RESUMO
Activation-induced deaminase (AID) deaminates deoxycytidine residues in immunoglobulin genes, triggering antibody diversification. Here, by use of two-hybrid and coimmunoprecipitation assays, we identify CTNNBL1 (also known as NAP) as an AID-specific interactor. Mutants of AID that interfere with CTNNBL1 interaction yield severely diminished hypermutation and class switching. Targeted inactivation of CTNNBL1 in DT40 B cells also considerably diminishes IgV diversification. CTNNBL1 is a widely expressed nuclear protein that associates with the Prp19 complex of the spliceosome, interacting with its CDC5L component. The results, therefore, identify residues in AID involved in its in vivo targeting and suggest they might act through interaction with CTNNBL1, giving possible insight into the linkage between AID recruitment and target-gene transcription.
Assuntos
Diversidade de Anticorpos , Proteínas Reguladoras de Apoptose/metabolismo , Citidina Desaminase/metabolismo , Proteínas Nucleares/metabolismo , Spliceossomos/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Galinhas , Humanos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ratos , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: To prepare high-affinity anti-aflatoxin M1 monoclonal antibodies by High Throughput Screening ELISA (HTS-ELISA) METHODS: Balb/C mice were immunized by aflatoxin M1-bovine serum albumin conjugate, and screen secret anti-aflatoxin M1 monoclonal antibody hybridoma by HTS-ELISA. The antibody was characterized. RESULTS: Fourteen hybridoma cell lines which could secret high activity anti-aflatoxin M1 monoclonal antibodies were obtained. The affinity of the purified monoclonal antibody was 5.5 x 10(-10) mol/L. The cross-reactivity of the monoclonal antibody clone against aflatoxin M1, aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol and BSA was 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%, 0%, respectively. The sensitivity of the anti-AFM1 monoclonal antibody binding to aflatoxin M1 was 0.01 microg/L and the linear range for developed indirect competitive ELISA was 0.1 - 10 microg/L aflatoxin M1. The binding inhibition IC50 of the anti-aflatoxin M1 monoclonal antibody was 0.82 microg/L. Assays of milk samples mixed with AFM1 ranging in concentration from 0.25 to 5.0 microg/L gave mean indirect competitive ELISA recovery of 60.3% - 152.8%. CONCLUSION: HTS-ELISA can be used for the preparation of the high-affinity anti-aflatoxin M1 monoclonal antibodies. The anti-aflatoxin M1 monoclonal antibody could be provided as the high quality material in the system of aflatoxin M1 immune detection.
Assuntos
Aflatoxina M1/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/imunologia , Aflatoxinas/imunologia , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Bovinos , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. METHODS: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. RESULTS: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. CONCLUSION: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.
Assuntos
Anticorpos Monoclonais/farmacologia , Melanoma/terapia , Animais , Anticorpos Monoclonais/imunologia , Antígeno CD146/imunologia , Linhagem Celular Tumoral , Humanos , Masculino , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Nus , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The activation-induced deaminase/apolipoprotein B-editing catalytic subunit 1 (AID/APOBEC) family comprises four groups of proteins. Both AID, a lymphoid-specific DNA deaminase that triggers antibody diversification, and APOBEC2 (function unknown) are found in all vertebrates examined. In contrast, APOBEC1, an RNA-editing enzyme in gastrointestinal cells, and APOBEC3 are restricted to mammals. The function of most APOBEC3s, of which there are seven in human but one in mouse, is unknown, although several human APOBEC3s act as host restriction factors that deaminate human immunodeficiency virus type 1 replication intermediates. A more primitive function of APOBEC3s in protecting against the transposition of endogenous retroelements has, however, been proposed. Here, we focus on mouse APOBEC2 (a muscle-specific protein for which we find no evidence of a deaminating activity on cytidine whether as a free nucleotide or in DNA) and mouse APOBEC3 (a DNA deaminase which we find widely expressed but most abundant in lymphoid tissue). Gene-targeting experiments reveal that both APOBEC2 (despite being an ancestral member of the family with no obvious redundancy in muscle) and APOBEC3 (despite its proposed role in restricting endogenous retrotransposition) are inessential for mouse development, survival, or fertility.
