Targeting T Cells for enhancing anti-tumor immunity in pediatric brain tumors. - PROJECT SUMMARY We will undertake hypothesis-driven, mechanistic studies to demonstrate the functional role of tumor neoantigen-specific T cells in pediatric brain tumors (PBT). Brain tumors represent the most common solid tumors in children, and a high proportion of patients have devastating outcomes despite multimodal therapy. Most notably, immunotherapies that show remarkable success in adult cancers are not approved for PBT. The notion that brain tumors are immune-cold with low mutational burden has precluded assessment of immunotherapies in children. Our recent studies (Nature Cancer, 2024) provide a compelling hypothesis that neoantigen-specific T cells are present in the brain tumor tissue, and therapies to boost their numbers and activity, like PD-1 blockers and neoantigen-directed vaccines, are likely to be beneficial. We established the TARGET BRAIN study (PI: Dr. Ganesan), a prospective observational cohort of children with brain tumors who undergo surgical resection (40 cases/year) at Rady Children’s Hospital, UC San Diego. Given the outcome disparities in PBT, a major focus of TARGET BRAIN is the inclusion of ethnically-diverse population and those under-represented in research (current enrollment−52% female, 58% Latino, 30% non- Hispanic White, 8% African-American, 4% Asian). From this cohort, we performed the first and in-depth survey of single-cell transcriptomes with paired T cell clonality studies using purified tumor-infiltrating CD8+ T cells (TILs) isolated from PBT (n=38). We found striking clonal expansion of CD8+ TILs which displayed effector program, tissue-residency, high levels of PD-1 expression and enrichment for gene signatures of immunotherapy response and neoantigen-specific T cells. Importantly, we experimentally validated tumor reactivity of TILs in PBT. We hypothesize that neoantigen-specific CD8+ T (TRM) cells are generated in a substantial fraction of pediatric brain tumors and are capable of mediating tumor control. We anchor our proposed studies to address this hypothesis systematically in two important and aggressive brain tumors – medulloblastoma (MB) and high-grade glioma (pHGG). In Aim 1, we will determine prevalence and transcriptional properties of neoantigen-specific CD8+ T cells in an ethnically-diverse cohort of children with MB (n=40) and pHGG (n=40). We will perform single-cell RNA-seq and TCR-seq analysis of CD8+ TILs isolated from brain tumor tissue. To determine TIL TCRs that are neoantigen-specific, we will test reactivity of T cells from donor-matched PBMC to neoantigens present in corresponding tumors. By assessing overlap of these experimentally-validated neoantigen-reactive TCRs with TIL TCR sequences, we will determine neoantigen-specific T cells in tumor. Given that tumor-associated antigens (TAA) may also drive functionally-relevant TIL responses, we will perform similar analysis to define TAA-directed T cells. We will couple single-cell TCR-seq with the transcriptomes of CD8+ TILs to determine the transcriptional properties of neoantigen-specific T cells and TAA-directed T cells. In Aim 2, we will determine function of neoantigen-specific T cells in controlling MB and pHGG. We will engineer primary T cells to express neoantigen TCRs and determine their epitope specificity and avidity in vitro. We will test the anti-tumor activity of neoantigen-specific T cells, by adoptively transferring T cell clones engineered to express neoantigen-reactive TCRs and assessing their effects on tumor growth in vivo in orthotopic PDX models. These studies will provide definitive evidence regarding prevalence and function of neoantigen-specific CD8+ T cells in pediatric brain tumors and propel clinical testing of neoantigen-directed vaccines in children.
