Discovery of a potent, selective, and tumor-suppressing antibody antagonist of adenosine A2A receptor

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A 2A receptor (A 2A R) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A 2A R could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A 2A R (hA 2A R) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries—two synthetic and two immunized—against hA 2A R and antagonist-stabilized hA 2A R. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA 2A R-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A 2A R but not human A 1 , A 2B , or A 3 receptors; functional antagonism of hA 2A R in hA 2A R-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.