Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin

Our plain-English summary of Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin, by S. Ogasawara, M.K. Kaneko, R. Honma, et al (Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2016).

The study

• Authors: S. Ogasawara, M.K. Kaneko, R. Honma, H. Oki, Y. Fujii, M. Takagi, H. Suzuki, Y. Kato
• Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
• Published: 2016;35(3):155-62
• Cited by: 19 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

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.

In plain English

This laboratory study from Japan described the making and testing of a new mouse antibody, called LpMab-13, against human podoplanin. Podoplanin is a protein found on many tumor cells and on some normal cells. The study did not examine asbestos exposure. Mice were immunized with glioblastoma cells engineered to carry human podoplanin. In lab tests, LpMab-13 recognized podoplanin made naturally by a glioblastoma cell line and by lymphatic endothelial cells. Using flow cytometry, it also detected podoplanin on a malignant mesothelioma cell line, NCI-H226, although Western blot did not detect it in that cell line. It did not react with cells whose podoplanin gene had been knocked out. Its binding strength was 2.9 times 10 to the minus 8 molar, which the authors called moderate. In tissue sections, it stained glioblastoma, oral and esophageal squamous cell carcinoma, osteosarcoma, seminoma, lymphatic vessels and lung type I alveolar cells. Its binding site was mapped to amino acids Ala42 to Asp49. The authors concluded that LpMab-13 could be useful for investigating podoplanin in cancers and normal tissues.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study developing and characterizing a mouse monoclonal antibody (mouse immunization, cell line tests, tissue staining)
• Who was studied: Human cell lines (including glioblastoma, oral cancer, lung squamous cell carcinoma, kidney, lymphatic endothelial cells and the malignant mesothelioma cell line NCI-H226), female BALB/c mice, and tumor tissue from cancer patients who had surgery at Sendai Medical Center and Yamagata University Hospital, Japan. The number of patients is not given.
• Exposure: No asbestos or other environmental exposure was studied. The study tested the antibody LpMab-13 on cells and tissues.
• Main results:
  • LpMab-13 recognized podoplanin made naturally by the glioblastoma cell line LN319 and by lymphatic endothelial cells, in Western blot and flow cytometry.
  • In flow cytometry, LpMab-13 detected podoplanin of the malignant mesothelioma cell line (printed 'HCI-H226', elsewhere NCI-H226). Podoplanin of NCI-H226 and HEK293T was not detected by Western blot.
  • LpMab-13 did not react with cell lines in which the podoplanin gene had been knocked out.
  • LpMab-13 detected all of the glycan-deficient podoplanin transfectants tested.
  • The binding constant (KD) on LN319 cells was 2.9 times 10 to the minus 8 molar, which the authors described as moderate affinity.
  • In tissue sections, LpMab-13 stained glioblastoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, osteosarcoma and seminoma. It also reacted with lymphatic endothelial cells and lung type I alveolar cells.
  • Binding was lost with the point mutations A42G, G45A, D48A and D49A. The minimum binding site was identified as Ala42 to Asp49 of human podoplanin.
• The authors' conclusion: The authors concluded that LpMab-13 could be useful for investigating the expression and function of human podoplanin in cancers and normal tissues.

Topics

Medical terms in this study, explained in our Lexicon: Monoclonal Antibody, Podoplanin (D2-40), Monoclonal, Lung Cancer, Malignant Mesothelioma, Immunohistochemistry.

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