An Fc–Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor

The adenosine A_2A receptor (A_2AR) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A_2AR have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A_2AR antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A_2AR effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein–small molecule conjugate, Fc–ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc–ZM was a potent A_2AR antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc–ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A_2AR. We found that Fc–ZM, compared with the small molecule ZM, was a superior A_2AR antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.