Abstract B034: Assessing the cytotoxic potential of novel CAR-NK cells targeting glioblastoma stem cell antigens

Abstract Engineering immune cells to express chimeric antigen receptors (CARs) allows the recognition of specific tumor-associated antigens, enhancing the immune cells’ potential for identification and elimination of tumor cells. In glioblastoma (GBM), the most common and aggressive primary central nervous system (CNS) tumor in adults, standard treatment—which includes maximal surgical resection followed by cycles of chemotherapy and radiotherapy—often leads to recurrence, with an average survival of about 15 months. This happens partly due to the presence of glioblastoma stem cells (GSCs), which are resistant to conventional treatments and possess tumor-initiating properties, highlighting the need for targeted therapies. CAR technology has been applied to various cell types, but Natural Killer (NK) cells, in particular, offer unique advantages, such as feasibility for allogeneic transplants and reduced cytotoxicity risks. Preclinical and some clinical studies have shown that CAR-NK cells can efficiently eliminate target cells with minimal side effects, though few studies have focused on GBM. Given the need for improvement in GBM therapy, we developed a third-generation CAR receptor adapted for expression in NK cells, incorporating two antigen-binding domains specific to selected GSC markers associated with GSC maintenance and tumorigenic potential. We evaluated the cytotoxic potential of CAR-NK cells vs. Mock-NK cells (control) against GBM cell lines through co-culture assays. After in silico analysis for relevant GSC markers, we verified antigen expression in GBM cell lines (A172, LN18, U87-MG, U138-MG, U251-MG, U343-MG) by flow cytometry (FC) and immunocytochemistry (ICC), in both tumor cell monolayer and tumor spheres. CAR-NK cells were produced by transducing NK-92 cells with a lentiviral vector carrying the CAR construct. Efficiency was confirmed via FC, neomycin selection, and RT-qPCR. CAR-NK and Mock-NK cells were co-cultured with ZsGreen+ U251-MG cells, and cytotoxicity was assessed using LDH release assays and fluorescence microscopy (FM). Results showed that the selected antigens were expressed in all six GBM cell lines, with increased expression in tumor spheres indicating their relevance as GSC markers. CAR-NK cells demonstrated superior cytotoxicity compared to Mock-NK cells, as indicated by higher death rates in LDH assays and greater fluorescence reduction detected by FM after co-cultivation with ZsGreen+ U251-MG. In conclusion, the selected GSC antigens are promising targets for designing CARs against GBM cells. The CAR-NK cells exhibited superior cytotoxic activity compared to Mock-NK cells, suggesting effective targeting of GBM cells. Further in vivo experiments are underway to confirm these findings and evaluate CAR-NK cell activity for potential clinical applications. Citation Format: Vitoria A Lima, Thiago G Mitsugi, Oswaldo K Okamoto. Assessing the cytotoxic potential of novel CAR-NK cells targeting glioblastoma stem cell antigens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B034.