Engineered PD1-NKG2D Dual-CAR NK92 cells broaden antitumor target recognition in preclinical tumor models

Abstract

To address challenges such as the complex manufacturing of CAR-T and the immunosuppressive tumor microenvironment (TME), CAR-NK cells offer greater potential as an “off-the-shelf” therapy. To broaden tumor recognition and reduce the risk of immune escape associated with single-target approaches, we developed a single-promoter-driven multicistronic CAR-NK92 system that employs NKG2D for broad recognition of stress ligands and combines a PD1-CAR to reverse PD-L1 inhibitory signaling, thereby significantly enhancing antitumor efficacy. A multicistronic construct co-expressing PD1-CAR and NKG2D-CAR was generated using a P2A peptide under the control of a single CMV promoter and introduced into NK92 cells. The expression of PD-L1 and MICA/B was screened across multiple tumor cell lines, and the functional robustness of PN-CAR-NK92 cells was evaluated in all models through in vitro cytotoxicity and cytokine secretion assays. The in vivo translational efficacy was further validated using an H1299 xenograft model, with a direct comparison between PN-CAR-NK92 cells and NK92 cells. The multicistronic design enabled stable surface co-expression of both receptor modules, providing a structural basis for dual-target functionality. In vitro cytotoxicity assays demonstrated that PN-CAR-NK92 cells maintained robust antitumor activity across tumor cell lines with distinct PD-L1 and MICA/B expression profiles, whereas single-target CAR-NK92 cells displayed more restricted target specificity. These findings suggest that dual-target CAR engineering broadens antigen recognition coverage and may help reduce the limitations associated with single-target antigen dependence. Furthermore, PN-CAR-NK92 cells demonstrated significantly enhanced tumor suppression in the H1299 xenograft model compared with control groups. Dual-target PD1/NKG2D CAR-NK92 cells exhibit broadened antitumor activity across tumor cells with distinct ligand-expression profiles and may represent a promising strategy to reduce the limitations associated with single-target CAR therapies.