T cells with split CARs specific for NKG2D ligands and PD-L1 exhibit improved selectivity towards monocyte-derived cells while effective in eliminating acute myeloid leukaemia in vivo

Purpose The expression of NKG2D ligands and PD-L1 has been detected on acute myeloid leukaemia (AML) cells, as well as normal cells of the myeloid lineage. To target leukemic cells while minimizing collateral damage to normal cells, we constructed a split dual CAR system based on the AND-gate logic. Methods The NKG2D extracellular domain linked with DAP12 without a co-stimulatory signal was used for the basal activation of T cells, and used together with the PD-L1-specific chimeric costimulatory receptor containing the 4-1BB activating domain for co-stimulatory signal 2 input. This dual CAR displayed cell-type specificity and activity similar as a 2nd generation NKG2D ligand-specific CAR. Results When compared to CD64 and PD-L1-specific 2nd generation CARs, we observed that the split dual CAR offered an improved myeloid cell type selectivity. For example, PD-L1-specific CAR-T cells lysed all tested myeloid cell types that expressed PD-L1, including M0 macrophages (Mø0), LPS-polarized Mø1, IFN-γ polarized Mø1, IL-4 polarized Mø2, monocytes, immature dendritic cells (imDCs), mature DCs, as well as KG-1 AML cells, while the dual CAR-T cells displaying killing activity only towards LPS polarized Mø1, mature DCs and KG-1 cells that expressed both NKG2D ligands and PD-L1. In a mouse liquid tumor model, the dual CAR-T cells were effective in eradicating established KG-1 AML xenografts. Conclusion The improved cell type specificity offered by our split dual CAR-T cell system targeting paired antigens would favour the reduction of the on-target off-tumor toxicity towards normal myeloid cells during the treatment of myeloid leukaemia.