Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody
- 31 October 1995
- journal article
- Published by Elsevier BV in Molecular Immunology
- Vol. 32 (14-15), 1065-1072
- https://doi.org/10.1016/0161-5890(95)00079-8
Abstract
We have developed a novel strategy to decrease the antibody:antigen off-rate which we call optimized residue substitution. This strategy employs alanine substitution to first identify residues non-optimal for binding, as evidenced by a decrease in off-rate upon alanine replacement. These positions are then individually randomized to all amino acids, and the best replacement for each position determined. Finally, a construct which combines all optimized substitutions is generated and evaluated. We applied this strategy to the heavy chain CDR3 of P5Q, a scFv antibody which recognizes an epitope on the V3 loop of HIV gp120. We identified two amino acid substitutions that together decrease the off-rate by nearly ten-fold. The contributions by the two substitutions were near additive, indicative of independent affects on binding. We suggest that this strategy can be generalized to strengthen protein:ligand and protein:protein interactions in other systems.Keywords
This publication has 24 references indexed in Scilit:
- By-passing immunisationJournal of Molecular Biology, 1992
- Selection of phage antibodies by binding affinityJournal of Molecular Biology, 1992
- Synergy Between Human Monoclonal Antibodies to HIV Extends Their Effective Biologic Activity Against Homologous and Divergent StrainsAIDS Research and Human Retroviruses, 1992
- Antibody framework residues affecting the conformation of the hypervariable loopsJournal of Molecular Biology, 1992
- By-passing immunizationJournal of Molecular Biology, 1991
- Making antibody fragments using phage display librariesNature, 1991
- Phage antibodies: filamentous phage displaying antibody variable domainsNature, 1990
- Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulinsJournal of Molecular Biology, 1990
- A novel genetic system to detect protein–protein interactionsNature, 1989
- Canonical structures for the hypervariable regions of immunoglobulinsJournal of Molecular Biology, 1987