Background and objective <p>Given the undeniable and promising outcomes of chimeric antigen receptor (CAR) technology-based immunotherapy reported in recent years, this study aimed to develop anti-HER2 CAR NK cells as a novel therapeutic strategy for cancer immunotherapy.</p> Materials and methods <p>The NK-92 cell line was transduced with a recombinant lentiviral vector encoding an anti-HER2 construct, either with or without IL-15 co-expression. This yielded two distinct CAR NK cell populations: (1) anti-HER2 CAR NK cells and (2) IL-15-secreting anti-HER2 CAR NK cells. The cytotoxic effects of these engineered cells against the HER2-positive SK-BR-3 target cells were then evaluated using the PE-Annexin V and 7-AAD assays. Flow cytometry analyses were performed to assess CAR NK cell activity by measuring the expression of degranulation marker CD107a and intracellular levels of granzyme B and perforin, following surface and intracellular staining.</p> Results <p>Our findings demonstrated that anti-HER2 CAR NK cells and IL-15-secreting anti-HER2 CAR NK cells induced significantly higher levels of total apoptosis in HER-positive SK-BR-3 cells compared to mock-transduced (control) and non-transduced NK cells (control). The mean percentage (± SD) of CD107a was significantly higher in CAR NK cells co-cultured with SK-BR-3 cells compared to both control groups. Moreover, the mean expression (based on MFI) of granzyme B and perforin was significantly elevated in both CAR NK cell types following co-culture with HER2-positive SK-BR-3 cells. Notably, IL-15-secreting anti-HER2 CAR NK cells exhibited superior cytotoxic potential compared to their non-secreting counterparts.</p> Conclusion <p>In summary, our findings demonstrate potent antitumor activity of anti-HER2 CAR NK cells. The promising results suggest that these engineered CAR NK cells, particularly those capable of IL-15 secretion, hold significant potential as a novel immunotherapeutic strategy for HER2-positive malignancies.</p>