Fig. 8: Applications of the cumate “safety switch” in regulating expression of potentially toxic cytokines.

a IL-2, IL-12 and IL-18 concentrations measured by ELISA from supernatants of U2OS cells 6 h after infection with VV-CymR-iIL12-2-18 (MOI 1) and treated with 100 µg/ml cumate or PBS. b IL-2, IL-12 and IL-18 concentrations measured by ELISA from supernatants of U2OS cells 6 h after infection with VV-CymR-iIL12-2-18 (MOI 1) and varying concentrations of cumate. c, d IFN-gamma and TNF-alpha levels measured by ELISA. Supernatants were taken from mouse splenocytes cultured ex vivo with conditioned media from U2OS cells treated with 100 µg/ml cumate or PBS following infection with VV-CymR-iIL12-2-18 (MOI 1). Neutralizing antibodies against IL-2, IL-12 and IL-18 were added to conditioned media for 1 h prior to transferring them to splenocytes. e–h Treatment-related mortality and toxicity were assessed 5 days after intratumorally injection of HT-29 tumors with control vaccinia virus, VV-IL12-2-18 or VV-CymR-iIL12-2-18 (1E7 PFU/tumor). Tumors were ~150 mm³ in size at the time of injection. Mice treated with VV-CymR-iIL12-2-18 were given a cumate (6000 mg/kg) or regular diet. Water in the lungs (an indication of pulmonary edema) (g) and alkaline phosphatase activity (h) in serum were measured as indicators of toxicity. i–m Treatment-related mortality and toxicity were assessed 5 days after intratumoral injection of HT-29 tumors with control vaccinia virus, VV-IL12-2-18 or VV-CymR-iIL12-2-18 (1E7 PFU/tumor). Tumors were ~150 mm³ in size at the time of injection. Mice treated with VV-CymR-iIL12-2-18 were given a regular diet, or a diet with varying amounts of cumate (1000, 2000 or 6000 mg/kg; j). Water in the lungs (k), and serum levels of alkaline phosphatase (l) and aspartate aminotransferase activity (m) were assessed. n Survival analysis of C57BL/6 mice injected intraperitoneally with 5E5 MC38 cells and treated with either PBS, control vaccinia virus, or VV-CymR-iIL12-2-18 at 3E7 pfu/mouse. Treatments were given at days 6, 8, and 10 post cell injection. A cumate diet (1000 mg/kg) was given to the VV-CymR-iIL12-2-18 treatment groups the same day, or 5 days after virus injection as indicated. o Crystal violet staining of U2OS and Vero cells 48 h after infection with VV-CymR-iIL12-2-18/TetR-iD13 at different MOIs in the presence of 100 µg/ml cumate (+Cu), 100 ng/ml doxycycline (+Dox), or both (+Dox +Cu). p IL-12, IL-2, and IL18 concentrations measured by ELISA in supernatants of U2OS cells 6 h following infection with VV-CymR-iIL12-2-18/TetR-iD13 (MOI 1) in the presence or absence of 100 ng/ml Dox, 100 µg/ml of cumate or both. q Survival analysis of C57BL/6 mice injected intraperitoneally with 5E5 MC38-WT cells and treated with either PBS, control vaccinia virus, or VV-CymR-iIL12-2-18/TetR-iD13 at 3E7 pfu/mouse. viruses were injected at days 6, 8, and 10 post cell injection, with mice receiving either Dox (625 mg/kg), cumate (1000 mg/kg) diets, or both for 5 days. Data indicate means ± SD of three (a–d, p) or five (e–n, q) biological replicates. ns P > 0.05, *P < 0.05 **P < 0.003841, ***P < 0.000125, ****P < 0.001 in unpaired two-samples t-test. Source data are provided as a Source Data file.