Podoplanin is a platelet aggregation-inducing mucin-like sialoglycoprotein that is highly expressed in brain tumors, malignant mesotheliomas, testicular tumors, and lung, esophageal, or head and neck squamous cell carcinomas. Podoplanin has also been reported to be a marker to enrich tumor-initiating cells. We previously established a neutralizing monoclonal antibody (mAb), NZ-1, which inhibits the association between podoplanin and C-type lectin-like receptor-2 (CLEC-2) and blocks the podoplanin-induced platelet aggregation and tumor metastasis. A human chimeric mAb, NZ-8 possesses extremely high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and showed high anti-tumor activities in several xenograft models. However, those mAbs also react with normal cells including lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells throughout the body. Recently, we established the platform to produce a cancer-specific mAb (CasMab) by combining glycobiology and cell engineering technologies. Herein, we report a novel cancer-specific anti-podoplanin mAb, LpMab-2. In Western-blot analyses, LpMab-2 did not react with sialic acid-deficient podoplanin and O-glycan deficient podoplanin, which were expressed in Lec2 and Lec8 cells, respectively. In enzyme-linked immunosorbent assay using several deletion mutants of podoplanin, the epitope of LpMab-2 was shown to be included in 55-88 amino acids of podoplanin, although almost all anti-podoplanin mAbs recognize platelet aggregation-stimulating (PLAG) domain near the N-terminus. Analyses of glycopeptides produced by Edman degradation revealed that O-glycans were attached to glycosylation sites at residues Thr65, Thr66, Thr70, Ser71, Ser74, and Thr76 around LpMab-2 epitope. LpMab-2 did not react with podoplanin-negative cell lines, indicating that not only glycans but also amino acids of podoplanin are necessary for LpMab-2 reaction. In flow cytometric and immunohistochemical analyses, LpMab-2 reacted with tumor cells, but not with lung type I alveolar cells and lymphatic endothelial cells, suggesting that LpMab-2 recognizes the cancer-type conformation of podoplanin. Furthermore, human chimeric LpMab-2 also showed cancer-specific reaction against podoplanin. Taken together, a cancer-specific mAb LpMab-2 should be useful for molecular targeting therapy against podoplanin without side effects such as respiratory or renal failures and lymphatic edema. The CasMab-producing platform could lead to establishment of the side effect-reducing mAbs.
Note: This abstract was not presented at the meeting.
Citation Format: Yukinari Kato, Mika K. Kaneko. Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2014-663