Abstract
“Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL 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 (Ka = 1.19 × 1010 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 Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M−1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-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.
Highlights
Revolutionary strategies are required for efficiently introducing functional mutations without unnecessarily enlarging the diversity and for reliably selecting rare and improved mutants without overlooking them
Essential for elevating the antigen-binding affinity. It would be of great help if it were possible to discover “hot spots” in the VH and/or VL domains of antibodies, where a wide range of mutations introduced thereto significantly improves the affinity
It is more desirable if fewer mutations introduced in a narrower region facilitate isolating improved species with high probability
Summary
Among the affinity-matured products reported, mutant scFvs or Fabs that showed Ka values greater than 1 × 109 M−1 (i.e., a standard value required for subpicomole-order analysis), owing to>10-fold enhancement, were selected and their structures are illustrated in Supplementary Fig. S1A. Because two of these four mutants (i.e., anti-fluorescein-biotin and anti-fluorescein antibody fragments) were generated via error-prone PCR18 or related methods, only some of the multiple substitutions introduced might have driven the enhanced affinity.
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