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1.
Vet Immunol Immunopathol ; 212: 9-14, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213252

RESUMO

Targeting antigens to endocytic receptors on the surface of dendritic cells is a new strategy for increasing the adaptive immune response. The objective of the current study was the construction and bacterial expression of a recombinant antibody single-chain fragment variable (ScFv) directed against chicken DEC 205, an endocytic receptor, for use in the genetic fusion of antigens. In particular, we use as antigen the hemagglutinin-neuraminidase (HN) of Newcastle disease virus. Our results show that inoculation of chickens with HN genetically fused to the ScFv anti-DEC 205 induced an evidently higher immune response against HN, in contrast to inoculation with unconjugated HN. In addition, neutralizing antibodies against Newcastle disease virus were detected only in the serum from chickens immunized with HN fused to ScFv anti-DEC 205. Inoculated fused antigens to ScFv against endocytic receptor DEC 205 resulted in a greater antibody-specific anti-HN production compared with antigens applied alone. The results of this study show that the strategy described here has the potential to be used in the development of more effective vaccines against infectious diseases in chickens.


Assuntos
Antígenos CD/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Vírus da Doença de Newcastle/enzimologia , Doenças das Aves Domésticas/prevenção & controle , Receptores de Superfície Celular/imunologia , Anticorpos de Cadeia Única/biossíntese , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Galinhas/imunologia , Escherichia coli/genética , Hemaglutininas Virais/imunologia , Neuraminidase/imunologia , Doenças das Aves Domésticas/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia , Vacinas Virais/imunologia
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.
Mol Biotechnol ; 61(6): 410-420, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963479

RESUMO

Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream protein validation pipelines.


Assuntos
Técnicas de Visualização da Superfície Celular , Clonagem Molecular/métodos , Vetores Genéticos/química , Ensaios de Triagem em Larga Escala , Anticorpos de Cadeia Única/genética , Códon de Terminação , Enzimas de Restrição do DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Anticorpos de Cadeia Única/biossíntese
4.
Toxicon ; 160: 38-46, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30802471

RESUMO

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.


Assuntos
Metaloendopeptidases/antagonistas & inibidores , Planticorpos/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/farmacologia , Animais , Antivenenos/biossíntese , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Testes de Neutralização , Planticorpos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Tabaco/genética , Tabaco/metabolismo
5.
MAbs ; 11(3): 532-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30735467

RESUMO

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.


Assuntos
Proteínas Aviárias , Técnicas de Visualização da Superfície Celular/métodos , Anticorpos de Cadeia Única , Animais , Proteínas Aviárias/biossíntese , Proteínas Aviárias/química , Proteínas Aviárias/genética , Bacteriófagos/genética , Galinhas , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
6.
MAbs ; 11(3): 516-531, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30663541

RESUMO

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/JH (H3J) fragments. One IGHV gene provided a universal VH scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal VH scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated KD values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.


Assuntos
Regiões Determinantes de Complementaridade , Biblioteca Gênica , Imunoglobulina G , Anticorpos de Cadeia Única , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Leucócitos Mononucleares/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
7.
Lett Appl Microbiol ; 68(3): 241-247, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30584665

RESUMO

scFv-BM3 is a single-chain variable fragment (scFv) against aflatoxin B1 (AFB1 ) engineered by affinity maturation and site-directed mutagenesis, and thus has a 31-fold higher affinity than its wild-type. To apply scFv-BM3 to immunological detection of AFB1 , periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv-BM3. scFv-BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv-BM3 secreted into the culture medium had binding activity to AFB1 . Expression conditions for scFv-BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv-BM3 was significantly improved by supplementation with Triton X-100 and optimization of expression conditions. The scFv-BM3 purified from the culture medium exhibited a typical antiparallel ß-sheet structure and adopted a proper conformation to bind AFB1 with high affinity and specificity in various biophysical and biochemical analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: Single-chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B1 (AFB1 ) (scFv-BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv-BM3 resulted in formation of inactive aggregates in E. coli, the scFv-BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB1 . This study promotes the application of functional scFv-BM3 to the immunological detection of AFB1 in biotechnology fields.


Assuntos
Aflatoxina B1/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos de Cadeia Única , Biotecnologia , Meios de Cultura/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
8.
Biochem Biophys Res Commun ; 499(1): 71-77, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29559238

RESUMO

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 106. Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity.


Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas Luminescentes/genética , Biblioteca de Peptídeos , Proteínas/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Antígenos/genética , Antígenos/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , Citometria de Fluxo , Expressão Gênica , Células HEK293 , Humanos , Integrases/genética , Integrases/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminescentes/metabolismo , Mutagênese , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Transdução Genética
9.
Bioprocess Biosyst Eng ; 41(5): 633-640, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29368032

RESUMO

Apoptosis has a negative impact on the cell survival state during cell cultivation. To optimize mammalian cell culture for production of biopharmaceuticals, one of the important approaches is to extend cell life through over-expression of anti-apoptotic genes. Here, we reported a cost-effective process to enhance cell survival and production of an antibody through transient co-transfection with anti-apoptotic genes Bcl-x L or Mcl-1 in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI). Under the optimal conditions, it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-x L or Mcl-1. The overall production yield of the antibody anti-PD1 increased approximately 82% in CHO cells co-transfected with Bcl-x L , and 34% in CHO cells co-transfected with Mcl-1. This work provides an effective way to increase viability of host cells through delaying apoptosis onset, thus, raise production yield of biopharmaceuticals without the process of generating stable cell lines and subsequent screening.


Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Receptor de Morte Celular Programada 1 , Anticorpos de Cadeia Única/biossíntese , Transfecção , Proteína bcl-X/biossíntese , Animais , Células CHO , Cricetulus , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Anticorpos de Cadeia Única/genética , Proteína bcl-X/genética
10.
Arch Virol ; 163(5): 1141-1152, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29356992

RESUMO

Enterovirus-71 (EV71) and coxsackievirus-A16 (CA16) frequently cause hand-foot-mouth disease (HFMD) epidemics among infants and young children. CA16 infections are usually mild, while EV71 disease may be fatal due to neurologic complications. As such, the ability to rapidly and specifically recognize EV71 is needed to facilitate proper case management and epidemic control. Accordingly, the aim of this study was to generate antibodies to EV71-virion protein-2 (VP2) by phage display technology for further use in specific detection of EV71. A recombinant peptide sequence of EV71-VP2, carrying a predicted conserved B cell epitope fused to glutathione-S-transferase (GST) (designated GST-EV71-VP2/131-160), was produced. The fusion protein was used as bait in in-solution biopanning to separate protein-bound phages from a murine scFv (MuscFv) phage display library constructed from an immunoglobulin gene repertoire from naïve ICR mice. Three phage-transformed E. coli clones (clones 63, 82, and 83) produced MuscFvs that bound to the GST-EV71-VP2/131-160 peptide. The MuscFv of clone 83 (MuscFv83), which produced the highest ELISA signal to the target antigen, was further tested. MuscFv83 also bound to full-length EV71-VP2 and EV71 particles, but did not bind to GST, full-length EV71-VP1, or the antigenically related CA16. MuscFv83 could be a suitable reagent for rapid antigen-based immunoassay, such as immunochromatography (ICT), for the specific detection and/or diagnosis of EV71 infection as well as epidemic surveillance.


Assuntos
Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/imunologia , Epitopos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Proteínas do Capsídeo/genética , Enterovirus Humano A/genética , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/virologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Escherichia coli/genética , Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/virologia , Humanos , Camundongos , Camundongos Endogâmicos ICR , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/biossíntese
11.
Appl Biochem Biotechnol ; 185(1): 233-247, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29124655

RESUMO

Single-chain variable fragment (scFv) antibodies as therapeutic agents have the potential to reduce the production cost and immunogenicity relative to monoclonal antibodies, but their monovalency and lack of a fragment crystallizable region can lead to reduced function. Multimerization is one strategy for recovering the function; however, their application is limited by the production of multimeric proteins. In our previous study, an anti-lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scFv showed potential use in diagnosis and therapy of atherosclerotic diseases, but is limited by its inherent low antigen-binding activity. In this study, to improve the efficacy of the anti-LOX-1 scFv, we constructed the anti-LOX-1 scFv multimers by modifying the linker length between the variable domains of the scFv or by fusing the scFv with self-merization domains and expressed these scFv multimers in Brevibacillus choshinensis hosts. After optimization, all of the scFv multimers obtained efficient secretion expression. Compared with the scFv monomer, the multimers that are successfully fractionated displayed increased neutralization activity and showed elevated antigen-binding avidity, especially the tetramer, which improved the antigen avidity by two orders of magnitude. Moreover, the scFv dimer and the tetramer both displayed better stability and longer half-life in serum, which can be attractive candidates for the next-generation anti-LOX-1 therapeutic antibody.


