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1.
Nat Commun ; 12(1): 1063, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594061

RESUMO

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I ß- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.


Assuntos
Anticorpos Antiprotozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Cinética , Camundongos Endogâmicos C57BL , Modelos Moleculares , Parasitos/imunologia , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
2.
Methods Mol Biol ; 2247: 77-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301113

RESUMO

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.


Assuntos
Cromatografia de Afinidade , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/isolamento & purificação , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Ordem dos Genes , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Modelos Moleculares , Plasmídeos/genética , Conformação Proteica , Proteólise , Células Sf9 , Relação Estrutura-Atividade
3.
Methods Mol Biol ; 2253: 175-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315224

RESUMO

We studied the molecular details of the recognition of antigens by the variable domain of their cognate antibodies in as well as those elicited by the constant domains, which do not directly interact with antigens. Such effects are difficult to study experimentally; however, molecular dynamics simulations and subsequent residue interaction network analysis provide insight into the allosteric communication between the antigen-binding CDR region and the constant domain. We performed MD simulations of the complex of Fab and prion-associated peptide in the apo and bound forms and follow the conformational changes in the antibody and cross-talk between its subunits and with antigens. These protocols could be generally applied for studies of other antigens-antibody recognition systems.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Príons/química , Príons/imunologia , Regulação Alostérica , Sítio Alostérico , Animais , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica
4.
PLoS One ; 15(12): e0239112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382708

RESUMO

Influenza virus A is a significant agent involved in the outbreak of worldwide epidemics, causing millions of fatalities around the world by respiratory diseases and seasonal illness. Many projects had been conducting to investigate recovered infected patients for therapeutic vaccines that have broad-spectrum activity. With the aid of the computational approach in biology, the designation for a vaccine model is more accessible. We developed an in silico protocol called iBRAB to design a broad-reactive Fab on a wide range of influenza A virus. The Fab model was constructed based on sequences and structures of available broad-spectrum Abs or Fabs against a wide range of H1N1 influenza A virus. As a result, the proposed Fab model followed iBRAB has good binding affinity over 27 selected HA of different strains of H1 influenza A virus, including wild-type and mutated ones. The examination also took by computational tools to fasten the procedure. This protocol could be applied for a fast-designed therapeutic vaccine against different types of threats.


Assuntos
Anticorpos Antivirais/química , Antígenos Virais/química , Desenho de Fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Fragmentos Fab das Imunoglobulinas/química , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Anticorpos Antivirais/genética , Antígenos Virais/genética , Antígenos Virais/imunologia , Sítios de Ligação , Simulação por Computador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/virologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
5.
PLoS One ; 15(9): e0232311, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915778

RESUMO

Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call "Crystal Kappa", enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Cadeias kappa de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica em Folha beta , Coelhos
6.
Int J Nanomedicine ; 15: 5575-5589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801705

RESUMO

Purpose: The overexpression of Her-2 in 25-30% breast cancer cases and the crosstalk between Her-2 and fatty acid synthase (FASN) establishes Her-2 as a promising target for site-directed delivery. The present study aimed to develop the novel lipid base formulations to target and inhibit the cellular proliferation of Her-2-expressing breast cancer cells through the silencing of FASN. In order to achieve this goal, we prepared DSPC/Chol and DOPE/CHEMS immunoliposomes, conjugated with the anti-Her-2 fab' and encapsulated FASN siRNA against breast cancer cells. Methods: We evaluated the size, stability, cellular uptake and internalization of various formulations of liposomes. The antiproliferative gene silencing potential was investigated by the cell cytotoxicity, crystal violet, wound healing and Western blot analyses in Her-2+ and Her-2¯ breast cancer cells. Results: The data revealed that both nanosized FASN-siRNA-encapsulated liposomes showed significantly higher cellular uptake and internalization with enhanced stability. The cell viability of Her-2+ SK-BR3 cells treated with the targeted formulation of DSPC/Chol- and DOPE/CHEMS-encapsulating FASN-siRNA reduced to 30% and 20%, respectively, whereas it was found to be 45% and 36% in MCF-7 cells. The wounds were not only failed to close but they became broader in Her-2+ cells treated with targeted liposomes of siRNA. Consequently, the amount of FASN decreased by 80% in SK-BR3 cells treated with non-targeted liposomes and it was 30% and 60% in the MCF-7 cells treated with DSPC/Chol and DOPE/CHEMS liposomes, respectively. Conclusion: In this study, we developed the formulation that targeted Her-2 for the suppression of FASN and, therefore, inhibited the proliferation of breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Ácido Graxo Sintase Tipo I/genética , Terapia de Alvo Molecular/métodos , Receptor ErbB-2/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Inativação Gênica , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Lipídeos/química , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/imunologia , Células MCF-7 , RNA Interferente Pequeno/genética , Receptor ErbB-2/imunologia
7.
Science ; 369(6505): 793-799, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792392

RESUMO

Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo-electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20.


