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CD73/Ecto-5'-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire-tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40-50 min.

Adenosine generated by CD73 is a key driver of immunosuppression in the hypoxic tumour microenvironment (TME) and particularly involved in angiogenic process and immunity. In immune-inflamed tumours, with CD8+ T-cell infiltrates, adenosine signalling is a cause of resistance to immune checkpoint therapies (ICTs) through the inhibition of T-cells, NK cells and more largely of the overall antitumor immunity. Evotec and Exscientia are collaborating to develop an innovative drug discovery platform for accelerating small molecule development in Immuno-Oncology targeting the adenosine pathway. The platform has integrated a unique biophysical screening approach for the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade. We have initiated research of CD73 specific inhibitory molecules based on SPR fragment screening and have identified lead compounds which fully bind in the active site of the CD73 protein in both the open and closed states. In vitro functional potency has been demonstrated for the CD73 inhibitors for inhibiting adenosine production by Rapid Fire technology performed using CD73 recombinant protein. Moreover, we demonstrated that CD73 inhibitors induce recovery of AMP-induced inhibition of T-cells activation as measured through IL-2 production. The series are under lead optimization to improve ADME/DMPK parameters in order to enable evaluation in PK/PD assays and in vivo efficacy studies specifically developed for the project. Finally, from the perspective of developing a dual pharmacological profile for A2AR and CD73 inhibition, we have assessed, using primary human CD3+ T-lymphocytes isolated from healthy donors, the synergy between the inhibition of the two targets in order to enhance the recovery of T-cell activation. We have demonstrated that EVOEXS21546, our pre-development A2AR inhibitor candidate, and our lead compound for CD73 inhibition act in synergy in functional human T-cell assays. Our SPR screening approach has also identified hit compounds with dual bispecific pharmacological activity against both A2AR and CD73. Citation Format: Pierre Fons, Andrew Bell, Michael Esquerre, Stephanie Versluys, Florie Bertrand, Adrian Schreyer, Iva Hopkins-Navratilova, Leonardo-Silvestre Hernani, Celia Bergeaud, Celine Poussereau-Pomie, Ghislaine Marchand, Sean Robinson, Simone Culurgioni, Richard Cox, Jeremy Besnard, Andrew Payne, Peter Ray, Emilie Pelissier, Michael Paillasse, Joanna Lisztwan, Craig Johnstone, Mark Whittaker, Andrew Hopkins. Identification of CD73 and A2AR/CD73 small molecule inhibitors for cancer immunotherapy as single agents or in combination with Immune-checkpoint therapies [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 507.