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1.
Nat Commun ; 12(1): 1359, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649336

RESUMO

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an 'adaptor' while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.


Assuntos
Anticorpos Monoclonais/metabolismo , Imunomodulação , Imunoterapia , Nanopartículas/química , Neoplasias/imunologia , Neoplasias/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Proteínas Imobilizadas/metabolismo , Imunidade , Células Matadoras Naturais/imunologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Linfócitos T/imunologia
2.
J Vis Exp ; (168)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33616089

RESUMO

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos CD/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Apresentação Cruzada , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/metabolismo , Ovalbumina/imunologia , Receptores de Superfície Celular/metabolismo
3.
J Vis Exp ; (167)2021 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-33522504

RESUMO

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.


Assuntos
Imunoglobulina G/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , Agrobacterium tumefaciens/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Cromatografia , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Humanos , Canamicina/farmacologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/crescimento & desenvolvimento , Tabaco/microbiologia , Raios Ultravioleta
4.
Nat Commun ; 12(1): 583, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495445

RESUMO

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Assuntos
Anticorpos Monoclonais/metabolismo , Complexos Multiproteicos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Necrose Tumoral alfa/química
5.
Methods Mol Biol ; 2270: 419-435, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479911

RESUMO

Type 1 diabetes is an organ-specific autoimmune disease characterized by immune-mediated beta cell destruction in pancreatic islets, which results in deficient insulin production. B cells have a dual role in type 1 diabetes pathogenesis. A pathogenic role for B cells has been widely described and is supported by the observation of a delay in the loss of C-peptide following B-cell depletion by Rituximab, in the first year after diagnosis. However, it is now clear that B cells, under certain conditions, can delay and prevent the onset of type 1 diabetes as demonstrated in mouse models. In this chapter, we describe the methods required to study the phenotype and function of regulatory B cells in the context of diabetes.


Assuntos
Linfócitos B Reguladores/patologia , Diabetes Mellitus Tipo 1/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD20/metabolismo , Doenças Autoimunes/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B Reguladores/citologia , Linfócitos B Reguladores/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD
6.
Science ; 371(6531): 823-829, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33495307

RESUMO

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , /terapia , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Epitopos/imunologia , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Domínios Proteicos , Engenharia de Proteínas , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
Adv Drug Deliv Rev ; 169: 100-117, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33309815

RESUMO

To address the COVID-19 pandemic, there has been an unprecedented global effort to advance potent neutralizing mAbs against SARS-CoV-2 as therapeutics. However, historical efforts to advance antiviral monoclonal antibodies (mAbs) for the treatment of other respiratory infections have been met with categorical failures in the clinic. By investigating the mechanism by which SARS-CoV-2 and similar viruses spread within the lung, along with available biodistribution data for systemically injected mAb, we highlight the challenges faced by current antiviral mAbs for COVID-19. We summarize some of the leading mAbs currently in development, and present the evidence supporting inhaled delivery of antiviral mAb as an early intervention against COVID-19 that could prevent important pulmonary morbidities associated with the infection.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antivirais/uso terapêutico , Fatores Imunológicos/uso terapêutico , /efeitos dos fármacos , /antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antivirais/química , Antivirais/metabolismo , /metabolismo , Humanos , Imunização Passiva , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , /metabolismo , Eliminação de Partículas Virais/efeitos dos fármacos , Eliminação de Partículas Virais/fisiologia
8.
Int J Food Microbiol ; 339: 109014, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33333444

RESUMO

The objective of this study was to develop a method with improved sensitivity for Campylobacter jejuni detection in foods. Nitrogen-doped carbon nanodots (N-CNDs) were synthesized and added to an enrichment medium (Bolton broth) at a concentration of 10 mg/mL. A light-emitting diode (LED) at a wavelength of 425 nm was used to irradiate the N-CNDs-supplemented enrichment medium to induce an exothermic reaction for 1 h. Additionally, a monoclonal antibody specific to C. jejuni NCTC11168 was developed using hybridoma cells to aid detection. The C. jejuni detection capabilities of N-CNDs-supplemented enrichment medium and the conventional Bolton broth enrichment, were compared using duck samples. C. jejuni in the enrichment was detected with the monoclonal antibody based-indirect enzyme-linked immunosorbent assay (ID-ELISA). The N-CNDs-supplemented enrichment medium showed a better C. jejuni detection capability than the conventional Bolton broth enrichment. Additionally, data from ID-ELISA showed excellent detection efficiency and a shortened detection time in the N-CNDs-supplemented enrichment medium after LED irradiation at 425 nm. These results indicate that 1-h LED irradiation at 425 nm to Bolton broth supplemented with the N-CNDs increased the detection efficiency and shortened the detection time with the monoclonal antibody for C. jejuni in food.


