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Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library

Nature Biotechnology volume 14, pages 309–314 (1996)Cite this article

Abstract

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 × 1010 single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10−3 to 10−4s−1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomoiar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

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Author information

Author notes

  1. Tristan J. Vaughan: Corresponding author Cambridge Antibody Technology Ltd., Unit B3, The Science Park, Melbourn, Cambridgeshire, SG8 6JJ, U.K.

Authors and Affiliations

  1. Cambridge Antibody Technology Ltd., Unit B3, The Science Park, Melbourn, Cambridgeshire, SG8 6JJ, U.K.

    Andrew J. Williams, Kevin Pritchard, Jane K. Osbourn, Anthony R. Pope, John C. Earnshaw, John McCafferty, Jane Wilton & Kevin S. Johnson

  2. Centre of Protein Engineering, MRC, Hills Road, Cambridge, CB2 2QH, U.K.

    Regina A. Hodits

Authors
  1. Tristan J. Vaughan
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Vaughan, T., Williams, A., Pritchard, K. et al. Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library. Nat Biotechnol 14, 309–314 (1996). https://doi.org/10.1038/nbt0396-309

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