Affinity maturation of recombinant antibodies using E-coli mutator cells

Background: Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response. However, the selected antibodies often have low binding affinity to their target antigen or hapten (K-D below 10(-6) M), which is characteristic of the primary immune repertoire. There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins. Objective: To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E. coli cells, incorporating phage-display strategies. Study design: Unique human scFvs were selected from a naive Fd-phage library. These genes were mutated by propagation in mutD5 mutator E. coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes. Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen. Results: The in vivo mutation of phage-displayed scFvs in E. coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity. The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity. Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs. In one case the apparent affinity of an antiglycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10(3). However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates. Conclusions: A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E. coli and incorporates phage display of antibody repertoires (libraries).