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Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries
J Hanes1, L Jermutus, S Weber-Bornhauser
1Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Summary
Ribosome display technology rapidly selected high-affinity antibody fragments (scFv) against a GCN4 peptide. This method demonstrated inherent affinity maturation, yielding a potent antibody with a dissociation constant of 4 x 10(-11) M.
Area of Science:
- Molecular biology
- Immunotechnology
- Protein engineering
Background:
- Antibody single-chain fragments (scFv) are crucial for diagnostics and therapeutics.
- Developing high-affinity scFvs efficiently is a key challenge in biotechnology.
Purpose of the Study:
- To apply ribosome display for affinity selection of scFvs against a GCN4 peptide variant.
- To characterize the selected scFvs and investigate the affinity maturation process.
Main Methods:
- Ribosome display for library screening and selection.
- Immunization of mice with a GCN4 peptide variant.
- Characterization of scFv affinity using dissociation constants.
- Functional expression in Escherichia coli.
Main Results:
- Isolation of multiple distinct scFvs after three rounds of selection.
- Identification of a highly related group of scFvs, suggesting a common progenitor.
- Achieved a dissociation constant of (4 +/- 1) x 10(-11) M for the best scFv.
- A single amino acid change in CDR L1 conferred a 65-fold affinity increase.
Conclusions:
- Ribosome display is a powerful tool for in vitro antibody library screening and evolution.
- The method inherently facilitates affinity maturation, accelerating the development of high-affinity binders.
- Selected scFvs are suitable for functional expression in Escherichia coli.