Assuntos
Campylobacter jejuni/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Microbiologia de Alimentos/métodos , Carne/microbiologia , Nanopartículas/química , Animais , Anticorpos Monoclonais/metabolismo , Bactérias/efeitos dos fármacos , Carbono/química , Carbono/farmacologia , Meios de Cultura , Patos/microbiologia , Nitrogênio/química , Nitrogênio/farmacologia
9.
Methods Mol Biol ; 2236: 67-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237541

RESUMO

Myeloid-derived suppressor cells (MDSC) are known to inhibit functions of T and NK cells. MDSC have been shown to be generated and to accumulate under chronic inflammatory conditions that are typical for cancer. Therefore, it would be highly beneficial to find ways to diminish the number and immunosuppressive functions of these cells in tumor-bearing hosts. Here we describe current protocols to deplete MDSC or induce their maturation in preclinical tumor models that could lead to the attenuation of their immunosuppressive functions.


Assuntos
Diferenciação Celular , Células Supressoras Mieloides/patologia , Neoplasias/patologia , Animais , Anticorpos Monoclonais/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Supressoras Mieloides/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Tretinoína/farmacologia
10.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33375207

RESUMO

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.


Assuntos
Anticorpos Monoclonais/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Glicosilação , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/ultraestrutura , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina G/ultraestrutura , Estabilidade Proteica
11.
MAbs ; 12(1): 1854149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33319649

RESUMO

Monoclonal antibody (mAb) therapy has been previously exploited for viral infections, such as respiratory syncytial virus pneumonia and Ebolavirus disease. In the ongoing COVID-19 pandemic, early signals of efficacy from convalescent plasma therapy have encouraged research and development of anti-SARS-CoV-2 mAbs. While many candidates are in preclinical development, we focus here on anti-SARS-CoV-2 neutralizing mAbs (or mAb cocktails) that represent the late-stage clinical pipeline, i.e., those currently in Phase 2 or Phase 3 clinical trials. We describe the structure, mechanism of action, and ongoing trials for VIR-7831, LY-CoV555, LY-CoV016, BGB-DXP593, REGN-COV2, and CT-P59. We speculate also on the next generation of these mAbs.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , /terapia , /fisiologia , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Humanos , Imunização Passiva/métodos
12.
Nat Commun ; 11(1): 6435, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353951

RESUMO

Human ß-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the ß-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits ß-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive ß-tryptase monomers, and may provide an alternative strategy for antibody engineering.


Assuntos
Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Triptases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Heparina/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Modelos Moleculares , Proteínas Mutantes/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Triptases/química
13.
Trends Immunol ; 41(11): 1006-1022, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33041212

RESUMO

The 2019 coronavirus pandemic remains a major public health concern. Neutralizing antibodies (nAbs) represent a cutting-edge antiviral strategy. We focus here on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, and discuss current progress in antibody research against rampant SARS-CoV-2 infections. We provide a perspective on the mechanisms of SARS-CoV-2-derived nAbs, comparing these with existing SARS-CoV-derived antibodies. We offer insight into how these antibodies cross-react and cross-neutralize by analyzing available structures of spike (S) glycoprotein-antibody complexes. We also propose ways of adopting antibody-based strategies - such as cocktail antibody therapeutics against SARS-CoV-2 - to overcome the possible resistance of currently identified mutants and mitigate possible antibody-dependent enhancement (ADE) pathologies. This review provides a platform for the progression of antibody and vaccine design against SARS-CoV-2, and possibly against future coronavirus pandemics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Ligação Proteica , Vírus da SARS/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
14.
Sci Rep ; 10(1): 17698, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077899

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 µg/g and 130 µg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Tabaco/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Folhas de Planta/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
15.
J Chromatogr A ; 1633: 461635, 2020 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-33128974

