Fig 2: In vivo anti-myeloma activity of CD70-targeting CAR NK cells. A, Mice were first injected with MM.1S tumor cells and subsequently treated (day 0) with no additional cells (tumor alone), NT NK cells, CAR27/IL-15 NK cells, CAR27 NK cells without IL-15 (CAR27), and NK cells transduced to express only IL-15 (IL-15 NK). B, BLI was obtained weekly until day 54. Different radiance scales were used for early (days 0–14) and late (days 21–54) imaging due to signal intensities spanning several orders of magnitude. An ‘X’ denotes that all mice in the group have died. C, BLI signal for individual mice plotted over days post-CAR NK cell treatment. D, Percentage of human CD45+ cells (NK cells) detected in the blood at day 12 after NK cell infusion. Only three of the five mice per group were bled for this analysis. E, Kaplan–Meier survival plot. P value significance is denoted for the following comparisons: blue asterisks: NT NK vs. CAR27/IL-15 NK; green asterisks: CAR27 NK vs. CAR27/IL-15 NK; and magenta asterisks: IL-15 NK vs. CAR27/IL-15 NK. F, Weight of the mice in grams over time. Data are represented as mean ± SD. P values were calculated using the log-rank test (E). **, P ≤ 0.01.

Fig 3: Armoring NK cells with CAR27/IL-15 results in enhanced antitumor activity against multiple myeloma. A, Schematic of the retroviral vector used to transduce CB-NK cells. B, Flow cytometry histogram assay of CD70 surface protein levels in NCI-H929 (blue) and MM.1S (magenta) cells. C,51Cr release assay of NT or CAR27/IL-15 NK cells against CD70+ MM.1S or (D) CD70− NCI-H929 tumor cells at different E:T ratios and measured 4 hours after coincubation (n = 3). E, MM.1S tumor cells transduced to express mKATE were cultured alone or with NT, IL-15, or CAR27/IL-15 NK cells. The number of MM.1S cells was measured over time with sequential images from the Incucyte machine. Representative images from 0 Hours (baseline) and after 24 Hours after coculture are shown. White bar, 400 μm. F, The percentage of initial MM.1S cells from panel (E) is plotted for each group over 30 hours. Bar graph (right) shows the AUC analysis. G, The Incucyte killing assay was repeated for NT and CAR27/IL-15 NK cells against the CD70-positive U266B1 myeloma cell line. Bar graph (right) shows the AUC analysis. Data are represented as mean ± SD. Data for the AUC graphs are represented as mean ± SEM. The P values were determined by two-way ANOVA with Sidak’s multiple comparisons (C), one-way ANOVA with Tukey multiple comparison (F), and unpaired t test (G). ***, P ≤ 0.001. ICD, intracellular domain; LTR, long terminal repeats; TMD, transmembrane domain.

Fig 4: Loss of BCMA does not affect the anti-myeloma activity of CAR27/IL-15 NK cells. A, Whole-cell lysates from K562 (negative control), MM.1S wild-type (WT), and MM.1S TNFRSF17 KO cells (KO) were analyzed by Western blotting for BCMA expression. β-actin served as the loading control. A representative blot is shown. B,TNFRSF17 KO efficiency analyzed by flow cytometry. C, CD70 expression of MM.1S WT and MM.1S TNFRSF17 KO tumor cells. D,51Cr release assay of NT NK cells or CAR27/IL-15 NK cells against MM.1S TNFRSF17 KO cells. E, Incucyte real-time killing assay of MM.1S TNFRSF17 KO tumor cells by NT NK, IL-15 NK, and CAR27/IL-15 NK cells. Bar graph (right) shows the AUC analysis. Data are represented as mean ± SD. Data for the AUC graph are represented as mean ± SEM. The P values were determined by two-way ANOVA with Sidak’s multiple comparisons (D) and one-way ANOVA with Tukey multiple comparisons (E). ***, P ≤ 0.001.

Fig 5: Phenotypic characterization of CD70 expression in patients with multiple myeloma. A, Bone marrow aspirates from 30 patients with multiple myeloma were stained with anti-CD70 and anti-BCMA antibodies and assayed by flow cytometry. The percentage of positive cells from the FSC/SSC/Single cells/Live-Dead/CD3−/CD19−/CD20−/CD14−/CD138+ population is plotted, with each dot representing one patient sample. The gating strategy is shown in Supplementary Fig. S3. B, The percentage of positive expression (circle size) and the robust z-score calculated from the mean fluorescence intensity (color) for CD70, BCMA, CD38, CS1, CD56, and CD45 proteins from the same population were plotted in a dot plot for each of the patient samples in (A). C, An optimized stochastic neighbor embedding (opt-SNE) plot was generated for each marker in the CD138+, CD3−, CD19/20−, and CD14− populations from an example patient with amp 1q and loss of one copy of TP53. Representative images of IHC CD70 staining of the gingiva (D) and bone marrow (E) clot of a patient with high-grade myeloma. F, CD70 staining of the humerus of a patient who had not responded to the BCMA antibody–drug conjugate belantamab mafodotin. G, CD70 staining from the bone marrow of a patient without multiple myeloma as a control. Scale bar, 100 μm.