PROJECT SUMMARY/ABSTRACT The long-term goal of this R01 application is to develop an effective therapy for pediatric patients with solid tumors using T cells expressing chimeric antigen receptors (CAR T cells). Pediatric patients with relapsed or refractory solid tumors experience dismal outcomes despite aggressive management with multimodality therapies, and T cell immunotherapy has the potential to improve outcomes for these challenging diseases. We propose to target B7-H3 with CAR T cells, a tumor associated antigen that is expressed at high levels on multiple pediatric solid tumors, with limited expression in most normal tissues. We have developed B7-H3-CAR T cells expressing 41BB ligand (41BBL), and have demonstrated improved antitumor activity compared to standard B7- H3-CAR T cells in preclinical models. We now wish to evaluate our approach clinically and hypothesize that the administration of B7-H3-CAR T cells is safe and results in antitumor effects. Secondary hypotheses include that carefully orchestrated correlative studies are needed to decipher human CAR T cell biology and improve their potency. These hypotheses will be evaluated in two interrelated research aims. Aim 1 is focused on translation, implementing a Phase I clinical trial evaluating the safety and antitumor activity of B7- H3-CAR T cells expressing 41BBL for pediatric patients with relapsed or refractory B7-H3-positive solid tumors. Aim 2 is focused on reverse translation, assessing the in vivo expansion and persistence of B7-H3-CAR T cells. It will perform single cell (sc) TCR analysis, 10x scRNAseq analysis and whole genome bisulfite sequencing (WGBS) to determine the clonal structure and functional state of B7-H3-CAR T cells. Tumors will also be biopsied to determine B7-H3-CAR T cell presence and interactions within the tumor microenvironment. Based on results of these correlative studies, we will implement one strategy to improve the effector function/antitumor activity of B7-H3-CAR T cells and evaluate it in preclinical models. Successful completion of the proposed studies in this R01 application will provide critical information for the field of cell therapy for solid tumors. While this application is focused on targeting B7-H3-positive pediatric solid tumors, our findings could be readily applied to other B7- H3-positive pediatric and adult tumors, and CAR T cell therapies targeting different tumor associated antigens.