ML RR-S2 CDA shows enhanced type I IFN production over CDA in THP-1
human monocytes. In contrast, the dithio, mixed-linkage cyclic
dinucleotide (CDN) derivatives (ML RR-CDA, ML RR-S2 CDG, and ML RR-S2
cGAMP) potently activate all five hSTING alleles, including the
refractory hSTINGREF and hSTINGQ alleles. ML RR-S2
CDA induces the highest expression of IFN-β and the pro-inflammatory
cytokines TNF-α, IL-6, and MCP-1 on a molar equivalent basis, as
compared to endogenous ML cGAMP and the TLR3 agonist poly I:C. ML RR-S2
CDA is also found to induce aggregation of STING and induce
phosphorylation of TBK1 and IRF3 in mouse bone marrow macrophage (BMM).
ML RR-S2 CDA induces significantly higher levels of IFN-α when compared
to ML cGAMP.
ML RR-S2 CDA shows higher anti-tumor control than the endogenous ML
cGAMP. A dose response of the ML RR-S2 CDA compound is performed in B16
tumor-bearing mice, which identifies an optimal antitumor dose level
that also elicites maximum tumor antigen-specific CD8+ T cell responses, and improves long-term survival to 50%.
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°C with 5%
CO2. Cells are stimulated with 10 μM ML RR-S2 CDA 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.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.