Increased Fab thermoresistance via VH-targeted directed evolution

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Antibody aggregation, often driven by variable domains, reduces purification yields, shelf-life, and increases immunogenicity risks.
• Developing aggregation-resistant antibodies is crucial for improving therapeutic efficacy and safety.

Purpose of the Study:

• To engineer antibody fragments (Fabs) with enhanced stability and aggregation resistance using directed evolution.
• To identify specific mutations that improve biophysical characteristics of Fabs.

Main Methods:

• Constructed a Fab-phage display vector targeting the VH domain for error-prone PCR mutagenesis.
• Enriched for thermoresistant clones through heat treatment and selection for binding to an anti-light chain antibody.
• Expressed engineered Fabs and IgGs in Escherichia coli and mammalian cells, respectively, and analyzed their biophysical properties.

Main Results:

• Identified five unique Fab variants with 1-3 amino acid substitutions conferring higher expression yields in E. coli.
• Observed a 2-3°C increase in apparent melting temperature and improved aggregation resistance in engineered Fabs.
• Demonstrated additive improvements when combining mutations and confirmed enhanced expression and aggregation resistance in engineered human IgG1.

Conclusions:

• Directed evolution is an effective strategy for improving Fab stability and aggregation resistance.
• Specific amino acid substitutions can significantly enhance antibody fragment biophysical properties.
• This approach provides a valuable framework for future antibody stabilization efforts, potentially leading to safer and more effective antibody therapeutics.