Phosphatidylserine as a tumor target for CAR-T cell therapy.

BACKGROUND: Phosphatidylserine (PS) exposed on apoptotic cells promotes immune clearance of dead cells without inducing inflammation. Conversely, PS exposure on live tumor cells promotes an immunosuppressive tumor microenvironment that hinders antitumor immune responses. After confirming elevated PS levels in various tumor cell lines and cancer tissues, we aimed to investigate its potential as a target antigen for chimeric antigen receptor T cell (CAR-T) therapy. METHODS: We used two different approaches to target PS. First, we employed the adaptor proteins, EDAnnexin or BCMAnnexin comprising annexin V and EDA (extra domain A of fibronectin) or B-cell maturation antigen (BCMA) antigens, to redirect the lytic activity of EDA CAR-T or BCMA CAR-T cells toward PS-expressing tumor cells. In a second approach, we developed an annexin V-based CAR (Anxa CAR-T) to directly recognize PS-positive tumor cells. RESULTS: The adaptors proteins EDAnnexin and BCMAnnexin successfully redirected EDA CAR-T or BCMA CAR-T cell activity, leading to an efficient recognition of PS(+) tumor cells in vitro. However, the established immunological synapse differs significantly from that observed when CAR-T cells recognize the tumor cells directly. In vivo administration of the adaptor proteins, combined with the corresponding CAR-T cells, displayed antitumor activity in mice bearing PS(+) tumors. Regarding the second approach, Anxa CAR-T cells effectively recognized and killed PS(+) tumor cells in vitro. Nonetheless, PS exposure on T-cell membranes during T-cell activation impeded efficient Anxa CAR-T cell manufacturing due to fratricide. By optimizing retroviral dose to reduce Anxa CAR expression on the cell membrane, or by using the multikinase inhibitor dasatinib, the fratricide effect was mitigated, enabling successful Anxa CAR(Low)-T cell production. Remarkably, Anxa CAR(Low)-T cells demonstrated antitumor activity in in vivo murine models of PS(+) hepatocarcinoma and teratocarcinoma. No signs of toxicity were observed after Anxa CAR-T cell administration. CONCLUSIONS: PS holds promise as a target antigen for CAR-T cell therapy, underscoring the need to address fratricide as a key challenge in the development of PS-targeting CAR-T cells.