Chemically regulated STING-activating prodrugs of deoxyribose cyclic dinucleotides elicit robust immune activation and durable antitumor immunity

Background: Prodrugs derived from deoxyribose cyclic dinucleotides (dCDNs) exhibit enhanced cellular permeability, stability, and sustained STING activation. However, the impact of phosphotriester chirality on their bioactivity remains underexplored. This study systematically evaluates the stereochemistry-dependent pharmacology of alkyne-conjugated, esterase-sensitive dCDN prodrugs in vitro and in vivo. Methods: Three diastereoisomers of the dCDN prodrug: (Rp,Rp)-10, (Sp,Sp)-10, and (Rp,Sp)-10-were synthesized, purified by chiral HPLC, and confirmed by NMR and HRMS. Cellular uptake and stability were assessed in THP1-Lucia ISG cells. STING pathway activation was measured by IFN-beta reporter assays and phospho-TBK1/IRF3 immunoblots. In vivo efficacy was evaluated in CT26 colon carcinoma-bearing BALB/c mice following intravenous administration. Immune memory was assessed by tumor rechallenge. Results: Among the isomers, (Rp,Rp)-10 exhibited the most potent STING activation (EC50 = 1.7 nM), superior cellular uptake, and prolonged TBK1/IRF3 signaling. In mice, all three prodrugs induced stronger systemic cytokine responses than ADU-S100 or the parent CDN 3′,3′-c-di-dAMP. In the CT26 model, (Rp,Rp)-10 achieved complete tumor regression in 9/10 mice (90% CR), significantly improved survival (p < 0.001), and established long-term immunological memory with 100% rejection upon rechallenge. No overt toxicity was observed. Conclusion: Phosphotriester chirality is a critical determinant of dCDN prodrug activity. (Rp,Rp)-10 represents a promising next-generation STING agonist with potent systemic antitumor immunity and durable therapeutic effects. These findings underscore the importance of stereochemical control in the rational design of STING-targeted cancer immunotherapies.