Characterization of anti-podoplanin monoclonal antibodies: critical epitopes for neutralizing the interaction between podoplanin and CLEC-2.
Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that is known as a useful marker for lymphatic endothelium and tumor-initiating cells (TICs). Interaction between podoplanin and C-type lectin-like receptor-2 (CLEC-2) is reported to be critical for podoplanin-induced platelet aggregation and cancer metastasis. Recently, several anti-human podoplanin antibodies have been created; however, these anti-podoplanin antibodies have not been well characterized. Five anti-podoplanin antibodies (NZ-1, D2-40, AB3, 18H5, and a rabbit polyclonal antibody) were investigated using ELISA, Western blot, and flow cytometry with synthesized podoplanin peptides and deletion mutants of recombinant podoplanin. The epitope of NZ-1 is platelet aggregation-stimulating (PLAG) domain-2/3; the epitope of D2-40, AB3, and 18H5 is PLAG1/2. The epitopes of D2-40 and AB3 are quite similar, although 18H5 is different from D2-40 and AB3. Using flow cytometric analysis, NZ-1 partially inhibited the interaction between podoplanin and CLEC-2, although other antibodies did not. In conclusion, the two most frequently used anti-podoplanin antibodies, D2-40 and AB3, have similar properties, although several studies have reported differences. NZ-1 neutralizes the interaction between podoplanin and CLEC-2, which may lead to the development of therapeutic antibodies against podoplanin-dependent cancer metastasis.
Ogasawara
Satoshi
S
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, Japan.
Kaneko
Mika Kato
MK
Price
Janet E
JE
Kato
Yukinari
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D000098853:Podoplanin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C104778:CLEC2B protein, human; C117468:PDPN protein, human; D000098853:Podoplanin