Chimeric Antigen Receptor (CAR) therapy has emerged as a promising approach in cancer immunotherapy, with recent efforts extending its application to various immune cells such as T and NK cells, and even macrophages. One key aspect of enhancing CAR therapy's efficacy in NK cells involves targeting and modulating their regulatory receptors. NK cell function is regulated by various activating and inhibitory receptors, among which TIGIT (T cell Immunoreceptor with Ig and ITIM domains) plays a crucial role. TIGIT, expressed on T and NK cells, binds strongly to CD155, inhibiting immune cell function. However, our engineered TIGIT-CAR, unlike native TIGIT which transmits inhibitory signals, delivers activating signals to NK cells. Therefore, our research goal is to investigate whether these CAR-NK cells exhibit antitumor activity.

We designed the human TIGIT domain into a pALD vector and produced virus using 293FT cells. Next, we performed transduction with the viral vector into NK92 Mi cells. To confirm successful introduction, we initially assessed the GFP rate present in the CAR vector. However, recognizing that gene insertion does not guarantee surface protein expression, we additionally utilized flow cytometry analysis to quantify TIGIT levels on the cell surface. Subsequently, we sorted the NK92 Mi cells expressing TIGIT for use in the following experiments.
To confirm the anti-tumor efficacy of our engineered TIGIT-CAR-MI cells, we conducted killing assays using cancer cell lines that either overexpressed or did not express the ligand of TIGIT, CD155 (PVR). The LDH (lactate dehydrogenase) assay results demonstrated significantly enhanced cell killing by TIGIT-CAR NK compared to control NK92 mi cells. Specifically, CD155-expressing A549 cells exhibited markedly increased TIGIT-CAR cell cytotoxicity, higher CD107a expression, and increased interferon-gamma secretion compared to control, while CD155-low K562 cells showed minimal differences. These results indicate that our engineered TIGIT-CAR-NK specifically targets and kills cancer cells overexpressing CD155.
In future studies, we plan to conduct in vivo experiments and investigate the synergistic effects of combining our therapy with various immune checkpoint inhibitors. This is the first study to show that TIGIT-CAR-NK cells could provide an effective clinical treatment for CD155 (PVR)-positive tumors. Since TIGIT is often overexpressed in several types of cancers such as ovarian cancer and colorectal cancer, TIGIT-CAR-NK therapy can potentially be extended to other solid tumors.