Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity
AUTOR(ES)
Boder, Eric T.
FONTE
The National Academy of Sciences
RESUMO
Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd = 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 105–107 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=27086Documentos Relacionados
- Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.
- Antigen-binding mutants of mouse myeloma cells.
- Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells.
- Single amino acid substitution altering antigen-binding specificity.
- Demonstration That Antigen-Binding Cells Are Precursors of Antibody-Producing Cells After Purification with a Fluorescence-Activated Cell Sorter