RESUMO
Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (ß-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.
Assuntos
Peptídeos , Venenos de Escorpião , Escorpiões , Anticorpos de Cadeia Única , Animais , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Venenos de Escorpião/imunologia , Peptídeos/química , Anticorpos de Cadeia Única/química , Humanos , Camundongos , Sequência de Aminoácidos , Antivenenos/imunologia , Antivenenos/química , Antivenenos/farmacologia , Animais PeçonhentosRESUMO
A key aspect during the development of antivenoms is the evaluation of the efficiency and security of the therapeutic molecules. In this work, we report the pharmacokinetic analysis of a neutralizing single chain antibody fragment named LR (scFv LR) where three sheep were used as a large animal model. The animals were injected through i.v. route with 2 mg of scFv LR. Blood samples were drawn every minute within the first 15 min, the sampling continues at 20, 25, 30, 45, 60, 90, 120 min, subsequently at 1-h intervals, 3, 4, 5, 6 h, two more samples at 9 and 12 h and, two more samples at 24 and 48 h and finally at one-day intervals during 4 days. scFv LR levels were measured from blood serum and urine samples by an ELISA. The pharmacokinetics of the experimental data was analyzed using the three-exponential kinetics. The value of the fast initial component (τ1=0.409±0.258min) indicated that the scFv is distributed rapidly into the tissues. The mean residence time, MRT, was 45 ± 0.51 min and the clearance (CL), 114.3 ± 14.3 mL/min. From urine samples it was possible to detect significant amounts of scFv LR, which is evidence of renal elimination.
Assuntos
Venenos de Escorpião , Anticorpos de Cadeia Única , Animais , Anticorpos de Cadeia Única/farmacocinética , Ovinos , Venenos de Escorpião/farmacocinética , Antivenenos , EscorpiõesRESUMO
OBJECTIVES: Among the most promising antibody formats in terms of inhibiting carcinogenesis are single-stranded variable fragments, whose targeted binding to the Fzd7 receptor has been proven effective at suppressing tumorigenesis. In this study, we investigated the effectiveness of an anti-Fzd7 antibody fragment against both tumor growth and metastasis of breast cancer cells. METHODS: To develop anti-Fzd7 antibodies, bioinformatics approaches were used and the antibodies were expressed recombinantly in E. coli BL21 (DE3). The expression of anti-Fzd7 fragments was verified by Western blotting. Analysis of the antibody's binding capacity to Fzd7 was conducted by flow cytometry. Cell death and apoptosis were assessed by MTT and Annexin V/PI assays. The transwell migration and invasion assays, as well as the scratch method, were used to evaluate cell motility and invasiveness. RESULTS: The anti-Fzd7 antibody was expressed successfully as a single band of 31 kDa. It could bind to 21.5% of MDA-MB-231 cells, as opposed to only 0.54% of SKBR-3 cells as negative control. According to MTT assay, induced apoptosis was 73.7% in MDA-MB-231 cells, compared with 29.5% in SKBR-3 cells. Also, the antibody exerted a significant inhibitory effect of 76% and 58% on migration and invasion of MDA-MB-231 cells, respectively. CONCLUSION: The recombinantly developed anti-Fzd7 scFv of this study could exhibit significant antiproliferative and antimigratory properties, along with a high apoptosis-inducing potential, making it suitable for the immunotherapy of triple negative breast cancer.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/patologia , Escherichia coli , Western Blotting , Apoptose , Proliferação de Células , Movimento CelularRESUMO
Este texto traz um relato de experiência advindo das atividades de uma orientadora social realizado no Serviço de Convivência e Fortalecimento de Vínculos (SCFV) de um Centro de Referência de Assistência Social (Cras) situado na Grande Florianópolis (SC). O serviço integrou 20 jovens com idade entre 12 e 18 anos no espaço de uma instituição de formação policial. Este trabalho analisará: (1) um acontecimento ocorrido na instituição policial; (2) quais foram os seus desdobramentos; e (3) como a orientadora social atuou na mediação do grupo. A análise da do relato de experiência e a mediação da orientadora social foram embasadas no conceito de acontecimento de Gilles Deleuze. No decorrer dos encontros, a instituição parceira do projeto adotou medidas disciplinares que desqualificavam a cultura proveniente dos cotidiano dos(as) jovens participantes, o que levou o grupo a constituir uma identidade coletiva a partir da resistência à violência simbólica. Assim, diante da situação-problema, tornou-se possível uma experiência de fortalecimentos de laços comunitários
