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Related Experiment Videos

Probing a protein-protein interaction by in vitro evolution.

George Thom1, Alexis C Cockroft, Andrew G Buchanan

  • 1Cambridge Antibody Technology, Milstein Building, Granta Park, Cambridge CB1 6GH, United Kingdom.

Proceedings of the National Academy of Sciences of the United States of America
|May 11, 2006
PubMed
Summary

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This study optimized an asthma therapeutic antibody using ribosome and phage display, significantly enhancing its binding affinity and potency. Ribosome display proved superior for identifying beneficial mutations across the antibody sequence.

Area of Science:

  • Biotechnology
  • Immunology
  • Pharmacology

Background:

  • Asthma is a chronic respiratory disease driven by interleukin-13 (IL-13).
  • Neutralizing antibodies targeting IL-13 are potential therapeutics for asthma.
  • Optimizing antibody affinity and potency is crucial for therapeutic efficacy.

Purpose of the Study:

  • To enhance the affinity and potency of a human IL-13-neutralizing antibody using in vitro protein evolution.
  • To compare the effectiveness of ribosome display and phage display for antibody optimization.
  • To identify beneficial mutations and understand their impact on antibody function.

Main Methods:

  • Employed in vitro protein evolution techniques, specifically ribosome display and phage display.
  • Utilized affinity-driven stringency selections to optimize antibody binding.

Related Experiment Videos

  • Performed complementarity-determining region (CDR)-targeted mutagenesis and error-prone PCR.
  • Assessed antibody potency using a cell-based assay measuring inhibition of IL-13-dependent proliferation.

    • Main Results:

    • Achieved a >150-fold increase in antibody affinity, reaching 81 pM.
    • Enhanced antibody potency by 345-fold, with an IC50 of 229 pM.
    • Ribosome display identified a broader range of beneficial mutations compared to phage display.
    • In vitro evolution successfully optimized potency without mutating critical paratope residues.

    Conclusions:

    • Ribosome display is advantageous for exploring extensive sequence space and identifying beneficial mutations in antibody engineering.
    • Optimized antibody demonstrates significantly improved affinity and potency for IL-13 neutralization.
    • In vitro protein evolution is an effective strategy for developing potent therapeutic antibodies.
    • The study identified key mutation sites for enhancing antibody efficacy without compromising essential functional residues.