Cell experiment:

Cryopreserved hPBMCs are thawed and 1×106 cells per well are plated in a 96 well plate in RPMI media supplemented with 10% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin, L-glutamine, 10 mM HEPES buffer, 1 mM Sodium Pyruvate, 0.055 mM β-ME at 37℃ with 5% CO2. Cells are stimulated with 10 μM ADU-S100 or ML cGAMP for 6 hours and supernatants are harvested. Supernatants are diluted 1:2 and assayed for IFN-α protein using Cytometric Bead Array (CBA) Human Flex Set. Data is collected using a FACSVerse cytometer and analyzed by FCAP Array Software[1].

Animal experiment:

Mice[1] WT C57BL/6 mice are inoculated with 5×104 B16.F10 cells in the left flank (n=8). When tumor volumes are 100 mm3 mice receive three IT doses of either ML RR-S2 CDG (25 μg), ADU-S100 (50 μg), or HBSS as control. WT C57BL/6 mice are inoculated with 5×104 B16.F10 cells in the left flank (n=5). When tumor volumes are 100 mm3 they received three IT doses of ADU-S100 at 5, 25, 50 or 100 μg or HBSS as control. WT C57BL/6 mice are inoculated with 5×104 B16.F10 cells in the left flank (n=8). When tumor volumes are 100 mm3 they receive three IT doses of 100 μg ADU-S100 or HBSS as control. Treatments are administered on days 13, 17 and 20 and tumor measurements are taken twice weekly. Results are shown as percent survival by Log-rank (Mantel-Cox) test (A and C).

ML RR-S2 CDA is a synthetic cyclic dinucleotide (CDN) that contains non-canonical 2'5'-phosphodiester bonds and is an activator of stimulator of interferon genes (STING). It contains mixed linkages (ML) with both 2'5' and 3'5' linkages, which leads to increased thermal stability of human STING in a differential scanning fluorimetry (DSF) assay. ML RR-S2 CDA increases type I interferon production by THP-1 human monocytes relative to unmodified cyclic di-AMP , indicating the ML enhances its action at human STING. It induces expression of IFN-β and the pro-inflammatory cytokines TNF-α, IL-6, and Mcp-1 in murine bone marrow macrophages (BMM) isolated from wild-type, but not STING-/-, mice. ML RR-S2 CDA also induces IFN-β expression in peripheral blood mononuclear cells (PBMCs) isolated from donors carrying STINGWT/WT, STINGWT/REF, and STINGWT/HAQ alleles. In vivo, ML RR-S2 CDA initiates tumor regression and prevents tumor growth upon tumor cell reimplantation in 4T1 breast and CT26 colon cancer mouse xenograft models.

References:
[1]. Corrales, L., Glickman, L.H., McWhirter, S.M., et al. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity Cell Rep. 11(7), 1018-1030 (2015).