Enhanced PD-L1 targeting boosts the cytotoxic activity of FOLR1- CAR NK92 cells against ovarian cancer.

Chimeric antigen receptor (CAR)-T cells have achieved remarkable success against hematologic malignancies; however, their application is associated with risks such as cytokine release syndrome (CRS) and neurotoxicity. In contrast, CAR-NK cells not only avoid these toxicities but also retain the natural cytotoxic activity of NK cells, demonstrating great potential for cancer immunotherapy.Our previous study demonstrated the potent efficacy of folate receptor alpha (FOLR1)-targeted CAR-NK92 cells against ovarian cancer (OC). Nevertheless, the application of CAR-NK therapy in solid tumors, including OC, still faces challenges such as tumor antigen heterogeneity and the immunosuppressive tumor microenvironment (ITME). A key mediator of ITME is programmed death-ligand 1 (PD-L1), which not only correlates with poor prognosis in OC but also drives T cell exhaustion via the PD-1/PD-L1 axis. Moreover, its inducible upregulation under NK cell-based therapy supports PD-L1 as a viable therapeutic target in OC.To enhance the therapeutic potential of CAR technology for OC, we engineered two novel third-generation bispecific CAR-NK92 cells based on our prior FOLR1-CAR design. These constructs-Tandem PD-L1/FOLR1-CAR (Tan-CAR1) NK92 and Tandem FOLR1/PD-L1-CAR (Tan-CAR2) NK92-were designed to simultaneously target FOLR1 and PD-L1. Notably, we confirmed that PD-L1 expression was significantly upregulated in the co-culture supernatant of effector and target cells. In vitro, Tan-CAR2 NK92 cells exhibited markedly superior cytotoxicity against FOLR1