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1.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
2.
Talanta ; 201: 397-405, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122440

RESUMO

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.


Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Zearalenona/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Triticum/química , Zea mays/química , Zearalenona/imunologia , Zearalenona/metabolismo
3.
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
4.
J Biotechnol ; 298: 45-56, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30959136

RESUMO

In order to maximize cell growth and productivity for an inducible CHO cell line expressing rituximab, various fed-batch culture strategies were investigated. In each case, the performance was evaluated for cultures induced at moderate and high cell density conditions (4 × 106 and 10 × 106 cells/mL) to assess the impact of the timing of induction. We first demonstrate the importance of starting the feeding process during the growth phase, as this translated into significantly improved integral of viable cells and antibody concentration, when compared to post-induction feeding only. Secondly, we investigated the impact of the feed rate by maintaining different levels of glucose (25, 35 and 50 mM) via a dynamic feeding strategy. The highest antibody concentrations were achieved under a moderate feeding regime for both cell densities at induction, highlighting the risks of under- or over-feeding the cultures. We then evaluated the impact of performing a temperature shift at induction by testing different mild hypothermia conditions. At small-scale, the highest production yields (1.2 g/L) were achieved when the temperature was reduced from 37 to 30 °C during the production phase of a culture induced at high cell density. When the strategy was applied in bioreactor, the better controlled conditions led to even greater product concentrations (1.8 g/L). Furthermore, this production protocol was shown to promote a more galactosylated glycan profile than a bioreactor culture initiated at 34 °C during growth and downshifted to 30 °C during the production phase.


Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura Celular por Lotes/métodos , Proliferação de Células/genética , Rituximab/biossíntese , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Células CHO , Sobrevivência Celular/genética , Cricetulus , Glucose/metabolismo , Humanos , Rituximab/química , Rituximab/genética
5.
PLoS Comput Biol ; 15(4): e1006952, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933973

RESUMO

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons.


Assuntos
Anticorpos Monoclonais/farmacologia , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Antígenos CD4 , Simulação por Computador , Previsões/métodos , Glicosilação , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Ligação Proteica
6.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991723

RESUMO

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some 'fine tuning' may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.


Assuntos
Anticorpos Monoclonais/genética , Técnicas de Visualização da Superfície Celular/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutagênese , Biblioteca de Peptídeos
7.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884802

RESUMO

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease of unknown etiology. It is characterized by the presence of rheumatoid factor and anticitrullinated peptide antibodies. The orchestra of the inflammatory process among various immune cells, cytokines, chemokines, proteases, matrix metalloproteinases (MMPs), and reactive oxidative stress play critical immunopathologic roles in the inflammatory cascade of the joint environment, leading to clinical impairment and RA. With the growing understanding of the immunopathogenic mechanisms, increasingly novel marked and potential biologic agents have merged for the treatment of RA in recent years. In this review, we focus on the current understanding of pathogenic mechanisms, highlight novel biologic disease-modifying antirheumatic drugs (DMRADs), targeted synthetic DMRADs, and immune-modulating agents, and identify the applicable immune-mediated therapeutic strategies of the near future. In conclusion, new therapeutic approaches are emerging through a better understanding of the immunopathophysiology of RA, which is improving disease outcomes better than ever.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/terapia , Imunomodulação , Inflamação/terapia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Quimiocinas/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/imunologia , Fator Reumatoide/imunologia
8.
J Biotechnol ; 296: 32-41, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30885656

RESUMO

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHOBRI/rcTA) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells.


Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas de Cultura Celular por Lotes/métodos , Fragmentos Fc das Imunoglobulinas/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Regulação da Expressão Gênica/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Estresse Fisiológico/genética , Transfecção
9.
Fish Shellfish Immunol ; 88: 464-471, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30858097

RESUMO

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Esocidae/imunologia , Septicemia Hemorrágica Viral/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Anticorpos Monoclonais/genética , Ensaio de Imunoadsorção Enzimática , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Peixes/imunologia , Peixes/virologia , Genótipo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Novirhabdovirus
10.
Monoclon Antib Immunodiagn Immunother ; 38(1): 18-24, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30802179

RESUMO

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos/genética
11.
Proc Natl Acad Sci U S A ; 116(8): 3161-3170, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718392

RESUMO

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1-3 (HNP1-3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.


