Basics of Antibody Phage Display Technology

Ledsgaard, Line; Kilstrup, Mogens; Karatt-Vellatt, Aneesh; McCafferty, John; Laustsen, Andreas Hougaard

doi:10.3390/toxins10060236

Cited by 160 publications

(152 citation statements)

References 64 publications

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“…In vitro neutralization in IgG format. The 35 IgG antibodies were tested for their ability to neutralize DT in the Vero cell TNT assay 47 (Fig. 6).…”

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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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“…In vitro neutralization in IgG format. The 35 IgG antibodies were tested for their ability to neutralize DT in the Vero cell TNT assay 47 (Fig. 6).…”

mentioning

confidence: 99%

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Wenzel

Bosnak

Tierney

et al. 2020

Sci Rep

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“…One of the most commonly used directed evolution method is phage display technology which is recently awarded 2018 Nobel prize in Chemistry. Phage display allows the presentation of peptide and protein libraries “displayed” on the surface of filamentous phage . Antibodies are subsequently selected based on affinity and/or biophysical properties.…”

Section: Introductionmentioning

confidence: 99%

“…Phage display allows the presentation of peptide and protein libraries "displayed" on the surface of filamentous phage. 23 Antibodies are subsequently selected based on affinity and/or biophysical properties. This technique is used in a variety of applications including protein engineering, affinity maturation, and drug discovery.…”

Section: Introductionmentioning

confidence: 99%

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

2019

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Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self‐interaction and polyspecificity compared to non‐phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non‐phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation‐prone natures of antibodies which should be avoided in early development phases.

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“…The technique of phage display ( Figure 1B) is an important tool for the identification of protein-protein interactions. [6][7][8][9][10][11][12][13][14][15] The process begins with insertion of a DNA sequence that encodes a particular protein into the coat protein gene of a bacteriophage. The protein is expressed on the surface of the bacteriophage.…”

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confidence: 99%

DNA Encoded Libraries: A Visitor's Guide

Flood

Kingston

Vantourout

et al. 2020

Israel Journal of Chemistry

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In 1992, Brenner and Lerner hypothesized that individual chemical transformations could be encoded in DNA, allowing the rapid synthesis and screening of large collections of small molecules. Since their report, huge investments into the development of the DNA encoded library (DEL) technology have enabled the acceleration of the drug discovery process especially early phase discovery undertakings such as target validation and hit identification. As DEL lies at the nexus between chemistry and biology, there is an increasing need to expand the toolboxes of both organic transformations and biological methods. However, the myriad of techniques and reactions already reported can be difficult to digest for practitioners whose expertise resides outside the realm of DEL. This review therefore focuses on a stepwise presentation of DEL from the basic concepts to newest developments. The presentation includes the history, fundamentals, and successes of DEL, different methods for DEL synthesis and affinity selection, the conventional transformations, and finally the latest developments from a synthetic organic perspective.

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Basics of Antibody Phage Display Technology

Cited by 160 publications

References 64 publications

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

DNA Encoded Libraries: A Visitor's Guide

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