TIRTL-sequencing to interrogate the clonal repertoire of CARpos and CARneg TILs post CAR T-cell therapy for pediatric solid tumors 2259261

Introduction Post-immunotherapy, tumor infiltrating lymphocytes (TIL) expanded from solid tumors reflect immune responses within the TME. We are investigating B7-H3-CAR T cells for pediatric patients on two early phase clinical trials. CAR T cells expressed a 2nd generation B7-H3.CD28ζ CAR and cell surface 41BB ligand. Loc3CAR (NCT05835687) evaluates intracranial CAR T cells for brain tumors, and 3CAR (NCT04897321) evaluates intravenous CAR T cells after lymphodepleting chemotherapy for solid tumors. Methods Small tumor biopsies from 3 patients were evaluated for TILs: Loc3CAR-19 (ependymoma), 3CAR-17 and 3CAR-18 (synovial sarcoma). We evaluated scalability of the Throughput-Intensive Rapid TCR Library sequencing (TIRTL-seq) to gain insight into the clonal kinetics and paired αβ TCR. CD45/CD3+ sorted cells were dispensed as low as 1 cell per well into a 384-well plate for sequencing. Results Immunophenotyping demonstrated 1% CAR+, 40% CD4+, and 25% CD8+ cells. 60% percent of TILs had an effector memory phenotype. TIRTL-seq identified paired αβ TCR chains in all samples, even when input was limited. Loc3CAR-19 exhibited 1 β chain in 80% of the reads and 2 α chains in 90% of reads, suggesting expansion of a specific TCR pair. In contrast, 3CAR-17 revealed high diversity, with over 86,000 unique chains and over 4,000 inferred αβ pairs. However, we also observed evidence of a clonal expansion in 3CAR-18, where over 12% of cells had the same TCR; in addition, other clonal expansions in this sample shared v and j segment usage, potentially suggesting convergent epitope recognition by distinct TCRs. Conclusion TILs can be expanded from small tumor fragments post-CAR T cell infusion. TIRTL-seq is a scalable platform for αβ TCR-seq for limited cells recovered from cultured TILs. TIRTL-seq analyses suggest TIL clonal expansion and segment usage patterns vary significantly, whereas some show strong clonal expansion and others display high diversity. Funding Source American Lebanese Syrian Associated Charities (ALSAC) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)