This paper discusses a situation that occurred in a public youth service for young people living in vulnerable city areas. The service worked with 20 young people aged between 12 and 18 years and the meetings took place at a police training institution. Inspired by Gilles Deleuze's theory, this text analyzes 1) an event that occurred at the police institution, 2) their consequences and 3) how the professional responsible dealt with the event in a group context. At some point in the meetings, the police training institution adopted disciplinary measures that disqualified the participants' culture, leading the group to create a collective identity to resist symbolic violence. Thus, a community gathering experience emerged in the face of the conflicted situation
Le texte discute une situation survenue dans une fonction publique désignée pour accueillir des jeunes vivant dans des quartiers vulnérables. Le service comprenait 20 jeunes de 12 à 18 ans et les réunions se sont déroulées dans un établissement de formation policière. Inspirées par la théorie de Gilles Deleuze, ce texte analyse 1) un événement survenu dans l'institution policière, 2) qu'elles en ont été ses conséquences et 3) comment le professionnel responsable a agi pour faire face à l'événement dans un contexte de groupe. À un moment donné au cours des réunions, l'institution de formation a adopté des mesures disciplinaires qui disqualifiaient la culture des jeunes participants, ce qui a conduit le groupe à se forger une identité collective basée sur la résistance à la violence symbolique. Par conséquent, une expérience de rassemblement communautaire a émergé face à la situation conflictuelle
El texto se refiere a una situación ocurrida en un servicio público designado para trabajar con jóvenes que viven en zonas vulnerables de la ciudad. El servicio incluyó a 20 jóvenes de entre 12 y 18 años y las reuniones se ubicaron en una institución de capacitación policial. Este texto analiza 1) un suceso que ocurrió en la institución policial, 2) cuáles fueron sus consecuencias y 3) cómo actuó el profesional responsable del servicio para enfrentar el suceso en un contexto grupal. El análisis de esta situación y la referencia teórica que apoyó los actos profesionales se inspiraron en la teoría de Gilles Deleuze. En algún momento de los encuentros, la institución de capacitación policial adoptó medidas disciplinarias que descalificaron la cultura de los jóvenes participantes, lo que llevó al grupo a constituir una identidad colectiva basada en la resistencia contra la violencia simbólica. Por lo tanto, ante la situación conflictiva, fue posible una experiencia de reunión comunitaria
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Política Pública , Apoio Social , Proteção da Criança , Acontecimentos que Mudam a VidaRESUMO
Este artigo tem como objetivo analisar os dados produzidos por uma pesquisa qualitativa que investigou os sentidos atribuídos ao Serviço de Convivência e Fortalecimento de Vínculos (SCFV) por seus usuários(as) em Florianópolis/SC. Por meio da observação participante realizada em um SCFV durante três meses e da aplicação de uma entrevista coletiva com idosos(as), este artigo traz algumas análises sobre os processos de significação vivenciados por esses sujeitos. Apoiados na perspectiva sócio-histórica, elaboramos dois eixos de significação, que serão discutidos neste artigo: 1) Intersubjetividade e vínculo: o SCFV como "lugar acolhedor" e de "bons encontros"; 2) O(a) idoso(a) e os processos de exclusão: o SCFV como promotor da cidadania. Em termos de resultados, é possível afirmar que esse espaço se configura como um lugar importante na vida desses(as) idosos(as) - onde eles(as) se sentem acolhidos, ouvidos e reconhecidos como sujeitos. Além do fato de esse espaço ser marcado por afetos e "bons encontros", o grupo também o revelou como um lugar potente para processos de reflexão e questionamento contra as diversas formas de segregação e violências vividas cotidianamente por ele.(AU)
