Macromolecular Diamidobenzimidazole Conjugates Activate STING.

Pharmacologic activation of the stimulator of interferon genes (STING) pathway has broad potential applications, including the treatment of cancer and viral infections, which has motivated the synthesis and testing of a diversity of STING agonists as next generation immunotherapeutics. A promising class of STING agonists are the non-nucleotide, small molecule, dimeric-amidobenzimidazoles (diABZI), which have been recently used in the synthesis of polymer- and antibody-drug conjugates to improve pharmacokinetics, modulate biodistribution, and to confer other favorable properties for specific disease applications. These approaches have leveraged diABZI variants functionalized with reactive handles and enzyme-cleavable linkers at the 7-position of the benzimidazole for conjugation to 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 enzymatic release and are instead conjugated to polymers via a stable linker. By covalently ligating diABZI to 5 or 20 kDa mPEG chains via an amide bond, we surprisingly found that these conjugates could activate STING To further evaluate this phenomenon, we designed a diABZI-functionalized RAFT chain transfer agent that provided an enabling tool for synthesis of large, hydrophilic, dimethylacrylamide (DMA) polymers directly from a single agonist and we found that these conjugates also elicited STING activation with similar kinetics to highly potent small molecule analogs. We further demonstrated the activity of these macromolecular diABZI platforms, which inhibited tumor growth to a similar extent as small molecule variants. Using flow cytometry and fluorescence microscopy to evaluate intracellular uptake and distribution of Cy5-labeled analogs, 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 delivery of endocytosed macromolecular diABZI compounds to STING. In conclusion, we have described new chemical strategies for the synthesis of stable macromolecular diABZI conjugates with unexpectedly high immunostimulatory potency, findings with potential implications for the design of polymer-drug conjugates for STING agonist delivery that also further motivate investigation of endosomal and intracellular trafficking as an alternative route for achieving STING activation.