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Chemodynamic therapy (CDT), an emerging cancer treatment modality, uses multivalent metal elements to convert endogenous hydrogen peroxide (H2O2) to toxic hydroxyl radicals (â¢OH) via a Fenton or Fenton-like reaction, thus eliciting oxidative damage of cancer cells. However, the antitumor potency of CDT is largely limited by the high glutathione (GSH) concentration and low catalytic efficiency in the tumor sites. The combination of CDT with chemotherapy provides a promising strategy to overcome these limitations. In this work, to enhance antitumor potency by tumor-targeted and GSH depletion-amplified chemodynamic-chemo therapy, the hyaluronic acid (HA)/polydopamine (PDA)-decorated Fe2+-doped ZIF-8 nano-scaled metal-organic frameworks (FZ NMs) were fabricated and utilized to load doxorubicin (DOX), a chemotherapy drug, via hydrophobic, π-π stacking and charge interactions. The attained HA/PDA-covered DOX-carrying FZ NMs (HPDFZ NMs) promoted DOX and Fe2+ release in weakly acidic and GSH-rich milieu and exhibited acidity-activated â¢OH generation. Through efficient CD44-mediated endocytosis, the HPDFZ NMs internalized by CT26 cells not only prominently enhanced â¢OH accumulation by consuming GSH via PDA-mediated Michael addition combined with Fe2+/Fe3+ redox couple to cause mitochondria damage and lipid peroxidation, but also achieved intracellular DOX release, thus eliciting apoptosis and ferroptosis. Importantly, the HPDFZ NMs potently inhibited CT26 tumor growth in vivo at a low DOX dose and had good biosafety, thereby showing promising potential in tumor-specific treatment.
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The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) has emerged as a promising strategy for cancer treatment. However, the poor photostability and photothermal conversion efficiency (PCE) of organic small-molecule photosensitizers, and the intracellular glutathione (GSH)-mediated singlet oxygen scavenging largely decline the antitumor efficacy of PTT and PDT. Herein, a versatile nanophotosensitizer (NPS) system is developed by ingenious incorporation of indocyanine green (ICG) into the PEGylated chitosan (PEG-CS)-coated polydopamine (PDA) nanoparticles via multiple π-π stacking, hydrophobic and electrostatic interactions. The PEG-CS-covered NPS showed prominent colloidal and photothermal stability as well as high PCE (ca 62.8 %). Meanwhile, the Michael addition between NPS and GSH can consume GSH, thus reducing the GSH-induced singlet oxygen scavenging. After being internalized by CT26 cells, the NPS under near-infrared laser irradiation produced massive singlet oxygen with the aid of thermo-enhanced intracellular GSH depletion to elicit mitochondrial damage and lipid peroxide formation, thus leading to ferroptosis and apoptosis. Importantly, the combined PTT and PDT delivered by NPS effectively inhibited CT26 tumor growth in vivo by light-activated intense hyperthermia and redox homeostasis disturbance. Overall, this work presents a new tactic of boosting antitumor potency of ICG-mediated phototherapy by PEG-CS-covered NPS.
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Quitosana , Glutationa , Nanopartículas , Fotoquimioterapia , Fármacos Fotossensibilizantes , Terapia Fototérmica , Polietilenoglicóis , Quitosana/química , Fotoquimioterapia/métodos , Animais , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Glutationa/metabolismo , Polietilenoglicóis/química , Camundongos , Nanopartículas/química , Terapia Fototérmica/métodos , Linhagem Celular Tumoral , Verde de Indocianina/química , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oxigênio Singlete/metabolismo , Humanos , Apoptose/efeitos dos fármacos , Indóis/química , Indóis/farmacologia , Polímeros/químicaRESUMO
[This corrects the article DOI: 10.1371/journal.pntd.0001636.].
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Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.
