Fig 2: CD70 expression in patients with multiple myeloma. Comparison of CD70 mRNA levels based on (A) RNA levels from the MMRF CoMMpass dataset of different translocations, Amp1q and Del17p, in patients with NDMM and (B) the ISS (n = 561). C, Paired comparison of CD70 mRNA in NDMM and at first relapse (left). The difference between the expression levels is plotted (right, relapsed–NDMM) with a dotted line at the mean of differences (0.609, P = 0.0028, paired t test, n = 61). D, Patients were stratified into tertiles based on CD70 mRNA expression, and overall survival (OS) was plotted (n = 792). Color shading represents the 95% confidence interval. E, Bone marrow aspirates from patients with multiple myeloma (n = 46) from the MD Anderson Cancer Center with different translocations involving chromosome 14 were analyzed for CD70 mRNA using the pseudobulk approach on single cell (sc)-mRNA. The control group included samples from n = 3 healthy control donors. The fourth data point in the control group represents nontumor or polyclonal plasma cells (PC) from patients with multiple myeloma identified by the expression of B-cell receptor VDJ using scRNA-seq (see “Methods”); monoclonal plasma cells were similarly defined based on BCR VDJ sequences, and each remaining data point corresponds to an individual patient with multiple myeloma: no translocation (none; n = 20), t(11;14) (n = 16), t(14;16) (n = 2), and t(4;14) (n = 8). F, The sc-mRNA transcriptome data from all samples projected onto a Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP). Individual CD70 mRNA was mapped at single-cell resolution. G, Translocations involving the immunoglobulin heavy chain locus identified by FISH are mapped onto a UMAP. Box and whisker plots show the median and IQR (25th and 75th percentiles), and the whiskers extend to ±1.5 times the IQR. The P values were determined by the Wilcoxon test [A, B, D (left panel) and E]. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. UMI, unique molecular identifier.

Fig 3: 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 4: 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 5: 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.