PROJECT SUMMARY: U01 CA217959 Supplement Application This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 24-029. The parent Multiple Principal Investigator (MPI) Project proposal for the Pediatric Immunotherapy Network is focused on high-risk neuroblastoma, 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 is 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. The primary goal of this Project is to achieve improved outcomes for patients with high-risk neuroblastoma through the development of a personalized vaccination strategy targeting individualized neoantigens. The central hypothesis is that high-risk neuroblastomas, despite a low tumor mutation burden (TMB), harbor a sufficient number of neoepitopes through canonical and non-canonical mutations to identify, predict, and validate optimal neoantigen peptides to engineer effective multivalent personalized neuroblastoma vaccines. The motivation for the proposed research is the urgent need to improve survival and to decrease treatment-related morbidities for patients with high-risk neuroblastoma. Indeed, the majority of high-risk neuroblastoma patients achieve a remission with standard therapy, and here we seek to engage the adaptive immune system to eradicate residual disease and prevent relapse. We will test our hypothesis through the two Specific Aims: 1) define the neoantigen landscape of high-risk neuroblastoma patient and genetically engineered mouse model (GEMM) tumors; 2) develop and test a readily translatable personalized vaccination strategy. In this new supplemental application, we take advantage of the expertise of the expertise of Dr. Drew Weissman and his University of Pennsylvania RNA Innovation Institute to develop mRNA-based lipid nanoparticle personalized neuroblastoma vaccines to be tested in parallel with the multivalent peptide vaccines we are pursuing in the parent award. This will extend the potential impact of this U01 Project by providing an alternative path to clinical translation that may have advantages that we will define in this supplemental sub-Project.