Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity*
- Christian Zahnd‡,
- Silvia Spinelli§¶,
- Béatrice Luginbühl‡¶,
- Patrick Amstutz‡,
- Christian Cambillau§ and
- Andreas Plückthun‡∥
- ‡Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland and §Architecture et Fonction des Macromolécules Biologiques, CNRS, 31 Chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France
- ∥ To whom correspondence should be addressed. Tel.: 41-1-6355570; Fax: 41-1-6355712; E-mail: plueckthun{at}bioc.unizh.ch.
Abstract
We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.
Footnotes
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↵1 The abbreviations used are: scFv, single chain Fv fragment; VL, variable domain of the light chain; VH, variable domain of the heavy chain; 8-oxo-dGTP, 8-oxo-2′-deoxyguanosine 5′-triphosphate; dPTP, 6-(deoxy-β-d-erythro-pentofuranosyl)-3,4-dihydro-8H-pyrimido-[4,-5c][1,2]oxazine-7-one-5′-triphosphate; RIA, radioimmunoassay; CDR, complementarity-determining region.
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The atomic coordinates and structure factors (code 1P4I and 1P4B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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↵* This work was supported by Schweizerische Nationalfonds Grant 31-65344.01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Both authors contributed equally to this study.
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- Received August 19, 2003.
- Revision received January 6, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
