Generation of CCR4/CD7 Bispecific CAR-T Cells Resistant to Fratricide and Exhaustion.

The use of chimeric antigen receptor (CAR) T-cell therapy for T-cell malignancies is limited by fratricide, antigen-escape, lack of functional endurance and adverse events such as multilineage cytopenia. To address these limitations, we developed a simple single-step CD7-depletion process followed by transduction with a lentiviral vector encoding a CCR4/CD7 bispecific CAR. CD7-negative (CD7N) CCR4/CD7 CAR-T cells could expand without experiencing fratricide, unlike the bulk CCR4/CD7 CAR-T cells. The CD7N CAR-T exhibited robust cytotoxicity against malignant T-cell lines with heterogeneous CD7 and CCR4 expression in vitro and in vivo. Incorporation of EGFRt in the CAR construct allowed elimination by cetuximab in case of adverse events, whereas inclusion of c-Jun in the construct reduced functional exhaustion after repeated tumor challenges in vitro. In comparison to non-transduced CD7N cells and Bulk CAR-T cells, scRNA-seq analysis of CD7N CAR-T cells revealed a unique AQP3+ CD4+ T-cell subset following exposure to tumor cells. This cell subset exhibited broad activation of the Src/Ras/ERK and Bcl-2 pathways, high levels of SOS1 and KLF2 expression, and specific ligand-receptor interactions within the tumor necrosis factor superfamily. Collectively, these results suggest that further clinical development of CCR4/CD7 bispecific CD7N CAR-T cells is warranted, including the AQP3+ subset with SOS1/KLF2 modulation.