Fig. 2: Engineering a STING agonist-producing live therapeutic (SYNB1891).
From: Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity
a Cyclic di-AMP (CDA) abundance from bacterial cell pellets of the indicated strains cultured with PBS (non-induced) or 100 ng/mL aTc (induced) for 4 h. Data are representative of three independent experiments. b IFNβ1 production by RAW 264.7 cells co-cultured for 4 h with non-induced (+PBS) or pre-induced (+aTc) SYN-Ptet-dacA. c B16.F10 tumor-bearing mice were i.t.-injected with 1 × 106 CFUs of EcN or SYN-pTet-dacA bacteria, or saline control (n = 5 mice per group). Four hours later all mice received 10 μg aTc intraperitoneally. Intratumoral IFNβ1 is shown at 24 h post-injection (**P = 0.0011 (vs saline), **P = 0.0016 (vs EcN), one-way ANOVA with Tukey’s multiple comparisons tests). Additional data are shown in Supplementary Fig. 2a–e. d B16.F10 tumor-bearing mice were i.t.-injected with saline or 5 × 106 CFUs of bacterial strains containing a constitutively expressed mCherry (RFP) and inducible GFP (n = 4 mice per group except n = 2 for groups 16 h Ptet-gfp and 16 h Pcmt-gfp). To induce GFP mice were injected with either saline (control), aTc, sodium salicylate (Sal) or p-isopropylbenzoate (Cmt). Percentage of induced bacteria (GFP+ among RFP+ cells) in tumors is shown at indicated times post-induction. e CDA abundance from bacterial cell pellets of the indicated strains cultured under aerobic (uninduced) or anerobic (induced) conditions. f Total CFUs recovered from B16.F10 tumors i.t.-injected with 1 × 106 CFUs of either prototrophic EcN or a strain containing dual thyA and dapA auxotrophies at the indicated time-points (n = 3 mice per group per time point). g CDA abundance from bacterial cell pellets of the indicated strains cultured under aerobic (uninduced) or anerobic (induced) conditions. Data are representative of five independent experiments. h IFNβ1 production from 4-h macrophage and bacterial cell co-cultures, as described in (b), with the indicated bacterial strains. i Schematic of the finalized SYNB1891 bacteria strain containing all engineering components. a, b, g, h n = 2 biological replicates per group per time point. c, d, f Mean and s.d. shown. b–f, h Data are representative of two independent experiments. Each circle represents an individual animal or independent experimental replicate.
