Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR

Area of Science:

• Immunology
• Biotechnology
• Protein Engineering

Background:

• Affinity maturation is crucial for enhancing therapeutic monoclonal antibody properties like binding.
• Standardized protocols for generating and selecting variegated antibody libraries are lacking.

Purpose of the Study:

• To compare two mutagenesis methods for antibody affinity maturation: random V(D)J mutagenesis and combinatorial complementarity-determining region (CDR) mutagenesis.
• To evaluate the impact of these methods on antibody binding affinity and function.

Main Methods:

• Yeast-based single-chain variable fragment (scFv) expression system.
• Random mutagenesis via error-prone PCR across V(D)J regions.
• Combinatorial mutagenesis targeting complementarity-determining regions (CDRs).
• Application to four human antibodies against immuno-oncology targets.

Main Results:

• Both error-prone PCR and combinatorial CDR mutagenesis improved scFv affinity with comparable efficiency.
• Error-prone PCR resulted in even mutational distribution, while combinatorial method exclusively targeted CDRs.
• Full-length antibodies showed comparable affinity to scFvs, but reduced function for one target.
• Increased affinity did not consistently improve cell-surface antigen binding or immune checkpoint blocking.

Conclusions:

• Both random and combinatorial mutagenesis are effective for antibody affinity maturation.
• Screening with full-length antibodies or antigen-binding fragments may be more predictive of functional outcomes than scFv screening alone.