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1.
Nat Commun ; 10(1): 4328, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551421

RESUMO

Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines.


Assuntos
Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/prevenção & controle , Proteínas de Protozoários/imunologia , Anticorpos Antiprotozoários/química , Formação de Anticorpos , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Malária Falciparum/transmissão , Proteínas de Protozoários/química
2.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461846

RESUMO

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.


Assuntos
Afinidade de Anticorpos , Antígeno CD47/química , Simulação de Acoplamento Molecular , Anticorpos de Cadeia Única/química , Sítios de Ligação de Anticorpos , Antígeno CD47/genética , Antígeno CD47/imunologia , Humanos , Mutação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
3.
Anal Bioanal Chem ; 411(22): 5633-5639, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177333

RESUMO

Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from synthetic antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from synthetic antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL-1 and 32.4 pg mL-1 respectively. Graphical abstract.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Estradiol/sangue , Imunofluorescência/métodos , Sítios de Ligação de Anticorpos , Estradiol/imunologia , Estradiol/normas , Humanos , Limite de Detecção , Masculino , Padrões de Referência
4.
Nat Rev Drug Discov ; 18(8): 585-608, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175342

RESUMO

The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities - that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.


Assuntos
Anticorpos Biespecíficos , Desenho de Drogas , Neoplasias , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/uso terapêutico , Sítios de Ligação de Anticorpos , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/imunologia
5.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 554-563, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205018

RESUMO

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.


Assuntos
Anticorpos Monoclonais Humanizados/química , Complexo Antígeno-Anticorpo/química , Antineoplásicos Imunológicos/química , Neoplasias/terapia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Cristalização , Cristalografia por Raios X/métodos , Feminino , Humanos , Domínios Proteicos , Receptor ErbB-2/imunologia
6.
MAbs ; 11(5): 884-898, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31107136

RESUMO

The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant "solution" involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.


Assuntos
Anticorpos Monoclonais Humanizados/genética , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos/genética , Animais , Sítios de Ligação de Anticorpos/imunologia , Células CHO , Sistemas CRISPR-Cas , Regiões Determinantes de Complementaridade/genética , Cricetulus , Endodesoxirribonucleases , Citometria de Fluxo , Edição de Genes , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Mutagênese Sítio-Dirigida , Receptor de Morte Celular Programada 1/imunologia
7.
MAbs ; 11(5): 809-811, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31122133

RESUMO

We live in an era of rapidly advancing computing capacity and algorithmic sophistication. "Big data" and "artificial intelligence"find progressively wider use in all spheres of human activity, including healthcare. A diverse array of computational technologies is being applied with increasing frequency to antibody drug research and development (R&D). Their successful applications are met with great interest due to the potential for accelerating and streamlining the antibody R&D process. While this excitement is very likely justified in the long term, it is less likely that the transition from the first use to routine practice will escape challenges that other new technologies had experienced before they began to blossom. This transition typically requires many cycles of iterative learning that rely on the deconstruction of the technology to understand its pitfalls and define vectors for optimization. The study by Vasquez et al. identifies a key obstacle to such learning: the lack of transparency regarding methodology in computational antibody design reports, which has the potential to mislead the community efforts.


Assuntos
Anticorpos Monoclonais/farmacologia , Desenho de Drogas , Sítios de Ligação de Anticorpos , Simulação por Computador , Epitopos/química , Humanos , Engenharia de Proteínas
8.
Mol Immunol ; 112: 123-130, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31100550

RESUMO

Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Bovino/imunologia , Animais , Sítios de Ligação de Anticorpos/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/veterinária , Infecções por Vírus Respiratório Sincicial/virologia , Proteínas do Envelope Viral/imunologia
9.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003532

RESUMO

Antigen-mimicking peptide (mimotope)-based vaccines are one of the most promising forms of active-immunotherapy. The main drawback of this approach is that it induces antibodies that react poorly with the nominal antigen. The aim of this study was to investigate the molecular basis underlying the weak antibody response induced against the naïve protein after peptide vaccination. For this purpose, we analyzed the fine specificity of monoclonal antibodies (mAb) elicited with a 13-mer linear peptide, complementary to theantigen-combining site of the anti-CD20 mAb, Rituximab, in BALB/c mice. Anti-peptide mAb competed with Rituximab for peptide binding. Even so, they recognized a different antigenic motif from the one recognized by Rituximab. This explains their lack of reactivity with membrane (naïve) CD20. These data indicate that even on a short peptide the immunogenic and antigenic motifs may be different. These findings highlight an additional mechanism for epitope spreading and should be taken into account when designing peptides for vaccine purposes.