Assuntos
Anticorpos Monoclonais Humanizados/química , Complexo Antígeno-Anticorpo/química , Antígenos CD20/química , Antineoplásicos/química , Imunoterapia , Linfoma de Células B/terapia , Rituximab/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Complexo Antígeno-Anticorpo/imunologia , Antígenos CD20/imunologia , Antineoplásicos/imunologia , Linfócitos B/imunologia , Ativação do Complemento , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Ligação Proteica , Conformação Proteica , Rituximab/imunologia , Rituximab/uso terapêutico
8.
Nat Struct Mol Biol ; 27(10): 950-958, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32737466

RESUMO

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.


Assuntos
Anticorpos Antivirais/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Reações Cruzadas , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Pandemias , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
9.
Cell ; 182(4): 828-842.e16, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32645326

RESUMO

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.


Assuntos
Anticorpos Neutralizantes/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Betacoronavirus/imunologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Reações Cruzadas , Microscopia Crioeletrônica , Mapeamento de Epitopos , Epitopos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/ultraestrutura , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/sangue , Vírus da SARS/química , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Medicina (B Aires) ; 80 Suppl 3: 1-6, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658841

RESUMO

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.


Assuntos
Anticorpos Antivirais , Infecções por Coronavirus/terapia , Soros Imunes/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Pandemias , Pneumonia Viral , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Argentina , Betacoronavirus , Cavalos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização
11.
Science ; 369(6510): 1505-1509, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32703908

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. There are no approved vaccines or therapeutics for treating COVID-19. Here we report a humanized monoclonal antibody, H014, that efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nanomolar concentrations by engaging the spike (S) receptor binding domain (RBD). H014 administration reduced SARS-CoV-2 titers in infected lungs and prevented pulmonary pathology in a human angiotensin-converting enzyme 2 mouse model. Cryo-electron microscopy characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a previously uncharacterized conformational epitope, which was only accessible when the RBD was in an open conformation. Biochemical, cellular, virological, and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncovered broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19.


Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Neutralizantes/química , Betacoronavirus/imunologia , Peptidil Dipeptidase A/imunologia , Receptores Virais/imunologia , Vírus da SARS/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Chlorocebus aethiops , Infecções por Coronavirus/terapia , Mapeamento de Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Pulmão/imunologia , Camundongos , Pandemias , Pneumonia Viral/terapia , Domínios Proteicos , Multimerização Proteica , Células Vero
12.
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
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.
Biochem Biophys Res Commun ; 526(4): 941-946, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32284170

RESUMO

Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.


Assuntos
Anticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/sangue , Proteínas Recombinantes/uso terapêutico , Albumina Sérica Humana/química , Albumina Sérica Humana/imunologia , Anticorpos/química , Reações Cruzadas , Meia-Vida , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Ligação Proteica , Receptores Fc/metabolismo
15.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 3): 116-129, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133997

RESUMO

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.


Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Cristalização/métodos , Imunoglobulina E/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Omalizumab/metabolismo , Anticorpos Anti-Idiotípicos/química , Cristalografia por Raios X/métodos , Humanos , Imunoglobulina E/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Omalizumab/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
16.
Artigo em Inglês | MEDLINE | ID: mdl-32062365

RESUMO

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.


Assuntos
Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/análise , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulinas/metabolismo , Sulfato de Amônio/química , Animais , Western Blotting , Galinhas , Cromatografia por Troca Iônica , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulinas/química , Papaína/metabolismo
17.
Biochemistry (Mosc) ; 85(1): 80-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079519

RESUMO

Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH. The most stable low-molecular-weight complexes with a hydrodynamic radius up to 10 nm were formed by rSkp and protein substrates at acidic pH values. Under these conditions, rSkp exhibited the lowest propensity to self-association and the highest affinity for human IgG and its Fc and Fab fragments, as well as prevented their aggregation most efficiently (i.e., demonstrated the maximal chaperone activity). As the medium pH increased, the affinity of rSkp for IgG and its fragments decreased; rSkp was not able to completely prevent the aggregation of protein substrates, but significantly slowed it down. The obtained information may be of practical interest, since the stability of therapeutic IgG preparations affects their safety and efficacy in medical applications.


Assuntos
Proteínas de Bactérias/química , Chaperonas Moleculares/química , Proteínas Recombinantes/química , Proteínas Quinases Associadas a Fase S/química , Yersinia pseudotuberculosis/metabolismo , Clonagem Molecular , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Dobramento de Proteína
18.
Science ; 367(6483): 1224-1230, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32079680

RESUMO

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.


Assuntos
Antígenos CD20/química , Rituximab/química , Antígenos CD20/imunologia , Proteínas do Sistema Complemento/imunologia , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas/química , Conformação Proteica , Multimerização Proteica , Rituximab/imunologia
19.
J Biomed Sci ; 27(1): 12, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31907057

RESUMO

BACKGROUND: Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. METHODS: The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). RESULTS: A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. CONCLUSIONS: The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.


Assuntos
Anticorpos Monoclonais Murinos/química , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Fragmentos Fab das Imunoglobulinas/química , Polietilenoglicóis/química , Cristalografia por Raios X
20.
J Exp Med ; 217(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31757867

RESUMO

We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Domínios Proteicos/imunologia , Proteínas do Envelope Viral/química , Zika virus/imunologia , Aedes , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Reações Cruzadas , Cristalografia por Raios X , Dengue/imunologia , Dengue/virologia , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Ligação Proteica/imunologia , Conformação Proteica , Células Vero , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
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