Chimeric antigen receptor (CAR)-T cell therapy is effective in B-cell malignancies but remains limited by T-cell dysfunction and poor persistence resulting from ex vivo manufacturing. We hypothesised that generating CAR-T cells in vivo using haematopoietic stem and progenitor cells (HSPCs) would better recapitulate physiological immune memory and improve long-term fitness. To test this, we developed a CD19 CAR-HSPC platform where murine HSPCs were transduced and expanded ex vivo before direct injection into the thymus.
We first characterised the kinetics of T-cell reconstitution following intravenous versus intrathymic HSPC administration at single-clone resolution. HSPCs were barcoded using SPLINTR, a single-cell lineage tracing platform, enabling us to examine clonal differences in T-cell output and lineage bias between the two routes. We found that diverse HSPCs reprogrammed their fate to produce only T cells when injected into the thymus. HSPCs that failed to engraft intravenously successfully engrafted when injected into the thymus. This yielded higher circulating T-cell numbers after intrathymic injection compared with intravenous delivery.
To assess whether this approach could be harnessed for anticancer immunity, murine HSPCs were expanded ex vivo and transduced with a CD19-targeting CAR construct (19CAR). In C57BL/6 mice, intrathymic 19CAR-HSPCs produced abnormal CD4-CD8- CAR-T cells, indicative of diverted T-cell development in the thymus. This phenotype was driven by 19CAR engagement with CD19 on thymic B cells. In contrast, intrathymic 19CAR-HSPCs in NSG mice generated phenotypically normal CAR-T cells that persisted for over 12 months and accounted for ~10% of circulating white blood cells. In vitro, these 19CAR-T cells displayed potent cytotoxicity against B-ALL. In vivo, intrathymic 19CAR-HSPCs protected NSG mice from B-ALL challenge, with repeated exposures triggering CAR-T expansion and persistent resistance to leukaemic engraftment. Stem cell–based in vivo CAR-T generation represents a promising strategy to overcome the limitations of conventional CAR-T cell therapy.