PROJECT SUMMARY/ABSTRACT Immunotherapy has shown limited efficacy against pediatric sarcoma. In our recently completed phase I study, infusion of HER2-targeted chimeric antigen receptor T cells (CAR-T) was safe and showed encouraging indicators of antitumor activity, including three durable complete responses. The key observations from this study are: (1) lymphodepletion promotes CAR-T expansion but only marginally improves persistence, (2) adoptively transferred CAR-T are susceptible to immune inhibition, and (3) antigen downregulation/loss is associated with disease relapse. While the literature is lacking on immune inhibitory mechanisms and tumor adaptations in sarcoma, our findings are in line with observations made by others in early clinical and pre-clinical studies of cellular therapy for solid tumors. Our long-term goal is to develop new approaches that capitalize on expected tumor defenses which may be key to unleashing the potential of CAR-T against sarcomas and other solid tumors. Therapeutic approaches that integrate strategies to address these compensatory mechanisms up front may mitigate downstream tumor escape and relapse. The objective of this project is to conduct prospective clinical testing of HER2 CAR-T in combination with PD-1/PD-L1 checkpoint disruption strategies in a disease-specific cohort. The central hypothesis is that the combination immunotherapy will promote immune infiltration, remodel the tumor microenvironment, alleviate CAR-T dysfunction, and thereby improve antitumor responses. Our specific aims will test the following hypotheses: (Aim 1) The combination of anti-PD-1 antibody and HER2 CAR- T administered after lymphodepletion is safe in patients with sarcoma, will improve CAR-T kinetics, and will elicit antitumor responses; (Aim 2) Cellular and serum-based studies in treated patients will help identify biomarkers of toxicity and response; and (Aim 3) Engineering CAR-T to positively transform the PD-1/PD-L1 interaction in the tumor microenvironment will potentiate antitumor effects of the cellular product while incorporating design attributes to enhance its safety . We will evaluate Aims 1 and 2 through implementation of a phase I study of autologous HER2 CAR-T and anti-PD-1 antibody after cyclophosphamide and fludarabine lymphodepletion in patients with sarcoma (HEROS 3.0 clinical trial). In Aim 3, we will develop an investigational new drug (IND) for HER2 CAR-T co-expressing a PD-1 checkpoint reversal receptor (CPR). At the conclusion of the project, we will understand if CAR-T can be safely administered with immune checkpoint inhibition to improve treatment outcomes for children and young adults with sarcoma. The significance of these contributions are: (1) establishing the clinical utility of combination immunotherapy for sarcoma, (2) potentially identifying “modifiable” patient- and treatment-related factors to guide future improvements to CAR-T therapy, and (3) enabling clinical testing of an innovative CAR...