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Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity
Description
<jats:title>Abstract</jats:title><jats:p>“Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the V<jats:sub>H</jats:sub> and V<jats:sub>L</jats:sub> 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 (<jats:italic>K</jats:italic><jats:sub>a</jats:sub> = 1.19 × 10<jats:sup>10</jats:sup> M<jats:sup>−1</jats:sup>) 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 <jats:italic>K</jats:italic><jats:sub>a</jats:sub>s indicated that a revertant with four substitutions (V<jats:sub>H</jats:sub>-L100gQ, V<jats:sub>L</jats:sub>-I29V, -L36M, -S77G) exhibited somewhat higher affinity (<jats:italic>K</jats:italic><jats:sub>a</jats:sub> = 1.46 × 10<jats:sup>10</jats:sup> M<jats:sup>−1</jats:sup>). Finally, the V<jats:sub>H</jats:sub>-L100gQ substitution, occurring in V<jats:sub>H</jats:sub> complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and V<jats:sub>L</jats:sub>-I29V and/or V<jats:sub>L</jats:sub>-L36M cooperated significantly. These findings encouraged us to reconsider the potential of V<jats:sub>H</jats:sub>-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.</jats:p>
Journal
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- Scientific Reports
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Scientific Reports 10 (1), 4807-, 2020-03-16
Springer Science and Business Media LLC