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Interleucina-17 , Receptores de Interleucina-17 , Humanos , Camundongos , Animais , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Fibronectinas/metabolismo , Ligantes , Anticorpos Neutralizantes/metabolismo , Regulação Neoplásica da Expressão Gênica , Carcinogênese , Anticorpos Monoclonais/metabolismoRESUMO
OBJECTIVE: To study the association of α2,6-sialic acid (SIA) content in serum IgG anti-dsDNA with human systemic lupus erythematosus disease activity index (SLEDAI) and the effect of sialylated and desialylated (deSIA) IgG anti-dsDNA on lupus B cells. METHODS: Blood from lupus patients was collected to determine the ratio of SIA in isolated IgG anti-dsDNA over serum IgG anti-dsDNA (SIA/IgG anti-dsDNA) ratios, which were plotted against SLEDAI using a receiver-operating-characteristics curve. Lupus B cells were cultured in vitro with chimeric sialylated IgG anti-dsDNA and its deSIA form. Culture supernatants were assayed for anti-inflammatory IL-10 and SIA/IgG anti-dsDNA ratios, which were compared among different pre-treatment groups using t-tests. RESULTS: The area-under-the-curve (AUC) for anti-dsDNA levels against SLEDAI was 0.791 positively (95% confidence interval [C.I.]: 0.699-0.884) and SIA/IgG anti-dsDNA ratios against SLEDAI yielded an AUC of 0.705 inversely (95% C.I: 0.601-0.809): not significantly different. SIA/IgG anti-dsDNA ratios discriminated significantly between patients without and patients with proteinuria (p = .046). SIA/IgG anti-dsDNA ratios correlated significantly and positively with serum C3c and C4 levels. Pre-treatment with IgG anti-dsDNA and its immune complexes (dsDNA/IgG anti-dsDNA IC) induced higher IL-10 from lupus B cells than medium pre-treatment (most p < .01 from day 2 to day 5 culture). DeSIA IgG anti-dsDNA IC induced lower IL-10 (p < .05) and lower SIA/IgG anti-dsDNA ratios (p < .001) from lupus B cells than medium and dsDNA pre-treatment. CONCLUSION: α2,6-SIA/IgG anti-dsDNA ratios inversely forecasted SLEDAI scores. Possible mechanisms may be due to the different effects of sialylated and deSIA IgG anti-dsDNA on lupus B cells in terms of IL-10 secretion and SIA/IgG anti-dsDNA ratios.
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Lúpus Eritematoso Sistêmico , Ácido N-Acetilneuramínico , Anticorpos Antinucleares , DNA , Humanos , Imunoglobulina G , Interleucina-10 , Projetos PilotoRESUMO
OBJECTIVE: Stromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC. DESIGN: Nitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro-in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo. RESULTS: The delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy. CONCLUSION: The co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Humanos , Camundongos , Nanogéis , Óxido Nítrico , Neoplasias Pancreáticas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.
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Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Cricetinae , Modelos Animais de Doenças , Feminino , Masculino , CamundongosRESUMO
Epidermal growth factor receptor (EGFR) remains the sole druggable molecular target other than the PD1/PD-L1 pathway with meaningful clinical benefit in squamous cell carcinoma of head and neck (SCCHN). Human epidermal growth factor receptor 3 (HER3) confers the resistance to EGFR-targeted treatment in SCCHN. Thus, it is essential to determine the distribution and regulatory mechanisms of HER3 in SCCHN. We explored the prevalence of HER3 expression and its distribution within SCCHN by immunohistochemical staining and clinicopathological correlations were analyzed. The regulatory mechanism of HER3 expression was then dissected in vitro, using RT-PCR, Western blotting, and immunoprecipitation in a set of SCCHN cell lines. Subsequent in vivo validation in the murine model was also performed. We found that concomitant high expression of HER3 and its ligand NRG1 in SCCHN is associated with the increased presence of regional lymphatic metastasis and the majority of HER3 is located on the differentiated tumor cells. Further investigation revealed that HER3 is under positive control of NOTCH1 through transcriptional activation and inhibition of protein degradation through the polyubiquitination machinery via AKT pathway and USP8 deubiquitinating enzyme. In addition, loss of function of NOTCH1 suppresses HER3 expression through increased phosphorylation of serine 473 of AKT in SCCHN cells, and promotes the aggressiveness of the tumor cells. These data indicated that the level of HER3 is regulated by NOTCH1 in SCCHN both transcriptionally and post-translationally, and NOTCH1 is in a higher hierarchy in the regulatory system of the AKT pathway. Since NOTCH1 is inactivated in approximately 10% of SCCHN cases and this aberration strongly impacts the AKT pathway and diminishes HER3, exclusion of patients with NOTCH1-inactivated SCCHN may be beneficial for future clinical trials of HER3-targeting antibodies.
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BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.
