Project Summary Tumor-specific immunotherapy is a promising modality capable of improving clinical outcomes for children affected with high-grade glioma (HGG). We have pioneered a ‘first-generation’ adoptive cellular therapy (ACT) platform utilizing total tumor RNA-pulsed dendritic cells (DCs) to expand polyclonal tumor-specific T cells for the treatment of invasive and refractory brain cancers. We have demonstrated the safety, feasibility, and promising pre-clinical and early clinical efficacy of this approach, including prolonged disease-free remission in some treated subjects (>2 to 5 years) and radiographic and clinical response in first-in-human phase I/II clinical trials treating patients with medulloblastoma (MB), HGG, and brain stem glioma (FDA INDs 14058 and 17298). In this proposal, we will develop a ‘next-generation’ precision ACT approach using a combination of patient-specific antigen profiling using a novel cancer immunogenomics-based algorithm developed in our lab, a novel gene enrichment strategy that allows us to target hundreds of tumor-specific antigens in a single pool of RNA (tsRNA), and the SELEX-CTL (Selective Expansion of Cytotoxic T Lymphocytes) platform for the stimulation and selective enrichment of polyclonal tumor-reactive T cells from precursor frequency of 1-3% after stimulation with tsRNA- pulsed DCs. This powerful approach to generating enriched T cell populations recognizing a plurality of tumor antigens uniquely addresses the challenge of dealing with tumor antigenic heterogeneity and confronting the reality of patient-to-patient variation in antigen expression in the development of antigen targeting strategies. We hypothesize that given the preclinical and clinical data we have generated demonstrating the capacity to engender a polyclonal T cell response against HGGs and MB antigens using DCs pulsed with unrestricted total tumor RNA, the approach studied within this proposal of selectively identifying and amplifying patient-specific tsRNA and uniquely identifying, sorting, and expanding responding tumor-specific T cells for use in ACT will constitute a highly significant and highly effective precision immunotherapy approach for the treatment of pediatric patients with invasive HGG. Our SPECIFIC AIMS will be to: 1. Evaluate the safety, efficacy, and immunologic effects of ACT targeting neoantigens and uniquely expressed tumor-associated antigens (TAAs) in preclinical models of pediatric HGG; 2. Determine the capacity to selectively isolate and expand antigen-specific memory T cells in vitro from pediatric patients with HGG who have received ACT at our center; 3. Conduct a phase 1 clinical trial of precision ACT targeting tumor-specific antigens in children, adolescents, and young adults (AYAs) with invasive HGG.