Fig. 4: Development of a smart cytokine.
a, Colocalization of SMART-IL-1β (IL-1βN–anti-Ag1/anti-Ag2–IL-1βC) on a target-cell template ligation and release of the cytokine IL-1β, which activates IL-1R1/IL-1RAcP on neighbouring cells. b, OE19 cells (HER2hi, EGFRlow and EpCAMhi) were treated with SMART-IL-1β (eNrdJ-1cage, 20 nM) and the conditioned media supplemented to HeLa cells to stimulate IL-1R1 receptor signalling (top). Control experiments included: adding anti-HER2/anti-EpCAM DARPins (500 nM); use of splicing-deficient SMART-IL-1β (eNrdJ-1N(C1A)cage, 20 nM); and IL-1RA (IL-1R1 antagonist) pretreatment of HeLa cells. NF-κB localization was assessed by immunofluorescence imaging. c–f, HEK-Blue IL-1β cells, reporting IL-1β stimulation by SEAP, were exposed to conditioned media. SMART-IL-1β was supplied to OE19 (**P = 0.0072) (c), K562HER2+/EGFR+ (d) and individual cultures of the specified K562 cell lines (e) at the indicated concentrations. The conditioned media were then transferred to HEK-Blue IL-1β cells for 24 h before SEAP quantification. Controls are as in b. K562HER2+/EGFR+ cells were used in dose–response experiments with SMART-IL-1β using eNrdJ-1cage, eNrdJ-1cage_1–38 (strengthened cage) or eNrdJ-1cage_1–27 (weakened cage) (f). g, OE19 cells were co-cultured with HeLaeGFP+ cells, stably expressing eGFP, and treated with SMART-IL-1β (eNrdJ-1cage, 20 nM). IL-1R1 pathway signalling was determined as in b. h, HEK-Blue IL-1β cells co-cultured with K562HER2+/EGFR+ cells were treated with SMART-IL-1β (eNrdJ-1cage, 20 nM) and SEAP activity determined after 24 h (***P = 0.0003). Controls are as in b. Data are mean ± s.e.m. (n = 3 independent biological replicates). Statistical analysis: unpaired two-sided t-test. Data in b and g are representative of two independent experiments. Scale bars: b, 40 μm; g, 20 μm.
