Skip to main content
SpringerLink
Log in

Generation of Semi-Synthetic Shark IgNAR Single-Domain Antibody Libraries

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1701))

Abstract

Besides classical antibodies with the composition of heavy and light chains, sharks produce a unique heavy chain only isotype, termed Immunoglobulin New Antigen Receptor (IgNAR), in which antigen binding is solely mediated by a single domain, referred to as vNAR. Owing to their high affinity and specificity combined with their small size and high stability, vNAR domains emerged as promising target-binding scaffolds that can be tailor-made for biotechnological and biomedical applications. Herein, we describe protocols for the construction of semi-synthetic, CDR3-randomized vNAR libraries for the isolation of target-specific antibodies using yeast surface display or phage display as platform technology. Additionally, we provide information for affinity maturation of target-specific molecules through CDR1 diversification and sublibrary establishment.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173

    Article  CAS  PubMed  Google Scholar 

  2. Zielonka S, Empting M, Grzeschik J, Könning D, Barelle CJ, Kolmar H (2015) Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 7:15–25

    Article  CAS  PubMed  Google Scholar 

  3. Krah S, Schröter C, Zielonka S, Empting M, Valldorf B, Kolmar H (2016) Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol 38:21–28

    Article  CAS  PubMed  Google Scholar 

  4. Dooley H, Stanfield RL, Brady RA, Flajnik MF (2006) First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate. Proc Natl Acad Sci U S A 103:1846–1851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Diaz M, Stanfield RL, Greenberg AS, Flajnik MF (2002) Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR): identification of a new locus preferentially expressed in early development. Immunogenetics 54:501–512

    Article  CAS  PubMed  Google Scholar 

  6. Kovalenko OV, Olland A, Piche-Nicholas N, Godbole A, King D, Svenson K, Calabro V, Müller MR, Barelle CJ, Somers W, Gill DS, Mosyak L, Tchistiakova L (2013) Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis. J Biol Chem 288:17408–17419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Stanfield RL, Dooley H, Flajnik MF, Wilson IA (2004) Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science 305:1770–1773

    Article  CAS  PubMed  Google Scholar 

  8. Stanfield RL, Dooley H, Verdino P, Flajnik MF, Wilson IA (2007) Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding. J Mol Biol 367:358–372

    Article  CAS  PubMed  Google Scholar 

  9. Streltsov VA, Carmichael JA, Nuttall SD (2005) Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype. Protein Sci 14:2901–2909

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Simmons DP, Streltsov VA, Dolezal O, Hudson PJ, Coley AM, Foley M, Proll DF, Nuttall SD (2008) Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures. Proteins 71:119–130

    Article  CAS  PubMed  Google Scholar 

  11. Flajnik MF, Deschacht N, Muyldermans S (2011) A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol 9:e1001120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Streltsov VA, Varghese JN, Carmichael JA, Irving RA, Hudson PJ, Nuttall SD (2004) Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc Natl Acad Sci U S A 101:12444–12449

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Henderson KA, Streltsov VA, Coley AM, Dolezal O, Hudson PJ, Batchelor AH, Gupta A, Bai T, Murphy VJ, Anders RF, Foley M, Nuttall SD (2007) Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15:1452–1466

    Article  CAS  PubMed  Google Scholar 

  14. Barelle C, Porter A (2015) VNARs: an ancient and unique repertoire of molecules that deliver small, soluble, stable and high affinity binders of proteins. Antibodies 4:240

    Article  Google Scholar 

  15. Zielonka S, Weber N, Becker S, Doerner A, Christmann A, Christmann C, Uth C, Fritz J, Schäfer E, Steinmann B, Empting M, Ockelmann P, Lierz M, Kolmar H (2014) Shark attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation. J Biotechnol 191:236–245

    Article  CAS  PubMed  Google Scholar 

  16. Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A, Goldman ER (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naive shark library. Mol Immunol 44:1775–1783

    Article  CAS  PubMed  Google Scholar 

  17. Goodchild SA, Dooley H, Schoepp RJ, Flajnik M, Lonsdale SG (2011) Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries. Mol Immunol 48:2027–2037

    Article  CAS  PubMed  Google Scholar 

  18. Kovaleva M, Ferguson L, Steven J, Porter A, Barelle C (2014) Shark variable new antigen receptor biologics–a novel technology platform for therapeutic drug development. Expert Opin Biol Ther 14(10):1527–1539

    Article  CAS  PubMed  Google Scholar 

  19. Müller MR, Saunders K, Grace C, Jin M, Piche-Nicholas N, Steven J, O'Dwyer R, Wu L, Khetemenee L, Vugmeyster Y, Hickling TP, Tchistiakova L, Olland S, Gill D, Jensen A, Barelle CJ (2012) Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain. MAbs 4:673–685

    Article  PubMed  PubMed Central  Google Scholar 

  20. Simmons DP, Abregu FA, Krishnan UV, Proll DF, Streltsov VA, Doughty L, Hattarki MK, Nuttall SD (2006) Dimerisation strategies for shark IgNAR single domain antibody fragments. J Immunol Methods 315:171–184

    Article  CAS  PubMed  Google Scholar 

  21. Uth C, Zielonka S, Hörner S, Rasche N, Plog A, Orelma H, Avrutina O, Zhang K, Kolmar H (2014) A chemoenzymatic approach to protein immobilization onto crystalline cellulose nanoscaffolds. Angew Chem Int Ed Engl 53:12618–12623

    CAS  PubMed  Google Scholar 

  22. Streltsov VA, Varghese JN, Masters CL, Nuttall SD (2011) Crystal structure of the amyloid-beta p3 fragment provides a model for oligomer formation in Alzheimer's disease. J Neurosci 31:1419–1426

