Clonal kinetics and single cell transcriptional profiling of adoptively transferred CD19-specific CD8+ CAR-T cells in adults with B cell malignancies

Lymphodepletion chemotherapy followed by infusion of T cells modified to express a CD19-targeting chimeric antigen receptor (CAR) has produced remarkable anti-tumor responses in patients with B cell malignancies. However, little is known about the clonal composition and transcriptional heterogeneity of CAR-T cells in the infusion products (IP) and clonal kinetics after adoptive transfer. We performed single-cell RNA sequencing (scRNA-seq) on CD8+ CAR-T cells isolated from the IP and the blood of patients treated on a phase 1 clinical trial (NCT01865617) with lymphodepletion chemotherapy and a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells. Infused CD8+ CAR-T cells displayed transcriptional heterogeneity which declined after adoptive transfer, coincident with early expression of genes associated with activation. We identified four transcriptionally distinct CAR-T cell subsets in the IP and found that these subsets differed in their contributions to the CAR-T cell population detected in blood after infusion. Better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy. We performed scRNA-seq on CD8+ CAR-T cells isolated from the IP and blood at the early, late, and very late time points from four patients who received CD19-specific CAR-T cells for refractory B cell malignancies. Single CD8+ CAR-T cells were sorted as CD8+/CD4-/EGFRt+ events and scRNA-seq was performed using the Chromium Single Cell 5’ library and V(D)J enrichment kit (10x Genomics). Libraries were sequenced on HiSeq2500.