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 Journal Article
| Authors: | Krykbaev, R. A.; Liu, W. R.; Jeffrey, P. D.; Margolies, M. N. |
| Article Title: | 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 |
| Abstract: | The heavy-chain CDR3 region of the high affinity (Ka = 1.3 × 1010 M-1) anti-digoxin monoclonal antibody 26-10 was modified previously to shift its specificity, by substitution of tryptophan 100 by arginine, toward binding analogs of digoxin containing substitutions at position 16. To further change specificity, two 5-mer libraries of the randomly mutagenized phage-displayed 26-10 HCDR3 region (positions 94-98) were panned against digoxin-bovine serum albumin (BSA) as well as against 16-acetylgitoxin-BSA. When a mutant Fab that binds 16-substituted analogs preferentially was used as a parent sequence, clones were obtained with affinities for digoxin increased 2-4-fold, by panning on digoxin-BSA yet retaining the specificity shift. Selection 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 consensus mutation of SerH95 to GlyH95. The residues at both position H95 (serine) and position H100 (tryptophan) contact hapten in the crystal structure of the Fab 26-10-digoxin complex. Thus, by mutating hapten contact residues, it is possible to reorder the combining site of a high affinity antibody, resulting in altered specificity, yet retain or substantially increase the relative affinity for the cross-reactive ligand. |
| Keywords: | unclassified drug; proteins; amino acid substitution; digoxin; structure activity relation; structure-activity relationship; monoclonal antibodies; immunology; chemistry; immunoglobulin heavy chain; immunoglobulin heavy chains; molecular recognition; drug derivative; ligands; binding site; antibody specificity; crystal structure; mutagenesis, site-directed; binding sites; bovine serum albumin; serum albumin, bovine; antibodies; antigen binding; site directed mutagenesis; antibody; mutagenesis; phage display; antibody affinity; peptide library; cloning; glycoside; bovinae; complementarity determining region; complementarity determining regions; priority journal; article; digoxin antibody; 16 acetylgitoxin |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 11 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-03-16 |
| Start Page: | 8149 |
| End Page: | 8158 |
| Language: | English |
| DOI: | 10.1074/jbc.M008108200 |
| PUBMED: | 11060305 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
