USE OF A NOVEL MUTAGENESIS STRATEGY, OPTIMIZED RESIDUE SUBSTITUTION, TO DECREASE THE OFF-RATE OF AN ANTI-GP120 ANTIBODY

We have developed a novel strategy to decrease the antibody:antigen of f-rate which we call optimized residue substitution. This strategy emp loys alanine substitution to first identify residues non-optimal for b inding, as evidenced by a decrease in off-rate upon alanine replacemen t. These positions are then individually randomized to all amino acids , and the best replacement for each position determined. Finally, a co nstruct which combines all optimized substitutions is generated and ev aluated. We applied this strategy to the heavy chain CDR3 of P5Q, a sc Fv antibody which recognizes an epitope on the V3 loop of HIV gp120. W e identified two amino acid substitutions that together decrease the o ff-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:li gand and protein:protein interactions in other systems.