A Single-Step “Breeding” Generated a Diagnostic Anti-cortisol Antibody Fragment with Over 30-Fold Enhanced Affinity

et al. 2020

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Antibody-breeding" has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. typically, random point mutations are introduced into the V H and V L domains of parent antibodies to generate diverse libraries of single-chain fv fragments (scfvs), from which evolved mutants are selected. We produced an scFv against estradiol-17β with 11 amino acid substitutions and a >100-fold improved affinity constant (K a = 1.19 × 10 10 M −1 ) over the parent scfv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their K a s indicated that a revertant with four substitutions (V H -L100gQ, V L -I29V, -L36M, -S77G) exhibited somewhat higher affinity (K a = 1.46 × 10 10 M −1 ). finally, the V H -L100gQ substitution, occurring in V H complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and V L -I29V and/or V L -L36M cooperated significantly. These findings encouraged us to reconsider the potential of V H -CDR3-targeting mutagenesis, which has been frequently attempted. the substitution(s) wherein might enable a "high rate of return" in terms of selecting mutants with dramatically enhanced affinities. The "high risk" of generating a tremendous excess of "junk mutants" can be overcome with the efficient selection systems that we developed. substitution gained 10-fold higher affinity (based on the calculated equilibrium affinity constant K a ), whereas the anti-E 2 scFv with 11 substitutions exhibited a K a that was increased (improved) by >100-fold. The scFv mutants against cortisol and cotinine showed intermediate improvement, exhibiting >30-fold and >40-fold higher K a s as the results of three and five substitutions, respectively. However, a reasonable explanation for such correlations requires evidence that most or many (if not all) of these multiple substitutions participated in increasing the affinity to at least some extent.Regarding the positions of the substitution(s), a remarkable difference was found between the anti-cortisol scFvs (only in the V L ) and the anti-THC (only in the V H ) (Fig. 1). In contrast, with the anti-E 2 and anti-cotinine scFvs that showed more significant improvements, the substitutions were spread over both the V H and V L domains. The most successful example of in vitro affinity maturation for an antibody against a small compound was reported for an scFv against fluorescein derivative (K a > 1 × 10 12 M −1 ), which represented an affinity increase of>2,000-fold 19 . This "super" mutant, which is almost beyond native antibodies, was the product of 14 different substitutions, 12 of which were located in the V H domain ( Supplementary Fig. S1A).Recently, some approaches have been developed for improving antibody functions via computational analysis, in order to minimize the trial and error that is an inevitable aspect of the conventional strategies 20-23 . H...

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Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity

et al. 2020

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Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

et al. 2020

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"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 10 5 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved K a values (> 10 10 M −1 ), compared with the wild-type scFv ( K a = 3.8 × 10 8 M −1 ), none of which could be recovered via conventional panning procedures operated for the entire library.

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

et al. 2021

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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 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.