RESUMO

Viral clearance is an important performance metric for the downstream process of monoclonal antibodies (mAbs) due to its impact on patient safety. Anion exchange chromatography (AEX) has been well-accepted in the industry as one of the workhorse techniques for removing viruses, and is considered to be able to achieve high log clearance values under most operating conditions. However, it is not uncommon for viral clearance results on AEX to fall below the desired level despite operating under conditions that should achieve high clearance levels according to conventional wisdom of how this mode of chromatography operates. In this study, a design of experiment (DoE) approach was used to develop a more fundamental understanding of viral clearance during AEX chromatography using Minute Virus of Mice (MVM) on POROS HQ resin. Load pH, conductivity and virus concentration were evaluated as design factors for three mAbs with varying physical and chemical properties. The hydrophobicity and surface charge distributions of the molecules were found to be the most significant factors in influencing viral clearance performance, and the viral clearance trends did not seem to fit with conventional wisdom. To explain this seemingly unconventional behavior, we propose a new mechanism that suggests that interactions between the mAb and the virus have a major contribution on retention of the virus on the resin. This furthered understanding may help improve the predictability, performance and robustness of viral clearance during AEX chromatography.


Assuntos
Anticorpos Monoclonais/metabolismo , Cromatografia por Troca Iônica/normas , Vírus Miúdo do Camundongo/metabolismo , Vírus/metabolismo , Animais , Ânions/química , Anticorpos Monoclonais/química , Camundongos , Vírus/química
16.
Science ; 370(6518): 861-865, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33082294

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.


Assuntos
Betacoronavirus/fisiologia , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Células CACO-2 , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Furina/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
17.
Farm. hosp ; 44(5): 212-217, sept.-oct. 2020. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-195148

RESUMO

OBJETIVO: Recientemente se han desarrollado anticuerpos monoclonales contra la vía del péptido relacionado con el gen de la calcitonina para la prevención de la migraña. El objetivo de este estudio es comparar la eficacia de los fármacos anticuerpos monoclonales contra la vía del péptido relacionado con el gen de la calcitonina en migraña crónica a través de una comparación indirecta ajustada, y establecer si pueden considerarse alternativas terapéuticas equivalentes en esta patología. MÉTODO: Se realizó una búsqueda bibliográfica de ensayos clínicos aleatorizados en la base de datos PubMed el 26 de diciembre de 2019. Los criterios de inclusión fueron: ensayos clínicos aleatorizados fase II/III de anticuerpos monoclonales contra la vía del péptido relacionado con el gen de la calcitonina con similar población, duración de seguimiento y comparador. Se seleccionó la reducción de al menos un 50% de días de migraña/mes como variable de eficacia. Se definió migraña crónica como ≥ 15 días de dolor de cabeza/mes, de los cuales ≥ 8 fueron días de migraña (duración del evento ≥ 4 horas). Se excluyeron los ensayos clínicos aleatorizados con diferentes contextos clínicos de migraña crónica y definición de enfermedad. Se desarrolló una compa-ración indirecta ajustada utilizando el método de Bucher. Para la evaluación de la posible equivalencia terapéutica se siguieron las directrices de la guía de alternativas terapéuticas equivalentes de posicionamiento. El valor delta (Δ, máxima diferencia como criterio clínico de equivalencia) se calculó como la mitad de la reducción absoluta del riesgo obtenida en un metaanálisis de los ensayos clínicos aleatorizados incluidos en la comparación indirecta ajustada. RESULTADOS: Se encontraron 30 ensayos clínicos aleatorizados: erenumab (n = 12), fremanezumab (n = 7), galcanezumab (n = 10) y eptinezumab (n = 1). Se seleccionaron tres estudios: uno de erenumab, uno de fremanezumab y otro de eptinezumab. El resto no se incluyó en la comparación indi-recta ajustada por incumplimiento de los criterios de inclusión. Los resultados de la comparación indirecta ajustada entre las diferentes posologías de los fármacos estudiados no mostraron diferencias estadísticamente signifi-cativas, y la mayor parte del intervalo de confianza del 95% se encontró dentro de los márgenes delta calculados (Δ = 9,5%). No se encontraron diferencias de seguridad relevantes entre los tres medicamentos. CONCLUSIONES: La comparación indirecta ajustada no mostró diferencias estadísticamente significativas en la reducción de ≥ 50% de días de migraña/mes entre erenumab, fremanezumab y eptinezumab. Se encontró una probable equivalencia clínica entre estos fármacos en términos de eficacia y seguridad, por lo que podrían considerarse alternativas terapéuticas equivalentes en migraña crónica


