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1.
Signal Transduct Target Ther ; 5(1): 212, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963228

RESUMO

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Síndrome Respiratória Aguda Grave/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/imunologia , Vírion/ultraestrutura
2.
PLoS Negl Trop Dis ; 14(9): e0008557, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976512

RESUMO

Novel diagnostic tools are a major challenge for brucellosis research, especially in developing countries. Herein, we established a handheld quantum dot (QD) immunochromatographic device for the fast detection of brucellosis antibodies in the field. Total bacterial protein extracted from Brucella 104M served as labelling and coating antigen. QD labelling and immunochromatography methods were used to optimise reaction conditions, labelling conditions, reaction temperature and storage temperature. QD test strips were employed to test brucellosis serum to determine their sensitivity, specificity and stability. Test strips were compared with Rose Bengal test, standard agglutination test and colloidal gold immunochromatographic assay. Labelled Brucella total protein displayed good specificity and no cross-reactivity. The concentration of labelled total bacterial protein was 3.9 mg/ml, the coating concentration was 2.0 mg/ ml, and the serum titre with the lowest detection sensitivity was 1:25. The optimal reaction temperature for the test strip was 25-30°C. The test strip was stable after storage at room temperature and the repeatability was high, with a coefficient of variation of 4.0%. After testing 199 serum samples, the sensitivity of the QD test strip was 98.53%, the specificity was 93.57%, and the coincidence rate with the standard agglutination test was 96.98%. The developed QD immunochromatographic method can be used for rapid detection and preliminary screening of brucellosis in the field.


Assuntos
Brucella/imunologia , Brucelose/diagnóstico , Cromatografia de Afinidade/métodos , Pontos Quânticos , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/análise , Brucelose/imunologia , Humanos , Fitas Reagentes/análise , Sensibilidade e Especificidade
3.
Adv Exp Med Biol ; 1255: 221-230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32949403

RESUMO

Monoclonal antibodies from human sources are being increasingly recognized as valuable options in many therapeutic areas. These antibodies can show exquisite specificity and high potency while maintaining a desirable safety profile, having been matured and tolerized within human patients. However, the discovery of these antibodies presents important challenges, since the B cells encoding therapeutic antibodies can be rare in a typical blood draw and are short-lived ex vivo. Furthermore, the unique pairing of VH and VL domains in each B cell contributes to specificity and function; therefore, maintaining antibody chain pairing presents a throughput limitation. This work will review the various approaches aimed at addressing these challenges with an eye to next-generation methods for high-throughput discovery from the human B-cell repertoire.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Descoberta de Drogas , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Humanos
4.
PLoS One ; 15(7): e0235815, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673351

RESUMO

Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Galinhas , Células HEK293 , Humanos , Imunização , Macaca fascicularis , Camundongos , Modelos Moleculares
5.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32718020

RESUMO

The ongoing pandemic of coronavirus disease-2019 (COVID-19) is being caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The disease continues to present significant challenges to the health care systems around the world. This is primarily because of the lack of vaccines to protect against the infection and the lack of highly effective therapeutics to prevent and/or treat the illness. Nevertheless, researchers have swiftly responded to the pandemic by advancing old and new potential therapeutics into clinical trials. In this review, we summarize potential anti-COVID-19 therapeutics that block the early stage of the viral life cycle. The review presents the structures, mechanisms, and reported results of clinical trials of potential therapeutics that have been listed in clinicaltrials.gov. Given the fact that some of these therapeutics are multi-acting molecules, other relevant mechanisms will also be described. The reviewed therapeutics include small molecules and macromolecules of sulfated polysaccharides, polypeptides, and monoclonal antibodies. The potential therapeutics target viral and/or host proteins or processes that facilitate the early stage of the viral infection. Frequent targets are the viral spike protein, the host angiotensin converting enzyme 2, the host transmembrane protease serine 2, and clathrin-mediated endocytosis process. Overall, the review aims at presenting update-to-date details, so as to enhance awareness of potential therapeutics, and thus, to catalyze their appropriate use in combating the pandemic.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Pandemias , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Polissacarídeos/química , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
6.
Nat Commun ; 11(1): 3418, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647286

RESUMO

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.