Assuntos
Anticorpos Monoclonais/genética , Antígenos CD20/imunologia , Peptídeos/genética , Rituximab/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos/genética , Anticorpos Monoclonais Murinos/imunologia , Antígenos CD20/genética , Sítios de Ligação de Anticorpos/genética , Epitopos/genética , Epitopos/imunologia , Humanos , Camundongos , Biblioteca de Peptídeos , Peptídeos/imunologia , Rituximab/genética , Vacinação/métodos , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
10.
Viruses ; 11(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875741

RESUMO

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Filoviridae/imunologia , Nucleoproteínas/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Sítios de Ligação de Anticorpos , Ebolavirus/imunologia , Epitopos de Linfócito B/imunologia , Feminino , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/virologia , Imunização , Isotipos de Imunoglobulinas , Marburgvirus/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologia
11.
Viruses ; 11(3)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857192

RESUMO

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. New strains evolve every 2⁻4 years by escaping herd immunity and cause worldwide epidemics. In the US alone, noroviruses are responsible for ~20 million cases and more than 70,000 hospitalizations of infected children, annually. Efforts towards a vaccine have been hindered by a lack of detailed structural information about antibody binding and the mechanisms of antibody escape. Caliciviruses have 180 copies of the major capsid protein (VP1; ~58 kDa), that is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. There is increasing evidence that these viruses are extremely dynamic and this flexibility is critical for viral replication. There are at least two modes of flexibility; the entire P domain relative to the shell and within the P domain itself. Here, the details and possible roles for this remarkable flexibility will be reviewed.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Capsídeo/química , Norovirus/química , Animais , Sítios de Ligação de Anticorpos , Gastroenterite/virologia , Genoma Viral , Humanos , Conformação Proteica , Receptores Virais/química
12.
Mol Cell ; 73(5): 1075-1082.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849388

RESUMO

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.


Assuntos
Anticorpos/metabolismo , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Afinidade de Anticorpos , Especificidade de Anticorpos , Automação Laboratorial , Sítios de Ligação de Anticorpos , Catálise , Análise Mutacional de DNA/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Cinética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligopeptídeos/genética , Análise Serial de Proteínas/instrumentação , Ligação Proteica , Engenharia de Proteínas , Fluxo de Trabalho
13.
Fish Shellfish Immunol ; 88: 472-479, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30880232

RESUMO

The Polymeric Immunoglobulin Receptor (pIgR) gene has been proved to play an important role in transporting polymeric immunoglobulin (Ig) in the mucosal tissues of mammals. pIgR gene also exists in teleost, but the genetic diversity and functions of this gene still need to be further explored. We obtained seven grass carp pIgR splicing transcripts, a full-length pIgR (CipIgR-1) and six truncated variants (CipIgR-2 to CipIgR-7). The full-length pIgR contained two immunoglobulin-like domains (ILD), a transmembrane domain (TMD) and a cytoplasmic domain (CyD). The CipIgR-2 lacked a small part in CyD, and CipIgR-3 lost TMD and CyD. Partial cDNA sequences of the other four grass carp pIgR variants (CipIgR-4 to CipIgR-7) were also cloned. The total expression levels of CipIgR and its variants in different tissues were detected by real-time quantitative PCR. The highest expression was found in the intestine, followed by the spleen and the skin. The function of the two extracellular ILDs of CipIgR was investigated based on its combining capacity with grass carp immunoglobulin M (IgM) and aquatic pathogenic bacteria. The cDNA sequences of two ILDs were cloned and expressed in Escherichia coli BL21 (DE3). Recombinant ILDs protein was purified and incubated with different bacteria respectively. Results of Western blot showed the recombinant protein could combine Bacillus subtilis, Vibrio parahaemolyticus, and Escherichia coli. In addition, binding activity of rILDs with grass carp IgM was detected. Collectively, these results indicated that multiple variants of pIgR gene in grass carp might be involved in the antibacterial immunity.


Assuntos
Carpas/genética , Carpas/imunologia , Domínios de Imunoglobulina , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Animais , Bactérias/imunologia , Sítios de Ligação de Anticorpos , Clonagem Molecular , Escherichia coli/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Variação Genética , Imunoglobulina M/metabolismo , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
14.
PLoS One ; 14(2): e0210749, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30730999

