1. Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody.
- Author
-
Lewis CM, Hollis GF, Mark GE 3rd, Tung JS, and Ludmerer SW
- Subjects
- Alanine genetics, Amino Acid Sequence, Base Sequence, Binding Sites, Antibody, Binding, Competitive immunology, Immunoglobulin Heavy Chains chemistry, Immunoglobulin Heavy Chains genetics, Immunoglobulin Variable Region chemistry, Immunoglobulin Variable Region genetics, Molecular Sequence Data, Antibody Affinity, HIV Antibodies biosynthesis, HIV Antibodies genetics, HIV Envelope Protein gp120 genetics, HIV Envelope Protein gp120 immunology, Mutagenesis, Insertional immunology
- 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.
- Published
- 1995
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