Selection of phage antibodies by binding affinity. Mimicking affinity maturation

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Antibody selection is crucial for therapeutic development.
• Phage display technology offers a powerful platform for antibody engineering.
• Improving antibody affinity and kinetics is a key goal in antibody research.

Purpose of the Study:

• To develop and validate a phage display process for selecting antibodies based on affinity and dissociation kinetics.
• To engineer and isolate high-affinity antibody mutants using this selection method.
• To mimic affinity maturation observed in the immune system.

Main Methods:

• Antibody display on filamentous bacteriophage surfaces.
• Affinity selection using low concentrations of biotinylated antigen and streptavidin-coated paramagnetic beads.
• Off-rate selection by diluting pre-bound antigen-antibody complexes into excess unlabeled antigen.
• In vitro mutagenesis of antibody genes using error-prone polymerase.

Main Results:

• A novel phage display selection process for both antibody affinity and off-rate was established.
• The process successfully distinguished between antibodies with closely related affinities.
• Engineered antibody mutants showed significantly improved affinity (fourfold increase) compared to the parent antibody.
• The observed affinity improvement in engineered mutants mirrored natural immune responses.

Conclusions:

• The described phage display method is effective for selecting antibodies based on binding affinity and dissociation kinetics.
• This technique enables rapid engineering of antibodies with enhanced binding properties.
• The approach provides a valuable tool for antibody discovery and optimization in various applications.