RESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a rare but severe type of thrombotic microangiopathy that is triggered by the abnormal activation of the alternative complement pathway. Previous studies have reported that three completely linked coding variants of CFHR1 form two haplotypes, namely, CFHR1*A (c.469C, c.475C, c.523G) and CFHR1*B (c.469T, c.475G, c.523C). CFHR1*B is associated with susceptibility to aHUS. To explore the genetic mechanism by which CFHR1 isoforms contribute to aHUS, we compared the structures of FHR1*A and FHR1*B by homology modeling and found differences in the angles between SCR3 and SCR4-SCR5, as FHR1*B had a larger angle than FHR1*A. Then, we expressed FHR1*A and FHR1*B recombinant proteins and compared their functions in complement system regulation and inflammation. We found that FHR1*B presented a significantly higher capacity for binding C3b and necrotic cells than FHR1*A. In a cofactor assay, the FHR-1*B showed stronger influence on FH mediated cofactor function than the FHR-1*A, resulted in fewer C3b cleavage products. In the C3 convertase assays, FHR1*B showed more powerful effect compared with FHR1*A regarding to de-regulate FH function of inhibition the assembling of C3bBb. Additionally, we also found that FHR1*B triggered monocytes to secrete higher levels of IL-1ß and IL-6 than FHR1*A. In the present study, we showed that variants of CFHR1 might differently affect complement activation and sterile inflammation. Our findings provide a possible mechanism underlying the predisposition to aHUS caused by CFHR1 isoform CFHR1*B.
Asunto(s)
Síndrome Hemolítico Urémico Atípico/etiología , Síndrome Hemolítico Urémico Atípico/metabolismo , Proteínas Sanguíneas/metabolismo , Activación de Complemento/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Síndrome Hemolítico Urémico Atípico/diagnóstico , Unión Competitiva/inmunología , Biomarcadores , Proteínas Sanguíneas/química , Complemento C3b/inmunología , Complemento C3b/metabolismo , Susceptibilidad a Enfermedades , Células Endoteliales/metabolismo , Humanos , Inflamación/complicaciones , Modelos Moleculares , Necrosis/inmunología , Necrosis/metabolismo , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Relación Estructura-ActividadRESUMEN
The aims of this study was to investigate the influences of hsa_circ_0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has_circ_0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa_circ_0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1ß, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ_0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa_circ_0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa_circ_0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa_circ_0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/genética , MicroARNs/metabolismo , ARN Circular/metabolismo , Espondilitis Anquilosante/genética , Apoptosis/genética , Apoptosis/inmunología , Unión Competitiva/genética , Unión Competitiva/inmunología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Biología Computacional , Fibroblastos , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , ARN Circular/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Espondilitis Anquilosante/inmunología , Espondilitis Anquilosante/patologíaRESUMEN
Aim: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Results: Alemtuzumab-Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Conclusion: In this proof-of-concept study, a CHO-CD52 cell line has been developed and used to detect the presence of anti-alemtuzumab neutralizing antibodies. This platform provides the basis of an assay for routine screening of serum for neutralizing antibodies from patients treated with alemtuzumab.
Asunto(s)
Alemtuzumab/inmunología , Anticuerpos Neutralizantes , Técnicas Citológicas/métodos , Inmunoensayo/métodos , Alemtuzumab/uso terapéutico , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Unión Competitiva/inmunología , Antígeno CD52/inmunología , Antígeno CD52/metabolismo , Células CHO/química , Células CHO/metabolismo , Cricetulus , Fluoresceínas , Humanos , Depleción Linfocítica/métodos , Esclerosis Múltiple/tratamiento farmacológico , Ácidos SulfónicosRESUMEN
In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences is required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmarks, e.g., binding energy of -11 kcal/mol, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated in the analyses of two small molecule targets, biotin and digoxin. Low nanomolar limits of detection were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sondas de ADN/química , ADN/química , Colorantes Fluorescentes/química , Inmunoensayo/métodos , Unión Competitiva/inmunología , Biotina/análisis , Biotina/inmunología , ADN/genética , Sondas de ADN/genética , Digoxigenina/química , Digoxina/análisis , Digoxina/inmunología , Fluorescencia , Límite de Detección , Hibridación de Ácido NucleicoRESUMEN
Programmed death ligand 1 (PD-L1)-mediated immunosuppression regulates peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but also binds to B7-1 (CD80) to regulate T-cell function. The binding interaction of PD-L1 with B7-1 and its functional role need further investigation to understand differences between PD-1 and PD-L1 tumor immunotherapy. We examined the molecular orientation of PD-L1 binding to B7-1 using cell-to-cell binding assays, ELISA, and flow cytometry. As expected, PD-L1-transfected cells bound to PD-1-transfected cells, and B7-1 cells bound to CD28 or CTLA-4-transfected cells; however, PD-L1 cells did not bind to B7-1 cells. By ELISA and flow cytometry with purified proteins, we found PD-L1 and B7-1 had a strong binding interaction only when PD-L1 was flexible. Soluble PD-1 and B7-1 competed for binding to PD-L1. Binding of native PD-L1 and B7-1 in cis on the same cell surface was demonstrated with NanoBiT proximity assays. Thus, PD-L1-B7-1 interaction can occur in cis on the same cell but not in trans between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7-1 in cis, in a manner that can competitively block the binding of PD-L1 to PD-1 or of B7-1 to CD28. This binding orientation emphasizes the functional importance of coexpression of PD-L1 and B7-1 on the same cell. We found such coexpression on tumor-infiltrating myeloid cells. Our findings may help better utilize these pathways in cancer immunotherapy. Cancer Immunol Res; 6(8); 921-9. ©2018 AACR.
