Abstract B068: An in vitro cellular functional STING pathway assay to measure the potency of STING agonists, using interferon-β response

Introduction and Relevance: We have developed an in vitro cellular functional STING pathway assay that can be used to screen for potential therapeutic agents in drug discovery. Stimulator of interferon genes (STING, or TMEM173) is an endoplasmic reticulum-associated protein and cytosolic DNA sensor, which plays a critical role in the innate and adaptive arm of the immune system. It is highly expressed in antigen-presenting cells (APC’s), such as macrophages and dendritic cells, as well as plasmacytoid dendritic cells, myeloid-derived suppressor cells, T-cells, and various endothelial or epithelial subtypes. The STING pathway is spontaneously activated by cyclic dinucleotides, a product derived from the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), upon invasion by pathogens and exposure to self-DNA, which leads to the production of type I interferons and proinflammatory cytokines. Recent studies in the tumor microenvironment have suggested that STING pathway activation in APC’s leads to the production of interferon-β (IFNβ) and spontaneous generation of a strong antitumor CD8+ T-cell response. Preclinical models with STING agonists have shown significant regression of established tumors, which has subsequently resulted in phase I clinical trials of these agents. Based on the collective evidence, pharmacologic STING agonists are now being developed as a potential new approach for cancer immunotherapy. Within this context, we investigated whether STING pathway activation in peripheral blood mononuclear cells (PBMCs) and the monocytic tumor cell line, THP-1, could be used to develop an assay to assist the discovery of novel STING agonist therapeutics. Experimental Procedures: Freshly isolated human PBMCs, or THP-1 cells, are plated and treated with STING agonists (e.g., 2’3’-cGAMP), or test agents, over a range of concentrations. Cells are incubated for 24 hours at 37°C, 5% CO2. Cell culture supernatants are then harvested and analyzed for IFNβ secretion, using a conventional sandwich ELISA format. Results: Using IFNβ as a readout, we show the ability to activate the STING pathway in both human PBMCs and THP-1 cells, using the STING agonist, 2’3’-cGAMP, as well as its close analogs, 2’3’-cGAM(PS)2 (Rp/Sp) and 2’3’-c-di-AM(PS)2 (Rp/Rp). This assay is robust and capable of measuring the relative potencies of these agonists. Treatment with 2’3’-cGAMP significantly induced IFNβ secretion, in a dose-dependent manner, in both PBMCs and THP-1 cells, with EC50 values of ~70 μM and 124 μM, respectively. Assessment of the potency of different STING agonists in THP-1 cells resulted in EC50 values for 2’3’-cGAM(PS)2 (Rp/Sp) and 2’3’-c-di-AM(PS)2 (Rp/Rp) of 39.7 μM and 10.5 μM, respectively. Induction and measurement of other cytokines, or chemokines, with STING agonists may also be discussed. Conclusions: We have shown the ability to quantitatively measure STING pathway activation in a newly developed in vitro cellular functional assay, using human PBMCs or THP-1 tumor cells. This assay is robust and capable of demonstrating different potencies of different agonists, and thus provides a convenient method to screen for potential therapeutic agents. Citation Format: Satheesh K. Sainathan, Alyssa M. Cracchiolo, Usha Warrior, Alastair J. King. An in vitro cellular functional STING pathway assay to measure the potency of STING agonists, using interferon-β response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B068.