RESUMO
SARS-CoV-2 remains a health threat with the continuous emergence of new variants. This work aims to expand the knowledge about the SARS-CoV-2 receptor-binding domain (RBD) interactions with cell receptors and monoclonal antibodies (mAbs). By using constant-pH Monte Carlo simulations, the free energy of interactions between the RBD from different variants and several partners (Angiotensin-Converting Enzyme-2 (ACE2) polymorphisms and various mAbs) were predicted. Computed RBD-ACE2-binding affinities were higher for two ACE2 polymorphisms (rs142984500 and rs4646116) typically found in Europeans which indicates a genetic susceptibility. This is amplified for Omicron (BA.1) and its sublineages BA.2 and BA.3. The antibody landscape was computationally investigated with the largest set of mAbs so far in the literature. From the 32 studied binders, groups of mAbs were identified from weak to strong binding affinities (e.g. S2K146). These mAbs with strong binding capacity and especially their combination are amenable to experimentation and clinical trials because of their high predicted binding affinities and possible neutralization potential for current known virus mutations and a universal coronavirus.Communicated by Ramaswamy H. Sarma.
Assuntos
Anticorpos Monoclonais , COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2/genética , Anticorpos Monoclonais/genética , COVID-19/genética , Predisposição Genética para Doença , Ligação Proteica , SARS-CoV-2/genéticaRESUMO
COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas contra COVID-19/imunologia , COVID-19/diagnóstico , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/genética , COVID-19/imunologia , Vacinas contra COVID-19/genética , Ensaios Clínicos como Assunto , Feminino , Engenharia Genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios e Motivos de Interação entre Proteínas/genética , Desenvolvimento de Vacinas , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of "living" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.
Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Nicotiana/crescimento & desenvolvimento , Rotavirus/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Antígenos Virais/química , Antígenos Virais/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Modelos Moleculares , Peso Molecular , Engenharia de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Rotavirus/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
BACKGROUND: A major drawback in Alzheimer's disease (AD) is the lack of validated biomarkers for routine clinical diagnostic. We have reported earlier a novel blood biomarker, named Alz-tau®, based on variants of platelet tau. This marker evaluates the ratio of high molecular weight tau (HMWtau) and the low molecular weight (LMWtau) tau. OBJECTIVE: To analyze a potential novel source of antigen for Alz-tau®, plasma tau, detected by immunoreactivity with the novel monoclonal antibody, tau51. METHODS: We evaluated tau variants in plasma precipitated with ammonium sulfate from 36 AD patients and 15 control subjects by western blot with this novel monoclonal antibody. RESULTS: The HMW/LMWtau ratio was statistically different between AD patients and controls. CONCLUSIONS: Plasma tau variants are suitable to be considered as a novel antigen source for the Alz-tau® biomarker for AD.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Anticorpos Monoclonais/sangue , Variação Genética/fisiologia , Proteínas tau/sangue , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Anticorpos Monoclonais/genética , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas tau/genéticaRESUMO
OBJECTIVES: To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. RESULTS: Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. CONCLUSIONS: The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.
Assuntos
Anticorpos Monoclonais/imunologia , Medicamentos Biossimilares/farmacologia , Sítios Internos de Entrada Ribossomal/genética , Pró-Proteína Convertase 9/genética , Sequência de Aminoácidos/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Células CHO , Cricetulus/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/farmacologia , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/farmacologia , TransfecçãoRESUMO
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
In addition to the full-length beta-amyloid peptides (Aß 1-40/42), several Aß variants, truncated at their N- or C-termini and bearing different post-translational modifications, have been detected in the brain of Alzheimer´s disease (AD) patients. AßN3(pE), an Aß peptide bearing an amino-terminal pyroglutamate at position 3, is a significant constituent of intracellular, extracellular and vascular Aß deposits in brain tissue from individuals with AD and Down syndrome. Pioneering immunotherapy studies have primarily focused on the full-length Aß peptide, disregarding the presence of N-truncated/modified species. However, in recent years, increasing attention has been directed towards AßN3(pE), in both pre-clinical studies and clinical trials. In the present study, we generated and characterized an anti-AßN3(pE) mouse monoclonal antibody (3B8) that recognizes amyloid aggregates in brain tissue from AD patients and in 3xTg-AD transgenic mice. To identify the epitope recognized by 3B8, a library of random heptapeptides fused to the minor coat protein of M13 phage was screened. Results from screening, along with those from ELISA assays against distinct Aß fragments, suggest recognition of two conformational epitopes present in AßN3(pE) and Aß 3-42, regardless of the glutamate-pyroglutamate modification. The novel 3B8 antibody may be useful in future therapeutic and diagnostic applications for AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais/metabolismo , Encéfalo/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Anticorpos Monoclonais/genética , Epitopos/genética , Epitopos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fragmentos de Peptídeos/genéticaRESUMO
Plant-based platforms are extensively use for the expression of recombinant proteins, including monoclonal antibodies (mAbs). Generally, immunoglobulins (Igs) are sorted to the apoplast, which is often afflicted with intense proteolysis. Here, we describe methods to transiently express mAbs sorted to central vacuole in Nicotiana benthamiana leaves and to characterize the obtained IgG. Central vacuole is an appropriate compartment for the efficient production of Abs, consequently vacuolar sorting should be considered as an alternative strategy to obtain high protein yields.
Assuntos
Anticorpos Monoclonais/análise , Imunoglobulina G/análise , Nicotiana/genética , Vacúolos/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Espectrometria de Massas/métodos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Vacúolos/metabolismoRESUMO
Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.
Assuntos
Adalimumab/imunologia , Anticorpos Monoclonais/imunologia , Medicamentos Biossimilares , Proteínas Recombinantes de Fusão/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adalimumab/genética , Adalimumab/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Camundongos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. MATERIALS AND METHODS: Recombinant polymeric anti-G IgG1 (IgG1µtp) and IgG3 (IgG3µtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. RESULTS: The recombinant IgG1µtp and IgG3µtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. DISCUSSION: The enhanced opsonic properties of the polymeric anti-G IgG1µtp and IgG3µtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.