OBJECTIVE: New monoclonal antibodies against the calcitonin gene-related peptide pathway have recently been developed for the prevention of migraine. The aim of this study is to compare the efficacy of monoclonal antibodies against the calcitonin generelated peptide pathway drugs in chronic migraine through an adjusted indirect treatment comparison, and to establish whether they can be considered equivalent therapeutic alter-natives in this pathology. METHOD: A bibliographic search of randomized clinical trials was performed in PubMed database on December 26, 2019. The inclusion criteria were phase II/III randomized clinical trials of monoclonal anti-bodies against the calcitonin generelated peptide pathway with similar population, length of follow-up and treatment comparator. The reduction of at least 50% migraine-days/month was selected as efficacy endpoint. Chronic migraine was defined as ≥ 15 headache days/month, of which ≥ 8 were migraine-days (event duration ≥ 4 hours). Randomized clinical trials with different clinical chronic migraine context and definition of disease were excluded. An indirect treatment comparison was developed using Bucher's method. The equivalent therapeutic alternatives positioning guide was used for the evaluation of potentially equivalent alternatives. Delta value (Δ, maximum difference as clinical criterion of equivalence) was calculated as half of absolute risk reduction obtained in a meta-analysis of randomized clinical trials included in indirect treatment comparison. RESULTS: Thirty randomized clinical trials were found: erenumab (n = 12), fremanezumab (n = 7), galcanezumab (n = 10) and eptinezumab (n = 1). Three studies were selected: one of erenumab, one of fremanezumab and another of eptinezumab. The rest were not included in indirect treatment comparison for non-compliance of inclusion criteria. Results of indirect treatment comparison among different regimens of studied drugs showed no statistically significant differences, and the most part of 95% confidence interval was within calculated delta margins (Δ = 9.5%). No relevant safety differences among the three drugs were found. CONCLUSIONS: Indirect treatment comparison showed no statistically sig-nificant differences in reduction of ≥ 50% migraine days/month between erenumab, fremanezumab and eptinezumab. Probable clinical equiva-lence was found between these drugs in terms of efficacy and safety, therefore they could be considered equivalent therapeutic alternatives in chronic migraine


Assuntos
Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Peptídeo Relacionado com Gene de Calcitonina/administração & dosagem , Anticorpos Monoclonais/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Medicina Baseada em Evidências , Análise de Dados , Ensaios Clínicos Controlados Aleatórios como Assunto
18.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
19.
Nat Struct Mol Biol ; 27(10): 942-949, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32753755

RESUMO

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, with 10 million infections and more than 500,000 fatalities by June 2020. To initiate infection, the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface and fusion of the viral and host membranes. Prefusion SARS-CoV-2 S is the main target of neutralizing antibodies and the focus of vaccine design. However, its limited stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a construct corresponding to the prefusion SARS-CoV-2 S ectodomain trimer, covalently stabilized by a disulfide bond in the closed conformation. Structural and antigenicity analyses show we successfully shut S in the closed state without otherwise altering its architecture. We demonstrate that this strategy is applicable to other ß-coronaviruses, such as SARS-CoV and MERS-CoV, and might become an important tool for structural biology, serology, vaccine design and immunology studies.


Assuntos
Betacoronavirus/química , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Microscopia Crioeletrônica , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas , Multimerização Proteica , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
20.
Biomed Pharmacother ; 130: 110559, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32768882

RESUMO

As the number of people infected with the newly identified 2019 novel coronavirus (SARS-CoV2) is continuously increasing every day, development of potential therapeutic platforms is vital. Based on the comparatively high similarity of receptor-binding domain (RBD) in SARS-CoV2 and SARS-CoV, it seems crucial to assay the cross-reactivity of anti-SARS-CoV monoclonal antibodies (mAbs) with SARS-CoV2 spike (S)-protein. Indeed, developing mAbs targeting SARS-CoV2 S-protein RBD could show novel applications for rapid and sensitive development of potential epitope-specific vaccines (ESV). Herein, we present an overview on the discovery of new CoV followed by some explanation on the SARS-CoV2 S-protein RBD site. Furthermore, we surveyed the novel therapeutic mAbs for targeting S-protein RBD such as S230, 80R, F26G18, F26G19, CR3014, CR3022, M396, and S230.15. Afterwards, the mechanism of interaction of RBD and different mAbs were explained and it was suggested that one of the SARS-CoV-specific human mAbs, namely CR3022, could show the highest binding affinity with SARS-CoV2 S-protein RBD. Finally, some ongoing challenges and future prospects for rapid and sensitive advancement of therapeutic mAbs targeting S-protein RBD were discussed. In conclusion, it may be proposed that this review may pave the way for recognition of RBD and different mAbs to develop potential therapeutic ESV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pandemias , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos Virais/metabolismo , Sítios de Ligação de Anticorpos , Coronavirus/química , Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/imunologia , Humanos , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais/imunologia
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