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COVID-19 , Epitopos de Linfócito B , Biblioteca de Peptídeos , SARS-CoV-2 , Proteínas Virais , COVID-19/genética , COVID-19/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Humanos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.
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Neoplasias da Mama/diagnóstico , Radioisótopos de Gálio/farmacocinética , Radioisótopos de Índio/farmacocinética , Oligopeptídeos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imagem Multimodal , Oligopeptídeos/química , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND OBJECTIVES: Squamous cell carcinoma of head and neck (SCCHN) is the fifth most prevalent cancer worldwide. Because the anatomical complexity of this region, complete surgical resection is often not achievable and conventional chemotherapy would aid locoregional control and mitigate distant metastasis. Nonetheless, the nonspecific cytotoxicity and short in vivo half-life of conventional chemotherapeutic drugs limit their effects. Given the high frequency of overexpression of wild type epidermal growth factor receptor (EGFR), we exploit EGFR as a homing beacon for drug delivery system with cytotoxic payloads. MATERIALS AND METHODS: We generated fully human anti-EGFR single chain variable fragment (scFv)-conjugated immunoliposomes (IL) containing doxorubicin and vinorelbine and tested their anti-neoplastic efficacy in vitro and in vivo. RESULT: Our IL enhanced endocytosis and significantly reduced the half maximal inhibitory concentrations of the therapeutic payloads when compared to non-targeting liposomal counterparts in various cell lines of SCCHN. Furthermore, median survival time was significantly prolonged in subcutaneous and orthotopic SCCHN xenograft murine models treated with our IL formulations than those treated with non-targeting counterparts (94 days versus 60 days and 72 days versus 56 days, respectively) without evident increased systemic toxicity. CONCLUSION: The therapeutic index of the chemotherapeutic payloads was augmented by our EFGR-targeting IL formulation and they are warranted for further development and preclinical trial.
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Receptores ErbB/antagonistas & inibidores , Lipossomos/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Animais , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCID , Transplante HeterólogoRESUMO
It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.
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Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Transmissíveis/tratamento farmacológico , Doenças Metabólicas/tratamento farmacológico , Neoplasias/tratamento farmacológico , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti-VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti-VEGFR2-AF, which shows excellent VEGFR2 binding activity. Anti-VEGFR2-AF bound Ig-like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF-A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti-VEGFR2-AF inhibited capillary structure formation and exerted Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC-3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC-3 xenograft mouse model, treatment with anti-VEGFR2-AF repressed tumor growth and angiogenesis as effectively and safely as US FDA-approved anti-VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti-VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL-60 human leukemia-xenografted mice, anti-VEGFR2-AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti-VEGFR2-AF has strong potential as a cancer therapy that could directly target VEGFR2-expressing tumor cells in addition to its anti-angiogenic action.
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Inibidores da Angiogênese/uso terapêutico , Leucemia/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos/uso terapêutico , Linhagem Celular Tumoral , Epitopos de Linfócito B , Humanos , Masculino , Camundongos , Fosforilação , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epithelial cell adhesion molecule (EpCAM) is highly expressed in colon cancers, but its role in cancer progression remains to be elucidated. In this work, we found that the extracellular domain of EpCAM (EpEX) activated EGFR and downstream ERK1/2 signaling to promote colon cancer cell migration and proliferation, as well as tumor growth. Mechanistically, we discovered that EpEX-EGFR-ERK1/2 signaling positively regulated intramembrane proteolysis (RIP) of EpCAM and shedding of the intracellular domain (EpICD). Treatment with an EGFR inhibitor ablated the EpEX-induced phosphorylation of ERK1/2 and AKT. Additionally, treatment with inhibitors of either EGFR or MEK decreased EpEX-induced EpICD shedding and further revealed that EpICD is necessary for nuclear accumulation of ß-catenin and the induction of HIF1α target gene expression in vitro and in vivo. Moreover, an anti-EpCAM neutralizing monoclonal antibody, EpAb2-6, inhibited the nuclear translocation of EpICD and ß-catenin and induced apoptosis in colon cancer cells. Importantly, analysis of colorectal cancer tissues showed that nuclear accumulation of EpICD was highly correlated with metastasis and poor prognosis, suggesting that it may play an important functional role in cancer progression. Thus, we provide novel insights into the mechanisms and functions of EpEX-mediated signaling, which may be considered as a promising target for the treatment of colon cancer.
