Abstract 1328: AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect

Abstract Natural Killer (NK) cells hold great promise for cancer therapy owing to their innate ability to detect cells expressing signs of stress or infection. However, NK cell-based therapeutics face significant challenges: limited expansion and genetic modification resistance. In response to these challenges, we present our pioneering work in the development of Base Editing-Directed NK (BED-NK) cell products, referred to as NK510, which is an off-the-shelf allogeneic cell therapy product containing peripheral blood mononuclear cell (PBMC)-derived TIGIT knock-out NK cells. Through a meticulously planned optimization process, we have achieved over 80% editing efficiency in NK510s using our high-fidelity Base Editor, AccuBase™. By knocking out TIGIT (an inhibitory receptor in NK cells), we have prompted tumor cell-derived CD155 (an inhibitory ligands in tumor cells) to engage with CD226 (an activating receptor in NK cells), triggering activating signals in NK510s. Our results showed that CD155 positive tumor cells were more susceptible to NK510s than CD155 negative tumor cells. And target killing percentage is positively related to CD155 expression level in tumor cells. This transformation effectively converted an inhibitory ligand into an activating one, resulting in tumor regression in multiple mouse models, including non-small cell lung cancer (NSCLC) and sarcoma. We developed a GMP-compliant NKBoost+ expansion system to generate a millionfold expansion of NK510 within a 21-day expansion protocol. To investigate the safety of NK510 product, we conducted repeated dosing toxicity, tumorigenicity, and oncogenicity study in a GLP facility, and the results validated the safety of NK510. Additionally, we developed an analytical protocol based on Droplet Digital PCR (ddPCR) technology to investigate the pharmacokinetics (PK) and biodistribution of NK510s in NSCLC tumor-bearing mice. We observed an enrichment of NK510-derived genomic DNA (gDNA) in lung tissues of tumor-bearing mice at day 1. The concentration of NK510-derived gDNA was up to 2000 copies/ug DNA. The concentration of NK510-derived gDNA sharply decreased from day 2 to day 6, and then decreased to below the lower limit of quantification (LLOQ). Distinct from the PK data of NK510s, the concentration of NK510-derived gDNA started low at day 1 and reached peak concentration at day 2 (in blood samples) and at day 4 (in liver tissues), respectively. These results demonstrated the efficacy and safety of BED-NK product and underscore the feasibility of combining base editing with ex vivo expansion of NK cells, significantly enhancing their therapeutic potential in clinical settings. Citation Format: Han Ying, Chao Yao, Cheng Fang, Guanglei Li, Lixin Wang, Yufu Zhou, Chenyuan Gong, Juewan Li, Yiming Wang, Wenwen Fan, Fenghua Lv, Xiaohu Shao, Haiyang Zhao, Liling Wang, Jun Yan, Tianhong Xu, Shiguo Zhu. AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1328.