Skip to main content
SpringerLink
Log in

Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2702))

  • 1243 Accesses

Abstract

The adaptive immune system of sharks comprises a unique heavy chain-only antibody isotype, termed immunoglobulin new antigen receptor (IgNAR), in which antigen binding is mediated by a single variable domain, referred to as vNAR. In recent years, efforts were made to harness these domains for biomedical and biotechnological applications particularly due to their high affinity and specificity combined with a small size and high stability. Herein, we describe protocols for the construction of semisynthetic, CDR3-randomized vNAR libraries for the isolation of target-specific paratopes by yeast surface display. Additionally, we provide guidance for affinity maturation of a panel of antigen-enriched vNAR domains through CDR1 diversification of the FACS-selected, antigen-enriched population 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

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 229.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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

Similar content being viewed by others

References

  1. Grzeschik J, Könning D, Hinz SC et al (2018) Generation of semi-synthetic shark IgNAR single-domain antibody libraries. In: Hust M, Lim TS (eds) Phage display. Springer New York, New York, pp 147–167

    Chapter  Google Scholar 

  2. Greenberg AS, Avila D, Hughes M et al (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173. https://doi.org/10.1038/374168a0

    Article  CAS  PubMed  Google Scholar 

  3. Zielonka S, Empting M, Grzeschik J et al (2015) Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 7:15–25. https://doi.org/10.4161/19420862.2015.989032

    Article  CAS  PubMed  Google Scholar 

  4. Krah S, Schröter C, Zielonka S et al (2016) Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol 38:21–28. https://doi.org/10.3109/08923973.2015.1102934

    Article  CAS  PubMed  Google Scholar 

  5. Könning D, Zielonka S, Grzeschik J et al (2017) Camelid and shark single domain antibodies: structural features and therapeutic potential. Curr Opin Struct Biol 45:10–16. https://doi.org/10.1016/j.sbi.2016.10.019

    Article  CAS  PubMed  Google Scholar 

  6. 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 103:1846–1851. https://doi.org/10.1073/pnas.0508341103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Simmons DP, Streltsov VA, Dolezal O et al (2008) Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures. Proteins Struct Funct Bioinf 71:119–130. https://doi.org/10.1002/prot.21663

    Article  CAS  Google Scholar 

  8. Streltsov VA, Carmichael JA, Nuttall SD (2005) Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype: structure of a shark ignar antibody variable domain and modeling of an early-developmental isotype. Protein Sci 14:2901–2909. https://doi.org/10.1110/ps.051709505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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. https://doi.org/10.1371/journal.pbio.1001120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Henderson KA, Streltsov VA, Coley AM et al (2007) Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15:1452–1466. https://doi.org/10.1016/j.str.2007.09.011

    Article  CAS  PubMed  Google Scholar 

  11. Li Z, Krippendorff B-F, Sharma S et al (2016) Influence of molecular size on tissue distribution of antibody fragments. MAbs 8:113–119. https://doi.org/10.1080/19420862.2015.1111497

    Article  CAS  PubMed  Google Scholar 

  12. 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 191:236–245. https://doi.org/10.1016/j.jbiotec.2014.04.023

    Article  CAS  PubMed  Google Scholar 

  13. 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 4:240–258. https://doi.org/10.3390/antib4030240

    Article  CAS  Google Scholar 

  14. Kovaleva M, Ferguson L, Steven J et al (2014) Shark variable new antigen receptor biologics – a novel technology platform for therapeutic drug development. Expert Opin Biol Ther 14:1527–1539. https://doi.org/10.1517/14712598.2014.937701

    Article  CAS  PubMed  Google Scholar 

  15. Bannas P, Hambach J, Koch-Nolte F (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol 8. https://doi.org/10.3389/fimmu.2017.01603

