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A roadmap to generate renewable protein binders to the human proteome

Nature Methods 8, 551–558 (2011) | Download Citation

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Abstract

Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.

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Acknowledgements

We acknowledge B. Liu and P. Nash for providing SH2 domain alignments; N. Bisson (Samuel Lunenfeld Research Institute) for providing a stable cell line encoding Flag-tagged Grb2; M. Taussig and M. Sundstrom for helpful discussions; and N. Bisson and B. Liu for critically reading the manuscript. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. This work was also supported by funds from Genome Canada through the Ontario Genomics Institute and Ontario Research Fund Global Leadership Round in Genomics and Life Sciences (to T.P.), The Human Frontier Science Program (O.R.), the European Commission 6th framework program coordination action 'Proteome Binders' (S.D.), from the Systematisch-Methodischen Platform Antibody Factory, within the German Nationales Genomforschungsnetz (S.D.), the Land Niedersachsen (S.D.), the Wellcome Trust (J.Mc.), the US National Institutes of Health (GM082288-09A1 and EY016094-01A1 to B.K.K. and R01-GM72688 and U54-GM74946 to A.A.K. and S.K.), to the Swedish Research Council (524-2008-617) (H.P.), and the Victorian State Government (The Department of Innovation, Industry and Regional Development), the Australian Federal Government (Bioplatforms Australia) and Monash University for financial support for the Monash Antibody Technologies Facility.

Author information

    • Helena Persson
    • , Alan Sawyer
    •  & Lars-Göran Dahlgren

    Present addresses: Department of Immunotechnology, Lund University, Lund, Sweden (H.P.); European Molecular Biology Laboratory Monoclonal Core Facility, Monterotondo-Scalo, Lazio, Italy (A.S.); School of Biological Sciences, Nanyang Technological University, Lab 7-01 Institute of Molecular and Cell Biology, Proteos, Singapore (L.-G.D.).

Affiliations

  1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

    • Karen Colwill
    • , Anna Dai
    • , Oliver Rocks
    • , Kelly Williton
    • , Frederic A Fellouse
    • , Kadija Hersi
    •  & Tony Pawson

  2. Structural Genomics Consortium, Department of Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Lars-Göran Dahlgren
    • , Alex Flores
    • , Ida Johansson
    • , Johan Weigelt
    •  & Susanne Gräslund

  3. Banting and Best Department of Medical Research, Department of Molecular Genetics, and the Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

    • Helena Persson
    • , Nicholas E Jarvik
    •  & Sachdev Sidhu

  4. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.

    • Arkadiusz Wyrzucki
    • , John Wojcik
    • , Akiko Koide
    • , Anthony A Kossiakoff
    •  & Shohei Koide

  5. Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, UK.

    • Michael R Dyson
    • , Aneesh Karatt-Vellatt
    • , Darren J Schofield
    •  & John McCafferty

  6. Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

    • Kritika Pershad
    • , John D Pavlovic
    •  & Brian K Kay

  7. Technische Universität Braunschweig, Institute of Biochemistry and Biotechnology, Braunschweig, Germany.

    • Michael Mersmann
    • , Doris Meier
    • , Jana Mersmann
    • , Saskia Helmsing
    • , Michael Hust
    •  & Stefan Dübel

  8. Monash Antibody Technologies Facility, Faculty of Medicine, Nursing and Health Sciences, Monash University, Victoria, Australia.

    • Susie Berkowicz
    • , Alexia Freemantle
    • , Michael Spiegel
    • , Alan Sawyer
    • , Daniel Layton
    •  & Edouard Nice

  9. Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.

    • Tony Pawson

  10. Department of Proteomics, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.

    • Peter Nilsson
    • , Mårten Sundberg
    • , Ronald Sjöberg
    • , Åsa Sivertsson
    • , Jochen M Schwenk
    • , Jenny Ottosson Takanen
    • , Sophia Hober
    •  & Mathias Uhlén

  11. Structural Genomics Consortium, Toronto, Ontario, Canada.

    • Lissette Crombet
    • , Peter Loppnau
    • , Ivona Kozieradzki
    • , Doug Cossar
    • , Cheryl H Arrowsmith
    •  & Aled M Edwards

Consortia

  1. Renewable Protein Binder Working Group

    A full list of authors appears at the end of this paper.

    In vitro Antibody Consortium

    Monash Antibody Technologies Facility

    Pawson Laboratory

    Human Protein Atlas

    Structural Genomics Consortium

Authors

  1. Search for Karen Colwill in:

  2. Search for Susanne Gräslund in:

Contributions

K.P., J.D.P., A.K.-V., D.J.S., N.E.J., A.W., J.Wo., A.K., M.M., D.M., J.M., S.H., S.B., A.F., M.S., K.W., A.D., K.H., M.S., R.S., J.M.S., A.S., J.O., S.H., L.-G.D., A.F., I.J., L.C., P.L., I.K., D.L. and F.A.F. designed and performed experiments; M.R.D., M.H., H.P., O.R., P.N., E.N. and D.C. conceived, designed and performed experiments and wrote the paper; J.We., C.H.A., B.K.K., A.A.K. and M.U. oversaw the project; J.Mc., S.K., S.S., S.D., A.S., T.P. and A.M.E. conceived and oversaw the project and wrote the paper; K.C. and S.G. conceived, designed and performed experiments, oversaw the project and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Karen Colwill or Susanne Gräslund.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Table 2

Excel files

  1. 1.

    Supplementary Table 1

    Summary of the results at each step in this study.

  2. 2.

    Supplementary Table 3

    Construction and sequence of SH2 domains.

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DOI

https://doi.org/10.1038/nmeth.1607

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