Carriers Multimerize STING Protein Fragments to Activate Type I Interferon Signaling in STING-Deficient Cancer Cells

Therapeutic activation of the stimulator of interferon genes (STING) innate immune pathway shows promise for cancer immunotherapy; however, frequent loss of STING expression in cancer cells renders these cells unresponsive to existing agonists. We report that cytosolic delivery of a soluble STING protein fragment bypasses this challenge by interacting directly with downstream signaling molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) to activate a Type I interferon response, even in STING-deficient cells. Prior work demonstrated that this same STING fragment was not capable of activating signaling when overexpressed, leading us to investigate how the cytosolic delivery of the protein enables activity. Our results suggest that in addition to facilitating transport across the cell membrane, complexation by delivery vehicles can promote multimerization of the STING fragment, allowing it to remain in a multimeric active state even after escape from the cytosol. Activity remains even after truncation or unfolding of STING's crystallizable domain containing the interface of the protein involved in full-length STING oligomerization, showing that multimerization of STING fragments can proceed by nontypical means. Finally, we demonstrate that this strategy can induce a type I interferon response in multiple STING-proficient and -deficient cancer cell lines. Overall, this work shows how delivery vehicles can be used to modify a protein therapeutic into an active state and provides a proof of concept motivating further development of STING fragment delivery as an immunotherapy.