RESUMO
Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.
Assuntos
Anticorpos Monoclonais/farmacologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Toxina Shiga II/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Proteínas Recombinantes , Toxina Shiga I/imunologia , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/imunologia , Células VeroRESUMO
The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
Assuntos
Amiloide/imunologia , Amiloidose/diagnóstico , Síndromes Neurotóxicas/diagnóstico , Agregação Patológica de Proteínas/diagnóstico , Amiloide/antagonistas & inibidores , Amiloidose/imunologia , Amiloidose/patologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/patologia , Fragmentos de Peptídeos/imunologia , Agregação Patológica de Proteínas/imunologia , Anticorpos de Domínio Único , Relação Estrutura-AtividadeRESUMO
Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.
Assuntos
Conexina 26/química , Conexina 30/química , Microscopia de Força Atômica , Sequência de Aminoácidos , Conexina 26/genética , Conexina 26/imunologia , Conexina 26/metabolismo , Conexina 30/genética , Conexina 30/imunologia , Conexina 30/metabolismo , Microscopia Crioeletrônica , Junções Comunicantes/metabolismo , Células HeLa , Histidina/genética , Histidina/imunologia , Histidina/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Multimerização ProteicaRESUMO
Most antivenoms are produced by techniques developed over 50 years ago, with minor modifications. Herein we revise the core of traditional antivenom production processes aiming to optimize key determinants for both consistent antivenom production and the best balance between F(ab')2 quality and recovery. Factorial design analysis revealed that pepsin digestion of 1:3 saline diluted equine plasma for 60 min under pH: 3.20, 37 °C temperature and a 1:15 pepsin to protein ratio conditions, allowed to achieve maximal IgG to F(ab')2 conversion with minimal protein aggregate formation. Further downstream processing by salting out with ammonium sulfate was also studied by factorial analysis. The influence of ammonium sulfate (AS) concentration, temperature (T) and the albumin to total plasma protein ratio plasma (Alb:P) were assayed, revealing that both AS, T and their interaction have a significant impact in F(ab')2 quality and recovery. Taking into account the existing compromise between F(ab')2 monomer recovery and quality two alternative conditions were selected: 14 g/dl AS at 56 °C and, alternatively 16 g/dl AS at 30 °C. Reasonable yields (42%) and product quality (2.5% of aggregates) without significant changes in production cost of traditional methodologies was achieved under the optimized conditions found.
Assuntos
Antivenenos/imunologia , Cavalos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Pepsina A/metabolismo , Mordeduras de Serpentes/imunologia , Venenos de Serpentes/imunologia , Sulfato de Amônio/química , Sulfato de Amônio/metabolismo , Animais , Antivenenos/sangue , Antivenenos/metabolismo , Proteínas Sanguíneas/metabolismo , Caprilatos/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cavalos/sangue , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/metabolismo , Papaína/metabolismo , Albumina Sérica/metabolismo , Mordeduras de Serpentes/prevenção & controleRESUMO
CAR-T cell therapy emerged in the last years as a great promise to cancer treatment. Nowadays, there is a run to improve the breadth of its use, and thus, new chimeric antigen receptors (CAR) are being proposed. The antigen-binding counterpart of CAR is an antibody fragment, scFv (single chain variable fragment), that recognizes a membrane protein associated to a cancer cell. In this chapter, the use of human scFv phage display libraries as a source of new mAbs against surface antigen is discussed. Protocols focusing in the use of extracellular domains of surface protein in biotinylated format are proposed as selection antigen. Elution with unlabeled peptide and selection in solution is described. The analysis of enriched scFvs throughout the selection using NGS is also outlined. Taken together these protocols allow for the isolation of new scFvs able to be useful in the construction of new chimeric antigen receptors for application in cancer therapy.
Assuntos
Técnicas de Visualização da Superfície Celular , Biblioteca de Peptídeos , Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoterapia Adotiva/métodos , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genéticaRESUMO
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab')2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab')2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab')2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab')2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab')2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab')2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais Murinos , Proteínas de Bactérias/imunologia , Fragmentos Fab das Imunoglobulinas , Staphylococcus aureus Resistente à Meticilina/imunologia , Proteínas de Ligação às Penicilinas/imunologia , Pepsina A/química , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , CamundongosRESUMO
Anti-Tityus discrepans F(ab')2 ELISA recognition of T. discrepans toxins was measured with regression analysis and its slope called ELISA recognition value (ERv). Fractions containing toxins affecting mammal macrophages or Na(+)-channels have Ervs >19. Toxins affecting potassium channels or insect NaV channels have ERvs <10. Fractions including curarizing or antineoplasic peptides had ERvs <1. Erv increases in proportion to mammalian toxin toxicity rather than to toxin molecular mass.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fragmentos Fab das Imunoglobulinas/imunologia , Venenos de Escorpião/análise , Animais , Cavalos , Peso Molecular , Venenos de Escorpião/imunologiaRESUMO
Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.
Assuntos
Antivenenos/imunologia , Venenos de Abelha/imunologia , Abelhas/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Antivenenos/toxicidade , Relação Dose-Resposta Imunológica , Hemólise/imunologia , Cavalos , Imunização , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/imunologia , Masculino , Camundongos , Testes de NeutralizaçãoRESUMO
We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 µg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.
Assuntos
Antivenenos/imunologia , Cavalos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Animais , Antivenenos/biossíntese , Antivenenos/sangue , Ensaio de Imunoadsorção Enzimática , Fragmentos Fab das Imunoglobulinas/sangue , Imunoglobulina G/sangue , Imunoproteínas/farmacocinética , CoelhosRESUMO
In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.