BAFF-R/APRIL dual-CAR T cells with engineered IL-18 secretion eliminate antigen-low myeloma in a stress-dose model. (A) Schematic of BAFF-R/APRIL dual-CAR T cells and the corresponding CAR vector maps. (B) Incucyte cytotoxicity assay of BAFF-R/APRIL dual or single-CAR T cells vs 10 000 IRFP713⁺ MOPC315.BM^low. Near-infrared signal was measured over time with the Incucyte SX5. Statistical analysis was performed with the 1-way ANOVA test at 72-hour end point. Data are representative of at least 2 independent experiments. (C) Binding avidity force between MOPC315.BM^low and BAFF-R/APRIL dual or single-CAR T cells. Myeloma cells were seeded on poly-L-lysine coated plates before adding EGFP⁺ T cells. Lumicks C-trap optical tweezers were used to locate EGFP⁺ T cells, bring them in contact with myeloma cells, and then separate them, allowing separating force to be recorded. Statistical analysis was performed with the 1-way ANOVA test. Data are combined from at least 2 independent experiments. (D) Confocal microscopy of coexpressed BAFF-R-EGFP and APRIL-tagRFP CARs present at the synapse interface between CAR T cells and MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low). CAR T cells were cocultured with MOPC315.BM^low for 2 hours and then fixed and processed for imaging. Z-stack images were acquired with Leica Stellaris 8 at 63× magnification and room temperature (25°C), in the following channels: green, red, and DAPI. Data were analyzed on the Imaris software (Oxford Instruments) and 2D images containing a clear representation of the T-cell:MOPC315.BM^low synaptic interface were exported. (E) NFAT signaling of BAFF-R/APRIL dual or single-CAR T cells when exposed MOPC315.BM^WT or MOPC315.BM^low myeloma cells. A total of 20 000 CAR T cells containing an NFAT-EGFP response element were cocultured with 60 000 myeloma cells for 24 hours. NFAT-GFP signaling was measured with flow cytometry. Data are representative of at least 2 independent experiments. (F) NF-κB signaling of BAFF-R/APRIL dual or single-CAR T cells. 10 000 CAR T cells containing an NF-κB-EGFP response element were cocultured with 30 000 MOPC315.BM^low cells. NF-κB-EGFP signal was measured over time with the Incucyte SX5. Statistical analysis was performed with the 1-way ANOVA test at 120 hours. Data are representative of at least 2 independent experiments. (G) Antigen-low myeloma standard dosing schematic, tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with BAFF-R/APRIL dual or single APRIL or BAFF-R-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. Statistical analysis of survival was performed with the Mantle-Cox log-rank test. Data are combined from at least 2 independent experiments. (H) Schematic of IL-18-secreting BAFF-R/APRIL dual-CAR T cells and the corresponding vector maps. (I) Model setup of stress-dose model of MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) with a 5× increase in tumor dose and 20× decrease in T-cell dose. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E6 MOPC315.BM^low. 1E5 CAR T cells were injected 3 days later. (J) Tumor BLI and survival of mice bearing 2E6 MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with a low dose of 1E5 IL-18-secreting BAFF-R/APRIL dual-CAR T cells, BAFF-R/APRIL dual-CAR T cells alone, BAFF-R/IL-18 CAR T cells, or APRIL/IL-18 CAR T cells. Statistical analysis of survival was performed with the Mantel-Cox log-rank test. Data are combined from at least 3 independent experiments. (K) ELISA of IL-18 production from BAFF-R-28-1XX/APRIL-BB-1XX/IL-18 compared with BAFF-R-28-1XX/IL-18 and APRIL-BB-1XX/IL-18 CAR T cells when cocultured with MOPC315.BM^low. Statistical analysis was performed with the 1-way ANOVA test. DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.