Background ADORA2A, is an adenosine receptor that suppresses immune cell toxicity through sensing extracellular adenosine. ADORA2A belongs to a group of seven-transmembrane receptor proteins called G protein-coupled receptors (GPCRs) which act as detectors to various signals from outside the cell. We aim to reduce the immunosuppressive properties of adenosine by targeting ADORA2A with monoclonal antibodies (mAbs) to enhance the potency of established immunotherapies. mAbs are an attractive modality to therapeutically target complex multi-pass membrane proteins, but mAb development is hindered by many obstacles. Here we report the discovery and characterization of 26 ADORA2A specific monoclonal antibodies developed using Icosagen’s proprietary multi-pass membrane protein focused chicken immunization and mAb screening platforms.

Methods Monoclonal antibodies against ADORA2A were generated using DNA or mRNA and virus-like particle (VLP)-based immunization strategy. VLPs were generated using novel proprietary technology ensuring high density display of ADORA2A. Chickens were selected as immunization hosts due to greater evolutionary distance from mammals increasing the chance of obtaining therapeutic antibody candidates. Antibody discovery was performed with Icosagen’s HybriFree B-cell cloning technology, complemented with chicken derived phage display library screening platform. Antibody specificity was confirmed using VLP-based immunoassays and overexpression cell lines. Antagonistic function of the antibodies was confirmed on immune cells and reporter cell line-based assays.

Results Immunization of chicken and dynamic antibody discovery methods resulted in discovery of 26 unique ADORA2A specific antibody clones. These antibodies exhibit high affinity binding to ADORA2A, as indicated by low nanomolar EC50 values determined through cell-based assays. Antibody clones with antagonistic function show enhancement of T cell activation upon ADORA2A agonist treatment, highlighting their potential to boost anti-tumour immune response.

Conclusions This study presents highly specific monoclonal antibodies targeting the adenosine receptor ADORA2A. The lead antibody clones exhibiting antagonistic function will undergo further optimization using Icosagen’s phage display-based affinity maturation platform. This process aims to enhance their properties for development into clinical candidates, advancing therapeutic interventions targeting ADORA2A and supporting their potential in immunotherapy applications.