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Directed evolution of human scFvs in DT40 cells.

Alfred W Y Lim1, Gareth T Williams1, Cristina Rada1

  • 1Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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Summary

This study adapted the chicken DT40 system to evolve human single-chain variable fragments (scFvs) for antibody discovery. The system efficiently generates and refines scFvs, leading to human IgG antibodies with high affinity.

Keywords:
affinity maturationin vitro selectionmonoclonal antibodiesscFvsomatic hypermutation

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • Somatic hypermutation in immunoglobulin genes allows antibody diversification.
  • The chicken DT40 cell line offers a system for genetic manipulation and antibody engineering.

Purpose of the Study:

  • To adapt the chicken DT40 system for evolving human single-chain variable fragments (scFvs).
  • To generate self-diversifying scFv libraries for antibody selection and affinity maturation.

Main Methods:

  • Engineered DT40 cells to express membrane-anchored scFvs within the Igλ locus.
  • Utilized somatic hypermutation for scFv diversification and iterative selection.
  • Overexpressed a hyperactive activation-induced deaminase variant to enhance mutation rates.

Main Results:

  • Successfully generated scFv libraries and selected for desired specificities.
  • Evolved scFvs for improved affinity and specificity, yielding human IgG antibodies with nanomolar affinities.
  • Isolated scFvs targeting CWC15 and human IFNγ.

Conclusions:

  • The adapted DT40 system provides a flexible platform for rapid generation and evolution of human antibody repertoires.
  • This method enables efficient selection, affinity maturation, and generation of high-affinity antibodies.