GENETICALLY-ENGINEERED ANTIBODIES WHICH HAVE REDUCED BINDING-AFFINITYFOR THYROXINE BUT RETAIN THE SAME AFFINITY FOR TRIIODOTHYRONINE

Based on a three-dimensional molecular model of the variable region of a monoclonal antibody (Ab) TT1, in which the complementarity determin ing regions (CDRs) associate to form a cavity large enough to accommod ate a single molecule of tri-iodothyronine (T3) or thyroxine (T4), we designed TT1 mutants with one amino acid substitution as candidates wh ich have their binding affinity for T4 reduced but retain the same aff inity for T3. Each candidate was subsequently tested by site-directed mutagenesis, transient expression in COS cells, and surface plasmon re sonance (SPR) analysis for its binding ability for T3- or T4-conjugate s with alkaline phosphatase (AP). Of the candidates, the Ab with serin e in place of glycine at position 92 of the light chain (L;G92S) and t he Ab with alanine in place of leucine at position 47 of the heavy cha in (H;L47A) had the association constant (KA = kass / kdiss) for bindi ng to T4-AP decreased by 5-fold, but retained the same KA for T3-AP.