Assuntos
Predisposição Genética para Doença , Sepse/genética , alfa-Defensinas/genética , Alelos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Variações do Número de Cópias de DNA/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Camundongos , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/genética , Piroptose/imunologia , Receptores Purinérgicos P2X7/genética , Fatores de Risco , Sepse/sangue , Sepse/patologia , alfa-Defensinas/antagonistas & inibidores , alfa-Defensinas/imunologia
12.
Transgenic Res ; 28(2): 177-188, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30746589

RESUMO

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.


Assuntos
Anticorpos Monoclonais/metabolismo , Oryza/imunologia , Peptídeos Cíclicos/imunologia , Plantas Geneticamente Modificadas/imunologia , Agrobacterium tumefaciens/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Cultura de Células , Células Cultivadas , Vetores Genéticos , Humanos , Oryza/genética , Oryza/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
13.
Methods Mol Biol ; 1956: 105-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779032

RESUMO

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in chronic lymphocytic leukemia have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire at single-cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow-cytometric isolation of single human B cells to the reverse transcription-polymerase chain reaction (RT-PCR)-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.


Assuntos
Anticorpos Monoclonais/genética , Linfócitos B/metabolismo , Clonagem Molecular/métodos , Citometria de Fluxo/métodos , Imunoglobulinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Separação Celular/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Cadeias J de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Proteínas Recombinantes/genética , Análise de Célula Única/métodos
14.
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
15.
Biotechnol Lett ; 41(3): 335-346, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30684155

RESUMO

Monoclonal antibodies represent the major class of biopharmaceutical products (for therapeutics and diagnostics) with an increasing demand that reaches several tons per year worldwide. Traditional large-scale manufacturing processes are based on stirred tank bioreactors for the growth of Chinese Hamster Ovary cells (CHO) which requires high initial investments and production costs. Therefore, there is an urgent need for alternative production platforms that can at least act as a complement to the over-exploited mammalian fermentation systems. In this perspective, the use of plants for the large-scale production of biopharmaceuticals ('Molecular farming') represents an interesting and mature technology that has already proved its benefits in terms of safety, scalability, rapidity and reduced manufacturing costs. Here we discuss the recent advances in the production of monoclonal antibodies (mAbs) in plant-based platforms such as transgenic plants, tissue and cell cultures and transient expression systems.


Assuntos
Anticorpos Monoclonais/metabolismo , Produtos Biológicos/metabolismo , Biotecnologia/métodos , Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Tecnologia Farmacêutica/métodos , Anticorpos Monoclonais/genética , Biotecnologia/tendências , Proteínas Recombinantes/genética , Tecnologia Farmacêutica/tendências
16.
Monoclon Antib Immunodiagn Immunother ; 38(1): 30-36, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30681406

RESUMO

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Membrana/imunologia , Podócitos/metabolismo , Alvéolos Pulmonares/citologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Citometria de Fluxo , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/genética , Camundongos , Podócitos/química , Alvéolos Pulmonares/imunologia , Suínos
17.
Artigo em Inglês | MEDLINE | ID: mdl-30648914

RESUMO

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Epitopos/imunologia , Aminoácidos/imunologia , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Mapeamento de Epitopos/métodos , Epitopos/química , Células HeLa , Humanos , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/imunologia
18.
Int Immunopharmacol ; 66: 362-365, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30529500

