Epitope Analysis of an Antihorse Podoplanin Monoclonal Antibody PMab-219

Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression is associated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. In addition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In this study, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between 55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 of horPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.