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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 H  framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment ( K  a , 3.6 × 10 8  M −1 ). 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 9  M −1   K  a  values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB 1-3 #130 (with a single residue insertion) showed the greatest  K  a  value, 1.1 × 10 10  M −1 .

The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants