Adenosine is elevated in the tumor microenvironment and plays a critical role in suppressing T-cell function through high-affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumor burden in syngeneic models. These effects are mimicked by small-molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumors, particularly in combination with checkpoint inhibitors. However, high levels of adenosine in the tumor microenvironment may pose a challenge for the development of A2a antagonists unless they possess the appropriate pharmaceutical profile. A series of potent antagonists were identified that displayed sub-nanomolar binding activity and selectivity over other adenosine receptors. Within this series, compounds were identified that maintained activity across a broad range of adenosine concentrations. These compounds behaved as insurmountable antagonists in a functional assay utilizing CHO cells expressing human recombinant A2a and displayed slow dissociation kinetics in a FRET-based receptor-binding assay. This series is exemplified by ARX1598, which had a Ki of 0.08nM in the A2a receptor-binding assay and an estimated KB of 0.06 nM in the recombinant cell-based assay. Schild analysis revealed that this compound behaved as an insurmountable antagonist and had a markedly slower off-rate than comparator competitive antagonists. The adenosine receptor agonist NECA suppressed cytokine production by peripheral blood mononuclear cells activated by anti-CD3/anti-CD28. ARX1598 prevented NECA-mediated suppression of cytokine production by human peripheral blood mononuclear cells (EC50= 6 nM) and prevented elevation of pCREB induced by high concentrations of NECA (3μM) in human whole blood (IC50=22nM). The potency of ARX1598 in the human whole blood assay was 50-100 times higher than comparator compounds currently being evaluated in cancer trials. These data illustrate that it is possible to identify potent compounds that have the potential to modulate the adenosine pathway in the tumor microenvironment.

Citation Format: Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Pores, Marta Assuncao, Karolina Gherbi, K. Sengmany, Elisabeth Rosethorne, Peter Finan, Steven Charlton, Clive McCarthy. Identification of potent, insurmountable A2a antagonists for modulation of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A94.