Despite the remarkable response rates of B-cell malignancies to CD19 targeting chimeric antigen receptor T-cells (CAR-T), many responding patients ultimately relapse. Whilst some mechanisms leading to primary resistance to CAR-T have been identified, little is known about the tumour intrinsic mechanisms that lead to relapse following an initial deep response.
To explore this clinical phenomenon, we developed a novel immunocompetent mouse model in which murine CAR-T target CD19 on a syngeneic B-cell acute lymphoblastic leukaemia (B-ALL) driven by a BCR::ABL1 transgene (p190 cell line). Transplanted p190 cells rapidly establish detectible disease within 7 days of transplant, with untreated mice succumbing to fulminant disease within 3 weeks. Following lymphodepletion therapy and infusion of CD19-directed CAR T-cells 1 week after tumour transplant, all mice demonstrate undetectable levels of disease, followed by subsequent relapse in the majority.
To investigate whether post CAR-T relapse is a pre-determined and heritable fate of specific B-ALL clones, we applied SPLINTR, an expressed molecular barcoding strategy (Fennell & Vassiliadis et al. Nature 2022), to this model. Treatment with CAR-T resulted in a significant reduction in clonal diversity at relapse when compared with mock-T cell treated control mice, suggesting that not all clones have the capacity to overcome the significant therapeutic pressure applied by CAR T-cell therapy. Importantly, the dominant clones present in post CAR-T relapse samples were highly concordant across multiple recipients and were present only at low frequency in both the initial transplanted tumour pool and in relapse samples from mock-T treated control mice. Together, this suggests that the potential for CAR-T resistance is a cell intrinsic phenomenon, and clonally heritable property.
Leveraging the ability of SPLINTR barcodes to be captured using single cell RNA sequencing technologies, we are further exploring the transcriptional dynamics of these rare relapse fated clones prior to and following CAR-T therapy which the aim of identifying targetable clone-specific therapeutic vulnerabilities.