Phage display-selected sequences of the heavy-chain CDR3 loop of the anti-digoxin antibody 26-10 define a high affinity binding site for position 16-substituted analogs of digoxin

The heavy-chain CDR3 region of the high affinity (K-alpha = 1.3 x 10(10) M- 1) anti-digoxin monoclonal antibody 26-10 was modified previously to shift its specificity, by substitution of tryptophan 100 by arginine, toward bind ing analogs of digoxin containing substitutions at position 16. To further change specificity, two 5 mer libraries of the randomly mutagenized phage d isplayed 26-10 HCDR3 region (positions 94-98) were penned against digoxin-b ovine serum albumin (BSA) as well as against 16-acetylgitoxin-BSA. When a m utant Fab that binds 16-substituted analogs preferentially was used as a pa rent sequence, clones were obtained with affinities for digoxin increased 2 -4-fold, by panning on digoxin-BSA yet retaining the specificity shift. Sel ection on 16-acetylgitoxin-BSA, however, resulted in nine clones that bound gitoxin (16-OH) up to 150-fold higher than the wild-type 26-10, due to a c onsensus mutation of Ser(H95) to Gly(H95). The residues at both position H9 5 (serine) and position H100 (tryptophan) contact hapten in the crystal str ucture of the Fab 26-10-digoxin complex. Thus, by mutating hapten contact r esidues, it is possible to reorder the combining site of a high affinity an tibody, resulting in altered specificity, yet retain or substantially incre ase the relative affinity for the cross-reactive ligand.