RESUMEN
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.
Asunto(s)
COVID-19 , Coinfección , Genoma Viral , Recombinación Genética , SARS-CoV-2 , Secuenciación Completa del Genoma , Humanos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , COVID-19/virología , COVID-19/complicaciones , Coinfección/virología , Genoma Viral/genética , Glicoproteína de la Espiga del Coronavirus/genética , Masculino , Japón , Femenino , Filogenia , Mutación , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Anticuerpos Neutralizantes , COVID-19 , Infecciones por Coronavirus , Coronavirus , Niño , Preescolar , Humanos , Anticuerpos Antivirales , Coronavirus Humano 229E , Coronavirus Humano NL63 , Coronavirus Humano OC43 , COVID-19/epidemiología , COVID-19/inmunología , Filipinas/epidemiología , Proteínas Recombinantes , SARS-CoV-2 , Estudios Seroepidemiológicos , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Coronavirus/genética , Coronavirus/inmunología , Betacoronavirus , Anticuerpos Neutralizantes/inmunologíaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/inmunología , Neoplasias de la Mama/inmunología , Moléculas de Adhesión Celular/inmunología , Ratones Endogámicos BALB C/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino , Células CHO , Cricetulus , Femenino , Humanos , Células MCF-7 , RatonesRESUMEN
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.
RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Glioblastoma/tratamiento farmacológico , Lectinas Tipo C/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Células CHO , Línea Celular Tumoral , Cricetulus , Fucosa/genética , Fucosa/inmunología , Glioblastoma/genética , Glioblastoma/inmunología , Glioblastoma/patología , Humanos , Inmunoglobulina G/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Metástasis de la Neoplasia , Activación Plaquetaria/inmunología , Agregación Plaquetaria/inmunología , Unión Proteica/inmunología , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Molécula de Adhesión Celular Epitelial/antagonistas & inhibidores , Neoplasias de la Boca/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antineoplásicos Inmunológicos/uso terapéutico , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Cricetulus , Femenino , Humanos , Ratones , Neoplasias de la Boca/inmunología , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Receptores de Hialuranos/antagonistas & inhibidores , Neoplasias de la Boca/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/aislamiento & purificación , Antineoplásicos Inmunológicos/farmacología , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetulus , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Neoplasias de la Boca/inmunología , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Células CHO , Carcinoma de Células Escamosas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetulus , Femenino , Inmunoglobulina G/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Boca/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos , Glicoproteínas de Membrana/inmunología , Ovinos/inmunología , Animales , Western Blotting , Células CHO , Cricetulus , Células Endoteliales/inmunología , Mapeo Epitopo , Femenino , Citometría de Flujo , Inmunohistoquímica/métodos , Glicoproteínas de Membrana/genética , Ratones Endogámicos BALB C , MutaciónRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Receptor ErbB-2/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Células CACO-2 , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Femenino , Humanos , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Diacilglicerol Quinasa/inmunología , Mapeo Epitopo/métodos , Animales , Cerebelo/inmunología , Cerebelo/patología , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Epítopos/metabolismo , Humanos , Inmunohistoquímica , Ratones Endogámicos , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Leones/inmunología , Tigres/inmunología , Células Epiteliales Alveolares/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos/inmunología , Células CHO , Gatos , Bovinos , Cricetinae , Cricetulus , Mapeo Epitopo , Epítopos/biosíntesis , Caballos/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Podocitos/inmunología , Conejos , Ratas , Ovinos/inmunología , Porcinos/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Receptor ErbB-2/inmunología , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina G/inmunología , Ratones , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Receptor ErbB-2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Ovinos/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos/inmunología , Células CHO , Cricetinae , Cricetulus , Células Endoteliales/inmunología , Mapeo Epitopo , Epítopos/biosíntesis , Humanos , Podocitos/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Células Endoteliales/inmunología , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos/inmunología , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Células CHO , Gatos , Bovinos , Cricetinae , Cricetulus , Perros , Mapeo Epitopo , Citometría de Flujo , Cabras , Caballos/inmunología , Humanos , Ratones , Agregación Plaquetaria/inmunología , Podocitos/inmunología , Conejos , Ratas , Ovinos/inmunología , Porcinos/inmunología , TigresRESUMEN
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.