RESUMEN
BACKGROUND/AIM: To predict the efficacy of platinum-containing chemotherapy, ERCC1 expression levels were investigated. Studies have shown changes in the performance of anti-ERCC1 antibodies; therefore, predicting chemotherapy efficacy by immunohistochemical assessment of ERCC1 is controversial. PATIENTS AND METHODS: Twenty-eight patients who received platinum-containing chemotherapy and underwent computed tomography evaluation 6-9 weeks after therapy initiation were retrospectively identified. The tumor samples were evaluated in 2012 and 2018 using the latest anti-ERCC1 antibodies available at those times. RESULTS: In 2012, the ERCC1 H-score was significantly higher in patients with disease progression than in patients without disease progression (p=0.019). Although the same trend was shown in 2018, there were some inconsistent results between the 2012 and 2018 samples. CONCLUSION: Patients with tumors showing low ERCC1 expression had a better disease control rate on platinum-containing chemotherapy. However, since the performance of the antibody changed over time, standardized technology to evaluate ERCC1 expression is needed.
Asunto(s)
Anticuerpos Antiidiotipos/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Anciano , Anciano de 80 o más Años , Anticuerpos Antiidiotipos/aislamiento & purificación , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Platino (Metal)/administración & dosificaciónRESUMEN
The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Toxinas de Bacillus thuringiensis/química , Endotoxinas/química , Proteínas Hemolisinas/química , Región Variable de Inmunoglobulina/química , Anticuerpos de Cadena Única/química , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Toxinas de Bacillus thuringiensis/inmunología , Toxinas de Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Simulación del Acoplamiento Molecular , Mariposas Nocturnas , Mutación , Biblioteca de Péptidos , Conformación Proteica , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Especificidad de Anticuerpos , Antineoplásicos Inmunológicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Tiburones/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Especificidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular Tumoral , Expresión Génica , Biblioteca de Genes , Humanos , Inmunoconjugados/genética , Inmunoconjugados/farmacología , Inmunofenotipificación , Linfoma/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores de Antígenos de Linfocitos B/sangre , Receptores de Antígenos de Linfocitos B/genética , Proteínas Recombinantes de Fusión/genética , Tiburones/genéticaRESUMEN
The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.
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Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Linfocitos B/inmunología , Separación Celular/métodos , Epítopos/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Monoclonales/genética , Linfocitos B/clasificación , Células Cultivadas , Clonación Molecular/métodos , Epítopos/genética , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , ConejosRESUMEN
The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.
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Trastorno del Espectro Autista/genética , Autoanticuerpos/inmunología , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Trastorno del Espectro Autista/inmunología , Trastorno del Espectro Autista/fisiopatología , Autoanticuerpos/efectos adversos , Conducta Animal , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Hipocampo/inmunología , Hipocampo/patología , Humanos , Masculino , Herencia Materna/genética , Herencia Materna/inmunología , Relaciones Materno-Fetales , Proteínas de la Membrana/inmunología , Ratones , Proteínas del Tejido Nervioso/inmunología , Neurogénesis/inmunología , Problema de ConductaRESUMEN
BACKGROUND: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL. METHODS: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT. RESULTS: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression. CONCLUSION: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Terapia de Reemplazo Enzimático , Epítopos/genética , Enfermedad de Fabry/tratamiento farmacológico , alfa-Galactosidasa/genética , Adulto , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/genética , Mapeo Epitopo , Epítopos/inmunología , Enfermedad de Fabry/genética , Enfermedad de Fabry/inmunología , Enfermedad de Fabry/patología , Humanos , Masculino , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , alfa-Galactosidasa/administración & dosificación , alfa-Galactosidasa/inmunologíaRESUMEN
Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Dependovirus/genética , Terapia Genética , Vectores Genéticos/efectos adversos , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/inmunología , Cápside/inmunología , Dependovirus/inmunología , Endopeptidasas/inmunología , Vectores Genéticos/uso terapéutico , Humanos , Inmunoglobulina G/farmacología , Hígado/inmunología , Hígado/metabolismo , RatonesRESUMEN
Infant mortality from dengue disease is a devastating global health burden that could be minimized with the ability to identify susceptibility for severe disease prior to infection. Although most primary infant dengue infections are asymptomatic, maternally derived anti-dengue immunoglobulin G (IgGs) present during infection can trigger progression to severe disease through antibody-dependent enhancement mechanisms. Importantly, specific characteristics of maternal IgGs that herald progression to severe infant dengue are unknown. Here, we define ≥10% afucosylation of maternal anti-dengue IgGs as a risk factor for susceptibility of infants to symptomatic dengue infections. Mechanistic experiments show that afucosylation of anti-dengue IgGs promotes FcγRIIIa signaling during infection, in turn enhancing dengue virus replication in FcγRIIIa+ monocytes. These studies identify a post-translational modification of anti-dengue IgGs that correlates with risk for symptomatic infant dengue infections and define a mechanism by which afucosylated antibodies and FcγRIIIa enhance dengue infections.
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Anticuerpos Antiidiotipos/genética , Virus del Dengue/genética , Dengue Grave/virología , Femenino , Humanos , Lactante , Recién NacidoRESUMEN
Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.