  16. Chanier T, Chames P (2019) Nanobody engineering: toward next generation immunotherapies and immunoimaging of cancer. Antibodies 8:13. https://doi.org/10.3390/antib8010013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pekar L, Busch M, Valldorf B et al (2020) Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform. MAbs:1812210. https://doi.org/10.1080/19420862.2020.1812210

  18. Yanakieva D, Pekar L, Evers A et al (2022) Beyond bispecificity: controlled Fab arm exchange for the generation of antibodies with multiple specificities. MAbs 14. https://doi.org/10.1080/19420862.2021.2018960

  19. Lipinski B, Arras P, Pekar L et al (2023) NKp46 -specific single domain antibodies enable facile engineering of various potent NK cell engager formats. Protein Sci. https://doi.org/10.1002/pro.4593

  20. Ubah OC, Buschhaus MJ, Ferguson L et al (2018) Next-generation flexible formats of VNAR domains expand the drug platform’s utility and developability. Biochem Soc Trans 46:1559–1565. https://doi.org/10.1042/BST20180177

    Article  CAS  PubMed  Google Scholar 

  21. Kovaleva M, Johnson K, Steven J et al (2017) Therapeutic potential of shark anti-ICOSL VNAR domains is exemplified in a murine model of autoimmune non-infectious uveitis. Front Immunol 8. https://doi.org/10.3389/fimmu.2017.01121

  22. Ubah OC, Steven J, Porter AJ, Barelle CJ (2019) An anti-hTNF-α variable new antigen receptor format demonstrates superior in vivo preclinical efficacy to Humira® in a transgenic mouse autoimmune polyarthritis disease model. Front Immunol 10. https://doi.org/10.3389/fimmu.2019.00526

  23. Sehlin D, Stocki P, Gustavsson T et al (2020) Brain delivery of biologics using a cross-species reactive transferrin receptor 1 VNAR shuttle. FASEB J 34:13272–13283. https://doi.org/10.1096/fj.202000610RR

    Article  CAS  PubMed  Google Scholar 

  24. Camacho-Villegas T, Mata-González M, García-Ubbelohd W et al (2018) Intraocular penetration of a vNAR: in vivo and in vitro VEGF165 neutralization. Mar Drugs 16:113. https://doi.org/10.3390/md16040113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Könning D, Rhiel L, Empting M et al (2017) Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders. Sci Rep 7. https://doi.org/10.1038/s41598-017-10513-9

  26. Macarrón Palacios A, Grzeschik J, Deweid L et al (2020) Specific targeting of lymphoma cells using semisynthetic anti-idiotype shark antibodies. Front Immunol 11. https://doi.org/10.3389/fimmu.2020.560244

  27. Li D, English H, Hong J et al (2022) A novel PD-L1-targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer. Mol Ther Oncolytics 24:849–863. https://doi.org/10.1016/j.omto.2022.02.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ubah OC, Lake EW, Gunaratne GS et al (2021) Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography. Nat Commun 12. https://doi.org/10.1038/s41467-021-27611-y

  29. Gauhar A, Privezentzev CV, Demydchuk M et al (2021) Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro. FASEB J 35. https://doi.org/10.1096/fj.202100986RR

  30. Buschhaus MJ, Becker S, Porter AJ, Barelle CJ (2019) Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents. Protein Eng Des Sel 32:385–399. https://doi.org/10.1093/protein/gzaa002

    Article  CAS  PubMed  Google Scholar 

  31. Leow CH, Fischer K, Leow CY et al (2018) Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library. Malar J 17. https://doi.org/10.1186/s12936-018-2531-y

  32. Könning D, Kolmar H (2018) Beyond antibody engineering: directed evolution of alternative binding scaffolds and enzymes using yeast surface display. Microb Cell Factories 17. https://doi.org/10.1186/s12934-018-0881-3

