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

Mika K Kaneko,Shinji Abe,Yasuhiko Nishioka,Hiroyuki Harada,Ryusuke Honma,Yukinari Kato,Hiroyoshi Suzuki,Michiaki Takagi,Yuki Fujii,Satoshi Ogasawara,Takuro Nakamura

doi:10.1002/cam4.954

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 glycan‐deficient 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.

Highlights

IntroductionHuman podoplanin (hPDPN) is expressed in many cancers, including malignant gliomas, lung cancers, esophageal cancers, malignant mesotheliomas, testicular cancers, bladder cancers, and osteosarcomas [1,2,3,4,5,6,7,8,9,10,11,12,13], and the expression of hPDPN in cancer-associated fibroblasts contributes to poor prognosis [14,15,16,17,18,19]
Our comparative crystallographic studies of the Human podoplanin (hPDPN)–CLEC-2 complex [24] revealed that CLEC-2 binds to hPDPN through residues Glu47 and Asp48 within its platelet aggregation-stimulating (PLAG) domain as well as to the α2-6-linked sialic acid linked to Thr52
LpMab-21 detected endogenous hPDPN, which is expressed in the glioblastoma cell line LN319, but not in LN319/hPDPN-K O cells (PDIS - 6), indicating that LpMab-21 is specific against hPDPN (Fig. 1B)

Summary

Introduction

Human podoplanin (hPDPN) is expressed in many cancers, including malignant gliomas, lung cancers, esophageal cancers, malignant mesotheliomas, testicular cancers, bladder cancers, and osteosarcomas [1,2,3,4,5,6,7,8,9,10,11,12,13], and the expression of hPDPN in cancer-associated fibroblasts contributes to poor prognosis [14,15,16,17,18,19]. Human PDPN (known as the platelet aggregation-inducing factor Aggrus) is involved in cancer metastasis [11, 20, 21]. Our comparative crystallographic studies of the hPDPN–CLEC-2 complex [24] revealed that CLEC-2 binds to hPDPN through residues Glu and Asp within its platelet aggregation-stimulating (PLAG) domain as well as to the α2-6-linked sialic acid linked to Thr. Sensitive and specific anti-hPDPN mAb are required to clarify the physiological function of hPDPN in normal tissues and cancers. We generated a novel anti-hPDPN mAb designated LpMab-21 that recognizes a sialylated glycopeptide epitope

Methods

Results

Conclusion