Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Abstract

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

Figure 1

Dissociation kinetics of mutant scFv at 25°C. (A) Yeast displaying mutant scFv isolated from a random library was assayed for antigen dissociation rate. The 4-4-20 and V_HS95A values were calculated from fluorescence quenching data with soluble scFvs. Dissociation rates for barnase/barstar (45), streptavidin/biotin (46), avidin/biotin (46), and trypsin/bovine pancreatic trypsin inhibitor (BPTI) (47) are indicated for comparison. (B) Dissociation kinetics of purified, soluble 4 M5.3 and 4-4-20 scFvs were assayed by fluorescence quenching. Triplicate traces for 4-4-20 scFv in PBS (150 mM NaCl/10 mM sodium phosphate buffer, pH 8) are shown, and 12 independent replicates are shown for 4 M5.3 in PBS or LSB. τ_1/2, half-time for dissociation.

Figure 2

Natural conservation of mutated 4-4-20 residues. Comparison of occurrence frequencies in mouse V_H and V_L κ genes of wild-type (abscissa) and mutation (ordinate) amino acid identities are shown. Frequencies were calculated at mutated sequence positions from statistics in the Kabat database (25).

Figure 3

Sites of consensus mutations in the 4-4-20 Fv. Backbone structure of the first 118 heavy-chain residues (gray) and the first 112 light-chain residues (blue) are shown. Fluorescein ligand (green) and mutated residues are depicted by space-filling models. Mutation sites are color-coded by distance from the binding site: first-shell residues are magenta; second-shell residues are yellow; third-shell residues are cyan; and fourth-shell residues are white. Residues in the first shell were defined as those with one or more atoms directly contacting ligand; second-shell residues were defined as those with one or more atoms directly contacting any residue in the first shell; third- and fourth-shell residues contact second- and third-shell residues, respectively. Definitions of contact were interatomic distances (in Å) equal to or less than 4.1 C-C, 3.3 O-O, 3.4 N-N and N-O, 3.8 C-N, and 3.7 C-O (48). Atomic coordinates were from the high-resolution crystal structure of Whitlow, et al. (ref. ; PDB ID code 1FLR).