This study aims to analyze the data produced by qualitative research that investigated the meanings attributed to the Service of Coexistence and Strengthening of Bonds (Serviço de Convivência e Fortalecimento de Vínculos - SCFV) by its users in the municipality of Florianópolis. By participant observation carried out in an SCFV for three months and the application of a collective interview with older adults, this study will bring some analysis of the processes of meaning experienced by these subjects. Sustained by the socio-historical perspective, we elaborated two axes of meaning to be discussed in this study : 1. Intersubjectivity and bonding: the SCFV as a "warm place" and "good meetings;" and 2. Older adults and processes of exclusion: the SCFV as a enhancer of citizenship. Results show that this space configures an important place in the lives of these older adults — a place in which they feel welcomed, heard, and recognized as subjects. In addition to the fact this space is marked by affections and "good meetings," the group also shows itself as a potent place for processes of reflection and questioning against the various forms of segregation and violence daily experienced by these people.(AU)
Este artículo tiene como objetivo analizar los datos producidos por un estudio cualitativo sobre los significados atribuidos al Servicio de Convivencia y Fortalecimiento de Lazos (SCFL) por sus usuarios en Florianópolis (Santa Catarina, Brasil). Desde la observación participante realizada en un SCFL durante el período de tres meses y desde la aplicación de una entrevista colectiva con personas mayores, este artículo presenta algunos análisis sobre los procesos de significado vividos por estos sujetos. Apoyados en la perspectiva sociohistórica, elaboramos dos ejes de significado que serán discutidos: 1) Intersubjetividad y vinculación: el SCFL como "lugar de acogida" y "buenos encuentros"; 2) Las personas mayores y los procesos de exclusión: el SCFV como promotor de ciudadanía. Los resultados permiten afirmar que este espacio se configura como un lugar importante en la vida de estas personas mayores, un lugar donde se sienten acogidos, escuchados y reconocidos como sujetos. Además de que este espacio está marcado por afectos y "buenos encuentros", el grupo también se reveló como un lugar propenso a procesos de reflexión y cuestionamiento frente a las diversas formas de segregación y violencia que viven a diario estas personas.(AU)
Assuntos
Humanos , Masculino , Feminino , Idoso , Idoso de 80 Anos ou mais , Serviço Social , Idoso , Centros de Convivência e Lazer , Grupos Populacionais , Política de Saúde , Apego ao Objeto , Psicologia , Qualidade de Vida , Previdência Social , Seguridade Social , Sistema Único de Saúde , Envelhecimento , Exercício Físico , Entrevista , Cognição , Violência Doméstica , Observação , Vida , Autonomia Pessoal , Pesquisa Qualitativa , Abuso de Idosos , Prevenção de Doenças , Prazer , Inclusão Social , Vulnerabilidade Social , Cidadania , Acessibilidade aos Serviços de Saúde , Atividades de Lazer , Destreza MotoraRESUMO
The first toxic component identified against mammals in the venom from Centruroides tecomanus scorpion from Colima, Mexico was Ct1a toxin, which was neutralized by human single chain variable fragment (scFv) RAS27. Venom characterization from these scorpions collected on the Pacific coast of Colima, enabled the identification of a second component of medical importance named Ct71 toxin. Amino acid sequence of Ct71 shares a high identity with Chui5 toxin from C. huichol scorpion, which was neutralized by scFv HV. For this reason, the kinetic parameters of interaction between Ct71 toxin and scFv HV were determined by surface plasmon resonance. Results showed a significantly higher affinity for Ct71 as compared to Chui5. As expected, this toxin was neutralized by scFv HV. The injection of a mixture of scFvs HV and RAS27, resulted in the neutralization of C. tecomanus venom, corroborating that human recombinant antibody fragments can efficiently contribute to the neutralization of medically important toxins and their respective venoms from Mexican scorpions.
