Protein Engineering, Vol. 14, No. 4, 287-296,
April 2001
© 2001 Oxford University Press
A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs
Antibody Engineering Laboratory, Department of Surgery, Massachusetts General Hospital, MGH East, 149 13th Street, Box 31, Charlestown,MA 02129 and 2 Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted 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 showed increased affinity for digoxin compared with 26-10 and retained the wild-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 than to digoxin. This specificity change was unexpected, as C16 lies on the opposite 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.
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