The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4 + or CD8 + T cells (T convs ), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3 + CD4 − CD8 − double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-T convs , we evaluated the feasibility, safety, and efficacy of using healthy donor–derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti–CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-T convs . However, unlike CAR19-T convs , CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

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