Fig. 4. Freeze-thawed mACE2-CAR_sIL15 NK cells show effective anti-spike activity in vitro and in vivo.

a CAR expression in mACE2-CAR_sIL15 NK cells post-thaw, as determined by flow cytometry. Data are summarized for cells from three donors. P value was determined by two-tailed paired t test. Data are presented as mean ± SD. b Cell viability of mACE2-CAR_sIL15 NK cells as determined at the indicated time points post-thaw using the Muse Cell Analyzer. Data are summarized for NK cells from three donors. P values were determined by two-way ANOVA with repeated measures and adjusted by the Holm-Sidak method. Data are presented as mean ± SD. c Lysis of freeze-thawed control NK or mACE2-CAR_sIL15 NK cells that were co-cultured with A549-spike or parental A549 cells and analyzed with real-time cell analysis (RTCA). Data are summarized for NK cells from three donors. P values were determined with a two-tailed paired t test. Data are presented as mean ± SEM. d Scheme for in vivo studies using NSG mice. e Tumor growth in NSG mice inoculated with firefly luciferase-labeled A549-spike cells, as monitored by changes in tumor bioluminescence. Colors indicate intensity of luminescence. f Summary of tumor burden data from e (four mice/group). P values were determined by one-way ANOVA with with multiple comparisons and adjusted by the Holm-Sidak method on day 19. Data are presented as mean ± SEM. g After hematoxylin and eosin (H&E) staining of lung tissues, tumor burden was identified and evaluated as the number of tumor nodules, which is defined as ≥50 µm. Four fields of Regions of Interest (ROIs) per lung were selected, and the number of lung tumor nodules per four fields was counted. Summary data are shown (four mice/group). P values were determined by one-way ANOVA with multiple comparisons and adjusted by the Holm-Sidak method. Data are presented as mean ± SD. Source data are provided as a Source Data file.