Construction of Human Immune and Naive scFv Libraries

Kügler, Jonas; Tomszak, Florian; Frenzel, André; Hust, Michael

doi:10.1007/978-1-4939-7447-4_1

Cited by 14 publications

(10 citation statements)

References 54 publications

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“…Phage libraries generated from human rearranged V-gene repertoires are constructed from mRNA or RNA extracted from B cells of immunized or naïve donors ( Figure 2 ) ( 73 , 142 – 144 ). Construction of immunized or naïve libraries involves using reverse transcription polymerase chain reaction (RT-PCR) to prepare the cDNA template.…”

Section: Types Of Antibody Phage Display Librariesmentioning

confidence: 99%

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

et al. 2020

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Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage–derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.

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Section: Types Of Antibody Phage Display Librariesmentioning

confidence: 99%

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

et al. 2020

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“…The donors have given their consent for study publication. The library construction was performed as described previously with minor modifications 79 . In brief, the peripheral blood mononuclear cells (PBMC) were extracted from the blood via Ficoll (GE Healthcare, Freiburg, Germany) and RNA isolated with Trizol and a Trizol RNA purification kit (Zymo Research, Freiburg, Germany).…”

Section: Generation Of An Immune Anti-dt Antibody Gene Librarymentioning

confidence: 99%

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Wenzel

Bosnak

Tierney

et al. 2020

Sci Rep

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Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. the bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (Dt), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against Dt (diphtheria antitoxin; DAt). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three Dt domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. these recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAt.Diphtheria is an infectious disease and caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways but can also affect other body sites. The produced toxin can spread through the bloodstream and affect organs, such as the heart and kidneys. In severe cases, diphtheria toxin (DT) may cause myocarditis or peripheral neuropathy. Due to a membrane of dead tissue over the throat and tonsils, swallowing and breathing can be difficult. The disease is spread through direct physical contact or by coughing or sneezing of infected individuals 1-3 . Diphtheria is fatal in 5-10% of cases, but children under the age of five have a mortality rate of up to 20%. Treatment involves antibiotics to kill the bacteria (erythromycin or penicillin for 14 days) and administering of diphtheria antitoxin (DAT) to neutralize the effects of the toxin 4-6 . C. diphtheriae was identified as the causative agent of diphtheria in 1883 and in 1888 the diphtheria toxin was first described in the culture medium of C. diphtheriae 7 . The gene for DT is encoded on a coryn...

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“…donors showing a good antibody titer against RBD was used. The library construction was performed as described previously with minor modifications (Kügler et al, 2018). In brief, the peripheral blood mononuclear cells (PBMC) were extracted from the blood via Ficoll RNA was converted into cDNA using Superscript IV (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's instructions.…”

Section: Covid-19 Convalescent Patient Library Constructionmentioning

confidence: 99%

“…donors showing a good antibody titer against RBD was used. The library construction was performed as described previously with minor modifications (Kügler et al, 2018). In brief, the peripheral blood mononuclear cells (PBMC) were extracted from the blood via Ficoll (GE Healthcare, Freiburg, Germany) and RNA isolated with TRIzol LS reagent (Life Technologies, Carlsbad, USA) and Direct-zol RNA Miniprep Plus kit (Zymo Research, Freiburg, Germany).…”

Section: Covid-19 Convalescent Patient Library Constructionmentioning

confidence: 99%

“…Human antibodies can be selected from two kind of antibody gene libraries: universal libraries and immune libraries. Universal libraries which have a naïve, semisynthetic or synthetic antibody gene repertoire, allow -in theory -the selection of antibodies against any kind of molecule, while immune libraries derived from immunized donors typically facilitate the antibody generation against the pathogen that affected the donors (Bradbury et al, 2011;Frenzel et al, 2017;Kretzschmar and von Rüden, 2002;Kügler et al, 2018;Kuhn et al, 2016). Immune phage display libraries from convalescent patients or immunized human donors were used in the past for the successful generation of antibodies against infectious diseases (Duan et al, 2009;Rahumatullah et al, 2017;Trott et al, 2014;Wenzel et al, 2020a;Zhang et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations

Bertoglio

Fühner

Ruschig

et al. 2020

Preprint

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The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed an IC50 of 0.56 nM in a plaque-based live SARS-CoV-2 neutralization assay. The crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD was solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. In contrast to other published anti-SARS-CoV-2 antibodies, the binding of STE90-C11 is not blocked by known RBD mutations, endowing our antibody with higher intrinsic resistance to those possible escape mutants.

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Construction of Human Immune and Naive scFv Libraries

Cited by 14 publications

References 54 publications

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

Phage Display Derived Monoclonal Antibodies: From Bench to Bedside

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations

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