Mentor Name: Conrad Russell Cruz

Pediatric brain tumors (PBT) remain some of the leading causes of morbidity and mortality in children. Prognosis for several malignancies, including the almost universally fatal diffuse midline gliomas, remains poor, despite advances in surgery, radiation, and chemotherapy. While current cell-based immunotherapies, including chimeric antigen receptor T cell (CAR-T) and antigen-specific T cell (AST) therapies, have shown success against acute leukemia in children, they continue to have limited efficacy against PBT. Recently, we have identified several genes that currently regulate the function, proliferation, and tumor infiltration of T cells. These genes are involved in the interaction of T cells with PBT and their microenvironment. This proposed project will apply CRISPR-mediated gene editing to reprogram T cells for improved resistance to inhibitory signaling and metabolic stress encountered during interaction with PBT. We hypothesize that PBT are highly enriched for ligands that interact with key T cell regulatory proteins and that knocking out these regulatory proteins from immunotherapeutic cell products will improve their therapeutic efficacy. In Aim 1 of this work, we will look at the different inhibitory pathways expressed by PBT, starting with diffuse midline gliomas (DMG). We will examine our bank of DMG cell lines using RNAseq and surface flow cytometry, and identify highly expressed ligands to proteins we have identified with collaborators (including ligands to checkpoint molecules). In Aim 2 of this work, we will look at knocking down gene targets, beginning with the well studied PTPN1 and 2 genes, and examine their effects on both B7H3 CAR-T and PRAME AST. Both B7H3 and PRAME are expressed in DMG. Depending on progress, we will also extend examination to T cells targeting other antigens in DMG. Gene-modified CAR-T and AST will be evaluated for increased anti-tumor function in vitro. The proposed work will hope to establish a platform for improved T cell therapies directed against PBT. Successful completion of this work will establish a framework for improving current T cell therapies for PBT at Children’s National and future clinical translation.