aMSLN Nb CAR-NK cells(Graduate School of Dong-A University)

Chimeric Ag receptor (CAR)-engineered immune cells have demonstrated remarkable clinical efficacy, particularly in hematologic malignancies. Central to their success is the Ag-binding domain of the CAR, which governs both target specificity and therapeutic efficacy. Nanobodies (Nbs) possess a single-domain architecture and smaller molecular size, making them particularly amenable to the construction of tandem CARs that can co-target multiple Ags. This structural flexibility is advantageous for addressing tumor heterogeneity and reducing the risk of Ag escape in solid malignancies. Here, we developed mesothelin (MSLN)-specific nanobody-based chimeric Ag receptor-NK (Nb CAR-NK) cells using a synthetic nanobody identified from a phage display VHH library. The nanobody was selected after three rounds of biopanning and validated for high affinity and specificity using surface plasmon resonance and flow cytometry. The selected nanobody-based chimeric Ag receptor (Nb-CAR) construct was introduced into ex vivo expanded umbilical cord blood-derived NK cells via third-generation lentiviral transduction, resulting in stable expression and functional CAR-NK cells. The Nb CAR-NK cells exhibited potent cytotoxicity against MSLN-positive pancreatic cancer cells in vitro and significantly suppressed tumor growth in xenograft models. These findings support the clinical potential of Nb CAR-NK cells and highlight the value of Nb-CAR designs for targeting cell-surface Ags in solid tumors.