    Article  CAS  PubMed  Google Scholar 

  23. Könning D, Zielonka S, Sellmann C, Schröter C, Grzeschik J, Becker S, Kolmar H (2016) Isolation of a pH-sensitive IgNAR variable domain from a yeast-displayed, histidine-doped master library. Mar Biotechnol (NY) 18:161–167

    Article  Google Scholar 

  24. Zielonka S, Empting M, Könning D, Grzeschik J, Krah S, Becker S, Dickgiesser S, Kolmar H (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) 17:386–392

    Article  CAS  Google Scholar 

  25. Walsh R, Nuttall S, Revill P, Colledge D, Cabuang L, Soppe S, Dolezal O, Griffiths K, Bartholomeusz A, Locarnini S (2011) Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 411:132–141

    Article  CAS  PubMed  Google Scholar 

  26. Liu JL, Anderson GP, Goldman ER (2007) Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library. BMC Biotechnol 7:78

    Article  PubMed  PubMed Central  Google Scholar 

  27. Nuttall SD, Humberstone KS, Krishnan UV, Carmichael JA, Doughty L, Hattarki M, Coley AM, Casey JL, Anders RF, Foley M, Irving RA, Hudson PJ (2004) Selection and affinity maturation of IgNAR variable domains targeting plasmodium falciparum AMA1. Proteins 55:187–197

    Article  CAS  PubMed  Google Scholar 

  28. Nuttall SD, Krishnan UV, Doughty L, Pearson K, Ryan MT, Hoogenraad NJ, Hattarki M, Carmichael JA, Irving RA, Hudson PJ (2003) Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur J Biochem 270:3543–3554

    Article  CAS  PubMed  Google Scholar 

  29. Ohtani M, Hikima J, Jung TS, Kondo H, Hirono I, Takeyama H, Aoki T (2013) Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library. Fish Shellfish Immunol 34:724–728

    Article  CAS  PubMed  Google Scholar 

  30. Bojalil R, Mata-Gonzalez MT, Sanchez-Munoz F, Yee Y, Argueta I, Bolanos L, Amezcua-Guerra LM, Camacho-Villegas TA, Sanchez-Castrejon E, Garcia-Ubbelohde WJ, Licea-Navarro AF, Marquez-Velasco R, Paniagua-Solis JF (2013) Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock. BMC Immunol 14:17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

    Article  CAS  PubMed  Google Scholar 

  32. Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A, Kirsch MI, Schütte M, Helmsing S, Meier D, Schirrmann T, Dübel S (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159–170

    Article  CAS  PubMed  Google Scholar 

  33. Ewert S, Honegger A, Plückthun A (2003) Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42:1517–1528

    Article  CAS  PubMed  Google Scholar 

  34. Ewert S, Huber T, Honegger A, Plückthun A (2003) Biophysical properties of human antibody variable domains. J Mol Biol 325:531–553

    Article  CAS  PubMed  Google Scholar 

  35. Flajnik MF, Dooley H (2009) The generation and selection of single-domain, v region libraries from nurse sharks. Methods Mol Biol 562:71–82

    Article  CAS  PubMed  Google Scholar 

  36. Müller MR, O'Dwyer R, Kovaleva M, Rudkin F, Dooley H, Barelle CJ (2012) Generation and isolation of target-specific single-domain antibodies from shark immune repertoires. Methods Mol Biol 907:177–194

    Article  PubMed  Google Scholar 

  37. Benatuil L, Perez JM, Belk J, Hsieh CM (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159

    Article  CAS  PubMed  Google Scholar 

  38. Boder ET, Wittrup KD (2000) Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:430–444

    Article  CAS  PubMed  Google Scholar 

  39. Angelini A, Chen TF, de Picciotto S, Yang NJ, Tzeng A, Santos MS, Van Deventer JA, Traxlmayr MW, Wittrup KD (2015) Protein engineering and selection using yeast surface display. Methods Mol Biol 1319:3–36

    Article  PubMed  Google Scholar 

  40. Schirrmann T, Hust M (2010) Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol Biol 651:177–209

    Article  CAS  PubMed  Google Scholar 

  41. Diaz M, Greenberg AS, Flajnik MF (1998) Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers. Proc Natl Acad Sci U S A 95:14343–14348

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sambrook J, Russell DW (2006) Transformation of E. Coli by electroporation. CSH Protoc 2006(1). https://doi.org/10.1101/pdb.prot3933

Download references

Acknowledgments

We thank Michael Hust for discussion and advice related to the page-display section of this chapter. Furthermore, we gratefully acknowledge funding from Merck Lab@Technische Universität Darmstadt.

Author information

Authors and Affiliations

  1. Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Strasse 4, D-64287, Darmstadt, Germany

    Julius Grzeschik, Doreen Könning, Steffen C. Hinz, Simon Krah, Christian Schröter, Harald Kolmar & Stefan Zielonka

  2. Protein Engineering and Antibody Technologies, Merck-Serono, Merck KGaA, Frankfurter Straße 250, D-64293, Darmstadt, Germany

    Simon Krah, Christian Schröter & Stefan Zielonka

  3. Department Drug Design and Optimization, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Saarbrücken, Germany

    Martin Empting

Authors
  1. Julius Grzeschik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Doreen Könning
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steffen C. Hinz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simon Krah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christian Schröter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin Empting
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Harald Kolmar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan Zielonka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Harald Kolmar or Stefan Zielonka .

Editor information

Editors and Affiliations

  1. Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, Penang, Malaysia

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Grzeschik, J. et al. (2018). Generation of Semi-Synthetic Shark IgNAR Single-Domain Antibody Libraries. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7447-4_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7446-7

  • Online ISBN: 978-1-4939-7447-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation