Construction of pH-sensitive Her2-binding IgG1-Fc by directed evolution

Biotechnol J. 2014 Aug;9(8):1013-22. doi: 10.1002/biot.201300483.


For most therapeutic proteins, a long serum half-life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half-life. In this study, we aimed to investigate whether pH-dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C-terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A yeast-displayed Fcab-library was alternately selected for binding at pH 7.4 and non-binding at pH 6.0. Selected Fcab variants showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH-dependent interactions with Her2-positive cells whereas their conformational and thermal stability was pH-independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH-dependent binding sites in proteins.

Keywords: Antibody engineering; Directed evolution; Fcab; Yeast surface display; pH-depending binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Directed Molecular Evolution*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / metabolism*
  • Immunoglobulin G / chemistry*
  • Molecular Dynamics Simulation
  • Pichia / physiology
  • Protein Conformation
  • Protein Structure, Secondary
  • Receptor, ErbB-2 / chemistry
  • Receptor, ErbB-2 / metabolism*


  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • ERBB2 protein, human
  • Receptor, ErbB-2