The VH framework region 1 as a target of efficient mutagenesis for generating

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 VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (Ka, 3.6 × 108 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.


Fig: Backgrounds of the designed mutagenesis targeting the VH-FR1 performed in this study. (a) Selected data of our previous CAP-based antibody-breeding experiments with scFvs against cortisol7. A schematic illustration of the primary structures and Ka values of wt-scFv and improved scFvs having only a single amino acid substitution or insertion in the VH-FR1. In wt-scFv, the VH and VLdomains were combined via a linker sequence VSS(GGGGS)3T. (b) The most common amino acids found at the positions 130 in VH (i.e., the VH-FR1) in different subgroups as defined by Kabat et al. The frequency of appearance of each residue is shown with different colors: magenta, invariant (> 95%) and common in all subgroups; green, “subgroup-specific residues” that are invariant within the relevant subgroup(s). The VH-FR1 amino acid sequence of wt-scFv is also shown for comparison. (c) The frequency of amino acids at the positions 1–10, 21, 23, 28, and 29 in VH, compiled for 1,820 antibodies that were available in the Kabat database. The detailed data listing for the positions 1–30 is available in Supplementary Table.

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 > 109 M−1 Ka values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest Ka value, 1.1 × 1010 M−1.

Kiguchi, Y., Oyama, H., Morita, I. et al. The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants. Sci Rep 11, 8201 (2021).


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