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Elevated levels of adenosine found in the tumor microenvironment have been highlighted as a potential mechanism contributing to the restricted efficacy of immune checkpoint inhibitors. While the advent of these inhibitors has brought significant benefit to patients with cancer, there is still significant unmet need even in disease indications for which these agents are approved. Adenosine has an immune-suppressive effect in tumors, and pre-clinical and early clinical evidence indicates that antagonists of the GPCR family of adenosine receptors are able to relieve that suppression and restore anti-tumor immune cell activity. EXS21546 is a non-furan, selective antagonist of the A2A receptor with low brain penetration. Using EXS21546, we show that activity at the A2A receptor is sufficient for complete restoration of T-cell function in the presence of high concentrations of adenosine analogue. Translational studies with EXS21546 have been undertaken using patient samples to better understand its activity in signaling and disease-relevant endpoints. We will present data characterizing the activity of EXS21546 ex vivo in patient-derived whole blood samples. Interrogation of primary cancer cells from pancreatic and lung cancer patients treated with EXS21546 ex vivo by phenotypic image-based screening, demonstrated that EXS21546 reduces the relative fraction of viable cancer cells while inducing an expansion of the viable CD8+ T cell fraction in the presence of high concentrations of an adenosine analogue. Together, these data indicate the potential for EXS21546 may have a role in cancer therapy. EXS21546 has completed pre-IND studies in preparation for initiation of first-in-human clinical studies. Citation Format: Andrew Payne, Pierre Fons, Isabella Alt, Joost van Ham, Christina Taubert, Andy Bell, Stephanie Versluys, Florie Bertrand, Virgile Visentin, Emilie Mirey, Emilie Pelissier, Michael Paillasse, Sabrina Pagliarusco, Gregory Vladimer, Mark Whittaker. EXS21546, a non-CNS penetrant A2AR-selective antagonist for anti-cancer immunotherapy [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 1731.

Evotec and Exscientia are collaborating to develop an innovative drug discovery platform for accelerating small molecule development in Immuno-Oncology. The primary focus is to target the immunosuppressive adenosine pathway. We have firstly sought to generate a novel and selective, non-brain penetrant A2AR antagonist. EVOEX21546 has been selected as our lead candidate with in vitro potency on primary human CD3+ T-lymphocytes for inducing the recovery of IL-2 production after CADO-mediated inhibition of T-cell activation. In addition, we have demonstrated the compound’s effect on other key biological features of T-cell activation including IFN-gamma production and T-cell proliferation. ADME/DMPK data show that EVOEX21546 has a favourable pharmacological profile consistent with its evaluation in syngeneic tumour models. Furthermore, we are pursuing efforts to understand the Mechanism-of-Action of EVOEXS21546 in order to identify the most relevant biomarker of activity for clinical translation. Finally, from the perspective to accelerate the IND submission, EVOEXS21546 entered an INDiGO campaign, an integrated and rapid process to reach IND complemented by high-end integrated CMC. These results, generated in the frame of the A2AR inhibitor pathway, have paved the way to an optimized process for identifying, improving and accelerating the path from drug discovery to the entering into the clinic for Immuno-Oncology drugs. Citation Format: Pierre Fons, Andy Bell, Michael Esquerre, Stephanie Versluys, Florie Bertrand, Iva Hopkins-Navratilova, Sean Robinson, Virgile Visentin, Adrian Schreyer, Richard Cox, Emilie Mirey, Emilie Pelissier, Celia bergeaud, Simone Culurgioni, Jeremy Besnard, Andrew Payne, Jerome Menegotto, Frederic Machet, Celine Poussereau-Pomie, Eric Cogo, Michael Paillasse, Federica Ferigo, Sabrina Pagliarusco, Joanna Lisztwan, Mark Whittaker, Craig Johnstone, Andrew Hopkins. EVOEXS21546 is a novel non-brain penetrant A2AR inhibitor for cancer immunotherapy with accelerated drug development [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 519.

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.

