Improving antibody affinity through in vitro mutagenesis in complementarity determining regions

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• High-affinity antibodies are crucial for diagnostics and therapeutics.
• Phage display antibodies lack in vivo affinity maturation, necessitating in vitro optimization.
• Engineering antibody affinity is essential for improving their clinical utility.

Purpose of the Study:

• To optimize the binding affinity and function of two human monoclonal antibodies using in vitro methods.
• To investigate the impact of specific mutations in complementarity-determining regions (CDRs) on antibody affinity.
• To establish a safe and effective in vitro strategy for enhancing antibody affinity.

Main Methods:

• Phage display was used to isolate human monoclonal antibodies.
• Site-directed mutagenesis was employed to introduce specific amino acid substitutions in CDRs.
• Affinity was measured using in vitro assays, and functional activity was assessed.
• Combinatorial mutagenesis was used to identify synergistic mutations for enhanced binding.

Main Results:

• A T57H mutation in the CDR-H2 of the 42A1 antibody (targeting glypican-3) improved affinity by 2.6-fold and enhanced cell-binding.
• A combination of S53P-S98T mutations in the I4A3 antibody (targeting SARS-CoV-2) improved binding affinity by 3.7-fold and neutralizing activity by 12-fold.
• Single point mutations in key CDR residues were sufficient to increase binding affinity and improve antibody functions.

Conclusions:

• In vitro optimization of antibody affinity is feasible and effective.
• Mutagenic combinations of key residues in different CDRs can lead to additive enhancements in antibody affinity and function.
• This study presents a safe and effective in vitro strategy for antibody affinity engineering, applicable to therapeutic antibody development.