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Related Experiment Video

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Rapid Determination of Antibody-Antigen Affinity by Mass Photometry
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Rapid Chimerization of Antibodies.

Koji Hashimoto1, Kohei Kurosawa1, Hidetaka Seo2

  • 1Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|December 13, 2018
PubMed
Summary

We developed a new method to efficiently change antibody formats. This system allows seamless Fc region exchange for chicken IgM antibodies, creating diverse chimeric antibodies for research and therapeutic applications.

Keywords:
ADLib systemChimeric antibodyCre recombinaseDT40 cellFc engineeringMonoclonal antibody libraryRecombination-mediated cassette exchange (RMCE)Trichostatin A (TSA)

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • The ADLib system generates monoclonal antibodies (mAbs) in chicken IgM format using DT40 cells.
  • Therapeutic and diagnostic applications often require antibodies in different formats, such as human or murine IgG.
  • Conventional Fc region engineering is time-consuming and labor-intensive.

Purpose of the Study:

  • To develop a streamlined method for Fc region exchange in antibodies generated by the ADLib system.
  • To enable the rapid generation of chimeric antibodies with desired Fc regions.

Main Methods:

  • Utilized recombination-mediated cassette exchange (RMCE) for seamless Fc region replacement.
  • Engineered DT40 genome by inserting Cre recombinase recognition sites into the IgM Fc region.
  • Co-transfected DT40 cells with a donor sequence and Cre recombinase expression vector to facilitate Fc exchange.

Main Results:

  • Successfully established a method for efficient Fc region exchange in ADLib-generated antibodies.
  • Demonstrated the generation of chimeric mAbs with various Fc regions.
  • Provided a detailed protocol for constructing RMCE host strains and generating chimeric mAbs.

Conclusions:

  • The developed RMCE-based method significantly simplifies and accelerates the generation of engineered antibodies.
  • This technology offers a versatile platform for producing chimeric antibodies for diverse applications.
  • The protocol facilitates the creation of customized antibody formats from chicken IgM precursors.