LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Abstract

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.

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 .…”

A novel anti‑EGFR monoclonal antibody (EMab‑17) exerts antitumor activity against oral squamous cell carcinomas via antibody‑dependent cellular cytotoxicity and complement‑dependent cytotoxicity

“…Anti-EGFR mAbs possess several functional mechanisms, including ADCC, CDC, blocking dimerization, and EGFR endocytosis. For investigating ADCC and CDC in mouse xenograft models, the subclass of mAbs should be IgG 2a or IgG 2b of mouse IgG (22), IgG 2a or IgG 2b of rat IgG (23), IgG 1 of human IgG (24), or type B of canine IgG (25).…”

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

“…A mAb recognizing O -linked glycosylated amino acid sequences of a mucin-like protein is useful for detecting an antigen produced by abnormal cells 11 , 23 , 24 . These mAbs, such as LpMab-12 23 , UN1 24 , and KL-6 25 , can bind to peptides containing several types of glycans; i.e., they may recognize the conformational structure of amino acid residues that is affected by glycosylation rather than the structure of both the amino acid sequences and the attached glycan. Such glycopeptide-recognizing mAb has been used in clinical trials of antibody therapeutics against cancer.…”