RESUMO

Globally, human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in newborns, young children, and the elderly for which there is no vaccine. The RSV fusion (F) glycoprotein is a major target for vaccine development. Here, we describe a novel monoclonal antibody (designated as R4.C6) that recognizes both pre-fusion and post-fusion RSV F, and binds with nanomole affinity to a unique neutralizing site comprised of antigenic sites II and IV on the globular head. A 3.9 Å-resolution structure of RSV F-R4.C6 Fab complex was obtained by single particle cryo-electron microscopy and 3D reconstruction. The structure unraveled detailed interactions of R4.C6 with antigenic site II on one protomer and site IV on a neighboring protomer of post-fusion RSV F protein. These findings significantly further our understanding of the antigenic complexity of the F protein and provide new insights into RSV vaccine design.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Sítios de Ligação de Anticorpos , Fragmentos Fab das Imunoglobulinas/química , Vírus Sinciciais Respiratórios/química , Proteínas Virais de Fusão/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Spodoptera , Proteínas Virais de Fusão/imunologia
15.
Nat Commun ; 10(1): 873, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787293

RESUMO

The envelope protein of human immunodeficiency virus-1 (HIV-1) and its fusion peptide are essential for cell entry and vaccine design. Here, we describe the 3.9-Å resolution structure of an envelope protein trimer from a very early transmitted founder virus (CRF01_AE T/F100) complexed with Fab from the broadly neutralizing antibody (bNAb) 8ANC195. The overall T/F100 trimer structure is similar to other reported "closed" state prefusion trimer structures. In contrast, the fusion peptide, which is exposed to solvent in reported closed structures, is sequestered (buried) in the hydrophobic core of the T/F100 trimer. A buried conformation has previously been observed in "open" state structures formed after CD4 receptor binding. The T/F100 trimer binds poorly to bNAbs including the fusion peptide-specific bNAbs PGT151 and VRC34.01. The T/F100 structure might represent a prefusion state, intermediate between the closed and open states. These observations are relevant to mechanisms of HIV-1 transmission and vaccine design.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sítios de Ligação de Anticorpos/imunologia , Microscopia Crioeletrônica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
16.
Nat Commun ; 10(1): 893, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792391

RESUMO

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.


Assuntos
Antígeno HLA-A11/química , Antígeno HLA-A11/metabolismo , Isoanticorpos/química , Isoanticorpos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Antígeno HLA-A11/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Isoanticorpos/genética , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica
17.
Arthritis Rheumatol ; 71(4): 507-517, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30811898

RESUMO

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA. METHODS: Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA-producing B cells by flow cytometry. Plasmablasts were single-cell sorted and sequenced to obtain antibody repertoires. Sixty-nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme-linked immuosorbent assay and synovial antigen arrays. Thirty-six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities. RESULTS: Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity-determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes. CONCLUSION: These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Sítios de Ligação de Anticorpos/imunologia , Epitopos/imunologia , Peptídeos Cíclicos/imunologia , Adulto , Artrite Reumatoide/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Feminino , Humanos , Masculino , Plasmócitos/imunologia
18.
Biotechnol J ; 14(5): e1800647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30810268

RESUMO

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 n m and a dissociation rate that is 55- to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.


Assuntos
Afinidade de Anticorpos/imunologia , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sítios de Ligação , Sítios de Ligação de Anticorpos , Biotina , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Humanos , Imobilização , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Peptídeo Hidrolases , Ligação Proteica , Especificidade por Substrato , Leveduras
19.
Nucleic Acids Res ; 47(6): e34, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30715449

RESUMO

Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.


Assuntos
Afinidade de Anticorpos , Sítios de Ligação de Anticorpos/genética , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Técnicas de Síntese em Fase Sólida/métodos , Uracila/metabolismo , Afinidade de Anticorpos/genética , Clonagem Molecular/métodos , Enzimas de Restrição do DNA/metabolismo , Mapeamento de Epitopos , Sequenciamento de Nucleotídeos em Larga Escala , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genética , Staphylococcus/genética , Biologia Sintética/métodos , Uracila/química
20.
Nat Commun ; 10(1): 721, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760721

RESUMO

Broadly neutralizing antibodies (bNAbs) represent a promising alternative to antiretroviral drugs for HIV-1 prevention and treatment. Selected antibodies to the CD4-binding site bolster envelope trimer binding via quaternary contacts. Here, we rationally engraft a new paratope, i.e., the extended heavy-chain framework region 3 (FR3) loop of VRC03, which mediates quaternary interaction, onto several potent bNAbs, enabling them to reach an adjacent gp120 protomer. The interactive quaternary surface is delineated by solving the crystal structure of two FR3 loop-chimeric antibodies. Chimerization enhances the neutralizing activity of several potent bNAbs against a majority of global HIV-1 strains. Compared to unmodified antibodies, chimeric antibodies display lower autoreactivity and prolonged in vivo half-life in huFcRn mice and rhesus macaques. Thus, paratope engraftment may be used to expand the epitope repertory of natural antibodies, improving their functionality for disease prevention and treatment.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/genética , Sítios de Ligação de Anticorpos/genética , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos , Feminino , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Macaca mulatta , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
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