Assuntos
Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Desaminases APOBEC , Desaminase APOBEC-3G , Animais , Southern Blotting , Sobrevivência Celular , Citidina/metabolismo , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Fertilidade , Citometria de Fluxo , Marcadores Genéticos , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Modelos Genéticos , Músculos/metabolismo , Miocárdio/metabolismo , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Recombinação Genética , Retroelementos/genética , Distribuição TecidualRESUMO
We investigated the effect on rheumatoid arthritis (RA) of an anti-gp130 monoclonal antibody (mAb) and its mechanism using RA fibroblast-like synoviocytes (FLS) and a collagen antibody-induced arthritis (CAIA) mouse model. We determined the interleukin 6 (IL-6), IL-6 receptor α (IL-6Rα), gp130, receptor activator of nuclear factor κB ligand (RANKL), matrix metalloproteinase 3 (MMP3), TIMP metallopeptidase inhibitor 1 (TIMP1), and Bcl-2 levels in RA and osteoarthritis (OA) serum and synovial fluid. RA FLS were cultured with or without IL-6/IL-6Rα; WNT5A and RANKL levels were detected. We generated an anti-gp130 mAb (M10) with higher affinity and specificity, blocked IL-6 signaling with it, and assessed its effects on the CAIA model, WNT5A and RANKL expression, and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The IL-6 signaling system in patients with RA was increased; RANKL, MMP3, TIMP1, and Bcl-2 in RA bone were elevated. IL-6/IL-6Rα increased RA FLS WNT5A and RANKL expression. M10 ameliorated arthritis in the CAIA model, and inhibited RANKL, WNT5A, and Bcl-2 expression in RA FLS by blocking IL-6 signaling, likely via Janus kinase-STAT3 pathway downregulation. The IL-6-soluble IL-6Rα-gp130 complex is hyperactive in RA and OA. M10 may be the basis for a novel RA treatment drug.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and it is unclear whether its stromal infiltrate contributes to its aggressiveness. Here, we demonstrate that Dickkopf-3 (DKK3) is produced by pancreatic stellate cells and is present in most human PDAC. DKK3 stimulates PDAC growth, metastasis, and resistance to chemotherapy with both paracrine and autocrine mechanisms through NF-κB activation. Genetic ablation of DKK3 in an autochthonous model of PDAC inhibited tumor growth, induced a peritumoral infiltration of CD8+ T cells, and more than doubled survival. Treatment with a DKK3-blocking monoclonal antibody inhibited PDAC progression and chemoresistance and prolonged survival. The combination of DKK3 inhibition with immune checkpoint inhibition was more effective in reducing tumor growth than either treatment alone and resulted in a durable improvement in survival, suggesting that DKK3 neutralization may be effective as a single targeted agent or in combination with chemotherapy or immunotherapy for PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Quimiocinas , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inativação Gênica , Humanos , Imunoterapia , Camundongos Endogâmicos C57BL , Camundongos Nus , NF-kappa B/metabolismo , Testes de Neutralização , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Comunicação Parácrina/efeitos dos fármacos , Análise de Sobrevida , Gencitabina , Neoplasias PancreáticasRESUMO
BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is crucial for successful drug development. However, due to immune tolerance, making it difficult to generate antibodies using conventional approaches. METHODOLOGY/FINDINGS: Mixed four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; sixteen highly positive hybridoma colonies were selected via fluorescence-activated cell sorting-high throughput screening (FACS-HTS) using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus human PBMC controls). MS17-57 and control commercial Alkaline Phosphatase (ALP) mAbs were used to confirm the target antigens (Ags), which were identified as ALPs expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). MS identified the Ags recognized by MS17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules. Immunofluorescence staining using MS17-57 demonstrated higher staining of gastrointestinal (GI) cancer tissues compared to normal GI tissues (P<0.03), and confirmed binding of MS17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assays using the PALP/IALP-expressing GC cell lines demonstrated that MS17-57 inhibited cell growth by 32 ± 8%. Transwell cell migration assays documented that MS17-57 can inhibit PALP/IALP-expressing GI cancer cell migration by 25 ± 5%. MS17-57 mAb inhibited tumor growth in nude mice. CONCLUSIONS: Our findings indicate that PALP and IALP can be ectopically expressed on extracellular matrix of GI cancers, and that MS17-57 directed against PALP/IALP can inhibit GI cancer cells growth and migration in vitro and in vivo. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.
Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/imunologia , Anticorpos Monoclonais/imunologia , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular/métodos , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Neoplasias Gastrointestinais/genética , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Transdução de SinaisAssuntos
Biomarcadores Tumorais/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Feminino , Seguimentos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Melanoma/mortalidade , Melanoma/patologia , Mucina-5AC/genética , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Fatores de Risco , Pele/patologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologiaRESUMO
The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.
Assuntos
Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Movimento Celular/genética , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Receptor Notch3 , Receptores Notch/genética , Receptores Notch/imunologia , Receptores Notch/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The objective of this study was to investigate the effects of antifactor D monoclonal antibody (Mab) 166-32 on platelet activation during and after hypothermic cardiopulmonary bypass (CPB) in baboons. Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. Seven of them were treated with a single injection of antifactor D Mab 166-32 (5 mg/kg) and the other seven animals were given saline as control. Each baboon was sedated with an intramuscular injection of 10 mg/kg of ketamine hydrochloride. A 20-gauge angiocatheter was placed in the cephalic vein, and 5 mg of diazepam was administered intravenously. Anesthesia was maintained with 0.80% to 2.25% isoflurane, 100% O2, and an inspiratory tidal volume of 13 mL/kg at a rate of 13 breaths per minute throughout the surgical procedure except during CPB. Pancuronium bromide, 0.1 mg/kg, was administered to achieve adequate muscle paralysis. Blood samples were collected before CPB, during CPB, and 1, 2, 3, and 6 h after CPB. Assays were performed to measure platelet activation [CD62P (P-selectin)] using immunofluorocytometric methods. There were no significant differences on CD62P expression of platelets between control and antibody groups before CPB (105 +/- 12% vs. 99 +/- 8%, P=NS), during normothermic CPB (62 +/- 6% vs. 63 +/- 19%, P=NS), during hypothermic CPB (55 +/- 8% vs. 54 +/- 13%, P=NS), and 1, 3, or 6 h after CPB (74 +/- 20% vs. 81 +/- 11%, P=NS). Anesthetic induction with ketamine caused significant reduction in the platelet activation in both groups. Ketamine did not affect complement, neutrophil, and monocyte activation or cytokine production. Further studies on the mechanisms of platelet inhibition by ketamine are warranted.