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Antibody humanization using monovalent phage display

M Baca1, L G Presta, S J O'Connor

  • 1Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.

The Journal of Biological Chemistry
|April 18, 1997
PubMed
Summary
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This study introduces a phage display method to rapidly optimize humanized antibodies by random mutagenesis. The new technique significantly enhanced antibody-antigen binding affinity, offering an efficient alternative for antibody engineering.

Area of Science:

  • Biotechnology
  • Immunology
  • Protein Engineering

Background:

  • Antibody humanization is crucial for reducing immunogenicity but often requires framework region optimization.
  • Traditional methods for framework optimization involve extensive site-directed mutagenesis and analysis of numerous mutants.

Purpose of the Study:

  • To develop and validate a phage display method for rapid optimization of humanized antibody framework regions.
  • To improve the antigen-binding affinity of humanized antibodies through targeted random mutagenesis.

Main Methods:

  • A phage display method was employed for random mutagenesis of key framework residues in humanized antibodies.
  • Affinity panning was used to select variants with enhanced antigen binding.
  • The method was applied to the humanized anti-vascular endothelial growth factor antibody A4.6.1.

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Main Results:

  • A humanized antibody mutant with over 125-fold enhanced affinity for its antigen was selected.
  • A single additional mutation further improved binding affinity by 6-fold.
  • The optimized variant exhibited an affinity of 9.3 nM, only 6-fold weaker than a chimeric antibody.

Conclusions:

  • The phage display method enables rapid selection of beneficial framework mutations for humanized antibodies.
  • This approach offers an efficient alternative to conventional methods for antibody framework optimization.
  • The optimized humanized antibody demonstrates significantly improved binding characteristics for therapeutic applications.