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Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development

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Single-Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2446))

Abstract

The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a two-decade long commitment in time and research effort to realize the clinical potential of single-domain antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in sharks provides an alternative small binding domain to conventional monoclonal antibodies and their fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposition to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new (neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

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References

  1. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136

    Article  CAS  PubMed  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 1995:168–173

    Article  Google Scholar 

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

    Article  Google Scholar 

  4. Ubah OC, Steven J, Kovaleva M et al (2017) Novel, anti-hTNF-α variable new antigen receptor formats with enhanced neutralizing potency and multifunctionality, generated for therapeutic development. Front Immunol 8:1780

    Article  PubMed  PubMed Central  Google Scholar 

  5. Nuttall SD, Krishnan UV, Hattarki M et al (2001) Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol Immunol 38:313–326

    Article  CAS  PubMed  Google Scholar 

  6. 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 7:1–10

    Article  Google Scholar 

  7. Liu JL, Anderson GP, Delehanty JB et al (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 

  8. Ohtani M, Hikima J, Jung T et al (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 

  9. 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

    Article  CAS  PubMed  Google Scholar 

  10. Crouch K, Smith LE, Williams R et al (2013) Humoral immune response of the small-spotted catshark, Scyliorhinus canicula. Fish Shellfish Immunol 34:1158–1169

    Article  CAS  PubMed  Google Scholar 

  11. Camacho-Villegas T, Mata-Gonzalez T, Paniagua-Solis J et al (2013) Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use. MAbs 5:80–85

    Article  PubMed  PubMed Central  Google Scholar 

  12. Dooley H, Flajnik MF, Porter AJ (2003) Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 40:25–33

    Article  CAS  PubMed  Google Scholar 

  13. Nuttall SD, Krishnan UV, Doughty L et al (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 

  14. Streltsov VA, Varghese JN, Carmichael JA et al (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 

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

    Article  CAS  PubMed  Google Scholar 

  16. 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 8:1121

    Article  PubMed  PubMed Central  Google Scholar 

  17. Fennell B, Darmanin-Sheehan A, Hufton S et al (2010) Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms. J Mol Biol 400:155–170

    Article  CAS  PubMed  Google Scholar 

  18. Streltsov VA, Carmichael JA, Nuttall SD (2015) Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype. Protein Sci 14:2901–2909

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Müller MR, Saunders K, Grace C et al (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 

  22. 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

    Article  CAS  PubMed  Google Scholar 

  23. Cabanillas-Bernal O, Dueñas S, Ayala-Avila M et al (2019) Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3. PLoS One 14:e0213394

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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

    Article  CAS  PubMed  Google Scholar 

  25. Roux KH, Greenberg AS, Greene L et al (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci U S A 95:11804–11809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Liu JL, Zabetakis D, Brown JC et al (2014) Thermal stability and refolding capability of shark derived single domain antibodies. Mol Immunol 59:194–199

    Article  CAS  PubMed  Google Scholar 

  27. Nuttall SD, Krishnan UV, Doughty L et al (2002) A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis. FEBS Lett 516:80–86

    Article  CAS  PubMed  Google Scholar 

  28. Konning D, Rhiel L, Empting M et al (2017) Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders. Sci Rep 7:9676

    Article  PubMed  PubMed Central  Google Scholar 

  29. Müller MR, O’Dwyer R, Kovaleva M et al (2012) Generation and isolation of target-specific single-domain antibodies from shark immune repertoires. Methods Mol Biol 907:177–194

    Article  PubMed  Google Scholar 

  30. Feng M, Bian H, Wu X et al (2019) Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naive nurse sharks. Antib Ther 2:1–11

    PubMed  Google Scholar 

  31. Dooley H, Flajnik MF (2005) Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur J Immunol 35:936–945

    Article  CAS  PubMed  Google Scholar 

  32. Burgess SG, Oleksy A, Cavazza T et al (2016) Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain. Open Biol 6:160089

    Article  PubMed  PubMed Central  Google Scholar 

  33. Goodchild SA, Dooley H, Schoepp RJ et al (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 

  34. 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 17:383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. O’Dwyer R, Kovaleva M, Zhang J et al (2018) Anti-ICOSL new antigen receptor domains inhibit T cell proliferation and reduce the development of inflammation in the collagen-induced mouse model of rheumatoid arthritis. J Immunol Res 2018:4089459

    Article  PubMed  PubMed Central  Google Scholar 

  36. Shao C, Secombes CJ, Porter AJ (2007) Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library. Mol Immunol 44:656–665

    Article  CAS  PubMed  Google Scholar 

  37. Zielonka S (2015) The shark strikes twice: generation of mono-and bispecific high-affinity vNAR antibody domains via step-wise affinity maturation. PhD thesis, Technische Universität Darmstadt. http://tuprints.ulb.tu-darmstadt.de/4481/

  38. Walsh R, Nuttall S, Revill P et al (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 

  39. Nuttall SD, Humberstone KS, Krishnan UV et al (2004) Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. Proteins 55:187–197