Assuntos
Anticorpos Antivirais/imunologia , Resistência a Múltiplos Medicamentos/imunologia , Farmacorresistência Viral/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mutação/genética , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Antígenos Virais/metabolismo , Líquidos Corporais/virologia , Análise Mutacional de DNA , Cães , Epitopos/química , Epitopos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Imagem Óptica , Ligação Proteica , Análise Espectral Raman
7.
Nature ; 584(7821): 450-456, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32698192

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Epitopos de Linfócito B/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/análise , Anticorpos Antivirais/química , Anticorpos Antivirais/ultraestrutura , Betacoronavirus/química , Betacoronavirus/ultraestrutura , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/ultraestrutura , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Pulmão/patologia , Pulmão/virologia , Masculino , Mesocricetus , Modelos Moleculares , Testes de Neutralização , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/ultraestrutura
8.
Food Chem ; 331: 127368, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32569962

RESUMO

A novel strategy for AFB1 detection in grains was proposed based on DNA tetrahedron-structured probe (DTP) and horseradish peroxidase (HRP) triggered polyaniline (PANI) deposition. Briefly, the DNA tetrahedron nanostructures were assembled on the gold electrode, with carboxylic group designed on top vertex of them. The carboxylic group was conjugated with the AFB1 monoclonal antibody (mAb) to form DTP. The test sample and a known fixed concentration of HRP-labeled AFB1 were mixed and they compete for binding to DTP. The HRP assembled on the gold electrode catalyzed the polymerization of aniline on DTP. AFB1 in grains could be determined by using PANI as electrochemical signal molecules. Interestingly, DNA tetrahedron-structure, which has mechanical rigidity and structural stability, can improve antigen-antibody specific recognition and binding efficiency through the use of mAb ordered assembly. Meanwhile, nucleic acid backbone with a large amount of negative charge is good template for aniline polymerization under mild conditions.


Assuntos
Aflatoxina B1/análise , Compostos de Anilina/química , Técnicas Eletroquímicas/métodos , Contaminação de Alimentos/análise , Nanoestruturas/química , Aflatoxina B1/imunologia , Anticorpos Monoclonais/química , DNA/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Peroxidase do Rábano Silvestre/química , Polimerização , Sensibilidade e Especificidade
9.
Cell ; 181(7): 1458-1463, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32492407

RESUMO

The SARS-CoV-2 pandemic that causes COVID-19 respiratory syndrome has caused global public health and economic crises, necessitating rapid development of vaccines and therapeutic countermeasures. The world-wide response to the COVID-19 pandemic has been unprecedented with government, academic, and private partnerships working together to rapidly develop vaccine and antibody countermeasures. Many of the technologies being used are derived from prior government-academic partnerships for response to other emerging infections.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Humanos , Colaboração Intersetorial , Pneumonia Viral/imunologia , Vacinas Virais/química
10.
Nat Commun ; 11(1): 3183, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576815

RESUMO

High-diversity genetically-encoded combinatorial libraries (108-1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106-108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/ß-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of ß-amino acids in facilitating a key binding contact.


Assuntos
Biblioteca de Peptídeos , Peptídeos/química , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Aminoácidos , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Proteínas de Transporte/química , Cromatografia Líquida , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Modelos Moleculares , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo
11.
Science ; 369(6504): 650-655, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571838

RESUMO

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo-eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos Virais/imunologia , Linfócitos B/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/terapia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Genes de Cadeia Pesada de Imunoglobulina , Humanos , Memória Imunológica , Pessoa de Meia-Idade , Mutação , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/terapia , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Adulto Jovem
12.
Cell Host Microbe ; 27(6): 891-898.e5, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: covidwho-260276

RESUMO

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Modelos Moleculares , Domínios Proteicos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química
13.
Nature ; 583(7815): 290-295, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32422645

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Reações Cruzadas/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linfócitos B/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Reações Cruzadas/efeitos dos fármacos , Microscopia Crioeletrônica , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Células HEK293 , Humanos , Evasão da Resposta Imune/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Memória Imunológica/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Modelos Moleculares , Testes de Neutralização , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/química , Células Vero
14.
Cell ; 182(1): 73-84.e16, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32425270

