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

Abstract

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...

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

“…4 A), represented by the less substitutions (and insertions) that were concentrated more in the V H and in CDRs. It should be noted that no scFv mutant obtained with CAP, as well as that recovered from the panning, had substitution/insertion in the V H -CDR3 that is regarded as the most crucial among the six CDRs to generate affinity and specificity against a definite antigen structure 11 (Fig. 4 A).…”

“…1 B). To enable more sensitive immunochemical analyses 10 , 11 , we have generated affinity-matured scFv mutants against estradiol-17β 12 – 14 , cotinine 15 , and cortisol 16 , each of which gained an equilibrium affinity constant ( K a ) enhanced by > 150-fold, > 40-fold, and > 30-fold, respectively (Fig. 2 A–C).…”

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

“…However, there seems to be an “affinity ceiling” for the native antibodies obtained from immunized animals through conventional hybridoma-based methods 4 . Indeed, murine antibodies against small biomarkers, such as steroids or synthetic drugs categorized as haptens, rarely show equilibrium affinity constants ( K a s) that exceed the range of 10 10 M −1 , which hampers the development of immunoassay systems with subfemtomole-range sensitivities 5 , 6 .…”

Derivatization-assisted immunoassays: application for group-specific detection of potent methamphetamine and amphetamine enantiomers

“…37 Indeed, we have generated scFv mutants that showed approximately 10-150-fold increased K a s over parental antibodies against various haptens, enabling immunoassays with improved sensitivities. 9,15,[38][39][40][41][42][43] We have also found an alternative approach for enhancing sensitivity of immunoassay for hapten molecules. 44 For example, the hallucinogenic compound psilocin (the hapten of interest) was detected aer chemical derivatization into its tertbutyldimethylsilyl (TBS) ether that has a larger M r and a wider Connolly molecular area.…”

“…The latter challenge arises from the fact that the haptenic antigens as small as (S)-MAP and -AP usually equip with only a few functional groups that are available for interaction with antibody paratopes. [7][8][9] Owens et al, however, have generated hybridoma-derived monoclonal antibodies (mAbs) that bind to (S)-MAP with an equilibrium dissociation constant (K d ) of $10 nM by immunizing mice with systematically designed haptenic derivatives. 5,10 However, these antibodies unfortunately failed to recognize (S)-AP with similar affinities.…”