Identification of Various Recombinants in a Patient Coinfected With the Different SARS-CoV-2 Variants.

BACKGROUND: Viral recombination that occurs by exchanging genetic materials between two viral genomes coinfecting the same host cells is associated with the emergence of new viruses with different virulence. Herein, we detected a patient coinfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants and identified various recombinants in the SARS-CoV-2 full-length spike gene using long-read and Sanger sequencing. METHODS: Samples from five patients in Japan with household transmission of coronavirus disease 2019 (COVID-19) were analyzed using molecular assays for detection and identification of SARS-CoV-2. Whole-genome sequencing was conducted using multiplex PCR with short-read sequencing. RESULTS: Among the five SARS-CoV-2-positive patients, the mutation-specific assay identified the Delta variant in three, the Omicron variant in one, and an undetermined in one. The undermined patient was identified as Delta using whole-genome sequencing, but samples showed a mixed population of Delta and Omicron variants. This patient was analyzed for viral quasispecies by long-read and Sanger sequencing using a full-length spike gene amplicon. In addition to the Delta and Omicron sequences, the viral quasispecies analysis identified nine different genetic recombinant sequences with various breakpoints between Delta and Omicron sequences. The nine detected recombinant sequences in the spike gene showed over 99% identity with viruses that were detected during the Delta and Omicron cocirculation period from the United States and Europe. CONCLUSIONS: This study demonstrates that patients coinfected with different SARS-CoV-2 variants can generate various viral recombinants and that various recombinant viruses may be produced during the cocirculation of different variants.

Seroprevalence of four endemic human coronaviruses and, reactivity and neutralization capability against SARS-CoV-2 among children in the Philippines.

Four endemic human coronaviruses (HCoV), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are closely related to SARS-CoV-2. These coronaviruses are known to infect humans living in temperate areas, including children under 5 years old; however, the seroprevalence of four HCoVs among children in tropical areas, including the Philippines, remains unclear. This study aimed to assess the prevalence of antibodies against four HCoVs and to determine the reactivity and neutralization of these antibodies against SARS-CoV-2 among children in the Philippines. A total of 315 serum samples collected from 2015 to 2018, before the emergence of SARS-CoV-2, in Biliran island, Philippines, were tested for the presence of antibodies against four HCoVs and SARS-CoV-2 using recombinant spike ectodomain proteins by IgG-enzyme-linked immunosorbent assay (ELISA). Reactivity to and neutralization of SARS-CoV-2 were also investigated. The seroprevalence of the four HCoVs was 63.8% for HCoV-229E, 71.4% for HCoV-NL63, 76.5% for HCoV-HKU1, and 83.5% for HCoV-OC43 by ELISA. Age group analysis indicated that seropositivity to all HCoVs reached 80% by 2-3 years of age. While 69/315 (21.9%) of the samples showed reactive to SARS-CoV-2, almost no neutralization against SARS-CoV-2 was detected using neutralization assay. Reactivity of antibodies against SARS-CoV-2 spike protein obtained by ELISA may not correlate with neutralization capability.

Comparison of Rhinovirus A-, B-, and C-Associated Respiratory Tract Illness Severity Based on the 5'-Untranslated Region Among Children Younger Than 5 Years.

Background: Rhinoviruses (RVs) are among the most frequently detected viruses from hospitalized children with severe acute respiratory infections, being classified into RV-A, RV-B, and RV-C (4 clades: C, GAC1, GAC2, and A2). This study aimed to compare the clinical characteristics and respiratory tract illness severity between the RV species and RV-C clades in children in primary care and hospital settings in rural communities in the Philippines. Methods: Clinical samples and information of children <5 years old in the Philippines were collected from 2014 to 2016. The samples were tested by reverse-transcription polymerase chain reaction (RT-PCR) targeting the 5'-untranslated region. PCR-positive samples were sequenced, and RV species were identified by phylogenetic analysis. Results: Overall, 3680 respiratory tract illness episodes in 1688 cohort children were documented; 713 of those were RV positive and identified as RV-A (n = 271), RV-B (n = 47), and RV-C (n = 395: C [n = 76], GAG1 [n = 172], GAG2 [n = 8], A2 [n = 138], and unidentified [n = 1]). Severe illnesses, low oxygen saturation, cough, and wheezing were more common in patients with RV-C, especially with GAC1, than in those with RV-A or RV-B. Furthermore, severe illness was significantly more common in RV-C (GAC1)-positive cases than in RV-A-positive cases (odds ratio, 2.61 [95% CI, 1.17-4.13]). Conclusions: Children infected with RV-C had more severe illnesses than children infected with RV-A and RV-B. Moreover, emerging clades of RV-C were associated with increased severity.

Japanese encephalitis virus genotype III from mosquitoes in Tarlac, Philippines.

Objectives: The aim of this study was to investigate the presence of Japanese encephalitis virus (JEV) in a rice-farming community in the Philippines and to determine its implications regarding the epidemiology of viral encephalitides in the Asia-Pacific Region. Methods: Mosquitoes were collected monthly from animal-baited traps close to flooded rice fields in two barangays (villages) in the Municipality of San Jose, Tarlac Province in Luzon, from May 2009 to July 2010. Virus was detected by nested reverse transcription PCR. Phylogenetic analysis of the amplified virus envelope gene was done using the maximum-likelihood method. Results: A total of 28 700 known vector mosquitoes were collected, namely Culex vishnui, Culex fuscocephala, Culex tritaeniorhynchus, and Culex gelidus. JEV genotype III was detected in C. tritaeniorhynchus, belonging to the same genotype but form a different clade from those reported in the 1980s and in 2020 in this country. Conclusions: Japanese encephalitis is associated with rice cultivation and the presence of infected mosquitoes in Tarlac, Philippines. It remains to be seen whether the observed genetic shift of genotype III to genotype I in Asia will in time have an impact on the epidemiology of Japanese encephalitis in the Philippines. For long-term disease control, regular surveillance and Japanese encephalitis immunization in children and travelers in high risk areas are recommended.

Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis.

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.

Development and characterization of TrMab­6, a novel anti­TROP2 monoclonal antibody for antigen detection in breast cancer.

Trophoblast cell­surface antigen 2 (TROP2) is a type I transmembrane glycoprotein that is overexpressed in a number of cancer types, including triple­negative breast cancer. The current study aimed to develop a highly sensitive and specific monoclonal antibody (mAb) targeting TROP2, which could be used to evaluate TROP2 expression using flow cytometry, western blot analysis and immunohistochemistry by employing the Cell­Based Immunization and Screening (CBIS) method. The established anti­TROP2 mAb, TrMab­6 (mouse IgG2b, κ), detected TROP2 on PA­tagged TROP2­overexpressing Chinese hamster ovary­K1 (CHO/TROP2­PA) and breast cancer cell lines, including MCF7 and BT­474 using flow cytometry. Western blot analysis indicated a 40 kDa band in lysates prepared from CHO/TROP2­PA, MCF7 and BT­474 cells. Furthermore, TROP2 in 57/61 (93.4%) of the breast cancer specimens was strongly detected using immunohistochemical analysis with TrMab­6. In conclusion, the current study demonstrated that TrMab­6 may be a valuable tool for the detection of TROP2 in a wide variety of breast cancer types.

The anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody EpMab-16 exerts antitumor activity in a mouse model of colorectal adenocarcinoma.

