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

Single-chain antibody mutants have been evolved in vitro with antigen-bindi ng equilibrium dissociation constant K-d = 48 fM and slower dissociation ki netics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet r eported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast su rface-displayed antibodies enabled a >1,000-fold decrease in the rate of di ssociation after four cycles of affinity mutagenesis and screening. The con sensus mutations are generally nonconservative by com parison with naturall y occurring mouse Fv sequences and with residues that do not contact the fl uorescein antigen in the wildtype complex. The existence of these mutants d emonstrates that the antibody Fv architecture is not intrinsically responsi ble far an antigen-binding affinity ceiling during in vivo affinity maturat ion.