Asunto(s)
Antígeno B7-1/metabolismo , Antígeno B7-H1/metabolismo , Animales , Sitios de Unión , Unión Competitiva/inmunología , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Neoplasias del Colon/inmunología , Ratones , Modelos Inmunológicos , Células Mieloides/inmunología , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Epítopos/inmunología , Espectrometría de Masas/métodos , Receptores de Superficie Celular/inmunología , Resonancia por Plasmón de Superficie/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Unión Competitiva/inmunología , Medición de Intercambio de Deuterio , Mapeo Epitopo , Epítopos/química , Epítopos/metabolismo , Humanos , Modelos Moleculares , Unión Proteica/inmunología , Conformación ProteicaRESUMEN
T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.
Asunto(s)
Traslado Adoptivo/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Unión Competitiva/inmunología , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Citometría de Flujo , Células HEK293 , Células Hep G2 , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Jurkat , Ratones , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2ß). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Receptores de Prolactina/antagonistas & inhibidores , Receptores de Prolactina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Unión Competitiva/inmunología , Células CHO , Proliferación Celular/fisiología , Cricetinae , Cricetulus , Hibridomas/inmunología , Ratones , Ratones Endogámicos BALB C , Fosforilación/inmunología , Prolactina/inmunología , Unión Proteica/inmunología , Transducción de Señal/inmunologíaRESUMEN
Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of protein modifications might occur in diabetic patients in hyperglycaemic conditions that may be recognised as foreign molecules and can induce autoantibodies. Increased level of anti-Amadori-albumin autoantibodies may be used as a biomarker in disease diagnosis and its progression.
Asunto(s)
Epítopos/inmunología , Glucosa/inmunología , Productos Finales de Glicación Avanzada/inmunología , Albúmina Sérica/inmunología , Animales , Autoanticuerpos/inmunología , Unión Competitiva/inmunología , Reacciones Cruzadas/inmunología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Femenino , Glucosa/química , Productos Finales de Glicación Avanzada/química , Glicosilación , Humanos , Hiperglucemia/inmunología , Sueros Inmunes/inmunología , Inmunoglobulina G/inmunología , Unión Proteica/inmunología , Conejos , Albúmina Sérica/químicaRESUMEN
Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were determined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.
Asunto(s)
Unión Competitiva/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Inmunoadsorbentes/química , Péptidos/inmunología , Troponina I/análisis , Troponina I/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Simulación de Dinámica Molecular , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/inmunología , Péptidos/química , Troponina I/químicaRESUMEN
The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies.
