Epitope Mapping of an Anti-Chinese/Golden Hamster Podoplanin Monoclonal Antibody

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG₃, kappa), and further changed its subclass into IgG_2a (281-mG_2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG_2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG_2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG_2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG_2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG_2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.