AbstractPatient-specific CAR-T cells treatment is an innovative new drug that has shown excellent therapeutic effects against B cell blood cancer. However, side effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome and ultra-high treatment costs must be overcome, and off-the-shelf CAR-NK cells can be a good alternative. The purpose of this study was to combine cellular reprogramming, gene editing, and differentiation technologies to produce full-off-the-shelf NK cells derived from iPSCs and verify their efficacy and safety. To this end, somatic cell-derived iPSCs were produced, seven genes were edited using CRISPR/Cas9 technology (5 kinds of knockout and 2 kinds of overexpression), and differentiated into NK cells. upCAR (universal and potent chimeric antigen receptor)-iPSCs with seven genes edited showed bialleic INDEL in the coding sequence of five genes and no off-target effect. In addition, normal transcriptome and genetic stability were maintained. High-efficiency differentiated upCAR-NK cells showed a very high differentiation yield, in vitro proliferation, and freezing/thawing were possible. In addition, upCAR-NK cells secrete interferon-γ when they meet cancer cells, showing cytotoxic effects in vitro and in vivo. upCAR-NK cells themselves showed no obvious toxicity in vivo. In conclusion, this study developed upCAR-iPSCs and upCAR-NK cells platform technologies that are less likely to have side effects and can be economically developed for B cell blood cancer than CAR-T. In the future, this technology can be useful in developing full-off-the-shelf CAR-NK cells anti-cancer immune cell therapy with low side effects, high efficacy, and low price.Citation Format: Daekee Kwon, Bo Kyung Moon, Mijung Han, Tae-Wook Lee, Ji-Han Lee, Kyung-Sun Kang. Universal and potent chimeric antigen receptor-natural killer (upCAR-NK) cells for B-cell malignancy treatment [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 1330.