Platelet Aggregation-stimulating Research Articles

Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260.

Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.

Podoplanin is indispensable for cell motility and platelet-induced epithelial-to-mesenchymal transition-related gene expression in esophagus squamous carcinoma TE11A cells

BackgroundThe transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood.MethodsSubclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed.ResultsPDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-β-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-β-induced EMT induction.ConclusionsThese results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

Epitope Mapping of PMab-241, a Lymphatic Endothelial Cell-Specific Anti-Bear Podoplanin Monoclonal Antibody.

Anti-bear podoplanin (bPDPN) monoclonal antibodies (mAbs), including PMab-247 and PMab-241, have been previously established. Although PMab-247 has shown positive immunostaining for lymphatic endothelial cells (LECs), type I alveolar cells of the lung, and podocytes of the kidney, PMab-241 stains LECs but does not react with lung type I alveolar cells. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and the PLAG-like domain (PLD). The binding epitope of PMab-247 was previously determined to include bPDPN residues Asp76, Arg78, Glu80, and Arg82. Among these, Glu80 and Arg82 are included in PLD of bPDPN. The purpose of this study is to determine the binding epitope of PMab-241 and to clarify the difference between these two anti-bPDPN mAbs. Analysis of bPDPN deletion mutants revealed that the N-terminus of the PMab-241 epitope exists between amino acids (aa) 75 and 80 of bPDPN. In addition, analysis of bPDPN point mutants demonstrated that the critical epitope of PMab-241 includes Thr75, Asp76, and Arg78 of bPDPN. The binding epitopes of PMab-241 and PMab-247 seem to overlap, but this slight difference may be sufficient to provide the specificity of PMab-241 to discriminate LECs from type I alveolar cells of the lung.

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

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 aggregation-stimulating (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.

The Epitope of PMab-210 Is Located in Platelet Aggregation-Stimulating Domain-3 of Pig Podoplanin.

Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.

Epitope Analysis of an Antihorse Podoplanin Monoclonal Antibody PMab-219.

Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression is associated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. In addition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In this study, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between 55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 of horPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.

Epitope Mapping of Monoclonal Antibody PMab-233 Against Tasmanian Devil Podoplanin.

The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses three tandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAG-like domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to be clarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34 of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.

Epitope Mapping of PMab-225 an Anti-Alpaca Podoplanin Monoclonal Antibody Using Flow Cytometry.

Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.

Epitope Mapping of Anti-Bear Podoplanin Monoclonal Antibody PMab-247.

Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs.

Epitope Mapping of Antipig Podoplanin Monoclonal Antibody PMab-213.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on lymphatic endothelial cells, podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor, comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported a mouse antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developed by CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213 using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN.

Characterization of Anti-Goat Podoplanin Monoclonal Antibody PMab-235 Using Immunohistochemistry Against Goat Tissues.

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231.

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.

Epitope Mapping of the Antihorse Podoplanin Monoclonal Antibody PMab-202.

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2.

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1

Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression in cancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectin-like receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in flow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, the binding epitope of PMab-1 remains to be clarified. In the present study, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These findings can be applied to the production of more functional anti-mPDPN monoclonal antibodies.

Platelet-activating factor podoplanin: from discovery to drug development

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin–CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44.

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

Human podoplanin (hPDPN), a platelet aggregation‐inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C‐type lectin‐like receptor 2 (CLEC‐2). The overexpression of hPDPN is involved in invasion and metastasis. Anti‐hPDPN monoclonal antibodies (mAbs) such as NZ‐1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation‐stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti‐hPDPN mAb, LpMab‐2, using the cancer‐specific mAb (CasMab) technology. In this study we developed chLpMab‐2, a human–mouse chimeric anti‐hPDPN antibody, derived from LpMab‐2. chLpMab‐2 was produced using fucosyltransferase 8‐knockout (KO) Chinese hamster ovary (CHO)‐S cell lines. By flow cytometry, chLpMab‐2 reacted with hPDPN‐expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab‐2 exhibited high antibody‐dependent cellular cytotoxicity (ADCC) against PDPN‐expressing cells, despite its low complement‐dependent cytotoxicity. Furthermore, treatment with chLpMab‐2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab‐2 suppressed tumor development via ADCC. In conclusion, chLpMab‐2 could be useful as a novel antibody‐based therapy against hPDPN‐expressing tumors.

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin.

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.

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin.

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.