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. 1995 Oct;32(14-15):1065-72.
doi: 10.1016/0161-5890(95)00079-8.

Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody

C M Lewis  1 , G F Hollis, G E Mark 3rd, J S Tung, S W Ludmerer
Affiliations

Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody

C M Lewis et al. Mol Immunol. 1995 Oct.

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.

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