Adenosine, generated by CD73 is a key driver of immunosuppression in the hypoxic tumour microenvironment (TME). 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 a drug discovery platform for accelerating small molecule development in Immuno-Oncology targeting this adenosine pathway. The platform has integrated a unique biophysical screening approach to the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade. We have firstly sought to generate a novel non-brain penetrant A2AR antagonist. We have selected EVOEX21546 as our lead candidate and demonstrated its in vitro potency on primary human CD3+ T lymphocytes for inducing the recovery of IL-2 production after CADO-mediated inhibition of T-cell activation. In addition, we have demonstrated the compounds effect on other key biological features of T-cell activation including IFN-γ production and T-cell proliferation. ADME/DMPK data show that EVOEX21546 has a favourable pharmacological profile consistent with its evaluation in in vivo models resistant to ICTs before its entry into preclinical development. In parallel of this first drug discovery program, we have initiated research of CD73 specific inhibitory molecules and two series binding at orthogonal sites on CD73 have been identified. In vitro functional potency has been demonstrated for the CD73 inhibitors for inducing recovery of AMP-induced inhibition of T-cells activation as measured through IL-2 production. The series are under optimization to improve activity, solubility and ADME/DMPK parameters. Finally, from the perspective of developing dual pharmacological profile for A2AR and CD73 inhibition, we have assessed in a primary human CD3+ T-lymphocyte assay the synergy between the two targets in order to optimize the recovery of T-cell activation. Fragment screening has been performed for this approach to further aid design new molecules with a dual pharmacological profile. These results, have paved the way to an optimized process for identifying, improving and accelerating drug discovery in Immuno-Oncology in the frame of the adenosinergic immunosuppressive pathway. Citation Format: Pierre Fons, Andy Bell, Michael Esquerre, Stephanie Versluys, Florie Bertrand, Celine Poussereau-Pomie, Adrian Schreyer, Richard Cox, Jeremy Besnard, Michael Paillasse, Mark Swindells, Joanna Lisztwan, Craig Johnstone, Mark Whittaker, Andrew Hopkins. Identification of a novel non-brain penetrant A2AR inhibitor and proof-of-concept of CD73 and A2AR/CD73 small-molecule inhibitors for cancer immunotherapy [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 3768.

Standard Immune Checkpoint Therapies, such as anti-CTLA-4 or anti-PD-1/PD-L1 mAb, demonstrate long progression-free-survival for responding patients but with a low response rate. This can be due to additional immunosuppressive mechanisms developed by resistant tumors. The adenosine pathway is one of the main drivers of tumor progression by acting on two key features of the TME (Tumor Micro-Environment); immunity and angiogenesis. Expression of CD73 on tumor cells and on immunosuppressive cell subsets leads to the generation of adenosine from AMP. Adenosine inhibits the biological functions of T lymphocytes infiltrating the tumor by binding to the A2AR (receptor). Evotec and Exscientia are collaborating to accelerate small molecule drug discovery in the field of Immuno-Oncology by integrating a unique biophysical screening approach to the adenosine receptors and CD73 to drive automated medicinal chemistry design with a translational-focused screening cascade, to discover novel agents that target the adenosinergic pathway in immuno-oncology. We have firstly sought to generate novel A2AR antagonists and secondly to create A2AR/CD73 bi-specific inhibitory molecules with a dual pharmacological profile, from fragment screening and de novo design approaches. In vivo combination study demonstrate the advantages of targeting CD73 and A2AR. We have obtained active molecules illustrated by in vitro data on primary human T lymphocytes. These results, have paved the way to an optimized process to identify adenosinergic pathway inhibitors with properties optimized for immuno-oncology. Citation Format: Pierre Fons, Michael Esquerré, Stéphanie Versluys, Gigliola Mambrini, Michael Paillasse, Andy Bell, Adrian Schreyer, Richard Cox, Richard Bickerton, Joanna Lisztwan, Mark Whittaker, Françoise Bono, Craig Johnstone, Andrew Hopkins. Targeting the adenosine immunosuppressive pathway for cancer immunotherapy with small molecule agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2017-3970