Assuntos
Venenos de Escorpião , Anticorpos de Cadeia Única , Animais , Humanos , México , Proteínas Recombinantes/química , EscorpiõesRESUMO
Monoclonal antibodies are among the most effective tools for detecting tumor-associated antigens. The U.S. Food and Drug Administration (FDA) has approved more than 36 therapeutic antibodies for developing novel alternative therapies that have significant success rates in fighting cancer. However, some functional limitations have been described, such as their access to solid tumors and low interaction with the immune system. Single-chain variable fragments (scFv) are versatile and easy to produce, and being an attractive tool for use in immunotherapy models. The small size of scFv can be advantageous for treatment due to its short half-life and other characteristics related to the structural and functional aspects of the antibodies. Therefore, the main objective of this review was to describe the current situation regarding the mechanisms of action, applications, and limitations of monoclonal antibodies and scFv in the treatment of cancer.
RESUMO
Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies.
Assuntos
Venenos de Escorpião , Animais , Humanos , Sequência de Aminoácidos , Caenorhabditis elegans , Anticorpos Neutralizantes , Fragmentos de ImunoglobulinasRESUMO
The structural and dynamic changes introduced during antibody humanization continue to be a topic open to new contributions. For this reason, the study of structural and functional changes of a murine scFv (mu.scFv) anti-rhIFN-α2b after humanization was carried out. As it was shown by long molecular dynamics simulations and circular dichroism analysis, changes in primary sequence affected the tertiary structure of the humanized scFv (hz.scFv): the position of the variable domain of light chain (VL) respective to the variable domain of heavy chain (VH) in each scFv molecule was different. This change mainly impacted on conformation and dynamics of the complementarity-determining region 3 of VH (CDR-H3) which led to changes in the specificity and affinity of humanized scFv (hz.scFv). These observations agree with experimental results that showed a decrease in the antigen-binding strength of hz.scFv, and different capacities of these molecules to neutralize the in vitro rhIFN-α2b biological activity. Besides, experimental studies to characterize antigen-antibody binding showed that mu.scFv and hz.scFv bind to the same antigen area and recognize a conformational epitope, which is evidence of docking results. Finally, the differences between these molecules to neutralize the in vitro rhIFN-α2b biological activity were described as a consequence of the blockade of certain functionally relevant amino acids of the cytokine, after scFv binding. All these observations confirmed that humanization affected the affinity and specificity of hz.scFv and pointed out that two specific changes in the frameworks would be responsible.
Assuntos
Antígenos , Regiões Determinantes de Complementaridade , Aminoácidos , Animais , Regiões Determinantes de Complementaridade/química , Citocinas , Epitopos , CamundongosRESUMO
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.
Assuntos
Venenos de Escorpião , Escorpiões , Sequência de Aminoácidos , Animais , Fragmentos de Imunoglobulinas , Mamíferos , México , Camundongos , Proteínas Recombinantes , Venenos de Escorpião/toxicidadeRESUMO
A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10-9 and 6.1 × 10-11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.
Assuntos
Anticorpos Neutralizantes/imunologia , Venenos de Escorpião/imunologia , Escorpiões/química , Anticorpos de Cadeia Única/imunologia , Animais , Reações Cruzadas , Feminino , Humanos , CamundongosRESUMO
Strongyloidiasis is a helminthiasis of neglected condition that has no gold standard parasitological diagnosis due to the intermittent release of larvae in feces. This study aimed to use an scFv (single chain variable fragment) obtained by Phage Display, previously validated to detect immune complexes in serum samples from individuals infected with Strongyloides stercoralis by enzyme-linked immunosorbent assay (ELISA). Now the ability of scFv to detect the immune complexes was verified by immunofluorescence, flow cytometry using magnetic beads and surface plasmon resonance (SPR). As ELISA, the SPR, immunofluorescence and flow cytometry demonstrated the ability of scFv to detect immune complexes in sera from individuals with strongyloidiasis and discriminate them from sera of individuals with other parasitic diseases and healthy individuals. Besides de conventional ELISA, the novel approaches can also be promptly applied as auxiliary diagnostic tools to the existing parasitological method for accurate diagnosis of human strongyloidiasis.
