Affinity maturation of recombinant antibodies using E. coli mutator cells

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Phage libraries offer vast antibody repertoires exceeding mammalian immune responses.
• Selected antibodies often exhibit low binding affinity (KD > 10^-6 M), necessitating affinity maturation.
• Current methods lack efficient strategies to mimic somatic hypermutation for enhanced antibody affinity.

Purpose of the Study:

• To evaluate the efficacy of combining mutator E. coli strains with phage display for antibody gene mutation and affinity selection.
• To investigate antigen-driven affinity maturation of recombinant antibody genes.
• To enhance the binding affinity of selected immunoglobulins.

Main Methods:

• Selected unique human single-chain variable fragments (scFvs) from a naive phage library.
• Propagated scFv genes in mutD5 mutator E. coli cells to introduce point mutations (mutD5-FIT).
• Performed affinity selection of phage-displayed scFvs and assayed soluble scFvs using ELISA and BIAcore.

Main Results:

• In vivo mutation and affinity selection in E. coli mutD5-FIT significantly improved scFv binding activity.
• Single amino acid substitutions from point mutations led to up to tenfold increases in apparent binding affinity.
• Mutations occurred in complementarity-determining regions (CDRs) and adjacent framework regions, with one case showing a 1000-fold affinity increase.

Conclusions:

• Developed a rapid affinity maturation strategy for antibody fragments (scFvs and Fabs).
• The strategy effectively utilizes mutator E. coli strains and phage display technology.
• This method enhances antibody repertoires for improved antigen binding capabilities.