Background: The activation of innate and adaptive immunity via Stimulator of Interferon Genes (STING) signaling is a potentially transformative immuno-therapeutic strategy in cancer. Using structure-based drug design and focused library synthesis, we have discovered novel cyclic dinucleotides, that self-assemble into nanostructures, show potent STING agonist activity in vitro, and profound anti-tumor activity in syngeneic mouse tumor models when administered by i.v., i.p., and i.t., routes. Methods: (a) Synthesis. Focused libraries of cyclic dinucleotides (1) were prepared using phosphoramidite chem. (b) Binding affinity of compounds with human STING CTD was determined by SPR assay, (c) STING-dependent Induction of IRF and NF-KB was assessed as % fold-change in luminescence by treating cells, carrying reporter constructs, with compounds, (d) Self-assembly to nanostructures was determined using SEM, (e) In vivo efficacy was assessed by measurement of mean tumor volumes of lead compounds administered by i.v. (3 to 6mg/kg) or i.t. (10 to 100mg) in the A20 lymphoma, CT26 carcinoma, B16 melanoma, and 4T1 breast cancer models. Flow cytometry, multiplexing assays and immuno-histochem. of blood, and tissues were carried out to assess MOA. Results: Lead compounds (EC50 1 to 10 nM) were found to self-assemble into 1 mM nanostructures that facilitated their uptake by immune cells. Highly potent and durable antitumor response in multiple tumor models was observed with M.E.D 10 mg (i.t.), and 1 mg/kg (i.v.). IND-enabling studies are in progress.