Antibody affinity maturation using bacterial surface display

Abstract

A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (KD = 0.91+/-0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning. The relative rate constants estimated by FACS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries.

Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity

“…A variety of methods, including randomized mutations (33)(34)(35), designed mutations such as "hot spot" and "codon-based" mutations (guided by structural knowledge and/or computer modeling) (36 -38) and "CDR walking" (39) have been used to construct the mutagenized libraries. These libraries were then displayed on the surface of filamentous phage (40,41), bacterial (42)(43)(44), yeast (45,46), or ribosome (47-49) and selected on relevant targets under conditions specifically designed for the enrichment of variants with desired properties (i.e. high affinity) (for review, see Ref.…”

Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

“…Furthermore, if fluorescent reagents with chelated metal ions were available, the biopanning might be more conveniently performed using fluorescence-activated cell sorting of staphylococcal cells carrying a surface-displayed CBD library, as has been demonstrated in antibody maturation and enzyme evolution strategies (7,35). Such a strategy would obviously have the advantage of eliminating the initial phage display selection procedure.…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…A variety of other display methods have been reported such as ribosome display, yeast surface display, E. coli surface display, and mRNA display [262,[275][276][277]. However, these combinatorial approaches to antibody affinity maturation require a large library size, which is often limited by the transformation steps following assembly and ligation [278].…”