Assuntos
Strongyloides stercoralis , Estrongiloidíase , Animais , Anticorpos Anti-Helmínticos , Ensaio de Imunoadsorção Enzimática , Fezes , Humanos , Imunoglobulina G , Testes Sorológicos , Estrongiloidíase/diagnósticoRESUMO
The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.
RESUMO
ABSTRACT Strongyloidiasis is a helminthiasis of neglected condition that has no gold standard parasitological diagnosis due to the intermittent release of larvae in feces. This study aimed to use an scFv (single chain variable fragment) obtained by Phage Display, previously validated to detect immune complexes in serum samples from individuals infected with Strongyloides stercoralis by enzyme-linked immunosorbent assay (ELISA). Now the ability of scFv to detect the immune complexes was verified by immunofluorescence, flow cytometry using magnetic beads and surface plasmon resonance (SPR). As ELISA, the SPR, immunofluorescence and flow cytometry demonstrated the ability of scFv to detect immune complexes in sera from individuals with strongyloidiasis and discriminate them from sera of individuals with other parasitic diseases and healthy individuals. Besides de conventional ELISA, the novel approaches can also be promptly applied as auxiliary diagnostic tools to the existing parasitological method for accurate diagnosis of human strongyloidiasis.
Assuntos
Humanos , Animais , Estrongiloidíase/diagnóstico , Strongyloides stercoralis , Imunoglobulina G , Ensaio de Imunoadsorção Enzimática , Testes Sorológicos , Anticorpos Anti-Helmínticos , FezesRESUMO
Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.
Assuntos
Calicreínas/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Inibidores de Serina Proteinase/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Calicreínas/imunologia , Proteínas Recombinantes/toxicidade , Inibidores de Serina Proteinase/toxicidade , Anticorpos de Cadeia Única/toxicidade , Dermatopatias/terapia , Células VeroRESUMO
Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.
RESUMO
Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.
Assuntos
Anticorpos Monoclonais , Antivenenos , Anticorpos de Cadeia Única , Venenos de Aranha/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antivenenos/administração & dosagem , Antivenenos/imunologia , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Testes de Neutralização , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Picada de Aranha/terapia , Venenos de Aranha/efeitos adversos , Aranhas/imunologiaRESUMO
OBJECTIVES: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. RESULTS: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen-antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. CONCLUSIONS: Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.
Assuntos
Anticorpos Neutralizantes , Interferon-alfa/imunologia , Proteínas Recombinantes , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Afinidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Interferon alfa-2 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
The main systemic alterations present in bothropic envenomation are hemostasis disorders, for which the conventional treatment is based on animal-produced antiophidic sera. We have developed a neutralizing antibody against Bothrops pauloensis (B. pauloensis) venom, which is member of the genus most predominant in snakebite accidents in Brazil. Subsequently, we expressed this antibody in plants to evaluate its enzymatic and biological activities. The ability of single-chain variable fragment (scFv) molecules to inhibit fibrinogenolytic, azocaseinolytic, coagulant and hemorrhagic actions of snake venom metalloproteinases (SVMPs) contained in B. pauloensis venom was verified through proteolytic assays. The antibody neutralized the toxic effects of envenomation, particularly those related to systemic processes, by interacting with one of the predominant classes of metalloproteinases. This novel molecule is a potential tool with great antivenom potential and provides a biotechnological antidote to snake venom due to its broad neutralizing activity.
Assuntos
Bothrops/metabolismo , Testes de Neutralização , Nicotiana/metabolismo , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Venenos de Serpentes/toxicidade , Animais , Brasil/epidemiologia , Caseínas/metabolismo , Galinhas , Células Clonais , Reações Cruzadas/imunologia , Fibrinogênio/metabolismo , Geografia , Hemorragia/patologia , Camundongos , Mapas de Interação de Proteínas , Proteólise , Anticorpos de Cadeia Única/isolamento & purificação , Mordeduras de Serpentes/epidemiologiaRESUMO
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.