Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin

Monoclon Antib Immunodiagn Immunother. 2016 Apr;35(2):109-16. doi: 10.1089/mab.2015.0077. Epub 2016 Mar 3.

Abstract

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Epitope Mapping
  • Epitopes / immunology
  • Flow Cytometry
  • HEK293 Cells
  • Haplorhini / immunology
  • Humans
  • Membrane Glycoproteins / immunology*
  • Membrane Glycoproteins / isolation & purification
  • Mice
  • Protein Domains / immunology*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Membrane Glycoproteins
  • PDPN protein, human