RESUMO

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Análise de Célula Única , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Convalescença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Pandemias , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Éxons VDJ
15.
J Biosci Bioeng ; 130(3): 327-333, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32439286

RESUMO

N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) system to achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão/métodos , Polissacarídeos/química , Humanos
16.
Int J Nanomedicine ; 15: 3087-3098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431503

RESUMO

Purpose: Aldo-ketoreductase (AKR) 1C3 is crucial for testosterone synthesis. Abnormally high expression/activity of AKR1C3 can promote castration-resistant prostate cancer (CRPC). A mansonone derivative and AKR1C3 inhibitor, 6e, was combined with 4D5 (extracellular fragment of the monoclonal antibody of human epidermal growth factor receptor-2)-modified chitosan to achieve a nanodrug-delivery system (CS-4D5/6e) to treat CRPC. Materials and Methods: Morphologies/properties of CS-4D5/6e were characterized by atomic force microscopy, zeta-potential analysis, and Fourier transform-infrared spectroscopy. CS-4D5/6e uptake was measured by immunofluorescence under confocal laser scanning microscopy. Testosterone in LNCaP cells overexpressing human AKR1C3 (LNCaP-AKR1C3) and cell lysates was measured to reflect AKR1C3 activity. Androgen receptor (AR) and prostate-specific antigen (PSA) expression was measured by Western blotting. CS-4D5/6e-based inhibition of AKR1C3 was evaluated in tumor-xenografted mice. Results: CS-4D5/6e was oblate, with a particle size of 200-300 nm and thickness of 1-5 nm. Zeta potential was 1.39±0.248 mV. 6e content in CS-4D5/6e was 7.3±1.4% and was 18±3.6% for 4D5. 6e and CS-4D5/6e inhibited testosterone production significantly in a concentration-dependent manner in LNCaP-AKR1C3 cells, and a decrease in expression of AKR1C3, PSA, and AR was noted. Half-maximal inhibitory concentration of CS-4D5/6e on LNCaP-AKR1C3 cells was significantly lower than that in LNCaP cells (P<0.05). CS-4D5/6e significantly reduced growth of 22Rv1 tumor xenografts by 57.00% compared with that in the vehicle group (P<0.01). Conclusion: We demonstrated the antineoplastic activity of a potent AKR1C3 inhibitor (6e) and its nanodrug-delivery system (CS-4D5/6e). First, CS-4D5/6e targeted HER2-positive CRPC cells. Second, it transferred 6e (an AKR1C3 inhibitor) to achieve a reduction in intratumoral testosterone production. Compared with 6e, CS-4D5/6e showed lower systemic toxicity. CS-4D5/6e inhibited tumor growth effectively in mice implanted with tumor xenografts by downregulating testosterone production mediated by intratumoral AKR1C3. These results showed a promising strategy for treatment of the CRPC that develops invariably in prostate-cancer patients.


Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Quitosana/química , Sistemas de Liberação de Medicamentos/métodos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Naftoquinonas/química , Antígeno Prostático Específico/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptor ErbB-2/imunologia , Receptores Androgênicos/metabolismo , Sesquiterpenos/química , Testosterona/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
PLoS One ; 15(5): e0232713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379792

RESUMO

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Becaplermina/imunologia , Desenho Assistido por Computador , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Modelos Moleculares , Mutação , Conformação Proteica , Propriedades de Superfície , Viscosidade
18.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
19.
J Med Chem ; 63(10): 5360-5366, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32374601

RESUMO

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15N direct detection nuclear magnetic resonance (NMR) technique, the 15N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15N direct detection.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Composição de Medicamentos/métodos , Espectroscopia de Ressonância Magnética/métodos , Mapeamento de Peptídeos/métodos , Anticorpos Monoclonais/metabolismo , Armazenamento de Medicamentos/métodos , Células HEK293 , Humanos , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/metabolismo , Estrutura Secundária de Proteína
20.
Artigo em Inglês | MEDLINE | ID: mdl-32413276

RESUMO

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Modelos Moleculares , Domínios Proteicos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química
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