The epithelial cell adhesion molecule (EpCAM), which is a calcium-independent homophilic intercellular adhesion factor, contributes to cell signaling, differentiation, proliferation and migration. EpCAM is essential for carcinogenesis in numerous types of human cancer. The purpose of the present study was to establish an anti-EpCAM monoclonal antibody (mAb) for targeting colorectal adenocarcinomas. Thus, an anti-EpCAM mAb, EpMab-16 (IgG2a, κ), was established by immunizing mice with EpCAM-overexpressing CHO-K1 cells, and validated using flow cytometry, western blot, and immunohistochemical analyses. EpMab-16 reacted with endogenous EpCAM specifically in a colorectal adenocarcinoma cell line as determined by flow cytometry and western blot analyses. Immunohistochemical analysis demonstrated that EpMab-16 stained a plasma membrane-like pattern in clinical colorectal adenocarcinoma tissues. The dissociation constant (K D) for EpMab-16 in a Caco-2 colorectal adenocarcinoma cell line determined by flow cytometry was 1.8×10-8 M, suggesting moderate binding affinity of EpMab-16 for EpCAM. Whether the EpMab-16 induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Caco-2 or antitumor activity was then assessed in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC induction in Caco-2 cells by EpMab-16 treatment. In vivo experiments in a Caco-2 ×enograft model demonstrated that EpMab-16 treatment significantly reduced tumor growth compared with that in mice treated with the control mouse IgG. These results suggested that EpMab-16 may be a promising treatment option for EpCAM-expressing colorectal adenocarcinomas.

Development of Core-Fucose-Deficient Humanized and Chimeric Anti-Human Podoplanin Antibodies.

Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2 interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanized anti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27, named as P1027 and a core-fucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f against LN319 (a human glioblastoma cell line) are 1.1 × 10-8 and 3.9 × 10-9 M, respectively. ADCC reporter assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f may be a useful therapy for patients with PDPN-expressing cancers.

Anti­EpCAM monoclonal antibody exerts antitumor activity against oral squamous cell carcinomas.

The epithelial cell adhesion molecule (EpCAM) is a calcium­independent, homophilic, intercellular adhesion factor classified as a transmembrane glycoprotein. In addition to cell adhesion, EpCAM also contributes to cell signaling, differentiation, proliferation, and migration. EpCAM is an essential factor in the carcinogenesis of numerous human cancers. In the present study, we developed and validated an anti­EpCAM monoclonal antibody (mAb), EpMab­16 (IgG2a, kappa), by immunizing mice with EpCAM­overexpressing CHO­K1 cells. EpMab­16 specifically reacted with endogenous EpCAM in oral squamous cell carcinoma (OSCC) cell lines in flow cytometry and Western blot analyses. It exhibited a plasma membrane­like stain pattern in OSCC tissues upon immunohistochemical analysis. The KD for EpMab­16 in SAS and HSC­2 OSCC cells were assessed via flow cytometry at 1.1x10­8 and 1.9x10­8 M, respectively, suggesting moderate binding affinity of EpMab­16 for EpCAM. We then assessed whether the EpMab­16 induced antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC) against OSCC cell lines, and antitumor capacity in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC inducement against OSCC cells treated with EpMab­16. In vivo experiments on OSCC xenografts revealed that EpMab­16 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that EpMab­16 could be a promising treatment option for EpCAM­expressing OSCCs.

A defucosylated anti­CD44 monoclonal antibody 5­mG2a­f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti­CD44 monoclonal antibody (mAb), C44Mab­5 (IgG1, kappa) was established by immunizing mice with CD44­overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab­5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab­5 lacks antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC), the antitumor activity of C44Mab­5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab­5 into an IgG2a subclass antibody, 5­mG2a, and further produced a defucosylated version, 5­mG2a­f, using FUT8­deficient ExpiCHO­S (BINDS­09) cells. Defucosylation of 5­mG2a­f was confirmed using fucose­binding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5­mG2a­f against SAS and HSC­2 oral cancer cells were determined through flow cytometry to be 2.8x10­10 M and 2.6x10­9 M, respectively, indicating that 5­mG2a­f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5­mG2a­f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5­mG2a­f showed moderate ADCC and CDC activities against SAS and HSC­2 oral cancer cells. In vivo analysis revealed that 5­mG2a­f significantly reduced tumor development in SAS and HSC­2 xenografts in comparison to control mouse IgG, even after injection seven days post­tumor inoculation. Collectively, these results suggest that treatment with 5­mG2a­f may represent a useful therapy for patients with CD44­expressing oral cancers.

