Phage display of a catalytic antibody to optimize affinity for transition-state analog binding.
Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. "Humanized" 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH --> Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.
Baca
M
M
Department of Protein Engineering, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA.
Scanlan
T S
TS
Stephenson
R C
RC
Wells
J A
JA
D000818:Animals; D017151:Antibodies, Catalytic / Q000235:genetics* / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics*; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006241:Haptens / Q000378:metabolism; D006801:Humans; D051379:Mice; D016296:Mutagenesis
D017151:Antibodies, Catalytic; D006241:Haptens