    Article  CAS  PubMed  Google Scholar 

  40. Grzeschik J, Könning D, Hinz SC et al (2018) Generation of semi-synthetic shark IgNAR single-domain antibody libraries. Methods Mol Biol 1701:147–167

    Article  CAS  PubMed  Google Scholar 

  41. 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 (NY) 18:161–167

    Article  Google Scholar 

  42. Könning D, Kolmar H (2018) Beyond antibody engineering: directed evolution of alternative binding scaffolds and enzymes using yeast surface display. Microb Cell Fact 17:32

    Article  PubMed  PubMed Central  Google Scholar 

  43. Kopsidas G, Roberts AS, Coia G et al (2006) In vitro improvement of a shark IgNAR antibody by Qβ replicase mutation and ribosome display mimics in vivo affinity maturation. Immunol Lett 107:163–168

    Google Scholar 

  44. Steven J, Müller MR, Carvalho MF et al (2017) In vitro maturation of a humanized shark VNAR domain to improve its biophysical properties to facilitate clinical development. Front Immunol 8:1361

    Google Scholar 

  45. Stocki P, Szary J, Rasmussen CL et al (2021) Blood-brain barrier transport using a high affinity brain-selective VNAR antibody targeting transferring receptor 1. FASEB J 35:e21172

    Article  CAS  PubMed  Google Scholar 

  46. Ubah OC, Steven J, Porter AJ et al (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 10:526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ubah OC, Porter AJ, Barelle CJ (2020) In vitro ELISA and cell-based assays confirm the low immunogenicity of VNAR therapeutic constructs in a mouse model of human RA: an encouraging milestone to further clinical drug development. J Immunol Res 2020:7283239

    Google Scholar 

  48. Kovalenko OV, Olland A, Piché-Nicholas N et al (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 

  49. Griffiths K, Dolezal O, Cao B et al (2016) i-bodies, human single domain antibodies that antagonize chemokine receptor CXCR4. J Biol Chem 291:12641–12657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bojalil R, Mata-González MT, Sánchez-Muñoz F et al (2013) Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock. BMC Immunol 14:1–7

    Article  Google Scholar 

  51. Häsler J, Flajnik MF, Williams G et al (2016) VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry. Mol Immunol 75:28–37

    Article  PubMed  PubMed Central  Google Scholar 

  52. Leow HC, Fischer K, Leow YC et al (2019) Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli. Prep Biochem Biotechnol 49:315–327

    Article  CAS  PubMed  Google Scholar 

  53. 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

    Article  CAS  PubMed  Google Scholar 

  54. Griffiths K, Dolezal O, Parisi K et al (2013) Shark variable new antigen receptor (VNAR) single domain antibody fragments: stability and diagnostic applications. Antibodies 2:66–81

    Article  CAS  Google Scholar 

  55. Pepple KL, Wilson L, Van Gelder RN et al (2019) Uveitis therapy with shark variable novel antigen receptor domains targeting tumor necrosis factor alpha or inducible T-cell costimulatory ligand. Transl Vis Sci Technol 8:11

    Article  PubMed  PubMed Central  Google Scholar 

  56. Camacho-Villegas TA, Mata-González MT, García-Ubbelohd W et al (2018) Intraocular penetration of a vNAR: in vivo and in vitro VEGF165 neutralization. Mar Drugs 16:113

    Article  PubMed Central  Google Scholar 

  57. Matz H, Dooley H (2019) Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents. Dev Comp Immunol 90:100–107

    Article  CAS  PubMed  Google Scholar 

  58. Cotton G, Thom J, Trumper P et al (2020) Exploiting the properties of VNAR domains for the development of novel efficacious protein drug conjugates targeting the oncofetal protein ROR1. In: Proceedings of the annual meeting of the American Association for Cancer Research 2020; 2020 Apr 27–28 and Jun 22–24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl): Abstract nr 538

    Google Scholar 

  59. Leach A, Smyth P, Ferguson L et al (2020) Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature. Nanoscale 12:14751–14763

    Article  CAS  PubMed  Google Scholar 

  60. Jain S, Doshi AS, Iyer AK et al (2013) Multifunctional nanoparticles for targeting cancer and inflammatory diseases. J Drug Target 21:888–903

    Article  CAS  PubMed  Google Scholar 

  61. Buschhaus MJ, Becker S, Porter AJ et al (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

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Elasmogen Limited, Aberdeen, UK

    Samata S. Pandey, Marina Kovaleva, Caroline J. Barelle & Obinna C. Ubah

Authors
  1. Samata S. Pandey
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  2. Marina Kovaleva
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  3. Caroline J. Barelle
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  4. Obinna C. Ubah
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Corresponding author

Correspondence to Obinna C. Ubah .

Editor information

Editors and Affiliations

  1. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Greg Hussack

  2. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Kevin A. Henry

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Pandey, S.S., Kovaleva, M., Barelle, C.J., Ubah, O.C. (2022). Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2075-5_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2074-8

  • Online ISBN: 978-1-0716-2075-5

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