Chemically Regulated STING‐Activating Prodrugs of Deoxyribose Cyclic Dinucleotides Elicit Robust Immune Activation and Durable Antitumor Immunity

ABSTRACT Derivatives of deoxyribose cyclic dinucleotides (dCDNs) displayed notable advantageous properties in the activation of STING pathway. The chirality of the phosphotriester of dCDN prodrugs may influence the stability, cellular permeability, bioactivity and duration, which remains inadequately explored through systematic evaluations in cellular and animal models. Herein, we isolated and characterized all three diastereoisomers of alkyne‐conjugated esterase‐sensitive dCDN prodrugs, and meticulously examined their difference in stability, cellular uptake and bioactivity. Within THP1 cells, the ( Rp,Rp ) diastereoisomer demonstrated the highest level of activation of STING pathway (EC 50 = 1.7 nM), and induced more robust and prolonged activation pulse than the ( Rp,Sp ) and ( Sp,Sp ) diastereoisomers. In murine models, prodrugs elicited significantly stronger stimulation in the development of an antitumor immune response compared to the parent CDN 3′,3′‐c‐di‐dAMP, as well as the clinically relevant STING agonist ADU‐S100. The ( Rp,Rp ) diastereoisomer exhibited the most pronounced antitumor activity in the context of intravenous administration, significantly suppressing tumor proliferation, extending the survival with a complete response (CR) rate of 90% in a mouse CT26 tumor model, and establishing long‐lasting tumor‐specific immunological memory. These findings underscore the importance of considering the chirality of phosphotriesters in the development of more effective, safer, and sustainable STING agonist in tumor immunotherapy.