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Phage Display Technology for Human Monoclonal Antibodies

  • Marco Dal Ferro
  • Serena Rizzo
  • Emanuela Rizzo
  • Francesca Marano
  • Immacolata Luisi
  • Olga Tarasiuk
  • Daniele SblatteroEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)

Abstract

During the last 20 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection and screening procedures to identify lead candidates. One of the most successful methods is based on the use of filamentous phages. Functional Antibodies (Abs) fragments can be displayed on the surface of phages by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridoma technology can be isolated by a series of cycles of selection on the antigen of interest. In this system, antibody genes can be recovered simultaneously with selection and can be easily further engineered, for example by increasing their affinity to levels unobtainable in the immune system, or by modulating their specificity and their effector functions (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction and selection of binder with desired specificity.

Key words

Phage display Antigens Monoclonal antibody High-throughput scFv 

References

  1. 1.
    Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581–597CrossRefGoogle Scholar
  2. 2.
    Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249:386–390CrossRefGoogle Scholar
  3. 3.
    Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci U S A 97:10701–10705CrossRefGoogle Scholar
  4. 4.
    Boder ET, Wittrup KD (1997) Yeast surface dispay for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557CrossRefGoogle Scholar
  5. 5.
    Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317CrossRefGoogle Scholar
  6. 6.
    Bradbury A, Velappan N, Verzillo V, Ovecka M, Chasteen L, Sblattero D, Marzari R, Lou J, Siegel R, Pavlik P (2003) Antibodies in proteomics I: generating antibodies. Trends Biotechnol 21:275–281CrossRefGoogle Scholar
  7. 7.
    McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554CrossRefGoogle Scholar
  8. 8.
    Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19:4133–4137CrossRefGoogle Scholar
  9. 9.
    Sblattero D, Bradbury A (1998) A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3:271–278CrossRefGoogle Scholar
  10. 10.
    Marks JD, Griffiths AD, Malmqvist M, Clackson T, Bye JM, Winter G (1992) By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology 10:779–783PubMedGoogle Scholar
  11. 11.
    Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library [see comments]. Nat Biotechnol 14:309–314CrossRefGoogle Scholar
  12. 12.
    Sblattero D, Bradbury A (2000) Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18:75–80CrossRefGoogle Scholar
  13. 13.
    Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227:381–388CrossRefGoogle Scholar
  14. 14.
    Prassler J, Thiel S, Pracht C, Polzer A, Peters S, Bauer M, Nörenberg S, Stark Y, Kölln J, Popp A, Urlinger S, Enzelberger M (2011) HuCAL PLATINUM, a synthetic fab library optimized for sequence diversity and superior performance in mammalian expression systems. J Mol Biol 413:261–278CrossRefGoogle Scholar
  15. 15.
    Weber M, Bujak E, Putelli A, Villa A, Matasci M, Gualandi L, Hemmerle T, Wulhfard S, Neri D (2014) A highly functional synthetic phage display library containing over 40 billion human antibody clones. PLoS One.  https://doi.org/10.1371/journal.pone.0100000
  16. 16.
    Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao M, Dreier T, Fischer D, Hoss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Sohlemann P, Winter J, Wolle J, Kretzschmar T (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods 254:67–84CrossRefGoogle Scholar
  17. 17.
    Fellouse FA, Esaki K, Birtalan S, Raptis D, Cancasci VJ, Koide A, Jhurani P, Vasser M, Wiesmann C, Kossiakoff AA, Koide S, Sidhu SS (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373:924–940CrossRefGoogle Scholar
  18. 18.
    Rouet R, Jackson KJL, Langley DB, Christ D (2018) Next-generation sequencing of antibody display repertoires. Front Immunol.  https://doi.org/10.3389/fimmu.2018.00118
  19. 19.
    Bradbury A, Velappan N, Verzillo V, Ovecka M, Chasteen L, Sblattero D, Marzari R, Lou J, Siegel R, Pavlik P (2003) Antibodies in proteomics II: screening, high-throughput characterization and downstream applications. Trends Biotechnol 21:312–317CrossRefGoogle Scholar
  20. 20.
    Di Niro R, Sulic AM, Mignone F, D’Angelo S, Bordoni R, Iacono M, Marzari R, Gaiotto T, Lavric M, Bradbury AR, Biancone L, Zevin-Sonkin D, De Bellis G, Santoro C, Sblattero D (2010) Rapid interactome profiling by massive sequencing. Nucleic Acids Res 38:e110CrossRefGoogle Scholar
  21. 21.
    Lim TS, Mollova S, Rubelt F, Sievert V, Dubel S, Lehrach H, Konthur Z (2010) V-gene amplification revisited – an optimised procedure for amplification of rearranged human antibody genes of different isotypes. New Biotechnol 27:108–117CrossRefGoogle Scholar
  22. 22.
    Krebber A, Bornhauser S, Burmester J, Honeggar A, Willuda J, Bosshard HR, Pluckthun A (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201:35–55CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marco Dal Ferro
    • 1
  • Serena Rizzo
    • 1
  • Emanuela Rizzo
    • 1
  • Francesca Marano
    • 1
  • Immacolata Luisi
    • 1
  • Olga Tarasiuk
    • 2
  • Daniele Sblattero
    • 1
    Email author
  1. 1.Department of Life SciencesUniversity of TriesteTriesteItaly
  2. 2.Department of Health Sciences and IRCADUniversity of Eastern PiedmontNovaraItaly

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