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Núcleo Celular/metabolismo , Neoplasias do Colo/patologia , Molécula de Adesão da Célula Epitelial/química , Molécula de Adesão da Célula Epitelial/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Linhagem Celular Tumoral , Movimento Celular , Neoplasias do Colo/metabolismo , Progressão da Doença , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Transplante de Neoplasias , Fosforilação , Prognóstico , Domínios Proteicos , Regulação para CimaRESUMO
Dengue virus (DENV) circulates in tropical and subtropical areas around the world, where it causes high morbidity and mortality. There is no effective treatment of infection, with supportive care being the only option. Furthermore, early detection and diagnosis are important to facilitate clinical decisions. In this study, seven monoclonal antibodies (mAbs) recognizing nonstructural protein 1 (NS1) of DENV were generated by hybridoma techniques. These antibodies can be divided into two groups: serotype-specific (DB6-1, DB12-3, and DB38-1) and nonspecific (consisting of antibodies DB16-1, DB20-6, DB29-1, and DB41-2). The B-cell epitopes of DB20-6 and DB29-1 were identified by phage display and site-directed mutagenesis, and its binding motif, WXXWGK, was revealed to correspond to amino acid residues 115-120 of the DENV-2 NS1 protein. A diagnostic platform, consisting of a serotype-specific capture antibody and a complex detection antibody, exhibited a detection limit of about 1 ng/mL, which is sufficient to detect NS1 in clinical serum samples from dengue patients. This diagnostic platform displayed better specificity and sensitivity than two examined commercial NS1 diagnostic platforms. In summary, our results indicate that these newly generated mAbs are suitable for detection of NS1 protein of DENV-2 in clinical samples.
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Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Dengue/genética , Dengue/diagnóstico , Dengue/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SorogrupoRESUMO
Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.
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Pesquisa Biomédica/métodos , Epitopos de Linfócito B , Epitopos de Linfócito T , Biblioteca de Peptídeos , Animais , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , HumanosRESUMO
Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.
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Vírus da Dengue/fisiologia , Dengue/imunologia , Epitopos/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Mapeamento de Epitopos , Epitopos/administração & dosagem , Epitopos/química , Epitopos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Vacinas de DNA/efeitos adversos , Vacinas de DNA/química , Vacinas de DNA/genética , Proteínas do Envelope Viral , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
BACKGROUND: The survival rate of head and neck squamous cell carcinoma (HNSCC) at advanced stage is poor, despite contemporary advances in treatment modalities. Recent studies have indicated that astrocyte elevated gene-1 (AEG-1), a single transmembrane protein without any known functional domains, is overexpressed in various malignancies and is implicated in both distant metastasis and poor survival. RESULTS: High expression of AEG-1 in HNSCC was positively correlated with regional lymph node metastasis and a poor 5-year survival rate. Knockdown of AEG-1 in HNSCC cell lines reduced their capacity for colony formation, migration and invasion. Furthermore, decreased tumor volume and metastatic foci were observed after knockdown of AEG-1 in subcutaneous xenografts and pulmonary metastasis assays in vivo, respectively. We also demonstrated that AEG-1 increased phosphorylation of the p65 subunit of NF-κB, and regulated the expression of MMP1 in HNSCC cells. Moreover, compromised phosphorylation of the p65 (RelA) subunit of NF-κB at serine 536 was observed upon silencing of AEG-1 in both HNSCC cell lines and clinical specimens. CONCLUSION: High expression of AEG-1 is associated with lymph node metastasis and its potentially associated mechanism is investigated.
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Carcinoma de Células Escamosas/metabolismo , Moléculas de Adesão Celular/fisiologia , Metaloproteinase 1 da Matriz/metabolismo , Neoplasias Bucais/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição RelA/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Linhagem Celular Tumoral , Indução Enzimática , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Metaloproteinase 1 da Matriz/genética , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Transplante de Neoplasias , Fosforilação , Proteínas de Ligação a RNA , Carga Tumoral , Regulação para CimaRESUMO
BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/terapia , Imunoterapia/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Dengue/virologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Análise de Sobrevida , Ensaio de Placa ViralRESUMO
BACKGROUND: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. CONCLUSIONS/SIGNIFICANCE: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.