Asunto(s)
Neoplasias Encefálicas/inmunología , Glioma/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Anticuerpos de Cadena Única/inmunología , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Unión Competitiva/inmunología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Glioma/tratamiento farmacológico , Glioma/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Cinética , Masculino , Ratones Desnudos , Microscopía Confocal , Unión Proteica/inmunología , Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Cadena Única/farmacología , Resonancia por Plasmón de Superficie , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-1ß (IL-1ß) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1ß antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1ß from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1ß was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1ß is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1ß and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1ß, canakinumab binds IL-1ß on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1ß:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1ß binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos/inmunología , Interleucina-1beta/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/metabolismo , Unión Competitiva/inmunología , Callithrix , Reacciones Cruzadas/inmunología , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Ácido Glutámico/química , Ácido Glutámico/inmunología , Ácido Glutámico/metabolismo , Humanos , Interleucina-1beta/química , Interleucina-1beta/metabolismo , Macaca , Ratones , Modelos Moleculares , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Conejos , Ratas , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Especificidad de la EspecieRESUMEN
Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Bioensayo/métodos , Eritropoyetina/inmunología , Técnicas Inmunológicas/métodos , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Unión Competitiva/efectos de los fármacos , Unión Competitiva/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Eritropoyetina/metabolismo , Eritropoyetina/farmacología , Humanos , Interleucina-4/metabolismo , Interleucina-4/farmacología , Ligandos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Receptores de Eritropoyetina/metabolismo , Receptores de Interleucina-4/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Affinity proteins have advanced the field of targeted therapeutics due to their generally higher specificity compared to small molecular compounds. However, side effects caused by on-target binding in healthy tissues are still an issue. Here, we design and investigate a prodrug strategy for improving tissue specificity of Affibody molecules in future in vivo studies. The prodrug Affibody (pro-Affibody) against the HER2 receptor was constructed by fusing a HER2-specific Affibody (ZHER2) to an anti-idiotypic Affibody (anti-ZHER2). The linker was engineered to comprise a substrate peptide for the cancer-associated matrix metalloprotease 1 (MMP-1). The hypothesis was that the binding surface of ZHER2 would thereby be blocked from interacting with HER2 until the substrate peptide was specifically hydrolyzed by MMP-1. Binding should thereby only occur where MMP-1 is overexpressed, potentially decreasing on-target toxicities in normal tissues. The pro-Affibody was engineered to find a suitable linker and substrate peptide, and the different constructs were evaluated with a new bacterial display assay. HER2-binding of the pro-Affibody was efficiently masked and proteolytic activation of the best variant yielded over 1,000-fold increase in apparent binding affinity. Biosensor analysis revealed that blocking of the pro-Affibody primarily affected the association phase. In a cell-binding assay, the activated pro-Affibody targeted native HER2 on cancer cells as opposed to the non-activated pro-Affibody. We believe this prodrug approach with proteolytic activation is promising for improving tissue specificity in future in vivo targeting applications and can hopefully be extended to other Affibody molecules and similar affinity proteins as well.
Asunto(s)
Neoplasias/metabolismo , Péptido Hidrolasas/metabolismo , Profármacos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Unión Competitiva/inmunología , Línea Celular Tumoral , Citometría de Flujo , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Neoplasias/enzimología , Neoplasias/inmunología , Profármacos/química , Unión Proteica/inmunología , Proteolisis , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.
Asunto(s)
Autoantígenos/inmunología , Autoinmunidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Autoantígenos/metabolismo , Autoinmunidad/genética , Unión Competitiva/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Citometría de Flujo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Autotolerancia/genética , Autotolerancia/inmunología , Linfocitos T/metabolismoRESUMEN
The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Heparitina Sulfato/biosíntesis , N-Acetilglucosaminiltransferasas/metabolismo , Células del Estroma/metabolismo , Acondicionamiento Pretrasplante , Animales , Anticoagulantes/farmacología , Unión Competitiva/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Proteínas Fluorescentes Verdes/genética , Heparina/farmacología , Heparitina Sulfato/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Células del Estroma/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.
