<p>Lung cancer is the most common cancer and one of the leading causes of cancer-related deaths in the world, however, the treatment of non-small cell lung cancer (NSCLC) is still limited, and it is a clinically urgent problem. ROBO1 is an important surface receptor on tumor cells, but the role of humanized chimeric antigen receptor (CAR) modified natural killer (NK) cells targeting ROBO1 in NSCLC is rarely explored. Furthermore, the role of PD-1 in NK cell killing tumor cells remains controversial. In this study, we identified the expression pattern of ROBO1 in lung squamous cell carcinoma (LUSC) by searching biological information databases. We constructed hROBO1-CAR-NK-92 cells and performed functional identification.We inserted the hROBO1-CAR at the PD-1 locus and performed functional detection in vitro and in vivo. The results showed that ROBO1 expression was significantly increased in LUSC. After inserting the hROBO1-CAR sequence at the PD-1 locus, the PD-1-KO-hROBO1-CAR-NK-92 cells had the best long-term killing ability and cytokine secretion ability, and had a significant inhibitory effect on tumor growth in the mouse xenograft model. We also observed that the long-term killing ability of PD-1-KO-hROBO1-CAR-NK-92 cells was achieved by inhibiting cell senescence via knocking out PD-1. These studies proposed ROBO1 as a key target for CAR-NK therapy in NSCLC and integrated hROBO1 CAR in PD-1 locus in NK cells, resulting in synergistic tumor killing effects in NSCLC, presenting a new treatment strategy for solid tumor treatment.</p><p></p>