FUNCTIONAL METRICS OF EFFICACIOUS CAR T CELLS REVEALED BY MULTI-DIMENSIONAL SINGLE-CELL PROFILING

Chimeric antigen receptors (CAR) T cells for B-cell malignancies serve as a model for identifying T-cell subsets with superior clinical activity. We profiled the infusion products (IP) of patients with large B-cell lymphoma (LBCL) using multi-omic single-cell assays to reveal the therapeutic potential of CD19-CAR+ T cells. Functional profiling using timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed high migration and serial killing. Cellular profiling using confocal microscopy revealed that migration is correlated with both mitochondrial and lysosomal volume; and molecular profiling scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and in vivo persistence. T cells enriched for migratory capacity showed sustained serial killing and optimal in vivo efficacy. Our results demonstrate that migration is a cell-intrinsic biomarker desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.