Scientist St. Jude Children's Research Hospital, Tennessee, United States
Disclosure(s):
Tara Walhart, MPH, NP-C, PhD: No financial relationships to disclose
Introduction/Rationale:
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:
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:
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:
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.
