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

Area of Science:

• Protein Engineering
• Immunology
• Biotechnology

Background:

• Therapeutic proteins often require extended serum half-lives for efficacy.
• Reduced antigen binding at acidic pH is a known strategy to prolong protein half-life.
• Engineering pH-dependent binding into proteins is a promising approach for improved pharmacokinetics.

Purpose of the Study:

• To engineer pH-dependent binding sites into antibody fragments (Fcabs).
• To reduce binding to the human epidermal growth factor receptor 2 extracellular domain (Her2-ECD) at acidic pH (6.0) compared to neutral pH (7.4).
• To evaluate the utility of yeast surface display for directed evolution of pH-sensitive proteins.

Main Methods:

• Engineering of C-terminal structural loops in an Fcab targeting Her2-ECD.
• Alternating selection of a yeast-displayed Fcab library for binding at pH 7.4 and non-binding at pH 6.0.
• Characterization of Fcab variants' binding affinity to soluble Her2-ECD and Her2-positive cells at different pH values.
• Assessment of conformational and thermal stability of selected Fcab variants.

Main Results:

• Selected Fcab variants demonstrated significantly decreased binding affinity to soluble Her2-ECD at pH 6.0 compared to pH 7.4.
• Some Fcab variants exhibited pH-dependent interactions with Her2-positive cells.
• The conformational and thermal stability of the engineered Fcabs remained independent of pH.
• Key mutations were found adjacent to existing histidines in the original Fcab, with some variants lacking histidine mutations entirely.

Conclusions:

• Yeast surface display is an effective method for the directed evolution of proteins with pH-dependent binding characteristics.
• Engineered Fcabs can achieve reduced binding at acidic pH, offering a strategy for prolonging serum half-life of therapeutic proteins.
• The study provides a valuable platform for developing next-generation biologics with enhanced pharmacokinetic properties.