Asunto(s)
Anticuerpos Neutralizantes/química , Estructura Terciaria de Proteína , Toxina Shiga II/química , Anticuerpos de Dominio Único/química , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Unión Competitiva/inmunología , Camélidos del Nuevo Mundo/inmunología , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/inmunología , Subunidades de Proteína/química , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de Aminoácido , Toxina Shiga II/inmunología , Toxina Shiga II/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunologíaRESUMEN
CONTEXT: As a new antibody concept, natural bispecific antibodies (nBsAbs) have been detected in long-term passive immunization and some diseases, but their potential immunomodulatory role remains unclear. Hashimoto thyroiditis (HT) appears to fulfill the condition for nBsAb production but has not yet been characterized. OBJECTIVE: The objective of the study was to identify a new nBsAb against thyroid peroxidase (TPO) and thyroglobulin (Tg) in HT patients and to preliminarily explore its immunomodulatory role. DESIGN, SETTING, AND PATIENTS: Serum samples were obtained from 136 HT patients, 92 diseased controls, and 99 healthy controls for anti-TPO/Tg nBsAb detection. The relationship between anti-TPO/Tg nBsAb and other clinical parameters was also analyzed. MAIN OUTCOME MEASURES: The anti-TPO/Tg nBsAb was detected using a double-antigen sandwich ELISA. Higher nBsAb levels were found to be associated with decreased inflammation in HT patients. RESULTS: The prevalence of anti-TPO/Tg nBsAb in HT was 44.9% (61 of 136), significantly higher than that of diseased controls (2.2%, 2 of 92) (P < .0001) and healthy controls (0%, 0 of 99) (P < .0001). HT patients who were nBsAb positive were prone to have significantly lower levels of serum C-reactive protein and TNF-α compared with the nBsAb-negative individuals (P < .05). The serum amyloid A and interferon-γ levels also showed a similar trend in the two groups. The IgG subclass of anti-TPO/Tg nBsAb was IgG4. Further analysis showed a negative correlation between anti-TPO/Tg nBsAb and serum total IgG4 (r = -0.697, P = .025) in IgG4 thyroiditis patients. CONCLUSIONS: A new type of nBsAb against TPO and Tg in HT patients is identified. Our data also indicate a protective effect of anti-TPO/Tg nBsAb in the pathogenesis of HT and extend prior knowledge about nBsAb in diseases.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Autoanticuerpos/inmunología , Enfermedad de Hashimoto/inmunología , Yoduro Peroxidasa/inmunología , Tiroglobulina/inmunología , Adulto , Anciano , Anticuerpos Biespecíficos/sangre , Anticuerpos Biespecíficos/aislamiento & purificación , Especificidad de Anticuerpos , Autoanticuerpos/aislamiento & purificación , Unión Competitiva/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedad de Hashimoto/epidemiología , Enfermedad de Hashimoto/terapia , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Inmunoterapia/métodos , Masculino , Persona de Mediana Edad , Estudios Seroepidemiológicos , Adulto JovenRESUMEN
Autoreactive T cells infiltrating the target organ can possess a broad TCR affinity range. However, the extent to which such biophysical parameters contribute to T cell pathogenic potential remains unclear. In this study, we selected eight InsB9-23-specific TCRs cloned from CD4(+) islet-infiltrating T cells that possessed a relatively broad range of TCR affinity to generate NOD TCR retrogenic mice. These TCRs exhibited a range of two-dimensional affinities (â¼ 10(-4)-10(-3) µm(4)) that correlated with functional readouts and responsiveness to activation in vivo. Surprisingly, both higher and lower affinity TCRs could mediate potent insulitis and autoimmune diabetes, suggesting that TCR affinity does not exclusively dictate or correlate with diabetogenic potential. Both central and peripheral tolerance mechanisms selectively impinge on the diabetogenic potential of high-affinity TCRs, mitigating their pathogenicity. Thus, TCR affinity and multiple tolerance mechanisms converge to shape and broaden the diabetogenic T cell repertoire, potentially complicating efforts to induce broad, long-term tolerance.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Tolerancia Inmunológica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Unión Competitiva/inmunología , Trasplante de Médula Ósea/métodos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Citometría de Flujo , Insulina/deficiencia , Insulina/genética , Insulina/inmunología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
Human ficolin-2 (L-ficolin/p35) is a lectin-complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during chronic hepatitis C virus (HCV) infection. In this study, we found that ficolin-2 inhibits the entry of HCV at an early stage of viral infection, regardless of the viral genotype. Ficolin-2 neutralized and inhibited the initial attachment and infection of HCV by binding to the HCV envelope surface glycoproteins E1 and E2, blocking HCV attachment to low-density lipoprotein receptor (LDLR) and scavenger receptor B1, and weakly interfering with CD81 receptor attachment. However, no interference with claudin-1 and occludin receptor attachment was observed. The C-terminal fibrinogen domain (201-313 aa) of ficolin-2 was identified as the critical binding region for the HCV-E1-E2 N-glycans, playing a critical role in the anti-HCV activity. More importantly, we found that apolipoprotein E (ApoE)3, which is enriched in the low-density fractions of HCV RNA-containing particles, promotes HCV infection and inhibits ficolin-2-mediated antiviral activity. ApoE3, but not ApoE2 and ApoE4, blocked the interaction between ficolin-2 and HCV-E2. Our data suggest that the HCV entry inhibitor ficolin-2 is a novel and promising antiviral innate immune molecule, whereas ApoE3 blocks the effect of ficolin-2 and mediates an immune escape mechanism during chronic HCV infection. HCV may be neutralized using compounds directed against the lipoprotein moiety of the viral particle, and ApoE3 may be a new target to combat HCV infection.