Assuntos
Multimerização Proteica , Receptores Depuradores Classe E/imunologia , Anticorpos de Cadeia Única , Animais , Células CHO , Cricetulus , Humanos , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
12.
Biotechnol Bioeng ; 115(3): 565-576, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29178403

RESUMO

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential.


Assuntos
Interleucina-2 , Leucócitos Mononucleares/metabolismo , Plantas Geneticamente Modificadas , Polissacarídeos , Proteínas Recombinantes de Fusão , Anticorpos de Cadeia Única , Tabaco , Humanos , Interleucina-2/biossíntese , Interleucina-2/química , Interleucina-2/genética , Interleucina-2/farmacologia , Leucócitos Mononucleares/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/genética , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/farmacologia , Tabaco/genética , Tabaco/metabolismo
13.
Methods Mol Biol ; 1701: 3-24, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29116497

RESUMO

Antibody phage display is the most commonly used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The prerequisite for successful generation of antibodies using phage display is the construction of high-quality antibody gene libraries. Here, we give the detailed methods for the construction of human immune and naive scFv gene libraries.


Assuntos
Biblioteca Gênica , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Humanos
14.
Methods Mol Biol ; 1701: 447-461, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29116521

RESUMO

We have recently described a one-step zero-background IgG reformatting method that enables the rapid reformatting of phage-displayed antibody fragments into a single-mammalian cell expression vector for full IgG expression (Chen et al. Nucleic Acids Res 42:e26, 2014). The strategy utilizes our unique positive selection method, referred to as insert-tagged (InTag) positive selection, where a positive selection marker (e.g. chloramphenicol-resistance gene) is cloned together with the antibody inserts into the expression vector. The recombinant clones containing the InTag adaptor are then positively selected without cloning background, thus bypassing the need to plate out cultures and screen colonies. This IgG reformatting method is rapid and can be automated and performed in a high-throughput (HTP) format. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein.


Assuntos
Expressão Gênica , Biblioteca Gênica , Imunoglobulina G , Biblioteca de Peptídeos , Anticorpos de Cadeia Única , Animais , Linhagem Celular , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
15.
J Nat Med ; 72(1): 310-316, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29027080

RESUMO

The binding properties of recombinant antibody fragments, such as affinity and specificity, determine their usefulness for therapeutic and analytical applications. Anti-paclitaxel single-chain variable fragment clone 1C2 (anti-PT scFv1C2) was expressed using Escherichia coli cell and Bombyx mori larvae expression systems. The binding characteristics of the scFvs were evaluated using indirect competitive ELISA. The linear range of binding between anti-PT scFv1C2 and paclitaxel was significantly different between the anti-PT scFv1C2s derived from E. coli (1.056-5.700 µg/ml) and B. mori (7.813-1000 ng/ml), which indicated that different expression systems resulted in different sensitivities for paclitaxel determination. In addition, the binding specificity of anti-PT scFv1C2 varied between expression systems. This finding implied that the expression system significantly affects the binding properties of recombinant antibodies, especially antibodies against low-molecular-weight targets.


Assuntos
Paclitaxel/imunologia , Anticorpos de Cadeia Única/biossíntese , Animais , Especificidade de Anticorpos , Bombyx , Clonagem Molecular , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Paclitaxel/química , Ligação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
16.
PLoS One ; 12(11): e0188191, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29155887

RESUMO

Aß-Immunotherapy has long been studied in the treatment of Alzheimer's disease (AD), but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aß-induced cytotoxicity by withdrawing Aß oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE) and J (apoJ) concentrations to non-pathological values, and Aß internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aß aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs). Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aß were studied by CD. The conformational change induced by the interaction of Aß with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aß complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aß uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aß uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Adulto , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/farmacologia , Astrócitos/metabolismo , Astrócitos/patologia , Clonagem Molecular , Clusterina/genética , Clusterina/metabolismo , Clusterina/farmacologia , Endocitose/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mimetismo Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/genética , Peptídeos/metabolismo , Cultura Primária de Células , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
17.
Protein Eng Des Sel ; 30(10): 713-721, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29040754

RESUMO

As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.


