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 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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Antibody Engineering Laboratory, Department of Surgery, Massachusetts General Hospital, MGH East, 149 13th Street, Box 31, Charlestown, MA 02129, USA.
Jeffrey
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Demirjian
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Margolies
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D000595:Amino Acid Sequence; D000889:Anti-Arrhythmia Agents / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics*; D002316:Cardiotonic Agents / Q000276:immunology; D022801:Complementarity Determining Regions / Q000235:genetics*; D016384:Consensus Sequence; D004077:Digoxin / Q000031:analogs & derivatives* / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library
D000889:Anti-Arrhythmia Agents; D002316:Cardiotonic Agents; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; C050199:digoxin antibodies Fab fragments; D004077:Digoxin