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 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.
Lewis
C M
CM
Merck Research Laboratories, Rahway, NJ 07065, USA.
Hollis
G F
GF
Mark
G E
GE
Tung
J S
JS
Ludmerer
S W
SW
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D015483:HIV Antibodies / Q000096:biosynthesis* / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000276:immunology*
D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D000409:Alanine