Assuntos
Anticorpos Antineoplásicos/química , Antígenos B7/química , Biomarcadores Tumorais/química , Proteínas Mutantes Quiméricas/química , Receptores de Antígenos de Linfócitos T/química , Anticorpos de Cadeia Única/química , Substituição de Aminoácidos , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/genética , Antígenos B7/genética , Antígenos B7/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Citotoxicidade Imunológica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Células HEK293 , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Camundongos , Modelos Moleculares , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/imunologia , Mutação , Receptor 3 Desencadeador da Citotoxicidade Natural/química , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética
18.
J Immunoassay Immunochem ; 38(6): 652-662, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29035147

RESUMO

Polycyclic aromatic hydrocarbons are chemical carcinogens which could induce the development of human cancers. Anti-idiotypic antibodies against benzo[a]pyrene (BP) are perspective for human cancer immunoprophylaxis and tumor immunodiagnostic techniques. The purpose of this study was to isolate anti-idiotypic antibodies against BP from human lymphocytes naïve phage library. The anti-idiotypic antibody, named B5, was selected. Analysis of the nucleotide and amino acid sequences B5 showed no similarity to known protein databases antibodies. B5 bound idiotypic antibodies against BP in direct and competitive ELISA. It was suggested that the B5 carried an immunological image of BP and bound the idiotypic antibodies against BP. ABBREVIATIONS: scFv: single-chain variable fragment; Ab1: idiotypic antibodies; Ab2: anti-idiotypic antibodies; CBD: cellulose binding domain; BSA: bovine serum albumin; PBS: phosphate buffer; BP-BSA: benzo[a]pyrene-BSA conjugate; Cr-BSA: chrysene-BSA conjugate; Py-BSA: pyrene-BSA conjugate; Ac-BSA: anthracene-BSA conjugate; Ba-BSA: benz[a]anthracene-BSA conjugate; PAH: polycyclic aromatic hydrocarbons; pSh: mouse idiotypic single-chain variable fragment against benzo[a]pyrene; T72: human idiotypic single-chain variable fragment against benzo[a]pyrene.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/isolamento & purificação , Benzo(a)pireno , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Anti-Idiotípicos/biossíntese , Ensaio de Imunoadsorção Enzimática , Humanos , Linfócitos/imunologia , Anticorpos de Cadeia Única/biossíntese
19.
Protein Eng Des Sel ; 30(9): 639-647, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981720

RESUMO

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting. After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These highly specific, affinity-matured variants displayed nanomolar to picomolar binding affinities to the CSPG4 antigen. While each of the mutants harbored only two to six amino acid substitutions, they represented ~270-3000-fold improvement in affinity compared to the parental clone. Our study has generated affinity-matured scFvs for the development of antibody-based clinical therapeutics targeting CSPG4-expressing tumors.


Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais/biossíntese , Técnicas de Visualização da Superfície Celular/métodos , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Anticorpos de Cadeia Única/biossíntese , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/imunologia , Citometria de Fluxo , Expressão Gênica , Células HEK293 , Humanos , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mutação , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
20.
Hum Vaccin Immunother ; 13(11): 2726-2737, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-28949787

RESUMO

While broadly neutralizing antibodies (bnAbs) are a promising preventative and therapeutic tool for HIV infection, production is difficult and expensive. Production of antibody-like fragments in bacterial cytoplasm provides a cheaper alternative. This work explored the transplantation of the complementarity determining regions of the anti-HIV bnAbs PGT121 and 10E8 onto a single-chain variable fragment (scFv) scaffold, previously discovered through a novel screening platform. The scaffolded 10E8 scFv, but not the scaffolded PGT121 scFv, was soluble in bacterial cytoplasm, enabling efficient production in bacteria. Three additional multimeric constructs employing the scaffolded 10E8 scFv were also generated and soluble versions produced in bacteria. However, the constructs were found to have substantially lost anti-HIV binding function and had completely abrogated neutralizing activity. Overall, while this study provides a proof-of-concept for anti-HIV bnAb construct production in bacterial cytoplasm, future refinement of these technologies will be required to realize the goal of producing inexpensive and effective bnAb-like tools for the control of HIV.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Bactérias/imunologia , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/terapia , Anticorpos de Cadeia Única/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/biossíntese , Escherichia coli/genética , Escherichia coli/imunologia , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/química , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Testes de Neutralização , Estudo de Prova de Conceito , Anticorpos de Cadeia Única/biossíntese
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