A single H : CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in t he high-affinity anti-digoxin antibody 26-10 to determine structural constr aints on affinity and specificity for digoxin. Libraries of mutant Fabs ran domized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-ace tylgitoxin. The sequence data from 83 different mutant Fabs showed highly r estricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA sh owed increased affinity for digoxin compared with 26-10 and retained the wi ld-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs th an to digoxin. This specificity change was unexpected, as C16 lies on the o pposite side of digoxin from H:CDR3, Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.