Abstract 4154: EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies
Authors: Martinoli Chiara , Brouwer Margreet , Houthuys Erica , Mercier Marjorie , Wald Noemie , Gomes Bruno , Crosignani Stefano , Bodo Veronique 
Published: August 2019

Abstract
High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types.
To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples.
The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors).
The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (&gt;50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors).
Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics.
Citation Format: Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4154.