  33. Pekar L, Klausz K, Busch M et al (2021) Affinity maturation of B7-H6 translates into enhanced NK cell–mediated tumor cell lysis and improved proinflammatory cytokine release of bispecific immunoligands via NKp30 engagement. J Immunol 206:225–236. https://doi.org/10.4049/jimmunol.2001004

    Article  CAS  PubMed  Google Scholar 

  34. Valldorf B, Hinz SC, Russo G et al (2021) Antibody display technologies: selecting the cream of the crop. Biol Chem 0. https://doi.org/10.1515/hsz-2020-0377

  35. Uchański T, Zögg T, Yin J et al (2019) An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep 9. https://doi.org/10.1038/s41598-018-37212-3

  36. Roth L, Krah S, Klemm J et al (2020) Isolation of antigen-specific VHH single-domain antibodies by combining animal immunization with yeast surface display. Methods Mol Biol 2070:173–189. https://doi.org/10.1007/978-1-4939-9853-1_10

    Article  CAS  PubMed  Google Scholar 

  37. Klausz K, Pekar L, Boje AS et al (2022) Multifunctional NK cell–engaging antibodies targeting EGFR and NKp30 elicit efficient tumor cell killing and proinflammatory cytokine release. J Immunol 209:1724–1735. https://doi.org/10.4049/jimmunol.2100970

    Article  CAS  PubMed  Google Scholar 

  38. 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 17:386–392. https://doi.org/10.1007/s10126-015-9642-z

    Article  CAS  Google Scholar 

  39. Pekar L, Klewinghaus D, Arras P et al (2021) Milking the cow: cattle-derived chimeric ultralong CDR-H3 antibodies and their engineered CDR-H3-only Knobbody counterparts targeting epidermal growth factor receptor elicit potent NK cell-mediated cytotoxicity. Front Immunol 12:4378 . https://doi.org/10.3389/fimmu.2021.742418

    Article  CAS  Google Scholar 

  40. Klewinghaus D, Pekar L, Arras P et al (2022) Grabbing the bull by both horns: bovine ultralong CDR-H3 paratopes enable engineering of ‘almost natural’ common light chain bispecific antibodies suitable for effector cell redirection. Front Immunol 12. https://doi.org/10.3389/fimmu.2021.801368

  41. 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. https://doi.org/10.1021/bi026448p

    Article  CAS  PubMed  Google Scholar 

  42. Ewert S, Huber T, Honegger A, Plückthun A (2003) Biophysical properties of human antibody variable domains. J Mol Biol 325:531–553. https://doi.org/10.1016/S0022-2836(02)01237-8

    Article  CAS  PubMed  Google Scholar 

  43. Könning D, Zielonka S, Sellmann C et al (2016) Isolation of a pH-sensitive IgNAR variable domain from a yeast-displayed, histidine-doped master library. Mar Biotechnol 18:161–167. https://doi.org/10.1007/s10126-016-9690-z

    Article  CAS  Google Scholar 

  44. Benatuil L, Perez JM, Belk J, Hsieh C-M (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159. https://doi.org/10.1093/protein/gzq002

    Article  CAS  PubMed  Google Scholar 

  45. Boder ET, Wittrup KD (2000) Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:430–444. https://doi.org/10.1016/s0076-6879(00)28410-3

    Article  CAS  PubMed  Google Scholar 

  46. 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 95:14343–14348. https://doi.org/10.1073/pnas.95.24.14343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany

    Harald Kolmar, Julius Grzeschik & Stefan Zielonka

  2. Antibody-Drug Conjugates and Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany

    Doreen Könning

  3. Antibody Discovery & Protein Engineering, Merck KGaA, Darmstadt, Germany

    Simon Krah & Stefan Zielonka

Authors
  1. Harald Kolmar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julius Grzeschik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Doreen Könning
    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. 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. Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kolmar, H., Grzeschik, J., Könning, D., Krah, S., Zielonka, S. (2023). Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries . In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3381-6_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3380-9

  • Online ISBN: 978-1-0716-3381-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation