Macromolecular Diamidobenzimidazole Conjugates Can Activate Stimulator of Interferon Genes

A promising class of stimulator of interferon genes (STING) agonists is the non-nucleotide, small molecule, dimeric amidobenzimidazoles (diABZI), which have recently been incorporated into polymer- and antibody-drug conjugates to improve pharmacokinetics and modulate biodistribution for disease-specific applications. These approaches have leveraged diABZI variants functionalized at the 7-position of the benzimidazole for conjugation and tunable drug release from carriers. However, since this position does not interact with STING and is exposed from the binding pocket when bound in an "open lid" configuration, we sought to evaluate the activity of macromolecular diABZI conjugates that lack stimuli-responsive release and are instead conjugated to polymers via a stable amide linker. By synthesizing stable mPEG-diABZI conjugates and <i>N</i>,<i>N</i>-dimethylacrylamide (DMA) homopolymers from a diABZI-functionalized reversible addition-fragmentation chain-transfer (RAFT) agent, we found that these conjugates could activate STING <i>in vitro</i> with similar kinetics to highly potent diABZI analogues. Our data indicate that although diABZI-DMA conjugates enter cells via endocytosis, they can still colocalize with the ER, suggesting that intracellular trafficking processes can promote the delivery of endocytosed macromolecular diABZI compounds to STING. Furthermore, we demonstrated the <i>in vivo</i> activity of these macromolecular diABZI platforms, which inhibited tumor growth to a similar extent as small molecule variants. In conclusion, we have described new chemical strategies for the synthesis of stable macromolecular diABZI conjugates with unexpected immunostimulatory activity─findings that have potential implications for the design of polymer-diABZI conjugates and further motivate investigation of endosomal and intracellular trafficking as an alternative route for STING activation.