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Proteínas Adaptadoras Transductoras de Señales/genética , Anticuerpos Antiidiotipos/farmacología , Atrofia Geográfica/tratamiento farmacológico , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Degeneración Macular/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Anciano , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Biomarcadores/sangre , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Atrofia Geográfica/sangre , Atrofia Geográfica/genética , Atrofia Geográfica/inmunología , Serina Peptidasa A1 que Requiere Temperaturas Altas/antagonistas & inhibidores , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Degeneración Macular/sangre , Degeneración Macular/genética , Degeneración Macular/inmunología , Masculino , Polimorfismo de Nucleótido Simple/genética , Proteoma/genética , Proteoma/inmunología , Ratas , Retina/efectos de los fármacos , Retina/inmunología , Retina/patología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Rationale: Placental-like chondroitin sulfate A (pl-CSA) is known to be exclusively synthesized in multiple cancer tissues and associated with disease severity. Here, we aimed to assess whether pl-CSA is released into bio-fluids and can serve as a cancer biomarker. Methods: A novel ELISA was developed to analyse pl-CSA content in bio-fluids using pl-CSA binding protein and an anti-pl-CSA antibody. Immunohistochemical staining of tissue chips was used as the gold standard control. Results: The developed ELISA method was specific and sensitive (1.22 µg/ml). The pl-CSA content was significantly higher in lysates and supernatants of cancer cell lines than in those of normal cell lines, in plasma from mouse cancer models than in that from control mice, and in plasma from patients with oesophageal, cervical, ovarian, or lung cancer than in that from healthy controls. Similar to the tissue chip analysis, which showed a significant difference in pl-CSA positivity between cancer tissues and normal adjacent tissues, the plasma pl-CSA analysis had 100% sensitivity and specificity for differentiating oesophageal and lung cancer patients from healthy controls. Importantly, in oesophageal and lung cancer patients, the pl-CSA content was significantly higher in late-stage disease than in early-stage disease, and it dramatically decreased after surgical resection of the tumour. Conclusion: These data indicate a direct link between plasma pl-CSA content and tumour presence, indicating that plasma pl-CSA may be a non-invasive biomarker with clinical applicability for the screening and surveillance of patients with multiple types of solid tumours.
Asunto(s)
Sulfatos de Condroitina/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Neoplasias/genética , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/inmunología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Neoplasias/inmunología , Neoplasias/patología , Placenta/metabolismo , Embarazo , Unión Proteica/inmunologíaRESUMEN
Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.
Asunto(s)
Anticuerpos Antiidiotipos/aislamiento & purificación , Desmogleína 3/aislamiento & purificación , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/aislamiento & purificación , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Química Clic , Cicloparafinas/química , Desmogleína 3/genética , Desmogleína 3/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Luciferasas/química , Oligonucleótidos , Proteínas/genética , Proteínas/inmunologíaRESUMEN
The antibody repertoire of cartilaginous fish comprises an additional heavy-chain-only antibody isotype that is referred to as IgNAR (immunoglobulin novel antigen receptor). Its antigen-binding site consists of one single domain (vNAR) that is reportedly able to engage a respective antigen with affinities similar to those achieved by conventional antibodies. While vNAR domains offer a reduced size, which is often favorable for applications in a therapeutic as well as a biotechnological setup, they also exhibit a high physicochemical stability. Together with their ability to target difficult-to-address antigens such as virus particles or toxins, these shark-derived antibody domains seem to be predestined as tools for biotechnological and diagnostic applications. In the following chapter, we will describe the isolation of anti-idiotypic vNAR domains targeting monoclonal antibody paratopes from semi-synthetic, yeast-displayed libraries. Anti-idiotypic vNAR variants could be employed for the characterization of antibody-based therapeutics (such as antibody-drug conjugates) or as positive controls in immunogenicity assays. Peculiarly, when using semi-synthetic vNAR libraries, we found that it is not necessary to deplete the libraries using unrelated antibody targets, which enables a fast and facile screening procedure that exclusively delivers anti-idiotypic binders.
Asunto(s)
Anticuerpos Antiidiotipos , Proteínas de Peces , Biblioteca de Péptidos , Tiburones , Anticuerpos de Cadena Única , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Dominios Proteicos , Tiburones/genética , Tiburones/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.
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Antígenos de Histocompatibilidad Clase II/metabolismo , Neuropéptidos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Enfermedades Autoinmunes/metabolismo , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Inmunoglobulina G , Ratones , Ratones Endogámicos BALB CRESUMEN
An anti-idiotypic nanobody-phage display-mediated immuno-polymerase chain reaction (PD-IPCR) method was developed for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. Two phages, displaying the variable domain of the heavy chain anti-idiotypic nanobody that binds aflatoxin- or zearalenone-specific monoclonal antibody (1C11 or 2D3), were used as competitors for corresponding analytes. Specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification. The results indicated that detection limits for total aflatoxins and zearalenone in a sample were 0.03 and 0.09â¯ngâ¯mL-1, respectively. Recoveries of spiked aflatoxins and zearalenone were 80-118% and 76.7-111%, respectively. Validation results were in good agreement with the gold-standard high-performance liquid chromatography method. This report is the first to describe PD-IPCR for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals.
