Identification of PD-1–PD-L1 blockade epitopes in vitro utilizing porcine immunoglobulin and heterologous Fc-fused protein

Programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), function as pivotal immune checkpoints. Numerous studies have demonstrated the association between the malignant progression of various swine diseases and aberrant expression of PD-1 and PD-L1, hence the development and screening of high-affinity porcine PD-1 and PD-L1 monoclonal antibodies (mAbs) holds substantial significance for advancing research and therapeutic interventions. In this study, we produced porcine PD-1 and PD-L1 mAbs which exhibited robust reactivity in western blot (WB), indirect immunofluorescence assay (IFA), and flow cytometry (FCM). The newly identified B-cell epitope ⁹⁰GRDPRFHVTPL¹⁰⁰ and ¹⁸⁵REEKLFNVTST¹⁹⁵ of PD-1 and PD-L1 mAbs were linear and surface-exposed as illustrated by WB and structure analysis. Comparative sequence analysis demonstrated that the PD-L1 epitope is highly conserved across species, whereas the PD-1 epitope exhibits lower interspecies conservation. In addition, the blocking efficacy of the two PD-1 mAbs and six PD-L1 mAbs was predicted low via molecular docking. To further evaluate the blocking efficacy, we generated a flow cytometry-based assay by using a porcine PD-L1-rabbit Fc fusion protein, expressed via a eukaryotic system. In agreement with the prediction, our in vitro data demonstrated a blocking rate below 4% compared with the IgG group for PD-1 mAbs and PD-L1 mAbs. In summary, we herein generated porcine PD-1 and PD-L1 mAbs recognizing unreported B-cell epitopes, and established a reliable method for identifying the nonblocking mAb and epitopes, which may facilitate the development of novel diagnostic approaches and therapeutic agents.