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 (A2AR), suppresses the Th1 cytokine production of T cells and monocytes and cytolytic activity of T and NK cells. We demonstrated that A2AR antagonists initially designed for Parkinson9s disease but repurposed for immuno-oncology dramatically lost potency in a high adenosine environment. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. Using experimental conditions that mimic tumor environment, we have shown that EOS100850 potently inhibited A2AR signalling in human T lymphocytes independently of adenosine concentrations, and rescued cytokine production, even in the presence of high concentrations of A2AR agonists. iTeos A2AR antagonist potently rescued Th1 cytokine production in human whole blood treated by A2AR agonists, and increased CD8+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. An in vivo pharmacodynamic assay based on phosphorylation of CREB (pCREB) in mouse peripheral CD8+ and CD4+ T was developed and validated as a readout for A2AR activation. EOS100850, 30 minutes after oral gavage at doses ranging from 0,03 to 1mg/kg demonstrated 80 to 100% inhibition of pCREB induced by the ex-vivo addition of A2AR agonist. Remarkably, 12 hours after gavage at 1 and 3mg/kg, when the EOS100850 antagonist was no longer detectable in the plasma, more than 50% of inhibition of pCREB was still observed. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK based on a long residence time. iTeos9s A2AR antagonist, uniquely designed to address the challenge of counteracting elevated adenosine concentrations in tumors, was tested for the first time in a mouse A20 lymphoma model. iTeos9s A2AR antagonist in combination with anti-PD-L1 demonstrated significant tumor growth suppression (p=0.0008) compared with anti-PD-L1 alone, with a 6 fold decrease in tumor volume compared to anti-PD-L1 alone (median tumor volume =105±79 mm3 vs 696±427mm3 on day 23, respectively). EOS100850 represents a novel, potent, insurmountable and best-in-class A2AR blocker, specifically optimized for immuno-oncology indications, that deserves studies in Human. Citation Format: Erica Houthuys, Reece Marillier, Theo Deregnaucourt, margreet Brouwer, Paola Basilico, Romain Pirson, Joao Marchante, Shruthi Prasad, Annelise Hermant, Florence Nyawouame, Julie Preillon, Kim Frederix, Anne Catherine Michaux, Florence Lambolez, Jakub Swiercz, Gregory Driessens, Noemie Wald, Chiara Martinoli, Veronique Bodo, Michel Detheux, Xavier Leroy, Stefano Crosignani. EOS100850, an insurmountable and non-brain penetrant A2A receptor antagonist, inhibits adenosine-mediated T cell suppression, demonstrates anti-tumor activity and exhibits best-in class characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-291.