Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin

Kato, Yoshinori; Ogasawara, Satoshi; Oki, Hiroharu; Honma, Ryusuke; Takagi, Michiaki; Fujii, Yuki; Nakamura, Takuro; Saidoh, Noriko; Kanno, Hazuki; Umetsu, Mitsuo; Kamata, Satoshi; Kubo, Hiroshi; Yamada, Mitsuhiro; Sawa, Yoshihiko; Morita, Kei; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K.

doi:10.1089/mab.2015.0077

Cited by 31 publications

(18 citation statements)

References 37 publications

(48 reference statements)

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“…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 .…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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Podoplanin (PDPN) is expressed on many tumors and is involved in tumor metastasis. The objective of the present study was to develop an ELISA for determining soluble PDPN (sPDPN) levels as a potential novel tumor marker in plasma of patients with cancers for detection of tumor occurrence and metastasis. Mouse monoclonal antibodies (mAb) against human PDPN were developed and characterized. Two anti‐PDPN mAb, SZ‐163 and SZ‐168, were used in a sandwich ELISA to detect plasma sPDPN in patients with cancers and in normal individuals. The levels of sPDPN were detected in patients with adenocarcinoma (87 cases, 31.09 ± 5.48 ng/ml), squamous cell carcinoma (86 cases, 6.91 ± 0.59 ng/ml), lung cancer (45 cases, 26.10 ± 7.62 ng/ml), gastric cancer (38 cases, 23.71 ± 6.90 ng/ml) and rectal cancer (27 cases, 32.98 ± 9.88 ng/ml), which were significantly higher than those in normal individuals (99 cases, 1.31 ± 0.13 ng/ml) (P < .0001). Moreover, the sPDPN levels in patients with metastatic cancers were higher (192 cases, 30.35 ± 3.63 ng/ml) than those in non‐metastatic cancer patients (92 cases, 6.28 ± 0.77 ng/ml) (P < .0001). The post‐treatment sPDPN levels of cancer patients (n = 156) (4.47 ± 0.35 ng/ml) were significantly lower compared with those seen pre‐treatment (n = 128) (43.74 ± 4.97 ng/ml) (P < .0001). These results showed that an ELISA method was successfully established for quantitation of plasma sPDPN and plasma sPDPN levels correlate significantly with tumor occurrence and metastasis.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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“…(22) Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washing with PBS, the cells were treated with PMab-1 (1 μg/mL) or LpMab-17 (1 μg/mL) for 30 minutes at 4°C, followed by treatment with 1:1000 diluted Oregon Green 488 goat anti-rat or mouse IgG (Thermo Fisher Scientific, Inc.).…”

Section: Methodsmentioning

confidence: 99%

MAP Tag: A Novel Tagging System for Protein Purification and Detection

Fujii

Kaneko

Kato

2016

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Protein purification is an essential procedure in fields such as biochemistry, molecular biology, and biophysics. Acquiring target proteins with high quality and purity is still difficult, although several tag systems have been established for protein purification. Affinity tag systems are excellent because they possess high affinity and specificity for acquiring the target proteins. Nevertheless, further affinity tag systems are needed to compensate for several disadvantages of the presently available affinity tag systems. Herein, we developed a novel affinity tag system designated as the MAP tag system. This system is composed of a rat anti-mouse podoplanin monoclonal antibody (clone PMab-1) and MAP tag (GDGMVPPGIEDK) derived from the platelet aggregation-stimulating domain of mouse podoplanin. PMab-1 possesses high affinity and specificity for the MAP tag, and the PMab-1/MAP tag complex dissociates in the presence of the epitope peptide, indicating that the MAP tag system is suitable for protein purification. We successfully purified several proteins, including a nuclear protein, soluble proteins, and a membrane protein using the MAP tag system. The MAP tag system is very useful not only for protein purification but also in protein detection systems such as western blot and flow cytometric analyses. Taken together, these findings indicate that the MAP tag system could be a powerful tool for protein purification and detection.

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“…(23)(24)(25)27,28,(30)(31)(32) LN229/hPDPN cells were immunized into mice to develop novel anti-hPDPN MAbs. The culture supernatants were screened using ELISA for the binding to recombinant hPDPN purified from LN229/hPDPN cells.…”

Section: Production Of a Novel Anti-pdpn Mab Lpmab-13mentioning

confidence: 99%

“…The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein. This study examined cancer patients who underwent surgery at Sendai Medical Center (27) and Yamagata University Hospital. (6,28) Informed consent for obtaining samples and for subsequent data analyses was obtained from patients or the patient's guardian.…”

Section: Introductionmentioning

confidence: 99%

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin

OgasawaraSatoshi

Kaneko

HonmaRyusuke

et al. 2016

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many anti-hPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established; however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced a novel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was identified as Ala42-Asp49 of hPDPN using Western blot and flow cytometry. The combination of different epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.

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Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin

Cited by 31 publications

References 37 publications

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

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

MAP Tag: A Novel Tagging System for Protein Purification and Detection

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin

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