Advertisement

Isolation of Antigen-Specific VHH Single-Domain Antibodies by Combining Animal Immunization with Yeast Surface Display

  • Lukas Roth
  • Simon Krah
  • Janina Klemm
  • Ralf Günther
  • Lars Toleikis
  • Michael Busch
  • Stefan Becker
  • Stefan ZielonkaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

Abstract

In addition to conventional hetero-tetrameric antibodies, the adaptive immune repertoire of camelids comprises the so-called heavy chain-only antibodies devoid of light chains. Consequently, antigen binding is mediated solely by the variable domain of the heavy chain, referred to as VHH. In recent years, these single-domain moieties emerged as promising tools for biotechnological and biomedical applications. In this chapter, we describe the generation of VHH antibody yeast surface display libraries from immunized Alpacas and Lamas as well as the facile isolation of antigen-specific molecules in a convenient fluorescence-activated cell sorting (FACS)-based selection process.

Key words

Yeast surface display Antibody engineering Protein engineering Camelid antibodies Single-domain antibodies VHH (variable domain of the heavy chain of heavy chain-only antibodies) 

Notes

Acknowledgments

We like to thank Preclinics GmbH for collaborating on this project.

References

  1. 1.
    Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448CrossRefGoogle Scholar
  2. 2.
    Zielonka S, Empting M, Grzeschik J et al (2015) Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 7:15–25CrossRefGoogle Scholar
  3. 3.
    Arezumand R, Alibakhshi A, Ranjbari J et al (2017) Nanobodies as novel agents for targeting angiogenesis in solid cancers. Front Immunol 8:1746CrossRefGoogle Scholar
  4. 4.
    Könning D, Zielonka S, Grzeschik J et al (2017) Camelid and shark single domain antibodies: structural features and therapeutic potential. Curr Opin Struct Biol 45:10–16CrossRefGoogle Scholar
  5. 5.
    Wesolowski J, Alzogaray V, Reyelt J et al (2009) Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol 198:157–174CrossRefGoogle Scholar
  6. 6.
    Jähnichen S, Blanchetot C, Maussang D et al (2010) CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc Natl Acad Sci U S A 107:20565–20570CrossRefGoogle Scholar
  7. 7.
    Maussang D, Mujić-Delić A, Descamps FJ et al (2013) Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. J Biol Chem 288:29562–29572CrossRefGoogle Scholar
  8. 8.
    Nguyen VK, Hamers R, Wyns L et al (2000) Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire. EMBO J 19:921–930CrossRefGoogle Scholar
  9. 9.
    Muyldermans S, Smider VV (2016) Distinct antibody species: structural differences creating therapeutic opportunities. Curr Opin Immunol 40:7–13CrossRefGoogle Scholar
  10. 10.
    Krah S, Schröter C, Zielonka S et al (2016) Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol 38:21–28CrossRefGoogle Scholar
  11. 11.
    Conrath KE, Wernery U, Muyldermans S et al (2003) Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol 27:87–103CrossRefGoogle Scholar
  12. 12.
    Vincke C, Loris R, Saerens D et al (2009) General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 284:3273–3284CrossRefGoogle Scholar
  13. 13.
    Tijink BM, Laeremans T, Budde M et al (2008) Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol Cancer Ther 7:2288–2297CrossRefGoogle Scholar
  14. 14.
    Helma J, Cardoso MC, Muyldermans S et al (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644CrossRefGoogle Scholar
  15. 15.
    Alvarez-Cienfuegos A, Nuñez-Prado N, Compte M et al (2016) Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains. Sci Rep 6:28643CrossRefGoogle Scholar
  16. 16.
    Desmyter A, Spinelli S, Boutton C et al (2017) Neutralization of human interleukin 23 by multivalent nanobodies explained by the structure of cytokine–nanobody complex. Front Immunol 8:884CrossRefGoogle Scholar
  17. 17.
    Goldman E, Liu J, Bernstein R et al (2009) Ricin detection using phage displayed single domain antibodies. Sensors 9:542–555CrossRefGoogle Scholar
  18. 18.
    Yan J, Li G, Hu Y et al (2014) Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J Transl Med 12:343CrossRefGoogle Scholar
  19. 19.
    Bencurova E, Pulzova L, Flachbartova Z et al (2015) A rapid and simple pipeline for synthesis of mRNA–ribosome–V H H complexes used in single-domain antibody ribosome display. Mol BioSyst 11:1515–1524CrossRefGoogle Scholar
  20. 20.
    Romao E, Morales-Yanez F, Hu Y et al (2016) Identification of useful nanobodies by phage display of immune single domain libraries derived from camelid heavy chain antibodies. Curr Pharm Des 22:6500–6518CrossRefGoogle Scholar
  21. 21.
    Cavallari M (2017) Rapid and direct VHH and target identification by staphylococcal surface display libraries. Int J Mol Sci 18:1507CrossRefGoogle Scholar
  22. 22.
    Eden T, Menzel S, Wesolowski J et al (2018) A cDNA immunization strategy to generate nanobodies against membrane proteins in native conformation. Front Immunol 8:1989CrossRefGoogle Scholar
  23. 23.
    Wu Y, Jiang S, Ying T (2017) Single-domain antibodies as therapeutics against human viral diseases. Front Immunol 8:1802CrossRefGoogle Scholar
  24. 24.
    Pardon E, Laeremans T, Triest S et al (2014) A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693CrossRefGoogle Scholar
  25. 25.
    McMahon C, Baier AS, Pascolutti R et al (2018) Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol 25:289–296CrossRefGoogle Scholar
  26. 26.
    Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557CrossRefGoogle Scholar
  27. 27.
    Lu Z-J (2012) Frontier of therapeutic antibody discovery: The challenges and how to face them. World J Biol Chem 3:187CrossRefGoogle Scholar
  28. 28.
    Doerner A, Rhiel L, Zielonka S et al (2014) Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett 588:278–287CrossRefGoogle Scholar
  29. 29.
    Zielonka S, Weber N, Becker S et al (2014) Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation. J Biotechnol 191:236–245CrossRefGoogle Scholar
  30. 30.
    Zielonka S, Empting M, Könning D et al (2015) The shark strikes twice: hypervariable loop 2 of shark IgNAR antibody variable domains and its potential to function as an autonomous paratope. Mar Biotechnol (NY) 17:386–392CrossRefGoogle Scholar
  31. 31.
    Grzeschik J, Könning D, Hinz SC et al (2018) Generation of semi-synthetic shark IgNAR single-domain antibody libraries. Methods Mol Biol 1701:147–167CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Lukas Roth
    • 1
  • Simon Krah
    • 1
  • Janina Klemm
    • 1
  • Ralf Günther
    • 1
  • Lars Toleikis
    • 1
  • Michael Busch
    • 2
  • Stefan Becker
    • 1
  • Stefan Zielonka
    • 1
    Email author
  1. 1.Protein Engineering and Antibody Technologies (PEAT)Merck KGaADarmstadtGermany
  2. 2.Discovery PharmacologyMerck KGaADarmstadtGermany

Personalised recommendations