Engineering pH responsive fibronectin domains for biomedical applications

Area of Science:

• Biotechnology
• Protein Engineering
• Biochemistry

Background:

• Engineered antibodies with pH-responsive binding show improved pharmacokinetics and reduced degradation.
• Fibronectin (Fn3) domains are promising biopharmaceuticals with potential advantages over antibodies.
• Engineering pH-responsive Fn3 domains is crucial for enhancing their pharmacokinetic properties.

Purpose of the Study:

• To engineer Fn3 domains with pH-responsive antigen binding capabilities.
• To demonstrate the feasibility of creating Fn3s with tunable binding affinities at acidic endosomal pH.

Main Methods:

• Utilized a yeast surface-displayed Fn3 histidine (His) mutant library screening approach.
• Screened for Fn3 domains that bind the epidermal growth factor receptor (EGFR).
• Analyzed binding affinities at neutral (pH 7.4) and acidic (pH 5.5) conditions.

Main Results:

• Successfully engineered EGFR-binding Fn3 domains with significantly decreased binding affinity at endosomal pH (5.5).
• Histidine mutations in Fn3 domains increased dissociation constants (KDs) up to four-fold at pH 5.5 compared to pH 7.4.
• Demonstrated that pH responsiveness is due to reversible conformational changes, not irreversible unfolding.

Conclusions:

• Established a generalizable method for constructing and screening pH-responsive Fn3 His mutant libraries.
• This method facilitates the development of Fn3-based biopharmaceuticals with tailored pharmacokinetic profiles.
• The approach is applicable to a wide range of biomedical applications requiring pH-sensitive binding.