Abstract 4532A: Development of selective small molecule STING agonists suitable for systemic administration

Abstract

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.

STING Agonists as Cancer Therapeutics

“…In vitro studies on peripheral blood mononuclear cells and the THP1 monocytic cell line show that Selvita agonists induce the expression of inflammatory cytokines and upregulate maturation markers on the surface of APCs. A group of selective non-CDN non-macrocylic small molecule compounds ( Ryvu’s agonists ) have shown promising results in pre-clinical animal models [ 87 ]. These agonists bind to STING proteins of different species and are able to induce DC maturation and cytokine expression from human PBMCs irrespective of the STING haplotype.…”

“…These agonists bind to STING proteins of different species and are able to induce DC maturation and cytokine expression from human PBMCs irrespective of the STING haplotype. Systemic administration of these compounds (route not specified) in mice bearing CT26 colorectal cancer cells resulted in complete tumor regression and the development of immunological memory [ 87 ].…”

Beyond DNA sensing: expanding the role of cGAS/STING in immunity and diseases

“…Using FTS, MST, FP, and crystallographic studies, activators of Ryvu reportedly bind to recombinant STING proteins and selectively activate STING-dependent signaling in both mouse and human immune cells, promoting antitumor immunity. Systemic administration of these compounds in CT26 mouse models bearing colorectal cancer cells resulted in complete tumor regression and the development of immunologic memory (Chmielewski et al 2020 ).…”