Asunto(s)
Aflatoxinas/análisis , Anticuerpos Antiidiotipos/inmunología , Inmunoensayo/métodos , Zearalenona/análisis , Aflatoxinas/inmunología , Anticuerpos Antiidiotipos/genética , Cromatografía Líquida de Alta Presión , Límite de Detección , Oryza/química , Oryza/metabolismo , Biblioteca de Péptidos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Zea mays/química , Zea mays/metabolismo , Zearalenona/inmunologíaRESUMEN
Effective delivery of protein therapeutics into the brain remains challenging because of difficulties associated with crossing the blood-brain barrier (BBB). To overcome this problem, many researchers have focused on antibodies binding the transferrin receptor (TfR), which is expressed in endothelial cells, including those of the BBB, and is involved in receptor-mediated transcytosis (RMT). RMT and anti-TfR antibodies provide a useful means of delivering therapeutics into the brain, but the anti-TfR antibody has a short half-life in blood because of its broad expression throughout the body. As a result, anti-TfR antibodies are only maintained at high concentrations in the brain for a short time. To overcome this problem, we developed a different approach which slows down the export of therapeutic antibodies from the brain by binding them to a brain-specific antigen. Here we report a new technology, named AccumuBrain, that achieves both high antibody concentration in the brain and a long half-life in blood by binding to myelin oligodendrocyte glycoprotein (MOG), which is specifically expressed in oligodendrocytes. We report that, using our technology, anti-MOG antibody levels in the brains of mice (Mus musculus) and rats (Rattus norvegicus) were increased several tens of times for a period of one month. The mechanism of this technology is different from that of RMT technologies like TfR and would constitute a breakthrough for central nervous system disease therapeutics.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Barrera Hematoencefálica/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Receptores de Transferrina/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos Antiidiotipos/genética , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Epítopos/inmunología , Humanos , Ratones , Glicoproteína Mielina-Oligodendrócito/genética , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Ratas , Transducción de Señal/inmunología , Transcitosis/genética , Transcitosis/inmunologíaRESUMEN
Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.
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Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Diacilglicerol Quinasa/inmunología , Epítopos/inmunología , Aminoácidos/inmunología , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/uso terapéutico , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Mapeo Epitopo/métodos , Epítopos/química , Células HeLa , Humanos , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/inmunología , Unión Proteica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
The association of systemic sclerosis with anti-Topoisomerase 1 antibody (ATASSc) with specific alleles of human leukocyte antigen (HLA)-DR has been observed among various ethnics. The anti-Topoisomerase 1 antibody is a common autoantibody in SSc with diffuse cutaneous scleroderma, which is one of the clinical subtypes of SSc. On the other hand, an immunodominant peptide of topoisomerase 1 (Top1) self-protein (residues 349-368) was reported to have strong association with ATASSc. In this study, molecular dynamics simulation was performed on the complexes of Top1 peptide with various HLA-DR subtypes divided into ATASSc-associated alleles (HLA-DRB1*08:02, HLA-DRB1*11:01 and HLA-DRB1*11:04), suspected allele (HLA-DRB5*01:02), and non-associated allele (HLA-DRB1*01:01). The unique interaction for each system was compared to the others in terms of dynamical behaviors, binding free energies and solvation effects. Our results showed that three HLA-DR/Top1 complexes of ATASSc association mostly exhibited high protein stability and increased binding efficiency without solvent interruption, in contrast to non-association. The suspected case (HLA-DRB5*01:02) binds Top1 as strongly as the ATASSc association case, which implied a highly possible risk for ATASSc development. This finding might support ATASSc development mechanism leading to a guideline for the treatment and avoidance of pathogens like Top1 self-peptide risk for ATASSc.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , Cadenas HLA-DRB1/química , Cadenas HLA-DRB5/química , Esclerodermia Sistémica/genética , Alelos , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/inmunología , Epítopos/genética , Epítopos/inmunología , Predisposición Genética a la Enfermedad , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Cadenas HLA-DRB5/genética , Cadenas HLA-DRB5/inmunología , Humanos , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Unión Proteica/genética , Estabilidad Proteica , Factores de Riesgo , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patologíaRESUMEN
BACKGROUND: Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. MATERIALS AND METHODS: Recombinant polymeric anti-G IgG1 (IgG1µtp) and IgG3 (IgG3µtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. RESULTS: The recombinant IgG1µtp and IgG3µtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. DISCUSSION: The enhanced opsonic properties of the polymeric anti-G IgG1µtp and IgG3µtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.
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Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Eritrocitos/química , Inmunoglobulina G/química , Sistema del Grupo Sanguíneo Rh-Hr/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Eritroblastosis Fetal/inmunología , Eritroblastosis Fetal/prevención & control , Eritrocitos/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunologíaRESUMEN
The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Receptores de Antígenos de Linfocitos B/química , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Modelos Moleculares , Orthomyxoviridae/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.