Anti­EGFR monoclonal antibody 134­mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo­ or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti­EGFR monoclonal antibody (mAb), EMab­134, by immunizing mice with the ectodomain of human EGFR. EMab­134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab­134 was converted from IgG1 to IgG2a (134­mG2a) to facilitate antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab­134 and 134­mG2a against EGFR­expressing CHO­K1 (CHO/EGFR) cells were determined by flow cytometry to be 3.2x10­9 M and 2.1x10­9 M, respectively; these results indicate that 134­mG2a has a higher binding affinity than EMab­134. The 134­mG2a antibody was more sensitive than EMab­134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134­mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC­2, and SAS cells. Moreover, the in vivo administration of 134­mG2a significantly inhibited the development of CHO/EGFR, HSC­2, and SAS mouse xenografts in comparison to the results observed in response to EMab­134. Taken together, the findings of the present study demonstrate that the newly­formulated 134­mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR­expressing oral cancer.

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.

An Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody H2Mab-19 Exerts Antitumor Activity in Mouse Colon Cancer Xenografts.

Trastuzumab is a humanized antibody against human epidermal growth factor receptor 2 (HER2) that offers significant survival benefits to patients with HER2-overexpressing breast or gastric cancer. HER2 is also known to be overexpressed in colon cancers. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, κ) was characterized for its anticancer activity in colon cancers. H2Mab-19 showed both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against Caco-2, a colon cancer cell line. Furthermore, H2Mab-19 significantly reduced tumor development in a Caco-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing colon cancers.

Epitope Mapping of the Anti-Diacylglycerol Kinase Monoclonal Antibody DhMab-4 for Immunohistochemical Analysis.

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKη is classified as a type II DGK. Reports have shown that DGKη is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG2b, kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKη and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKη.

Detection of Lion Podoplanin Using the Antitiger Podoplanin Monoclonal Antibody PMab-231.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.

H2Mab-19 Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody Therapy Exerts Antitumor Activity in Pancreatic Cancer Xenograft Models.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast cancer, gastric, lung, colorectal, oral, and pancreatic cancers. HER2 expression is associated with poor clinical outcomes. An anti-HER2 humanized antibody, trastuzumab, has improved survival rates in patients with HER2-overexpressing breast and gastric cancers. Previously, we established a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa). It has also been characterized for breast, oral, and colon cancers. In this study, we investigated the antitumor activities of H2Mab-19 in pancreatic cancer xenograft models. We selected MIA PaCa-2, a pancreatic cancer cell line which expresses HER2. H2Mab-19 showed high binding affinity (KD: 1.2 × 10-8 M) against MIA PaCa-2 cells. Furthermore, H2Mab-19 significantly reduced tumor development in a MIA PaCa-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing pancreatic cancers.

Thr80 of Sheep Podoplanin Is a Critical Epitope of the Antisheep Podoplanin Monoclonal Antibody: PMab-256.

An antisheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established. PMab-256 shows positive immunostaining for lymphatic endothelial cells, lung type I alveolar cells, and kidney podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG domains or PLD. The purpose of this study is to determine the binding epitope of PMab-256. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids 75 and 80 of sPDPN. Furthermore, analysis of sPDPN point mutations demonstrated that the critical epitope includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.

Development of an Anti-Sheep Podoplanin Monoclonal Antibody PMab-256 for Immunohistochemical Analysis of Lymphatic Endothelial Cells.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the Cell-Based Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1, kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256 stained type I alveolar cells of lung, renal glomerulus and Bowman's capsule, and lymphatic endothelial cells of lung and colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.

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.