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Background: Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs are developing cyclic dinucleotides - analogues of endogenous STING ligands. However their chemical nature and stability limit their use as systemic immuno-therapeutics. Herein, we present potent and selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, structurally unrelated to known chemotypes and suitable for systemic administration. Methods: Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Human macrophages (HMDM) and dendritic cells (HMDC) were differentiated from monocytes (obtained from PBMC) in the presence of M-CSF and GM-CSF/IL-4 for HMDM and HMDC, respectively. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 or STING KO mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma cells and the compound was administered, followed by the regular tumor growth monitoring. Finally, the compound was administered to C57BL/6 WT and STING KO mice in several escalating doses. Results: Ryvu's agonists demonstrate a strong binding affinity to recombinant STING proteins across tested species. They trigger pro-inflammatory cytokine release from human PBMC and HMDC and induce dendritic cell maturation regardless of the STING haplotype. Systemic in vivo administration leads to dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, suggesting immune activation which translates into efficacy in vivo in CT26 mouse colorectal cancer model and complete tumor remissions. Furthermore, cured animals develop lasting immunological response demonstrated by diminished tumor growth or lack of palpable tumors in re-challenged mice. Conclusion: Ryvu's STING agonists selectively activate STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu's STING agonists leads to engagement of the immune system which results in complete tumor remission and development of immunological memory against cancer cells. The compounds show good selectivity and ADME properties enabling development for systemic administration as a single agent or in combinations with immunotherapies or targeted agents. Citation Format: Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Maciej K. Rogacki, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Grzegorz Ćwiertnia, Grzegorz Topolnicki, Maciej Kujawa, Eliza Zimoląg, Urszula Głowniak-Kwitek, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Marek Wronowski, Kinga Michalik, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Łukasz Dudek, Jose Alvarez, Anna Rajda, Faustyna Gajdosz, Aniela Gołas, Katarzyna Wnuk-Lipińska, Kamil Kuś, Ewelina Gabor-Worwa, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Monika Dobrzańska. Development of selective small molecule STING agonists suitable for systemic administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4532A.

Background. Stimulator of interferon genes, known as STING, is an intracellular sensor of nucleic acids and one of key regulators in activating the innate immune response. Employing synthetic STING agonists has been shown to promote immune-mediated antitumor response in preclinical animal models. Ryvu is developing small-molecule STING agonists suitable for systemic administration. Herein we present unpublished results from characterization of the new generation of our agonist series with significantly improved potency on human immune cells. Methods. Binding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation and was confirmed by X-ray crystallography. Primary screen was performed in THP-1 Dual reporter cells and selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing varying STING variants. T cell viability and proliferation was assessed by flow cytometry using activated, human T cells exposed to STING agonists. STING pathway activation pattern in cells treated with Ryvu's molecules was confirmed using Western blot analysis. BALB/c mice were injected with compounds and the levels of cytokine release were measured in the plasma. Mice were inoculated with CT26 or EMT6 tumor cells and the compound was administered intravenously followed by the regular monitoring of tumor growth. Results. New generation Ryvu STING agonists are strong binders of human STING protein. Ryvu's compounds show high cellular potency inducing cytokine production in human immune cells at low nM range. Moreover, high activity of developed agonists is maintained irrespective of the natural human STING variant as seen in THP-1 reporter cells as well as in human primary immune cells. High cellular potency of developed compounds also translates into efficacy observed in vivo, where systemic intravenous administration leads to significant tumor growth inhibition and complete tumor regressions in mouse syngeneic models. Conclusion. Ryvu has developed a new generation of potent, direct and selective small-molecule STING agonists. The compounds are characterized by drug-like properties and high in vitro potency on par or outperforming known references. Ryvu agonists are suitable for systemic administration and allow to achieve excellent antitumor efficacy. Taken together, the promising results suggest that the developed series holds high potential for improving immunotherapy in cancer patients. Citation Format: Maciej Krzysztof Rogacki, Stefan Chmielewski, Jolanta Mazurek, Magdalena Zawadzka, Katarzyna Wnuk-Lipińska, Kamil Kuś, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Łukasz Dudek, Wojciech Schonemann, Urszula Głowniak-Kwitek, Marcin Leś, Marek Wronowski, Tushar Mahajan, Urszula Kulesza, Magdalena Zastawna, David Synak, Karol Zuchowicz, Karolina Gluza, Katarzyna Banaszak, Karolina Wiatrowska, Izabela Strojny, Mirosława Gładysz, Justyna Jabłońska, Ewelina Gabor-Worwa, Monika Dobrzańska, Raghuram Tangirala, Peter Littlewood, Krzysztof Brzózka. New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1280.

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BackgroundSTimulator of INterferon Genes (STING) is a key signaling protein involved in activation of the immune system in response to self-DNA. In recent years, STING signaling has been demonstrated to play a major role in activating the antitumor immune response and therefore is considered an attractive drug target in immuno-oncology. The first wave of STING agonists, cyclic-dinucleotide analogues of the internal ligand cGAMP, were developed for local, intratumoral administration. Herein we present the most recent profiling results of our frontrunner molecule RVU-27065, a potent and selective systemic STING agonist with a favorable drug profile.MethodsBinding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation. Primary activity screen was performed in THP-1 Dual reporter cells. Selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing STING variants. T cell viability and proliferation was assessed by flow cytometry using activated human T cells. PBMCs were isolated by density gradient from whole blood of healthy donors. Downstream STING pathway activation in cells treated with RVU-27065 was confirmed using Western blot analysis. BALB/c mice were inoculated with EMT6 tumor cells and the compound was administered intravenously followed by regular monitoring of tumor growth. Cured animals were rechallenged by repeated inoculation of EMT6 cells.ResultsRVU-27065 binds and strongly thermostabilizes recombinant STING proteins of all tested species. Binding to the protein results in activation of downstream signalling pathway, confirmed by western blot analysis. The agonist is characterized by selectivity and excellent potency in THP-1 dual reporter cells as well as in human PBMCs and dendritic cells. Short term incubation of RVU-27065 has no impact on T cell viability, activation or proliferation. Furthermore, STING activation with RVU-27065 leads to repolarization of immunosuppressive M2 macrophages into pro-inflammatory M1-like phenotype. In vivo efficacy of RVU-27065 was confirmed, leading to significant tumor growth inhibition and complete tumor regressions in an EMT6 mouse breast cancer syngeneic tumor model.ConclusionsRVU-27065 is a novel representative of a 3rd generation of Ryvu STING agonists – small-molecule, non-macrocyclic molecules built around a unique chemotype. The compound is characterized by high in vitro potency which translates to efficacy in vivo in preclinical animal models. Drug-like properties, excellent selectivity and a good safety profile make RVU-27065 an attractive candidate for further development for standalone as well as targeted delivery, which holds high potential for improved immunotherapy in cancer patients.