Decade-Long Remissions of Leukemia Sustained By the Persistence of Activated CD4+ CAR T-Cells

The adoptive transfer of chimeric antigen receptor (CAR)-reprogrammed T lymphocytes has demonstrated significant potential in various malignancies. Functional persistence of CAR T-cells in chronic lymphocytic leukemia (CLL) is a key predictor of durable remissions, yet the characteristics of long-term persisting CAR-engineered T cells have not been extensively studied. We here studied the fate of CD19-specific (CAR19) T-cells in two leukemia patients who achieved and sustained a complete remission over a decade ago. Molecular fate mapping was carried out on long-term persisting CAR T cells using lentiviral vector integration site sequencing across multiple time points up to 9.0 and 7.2 years post-infusion in patients 1 and 2, respectively. This analysis revealed little if any CAR T-cell clonal stability in the first 1.6 years in patient 1. Close to year 2 the CAR T cell repertoire stabilized in the first patient, with a strong clonal focusing until the last follow-up. The second patient had episodes of repertoire stability from the first month to approximately 12 months later, as well as from year 1 to 5. The CAR T cell repertoire in both patients were dominated by a few clones which were detected across a multiyear time span. CAR T-cells were still detectable using flow and mass cytometry 10+ years post-infusion. Deep immunophenotyping using a 40-marker mass cytometry panel identified divergence in effector but convergence in memory CAR T cell characteristics, with a prominent highly activated effector-memory CD4+ population developing late after infusion, expressing immune regulatory molecules. The CD4+ CAR T-cells were notable for a subpopulation highly expressing Ki67, suggestive of a proliferative phenotype. Ki67[hi] CD4+ CAR T-cells steadily emerged as the dominant population in both patients: this population constituted 15.9% of CAR T-cells at month 1.8 in patient 1, increasing to 97.0% by year 9.3; and constituted 0.2% of CAR T-cells in patient 2 at month 2.4, increasing to 87.2% by year 7.2. We assessed Ki67 expression in the CD4+ CAR T-cells compared to the CAR-negative T cells from these patients at matched time points, finding that this level of Ki67 expression was strongly CAR T-cell specific. CD8+ CAR T-cells also exhibited a proliferative trend overall, but Ki67 expression was generally lower and less robustly observed compared to the CD4+ CAR T-cell subset. These Ki67[hi] CD4+ T cells expressed a distinct marker profile, including activation markers CD38, HLA-DR, and CD95; transcription factors EOMES and TOX; checkpoint markers CTLA-4, LAG-3, TIGIT; and memory markers CD27 and CCR7. Together, these data suggest two major phases of CAR T-cell therapy responses in these patients: an initial response phase dominated by cytotoxic CD8+ T cells and double-negative Helios[hi] CAR T-cells, and a long-term remission phase dominated by a uniquely proliferative CD4+ CAR T-cell phenotype.

To characterize these long-persisting CAR T-cells at a single-cell resolution, we performed CITE-Seq with single-cell T cell receptor-beta VDJ profiling on CAR T-cells obtained 9.3 years post-infusion. These long-persisting CAR T-cells exhibited strong evidence of ongoing activation, proliferation, and aerobic glycolysis despite evidence of chronic antigen-mediated signaling. Our data in aggregate provide important insight into and the development of a long-term memory anti-tumor response, necessary for sustained remission in leukemia following CAR T-cell therapy.