NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma

Abstract

CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA^- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

Fig. 1. NKG2D ligand expression in MM cells.

Expressions of MICA, MICA/B, ULBP-1, ULBP-2/5/6, and ULBP-3 were analyzed in plasma cells from bone marrow samples of MM patients. Representative histograms are shown. For each staining, the percentages of positive cells versus the fluorescence minus one (FMO) control are indicated.

Fig. 2. NKG2D-CAR transduction.

NKAE cells and CD45RA⁻ T cells were transduced with an NKG2D-CAR. Representative dotplots are shown for NKAE cells (A) and CD45RA⁻ T cells (B) transduction. Both cell types showed increased percentages of NKG2D-positive cells (over the percentage of untransduced cells) and median fluorescence intensity (MFI). The expression remained stable in CAR-expressing NKAE cells after 6 days; however, T cells showed more stable transduction (C). Results are shown as the mean ± SEM of five different experiments.

Fig. 3. Phenotype of NKAE cells and T cells.

The expression profile of different surface markers was analyzed by flow cytometry in untransduced and CAR-expressing (A) NKAE cells and (C) T cells. Data are represented as median and IQR of three different experiments. Statistical analysis was performed for each pair of untransduced and CAR-transduced cells. Representative density plots of (B) NK cells, NKAE cells, and CAR-NKAE cells and density plots of (D) CD45RA⁻ T cells and CD45RA⁻ CAR-T cells are shown.

Fig. 4. Antimyeloma activity of CAR-NKAE and memory CAR-T-cell products.

A Cytotoxic activity of untransduced NKAE cells and memory T cells (CD45RA⁻ T cells), CAR-NKAE cells, and memory CAR-T cells against different MM cell lines. Results are shown as mean ± SEM of four independent experiments. *P < 0.05 compared to untransduced cells (NKAE and memory T cells) and memory CAR-T cells. B Cytotoxicity of NKAE and CAR-NKAE against primary MM cells from bone marrow sample at different E:T ratios (triplicates). C Plots representing the relationship between the differences in the degree of killing (between NKAE cells and CAR-NKAE cells) and the expression of NKG2DL (MICA, ULBP-1, ULBP-2/5/6, and ULBP-3) in 3 MM cell lines with different expression of NKG2DL. D Time-lapse experiments were performed, U-266 MM cells (green) were destroyed by CAR-NKAE cells (red) with only 20 min of exposure. After 3 h, MM cells were destroyed and released the green fluorescent probe.

Fig. 5. Death cell mechanisms of NKAE and T-cell products.

A Cytokines and cytotoxic molecules release by NKAE cells, CAR-NKAE cells, memory T cells, and memory CAR-T cells. B Expression of apoptosis ligands: FasL and TRAIL, and CD16 (antibody-dependent cell-mediated cytotoxicity) and representative flow cytometry density plots. RNA-seq and differential gene expression analysis were performed in C NKAE/CAR-NKAE cells and D T /CAR-T cells.

Fig. 6. CAR-NKAE reduces clonogenicity of MM.

A L-363 MM cells were exposed to different concentrations of NKAE cells or CAR-NKAE cells (E:T ratios of 1:4 to 32:1) and then seeded in methylcellulose to evaluate the cytotoxicity against clonogenic MM cells. Results are shown as percentage of colonies grown (with autonomous MM growth set as 100%), and represented as mean ± SEM of triplicates. *P < 0.05. B Representative pictures of colonies grown at different E:T ratios.

Fig. 7. NKG2D-CAR-transduced NKAE cells exhibit potent efficacy in vivo.

A Imaging of tumor burden monitored by bioluminescence at the indicated timepoints in MM mice, NKAE-cell-treated mice, and CAR-NKAE-cell-treated mice (At day 73 mice 2 and 3 from NKAE group were accidentally interchanged). B Quantification of the bioluminescence signal in MM mice, NKAE-cell-treated mice, CAR-NKAE-cell-treated, CD45RA⁻ T-cell-treated mice and CD45RA⁻ T-cell-treated mice at the indicated timepoints and C Kaplan–Meier survival curves. D Representative flow cytometry dotplots showing bone marrow infiltration (GFP⁺ CD138⁺) of MM mice (top left), NKAE-cell-treated mice (top right), and the two CAR-NKAE-treated mice that were disease free at the end of the study (bottom). E Persistence of effector cells in peripheral blood (i) 10 days after infusion and (ii) 20 days after infusion. Representative flow cytometry density plots for NKAE/CAR-NKAE cells that were identified by human CD45 and CD16 labelling and for CD45RA⁻ T/CAR-T cells that were identified by human CD45 and CD45RO labelling.

Fig. 8. Toxicity of CAR-expressing cells.

A The cytotoxicity of CAR-NKAE and CAR-T cells against healthy tissues (lung, colon, and PBMCs) was evaluated and compared to that of untransduced NKAE and T cells. Results are represented as mean ± SEM of four replicates. B In vivo toxicity was evaluated by monitoring the weights of mice treated during treatment.