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


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.

Submission Details

ID: wLTG2sYt

Submitter: Stephanie Contreras

Submission Date: Dec. 15, 2020, 3:44 p.m.

Version: 1

Publication Details
Boder ET;Midelfort KS;Wittrup KD,Proc Natl Acad Sci U S A (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. PMID:10984501
Additional Information

Study Summary

Number of data points 39
Proteins 4-4-20 FAB fragment
Unique complexes 35
Assays/Quantities/Protocols Experimental Assay: kdiss (x10^-6)
Libraries single chain antibody mutants

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.7 L 4-4-20 FAB fragment P01631 KV2A7_MOUSE
100.0 L 4-4-20 FAB fragment P01837 IGKC_MOUSE
100.0 H 4-4-20 FAB fragment P01863 GCAA_MOUSE
100.0 H 4-4-20 FAB fragment P01865 GCAM_MOUSE
94.0 H 4-4-20 FAB fragment P01864 GCAB_MOUSE