Abstract 2916: CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells

Abstract NK (natural killer) cells have several advantages compared to T cells such as no cytokine release syndrome (CRS), neurotoxicity and GVHD in allogenic setting to kill tumors. We developed CAR-NK platform using RNA-LNP (lipid-nanoparticles) technology. High expansion of NK cells (up to 2000-fold) was achieved using K562 feeder cells with stable expression of 4-1BBL and IL-21. We showed efficient encapsulation of BCMA and CD19-CAR RNA into LNP that resulted in up to 99% expression of CAR+ CD56+ NK cells after transfection. The BCMA-CAR-NK cells more effectively than NK cells killed MM1S, H929 and RPMI8226 multiple myeloma cells and secreted IFN-gamma in a dose-dependent manner. In addition, frozen BCMA-CAR-NK cells were viable and expressed similar cytotoxic activity to not frozen expanded CAR-NK cells. In addition, frozen-thawed BCMA-CAR-NK cells showed 93% CAR expression at 24 hours post-thaw which remained >70% at 48 hours post-thaw. In a similar way, CD19-CAR-NK cells generated with CD19-CAR-LNP killed CD19-positive Raji and Daudi target cells and secreted high level of IFN-gamma. This, frozen/thawed CAR-NK cells demonstrated high in vitro cytotoxic activity against target cells. Manufacturing of NK cells using G-Rex system resulted in 1862-fold expansion at day 13 and 2450-fold expansion of 97% CD56+ NK cells at day 14. Transfection of BCMA-CAR RNA-LNP into 13 day expanded NK cells resulted in 95% BCMA-CAR-NK positive cells. Thus, CAR-NK cells generated with RNA-LNP technology can be used in future pre-clinical studies. Citation Format: Vita M. Golubovskaya, Jinying Sun, John Sienkiewicz, Yanwei Huang, Hua Zhou, Gongping Deng, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. CAR-NK cells generated with CAR-RNA-LNP express high cytotoxic activity against tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2916.