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Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines

Nature Biotechnology volume 20, pages 1129–1134 (2002)Cite this article

Abstract

We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein–ligand interactions.

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Figure 1: A lineage of streptavidin-specific IgMs from Ramos.
Figure 2: Mutations contributing to the affinity maturation of Ramos IgM to streptavidin.
Figure 3: A lineage of IgMs from DT40 specific for a rat immunoglobulin idiotype.
Figure 4: A lineage of protein A–specific IgMs from DT40.
Figure 5: Antigen selection of DT40 can yield polyreactive IgMs with the possibility of maturation into specificity.

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Acknowledgements

We thank Veronica Morea for the model of the Ramos structure, Andy Riddell and Andy Johnson for help with flow cytometry, Alan Weeds for advice, and the late CĂ©sar Milstein for many inspiring discussions. S.L.D. and F.D.B. were in part supported by grants from the Leukaemia Research Fund and Arthritis Research Campaign, respectively.

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  1. Sarah J. Cumbers and Gareth T. Williams: These authors contributed equally to the work.

Authors and Affiliations

  1. Medical Research Laboratory of Biology, Hills Road, Cambridge, CB2 2QH, UK

    Sarah J. Cumbers, Gareth T. Williams, Sarah L. Davies, Richard L. Grenfell, Facundo D. Batista, Julian E. Sale & Michael S. Neuberger

  2. CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, 606-8501, Kyoto, Japan

    Shunichi Takeda

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  1. Sarah J. Cumbers
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Correspondence to Michael S. Neuberger.

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Cumbers, S., Williams, G., Davies, S. et al. Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines. Nat Biotechnol 20, 1129–1134 (2002). https://doi.org/10.1038/nbt752

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