Nectin-4-targeted CAR-NK cells exhibit potent antitumor activity against bladder cancer

Background: Bladder cancer (BC) remains a major clinical challenge due to frequent recurrence and limited durable responses to current therapies such as Bacillus Calmette-Guérin (BCG) immunotherapy and cisplatin-based chemotherapy. Natural Killer (NK) cell therapy has recently emerged as a promising immunotherapeutic approach, particularly when combined with Chimeric Antigen Receptor (CAR) technology to enhance tumor recognition and cytotoxicity. Nectin-4, an adhesion molecule highly expressed in BC, represents a strong therapeutic target for CAR-engineered NK cells. In this study, we aimed to target bladder cancer using Nectin-4-specific CAR-NK cells. Methods: Nectin-4-specific CAR-NK cells were generated via lentiviral transduction, and CAR expression was confirmed by fluorescence microscopy. The cytotoxic activity of CAR-NK and unmodified NK cells was evaluated against Nectin-4-positive (SW780) and Nectin-4-negative (T24) bladder cancer cell lines using LDH release and CCK-8 viability assays at early time points. A 3D spheroid model was used to assess CAR-NK cell attachment, infiltration, and long-term cytotoxicity. Gene expression profiling following co-culture was performed to evaluate activation and apoptosis-related pathways. An in vivo xenograft model was used to validate the in vitro findings. Results: Nectin-4 CAR-NK cells demonstrated significantly enhanced cytotoxicity compared with parental NK cells within 4 hours of co-culture with SW780 cells, as assessed by LDH and CCK-8 assays. In 3D spheroid models, CAR-NK cells showed stronger attachment and affinity for Nectin-4-positive tumor spheroids during short-term co-culture and exhibited higher cytotoxicity after prolonged exposure compared with unmodified NK cells. Gene expression analysis revealed increased activation markers and cytotoxic mediators in CAR-NK cells, along with upregulation of apoptosis-related genes in target tumor cells following treatment. Conclusions: Engineering NK cells with a Nectin-4-specific CAR markedly enhances their ability to recognize and eliminate Nectin-4-positive bladder cancer cells. These findings support the potential of Nectin-4 CAR-NK therapy as a targeted immunotherapeutic strategy for bladder cancer. Future development of multi-targeted CAR-NK constructs designed to modulate the tumor microenvironment may further improve efficacy and overcome immune resistance.