Abstract

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. 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−1Ka 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.

Highlights

  • 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

  • Other affinity-improved single-chain Fv fragment (scFv) were identified that contained multiple mutations including substitutions in the ­VH-FR1 (Ka, 0.53–2.4 × 1­ 010 ­M−1) as follows: K23R and T28S, P7S, and S21P7

  • It is generally recognized that the amino acid sequences of framework region (FR) are considerably conserved despite being part of a “variable” domain and can be classified into subgroups based on sequence similarity as defined by Kabat et al (Fig. 1b)[18,27]

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Summary

Introduction

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. We have generated mutants of the single-chain Fv fragment (scFv) against estradiol-17β8–10, ­cotinine11, ­cortisol[12], and ∆9-tetrahydrocannabinol[13], with Ka > 150fold, > 40-fold, > 30-fold, and tenfold greater, respectively, than the corresponding wild-type scFvs (wt-scFvs; scFvs composed of the native ­VH and ­VL) These mutants were obtained through random mutagenesis based on the error-prone polymerase chain reaction (PCR)[8,14] performed on the wt-scFv genes, followed by phage-display of the randomized g­ enes[15,16,17] and subsequent panning-based selection of the target-specific scFv-displaying phage (scFv-phage) clones. In the first application of CAP, only two operations for a small (~ 1­ 05-order) library of anti-cortisol scFvs successfully allowed the discovery of eight scFv mutants showing approximately 14–63-fold increased Ka values (0.53–2.4 × ­1010 ­M−1) over the corresponding wt-scFv (Fig. 1a)

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