Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities

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 (Tconvs), 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-Tconvs, 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-Tconvs. However, unlike CAR19-Tconvs, 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. Description Allogeneic CAR-DNTs with minimal genetic modification show potent anti-tumor activity without off-tumor toxicities. A CAR that drives killing but not toxicity Chimeric antigen receptor (CAR)–T cell therapies are FDA-approved for treating blood cancers but are often limited by toxicities, and difficult and expensive production. Thus, developing more streamlined CAR-T cell therapies is crucial. Here, Vasic et al. used mouse and human double-negative (DN) T cells as a CAR-T cell therapy, showing robust efficacy in mouse models of blood and lung cancers. They found that the DN CAR-T cells were just as effective as conventional CAR-T cells but induced no toxicity. The double-negative CAR-T cells could be produced from mixed donors, were not rejected from hosts, and could be frozen for long periods of time. Thus, DN CAR-T cells are an attractive “off-the-shelf” CAR-T cell therapy option.