Genetically modified NK cells equipped with a switchable CAR for the treatment of HER2-positive cancers.

Abstract

This study is focused on the development and characterization of NK-92 cells bearing a switchable chimeric antigen receptor (CAR) system for targeting HER2-positive breast cancers. The system employs a universal CAR framework based on the high-affinity interaction between barstar within the CAR (BsCAR) and barnase in the antigen-specific module. The barnase component was fused with an engineered ankyrin repeat protein (DARPin) specifically recognizing the HER2 tumor antigen. NK-92 cells were successfully modified to express BsCAR, while control mock-NK92 cells were generated with a variant of BsCAR incapable of surface expression. Flow cytometry analysis confirmed successful transduction and proper surface expression of the BsCAR construct. The cytotoxic potential of the modified cells was evaluated through multiple approaches, including degranulation activity measurements, target cell lysis assays, and three-dimensional spheroid models. In the presence of the HER2-specific targeting module (Da-9.29-Bn), BsCAR-NK92 cells demonstrated significant and specific cytotoxicity against HER2-positive tumor cells, particularly those with high HER2 expression (SKBR3, SKOV-Kat, BT-474). The specificity of the system was confirmed using MCF7 cells expressing low levels of HER2 as controls. In three-dimensional models, BsCAR-NK92 cells maintained their cytotoxic activity. These findings demonstrate the potential of BsCAR-NK92 cells as an "off-the-shelf" therapeutic approach for targeting HER2-positive cancers, offering a flexible platform that can be adapted to target different tumor antigens through the modular barnase-barstar system.