PROJECT SUMMARY While chimeric antigen receptor T cells (CAR-Ts) have provided impressive responses in hematologic malignancies, children and young adults with metastatic or relapsed solid tumors have not yet benefited from CAR-Ts and continue to suffer dismal outcomes. A major barrier to CAR-T efficacy in solid tumors is inadequate CAR-T expansion, driven in part by CAR-T exhaustion and poor memory potential. Preclinically, CAR-T exhaustion results from excessive CAR signaling, and can be rescued by eliminating the CD28 T cell costimulatory domain from CAR design. Clinically, in samples from general pediatric oncology patients, poor memory potential in T cells is associated with chemotherapy exposure. Preclinical models further suggest that CAR-T exhaustion can be reduced by overexpressing a transcription factor, cJun, and that memory potential can be enhanced by exposing T cells to a drug, Ibrutinib. Based on these observations, Dr. Ramakrishna will apply novel single-cell and multi-dimensional technologies to CAR-T patient samples to assess the impact of CAR costimulatory domain or pre-apheresis chemotherapy exposure on CAR-T exhaustion and memory potential and ultimately on patient CAR-T fitness, defined as CAR-T molecular signature paired with functionality. To accomplish her aims, Dr. Ramakrishna will innovatively compare CAR-T samples across three GD2 CAR-T clinical trials. In Aim 1, with training in multi-dimensional data analysis, Dr. Ramakrishna will integrate phenotypic (CyTOF), epigenetic (ATACseq), and transcriptomic (RNAseq) molecular signature with CAR-T functional assessments to determine whether GD2.Ox40.CD28.z CAR-Ts (NCT02107963) confer exhaustion, thereby affecting CAR-T fitness, as compared to GD2.41BB.z CAR-Ts (NCT04539366) in osteosarcoma patients. In Aim 2, with training on cancer immune biology, Dr. Ramakrishna will use CyTOF, ATACseq, and in vitro cytokine production, to test the hypothesis that apheresis and CAR-T products from chemotherapy-naïve patients (diffuse midline glioma; NCT04196413) have improved CAR-T fitness as compared to those from chemotherapy-treated patients (osteosarcoma; NCT04539366), followed by single-cell RNAseq to track persistent CAR-T transcriptional profiles in patients. In Aim 3, with training in immune regulation, Dr. Ramakrishna will evaluate whether CAR-T fitness in chemotherapy-treated patient aphereses can be enhanced through modulating molecular pathways by cJun or Ibrutinib. To build upon her substantial prior CAR-T research and clinical experience, Dr. Ramakrishna has developed a strong training plan and mentorship team, including her primary mentor, Dr. Crystal Mackall, a pioneer in translational immunotherapy research; co-mentor, Dr. Sean Bendall, an innovator in multi-dimensional immune assays; advisory committee, Dr. David Miklos, Dr. Holden Maecker, and Dr. Michelle Monje; and collaborators, Dr. Rosie Kaplan and Dr. Steven Feldman. In completing her proposed plan with this team, ...