Development and characterization of anti‐glycopeptide monoclonal antibodies against human podoplanin, using glycan‐deficient cell lines generated by CRISPR/Cas9 and TALEN

Abstract

Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycandeficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. Cancer Medicine Open Access 383

Plasma soluble podoplanin is a novel marker for the diagnosis of tumor occurrence and metastasis

“…Therefore, the extracellular domain of PDPN may be shed through the action of these proteolytic enzymes, especially in the tumor environment where O ‐glycosylation of PDPN is known to be misregulated. This speculation is supported by recent progress in production of a series of PDPN cancer‐specific monoclonal antibodies (CasMab) by Kato group . For example, the CasMabLpMab‐2 recognizes cancer‐type aberrant glycosylation ( O ‐glycosylation or sialylation, not keratan sulfate) of Thr55 and/or Ser56; therefore, it reacts with hPDPN‐expressing cancer cells but not with normal cells .…”

“…For example, the CasMabLpMab‐2 recognizes cancer‐type aberrant glycosylation ( O ‐glycosylation or sialylation, not keratan sulfate) of Thr55 and/or Ser56; therefore, it reacts with hPDPN‐expressing cancer cells but not with normal cells . Another CasMab LpMab‐21, the epitope of which contains sialyated Thr76, detects PDPN in glioblastomas, oral cancers and seminomas as well as in normal cells such as lymphatic endothelial cells, but does not recognize PDPN in the renal glomerulus or type I alveolar cells of lung . LpMab‐7, an anti‐non‐PLAG hPDPN mAb, can be used for detecting different glycan profiles of hPDPN, including the poly LacNAc structure .…”

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231

“…(40) Almost all mAbs against PDPNs reportedly react with PLAG domains or PLDs. (40,41) As summarized in Figure 1, PMab-231 also detected PLAG1 (Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, and Gly36) and a part of PLAG2 (Glu38) of tigPDPN. Therefore, PLAG1 of tigPDPN was clarified to be the advantageous epitope for several applications, such as flow cytometry, Western blotting, and immunohistochemical analyses.…”

Antitumor activity of chLpMab‐2, a human–mouse chimeric cancer‐specific antihuman podoplanin antibody, via antibody‐dependent cellular cytotoxicity

“…Previous studies have shown that hPDPN is O ‐glycosylated and not N ‐glycosylated . We used a GnT‐1‐KO cell line (CHO‐S/GnT‐1‐KO, PDIS‐9) and a CMP‐sialic acid transporter (SLC35A1)‐KO cell line (CHO‐S/SLC35A1‐KO, PDIS‐14) to characterize chLpMab‐2. As shown in Figure B, chLpMab‐7 reacted with CHO‐S/hPDPN, PDIS‐9/hPDPN, and PDIS‐14/hPDPN cells transfected with the hPDPN expression vector.…”