Engineered IL-18 secretion by CAR T cells improves therapeutic activity against antigen-high and antigen-low myeloma. (A) Schematic of APRIL, BCMA, or BAFF-R CAR T cells engineered to secrete IL-18. Vector maps for CAR constructs used in this figure are depicted to the right. (B) Engineered IL-18 secretion levels by APRIL-CAR T cells ± engineered IL-18 secretion. Approximately 100 000 CAR T cells were cultured for 24 hours in IL-2 and the supernatant was collected for ELISA. Concentration was determined based on standard curve measurements. Statistical analysis performed with the Student t test. (C) IFN-γ secretion by APRIL-CAR T cells ± engineered IL-18 secretion. Supernatant from setup in panel B was analyzed using an IFN-γ flow cytometric bead array kit. Statistical analysis performed with the Student t test. (D) Tumor BLI and survival of mice bearing MOPC315.BM^WT and treated with CD28-costimulated, IL-18-secreting APRIL-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. Data are combined from at least 2 independent experiments. (E) Tumor BLI and survival of mice bearing MOPC315.BM^WT and treated with CD28-4-1BB-costimulated IL-18-secreting APRIL-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. (F) Schematic of syngeneic antigen-low MOPC315.BM^low in vivo model (used in panels G-I). 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. (G) Tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with IL-18-secreting APRIL or BCMA-CAR T cells. Tumor BLI was performed weekly starting on day 7. Data are combined from at least 2 independent experiments. (H) Tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with BAFF-R-28-1XX CAR T cells ± IL-18. Tumor BLI was performed weekly starting on day 7. (I) Tumor BLI, T-cell BLI, and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with APRIL-BB-1XX/IL-18 T cells compared with control APRIL-BB-1XX T cells without engineered IL-18 expression. T-cell BLI was performed weekly starting on day 6. Tumor BLI was performed weekly starting on day 7. Data are combined from at least 2 independent experiments. (J) Survival of APRIL-BB-1XX/IL-18 treated complete responder mice that were rechallenged with 4E5 MOPC315.BM TACI^KO(BCMA^lowBAFF-R^low) 93 days after original experiment start date. Mice were not irradiated before re-challenge. Statistical analysis of all survival curves was performed with the Mantel-Cox log-rank test. ELISA, enzyme-linked immunosorbent assay. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.