Tumors utilize many different escape mechanisms to evade anti-tumor immune responses. Xios Therapeutics has screened X-Chem’s proprietary 200-billion molecule DNA-encoded library against several immuno-oncology (IO) targets addressing T-cell centric, myeloid immunity and onco-metabolite pathways. Adenosine, for example, is a potent immunosuppressive metabolite, and the ecto-5-nucleotidase (a.k.a. CD73), which catalyzes the conversion of AMP to adenosine, is the rate limiting enzyme for the production of extracellular adenosine in the tumor microenvironment. Hence, CD73 and/or the downstream adenosine receptors are considered attractive targets for IO drug discovery. Likewise, the characteristics of tumor-associated macrophages (TAMs) have fueled interest in therapeutically targeting the colony-stimulating factor 1 axis (CSF1R). Here, we exemplify and enumerate the diversity, selectivity and physiochemical properties of selected hit-to-lead compounds identified from our DNA-encoded library screens of CD73, the adenosine A2A receptor and CSF1R.

In the context of the immune-suppressive purinergic pathway, we have integrated structural biology, medicinal chemistry and clinical pathology evaluation of target expression across tumor types and developed both A2A selective and dual A2A/A2B selective receptor antagonists. From a DNA-encoded library screen, we have identified novel sub-micromolar ligands, which binds to CD73 in an ‘open conformation’ revealed by the co-crystal structure of X6034 (EC50 = 310 nM) in complex with CD73. Interestingly, an inorganic phosphate molecule (Pi) that is structurally shown to be co-present in the active site, illustrates the novelty of these inhibitors, which are chemically distinct from currently reported ADP/AMP substrate analogs. Finally, using tumor tissue microarrays and in situ cell hybridization (RNA-Scope; Advanced Cell Diagnostics), we have explored the co-expression pattern of pathway targets across a subset of immune cells. Informed by the tissue expression pattern of A2AR (primarily CD3+ T-Cells) and A2BR (primarily CD33+ myeloid cells), we compared the ability of equipotent A2A and dual A2A/A2B adenosine receptor antagonists to reverse the effect of NECA, a nonhydrolyzable analog of adenosine, on the maturation and activation of dendritic cells (DC). Starting with the differentiation of human immature DC, we demonstrate the added benefit of dual A2A/A2B inhibitors vs A2A selective inhibitors on relieving immunosuppression of myeloid cells. To conclude, the combination of all these data, together with the productivity of the DNA-encoded library screens for identifying novel, drug like chemical matter on challenging targets like CD73, provide a unique opportunity for potentially harnessing the full power of immunotherapy for cancer.

Citation Format: Andrew J. McRiner, Jannik N. Andersen, Lynette A. Fouser, Junyi Zhang, Kaan Certel, John Cuozzo, Betty Chan, Ragunath Chandran, Matt Clark, Diana Gikunju, Christopher D. Hupp, Anthony D. Keefe, Julie Liu, Yanbin Liu, Michael Monteiro, Allison Olszewski, Moritz Von Rechenberg, Daniel Resnicow, Heather A. Thomson, Dawn M. Troast, Zooey Wang, Neil Westlund, Ying Zhang, Fei Zhou, Xiaotian Zhu, Michael Briskin, Diala Ezzeddine. Novel, potent, and selective small-molecule inhibitors modulating immuno-oncology targets CD73, A2A/A2B adenosine receptors and CSF1R discovered via DNA-encoded library screening [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 4453.