ONM-501-A synthetic polyvalent STING agonist for cancer immunotherapy

Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Naturally, the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-STING pathway adopts polyvalent interactions to form higher-order assemblies to achieve a specific and rapid response while limiting unnecessary stimulation by host DNA. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have been studied in clinical trials showing limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation. Mechanistically, PC7A binds to STING through a non-competitive surface site that is distinct from the binding pocket of CDN and non-CDN small molecule STING agonists. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst9 and ‘sustained9 STING activation. Effectiveness of using ONM-501 for immunotherapy against solid tumors has been demonstrated. Methods: Polyvalent interaction between PC7A and STING was studied and characterized by several different methods including ITC, a FRET assay between fluorophore labeled STING and PC7A and Nile-Red assay. The binding valency was investigated using a series of PC7A polymers with an increasing number of repeating units. The PC7A-STING binding site was elucidated using STING mutants produced by site-directed mutagenesis. STING activation was evaluated by measuring Ifnb1 and Cxcl10 expression using RT-qPCR. STING activation by PC7A and ONM-501 was also investigated using freshly resected human tissue. ONM-501 antitumor efficacy was evaluated in 6 different murine syngeneic tumor models. Results: PC7A activates STING through a non-canonical biomolecular condensation. It binds to a non-competitive surface site on the α5 helix of STING which is different from the CDN binding pocket. This binding was also retained with cGAMP-resistant STING variants (e.g., R232H). The formation of STING-PC7A condensate and the downstream activation were dependent on polymer repeating units. ONM-501 achieves a synergistic, rapid and sustained (6-24h) STING activation in vivo compared to cGAMP which peaked at 6h. Injection of ONM-501 in fresh human tissue resulted in >100-fold increase in cytokine expression while free CDN only showed marginal effect. Antitumor efficacy was demonstrated in MC38, CT26, B16F10, 4T1, A20 and TC-1 models after ONM-501 treatment. Complete response was observed when combined with anti-PD1 checkpoint blockade therapy. Conclusions: PC7A polymer achieves prolonged innate immune activation by polyvalent STING condensation through a distinct binding site. ONM-501 combines endogenous cGAMP with PC7A that potentially offers a synergistic strategy in spatiotemporal orchestration of immune environment for a highly effective immunotherapy against cancer. Citation Format: Suxin Li, Min Luo, Zhaohui Wang, Qiang Feng, Jonathan Wilhelm, Xu Wang, Wei Li, Jian Wang, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Katy Torres, Stephen Gutowski, Agnieszka Cholka, Yang-xin Fu, Tian Zhao, Baran Sumer, Hongtao Yu, Jinming Gao. ONM-501 ― A synthetic polyvalent STING agonist for cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P049.