro. PC-CAR proliferation and memory formation will be assessed by flow cytometry. In parallel, cytokine production and cytotoxicity against A24+ NB cell lines will be assessed. We will then test the ability for PHOX2B-mRNA-LNP vaccination to modulate PC-CAR T cells in vivo. Then, we will create humanized mice, engrafted with both PC-CAR T cells and donor-matched DC cells and treat with PHOX2B-mRNA-LNP to measure CAR T cell expansion, persistence, and memory formation and ex vivo cytotoxicity.