title: Construction of pH-sensitive Her2-binding IgG1-Fc by directed evolution.
abstract: 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.
authors: Christian Doppler Laboratory for Antibody Engineering at Department of Chemistry and Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria; Department of Chemistry, Vienna Institute of BioTechnology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Michael W
authors: MW
authors: Traxlmayr
authors: Elisabeth
authors: E
authors: Lobner
authors: Christoph
authors: C
authors: Hasenhindl
authors: Gerhard
authors: G
authors: Stadlmayr
authors: Chris
authors: C
authors: Oostenbrink
authors: Florian
authors: F
authors: Rüker
authors: Christian
authors: C
authors: Obinger
mesh_terms: D001665:Binding Sites; D019020:Directed Molecular Evolution; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D010843:Pichia / Q000502:physiology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human
keywords: Antibody engineering; Directed evolution; Fcab; Yeast surface display; pH-depending binding