Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

Furusawa, Yoshikazu; Takei, Junko; Sayama, Yusuke; Yamada, Shinji; Kaneko, Mika K.; Kato, Yukinari

doi:10.1016/j.bbrep.2019.100644

Cited by 42 publications

(17 citation statements)

References 25 publications

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“…(20) We successfully determined the binding epitope of those mAbs. (22,34,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) These epitope mapping results showed that almost all anti-PDPN mAbs react with PLAG domains or PLDs. (7,(39)(40)(41)43,44,(49)(50)(51) The critical epitope of PMab-237 was also shown to be located in PLD (Fig.…”

Section: Discussionmentioning

confidence: 75%

“…We have demonstrated that the critical epitope of PMab-237 could include Leu82 and Thr84 of wPDPN using the deletion mutants and point mutants of wPDPN in CHO-K1 cells by flow cytometry. In our previous studies, we developed many mAbs against PDPNs of human, (24) mouse, (25) rat, (26) rabbit, (27) dog, (28) cat, (29) bovine, (30) horse, (31) Tasmanian devil, (32) alpaca, (33) bear, (34) tiger, (35) goat, (36) pig, (37,38) and whale. (20) We successfully determined the binding epitope of those mAbs.…”

Section: Discussionmentioning

confidence: 99%

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Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

Sayama

Sano

Kaneko

et al. 2020

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregationstimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

Sayama

Sano

Kaneko

et al. 2020

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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“…(11) In this study, we selected a novel anti-bPDPN mAb (PMab-241) of IgG 2b subclass from previously established anti-bPDPN mAbs. (10) PMab-241 reacted with CHO/bPDPN, not parental CHO-K1 cells in flow cytometry (Fig. 1).…”

Section: Resultsmentioning

confidence: 93%

“…Although we have already established an anti-bPDPN mAb (PMab-247), which is sensitive and specific for immunohistochemical analyses, (10) it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung using PMab-247. (11) In this study, we selected a novel anti-bPDPN mAb (PMab-241) of IgG 2b subclass from previously established anti-bPDPN mAbs.…”

Section: Resultsmentioning

confidence: 99%

“…CHO/bear PDPN (bPDPN) was previously established. (10) CHO-K1 and CHO/bPDPN were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% of heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.). The cells were grown in an incubator at 37°C with humidity and 5% CO 2 and 95% air atmosphere.…”

Section: Cell Linesmentioning

confidence: 99%

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PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear

Takei

Yamada

Konnai

et al. 2019

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

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A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts antitumor effects in mouse xenograft models of pancreatic carcinoma

Kaneko

Ohishi

Kawada

et al. 2020

Biochemistry and Biophysics Reports

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Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG 2a -type mAb, named as 60-mG 2a , to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG 2a , named as 60-mG 2a -f, to augment its ADCC activity. In vivo analysis revealed that 60-mG 2a -f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG 2a -f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.

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Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

Cited by 42 publications

References 25 publications

Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear

A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts antitumor effects in mouse xenograft models of pancreatic carcinoma

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