The V Framework Region 1 As a Target of Efficient Mutagenesis for Generating a Variety of Affinity-matured ScFv Mutants

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two "designed" site-directed mutagenesis (A and B) targeted the N-terminal 1-10 positions of the V framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K, 3.6 × 10 M). Mutagenesis A substituted the amino acids at the position 1-3, 5-7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1-6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17-31-fold increased affinity with > 10 M K values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB#130 (with a single residue insertion) showed the greatest K value, 1.1 × 10 M.