CAR-NK Cells Generated with mRNA-LNPs Kill Tumor Target Cells In Vitro and In Vivo
Authors: Vita Golubovskaya 1,*, John Sienkiewicz 1 , Jinying Sun 1 , Shiming Zhang 1 , Yanwei Huang 1 , Hua Zhou 1 , Hizkia Harto 1 , Shirley Xu 1 , Robert Berahovich 1 and Lijun Wu 1,2,*
Abstract
Natural killer (NK) cells are cytotoxic lymphocytes that are critical for the innate immune system. Engineering NK cells with chimeric antigen receptors (CARs) allows CAR-NK cells to target tumor antigens more effectively. In this report, we present novel CAR mRNA-LNP (lipid nanoparticle) technology to effectively transfect NK cells expanded from primary PBMCs and to generate functional CAR-NK cells. CD19-CAR mRNA and BCMA-CAR mRNA were embedded into LNPs that resulted in 78% and 95% CAR expression in NK cells, respectively. BCMA-CAR-NK cells after transfection with CAR mRNA-LNPs killed multiple myeloma RPMI8226 and MM1S cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner in vitro. In addition, CD19-CAR-NK cells generated with CAR mRNA-LNPs killed Daudi and Nalm-6 cells and secreted IFN-gamma and Granzyme B in a dose-dependent manner. Both BCMA-CAR-NK and CD19-CAR-NK cells showed significantly higher cytotoxicity, IFN-gamma, and Granzyme B secretion compared with normal NK cells. Moreover, CD19-CAR-NK cells significantly blocked Nalm-6 tumor growth in vivo. Thus, non-viral delivery of CAR mRNA-LNPs can be used to generate functional CAR-NK cells with high anti-tumor activity.
Selected Figure
CD19-CAR-NK decreased Nalm-6-luciferase tumor growth significantly more than NK cells using NSG mouse model. (A). Scheme of the mouse experiment. 1 × 105 of human leukemia, Nalm-6-luciferase cells were intravenously injected into immunodeficient NSG mice on day 0. Then, 5 × 106 frozen CD19-CAR-NK and NK cells were intravenously injected at days 1, 3, 6, and 8. Imaging was performed with the Xenogen Ivis system on days 6, 9, 12, and 15. N = 5 mice per group. (B). CD19-CAR-NK cells significantly decreased Nalm-6 xenograft tumor growth compared with NK cells. The images of mice treated with CAR-NK or NK cells are shown in the Nalm-6 xenograft model. (C). Quantification of imaging is shown. Average bioluminescence (photons/s) ± standard deviations is shown on the Y-axis and days on the X-axis. * p < 0.00002, CD19-CAR-NK cells vs. NK cells by Student’s t-test.
Keywords: natural killer cells; chimeric antigen receptor; lipid nanoparticles; mRNA; leukemia; multiple myeloma; tumor; immunotherapy
Int. J. Mol. Sci.2023, 24(17), 13364; https://doi.org/10.3390/ijms241713364