RESUMO

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by pathogenic immunoglobulin G (IgG) autoantibodies that bind to platelets, causing their phagocytic removal and leading to reductions in platelet number. The neonatal Fc receptor (FcRn) selectively salvages and recycles IgG, including pathogenic IgG, thereby extending the half-life of IgG in plasma. Two anti-mouse FcRn monoclonal antibodies (mAb) (4470 and 4464) were generated to evaluate the effect of inhibiting IgG recycling. Statistically significant reductions in plasma IgG concentration were observed upon administration of 4470 (10, 30 and 100 mg/kg) in wild-type mice. In a passive mouse model of ITP, 4464 alleviated the reduction in platelet number and/or preserved newly produced platelets when dosed prophylactically as well as in a therapeutic dosing regimen once platelet numbers had already been reduced. These results support the investigation of anti-FcRn therapy as a potential treatment for ITP.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Plaquetas/imunologia , Imunoglobulina G/sangue , Imunoglobulinas Intravenosas/uso terapêutico , Imunoterapia/métodos , Anticorpos de Cadeia Única/uso terapêutico , Trombocitopenia/terapia , Animais , Anticorpos Monoclonais/genética , Autoanticorpos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Receptores Fc/imunologia , Anticorpos de Cadeia Única/genética , Trombocitopenia/imunologia
19.
Dev Comp Immunol ; 90: 100-107, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30236879

RESUMO

Many of the most successful drugs generated in recent years are based upon monoclonal antibodies (mAbs). However, for some therapeutic and diagnostic applications mAbs are far from ideal; for example, while their relatively large size and inherent receptor binding aids their longevity in vivo it can also limit their tissue penetration. Further, their structural complexity makes them expensive to produce and prone to denaturation in non-physiological environments. Thus, researchers have been searching for alternative antigen-binding molecules that can be utilized in situations where mAbs are suboptimal tools. One potential source currently being explored are the shark-derived binding domains known as VNARs. Despite their small size VNARs can bind antigens with high specificity and high affinity. Combined with their propensity to bind epitopes that are inaccessible to conventional mAbs, and their ability to resist denaturation, VNARs are an emerging prospect for use in therapeutic, diagnostic, and biotechnological applications.


Assuntos
Anticorpos Monoclonais/metabolismo , Doenças dos Peixes/diagnóstico , Proteínas de Peixes/metabolismo , Imunoterapia/métodos , Tubarões/imunologia , Imunidade Adaptativa , Animais , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Especificidade de Anticorpos/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/terapia , Proteínas de Peixes/genética , Humanos , Domínios Proteicos/genética , Estabilidade Proteica
20.
Methods Mol Biol ; 1904: 1-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539464

RESUMO

The major reasons for developing human monoclonal antibodies were to be able to efficiently manipulate their effector functions while avoiding immunogenicity seen with rodent antibodies. Those effector functions involve interactions with the complement system and naturally occurring Fc receptors on diverse blood white cells. Antibody immunogenicity results from the degree to which the host immune system can recognize and react to these therapeutic agents. Thus far, there is still no generally applicable technology guaranteed to render therapeutic antibodies antigenically silent. This is not to say that the task is impossible, but rather that we need to train the immune system to help us. This can be achieved if we take advantage of natural mechanisms by which an individual can be rendered tolerant of "foreign" antigens, and as a corollary minimize the potential immunogenicity of any contaminating protein aggregates, or "aggregates" arising from antibodies complexing with their antigen. I here summarize our efforts to engineer antibodies to harness optimal effector functions, while also minimizing their immunogenicity. Potential avenues to achieve the latte are predicted from classical work showing that monomeric "foreign" immunoglobulins are good tolerogens, while aggregates of immunoglobulins ate intrinsically immunogenic. Consequently, I argue that one solution to the immunogenicity problem lies in ensuring a temporal quantitative advantage of tolerogenic non-cell-bound monomer over the cell-binding immunogenic form.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Afinidade de Anticorpos/imunologia , Antígenos/imunologia , Engenharia Genética , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunoglobulina G/imunologia , Mutagênese , Receptores Fc/metabolismo
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