Skip to main content
SpringerLink
Log in

Design and Construction of Antibody Fusion Proteins Incorporating Variable New Antigen Receptor (VNAR) Domains

  • Protocol
  • First Online:
Therapeutic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2720))

  • 813 Accesses

Abstract

Antibody therapeutics have become a cornerstone of the pharmaceutical market due to their precise molecular targeting, favorable pharmacokinetic properties, and multitiered mechanisms of action. Since the first monoclonal antibody was clinically approved 35 years ago, there have been considerable advances in antibody technology. A major breakthrough has been the design of multispecific antibodies and antibody fusion proteins, which introduce the possibility of recognizing two or more targets with a single molecule. However, despite tremendous progress in the antibody engineering field, challenges in formulation, stability, and tissue penetration necessitate the design of novel antibody formats with improved pharmaceutical properties. There is a growing interest in development of single-domain antibodies, which harbor advantages such as high solubility, robust thermostability, and unique geometries that allow for access to cryptic epitopes. Chondrichthyes such as sharks and rays provide a source of single-domain antibody fragments known as variable new antigen receptors (VNARs), which have been exploited as molecular targeting agents. Here, we describe methods to engineer antibody fusion proteins that incorporate VNARs. We present multiple fusion topologies, and detail the design, expression, and purification for each format. These novel antibody fusion proteins hold great promise for a range of applications in biomedical research and therapeutic design.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
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. Leavy O (2010) Therapeutic antibodies: past, present and future. Nat Rev Immunol 10:297–297. https://doi.org/10.1038/nri2763

    Article  CAS  PubMed  Google Scholar 

  2. Liu JKH (2014) The history of monoclonal antibody development – progress, remaining challenges and future innovations. Ann Med Surg 3:113–116. https://doi.org/10.1016/j.amsu.2014.09.001

    Article  Google Scholar 

  3. Cheong WS, Leow CY, Abdul Majeed AB, Leow CH (2020) Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody. Int J Biol Macromol 147:369–375. https://doi.org/10.1016/j.ijbiomac.2020.01.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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 101:12444–12449. https://doi.org/10.1073/pnas.0403509101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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 

  6. Liu JL, Anderson GP, Delehanty JB et al (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library. Mol Immunol 44:1775–1783. https://doi.org/10.1016/j.molimm.2006.07.299

    Article  CAS  PubMed  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. https://doi.org/10.1126/science.1101148

    Article  CAS  PubMed  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. Protein Sci Publ Protein Soc 14:2901–2909. https://doi.org/10.1110/ps.051709505

    Article  CAS  Google Scholar 

  9. 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. https://doi.org/10.1016/j.jmb.2006.12.045

    Article  CAS  PubMed  Google Scholar 

  10. Matz H, Dooley H (2019) Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents. Dev Comp Immunol 90:100–107. https://doi.org/10.1016/j.dci.2018.09.007

    Article  CAS  PubMed  Google Scholar 

  11. 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. https://doi.org/10.4161/mabs.22242

    Article  PubMed  PubMed Central  Google Scholar 

  12. 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. https://doi.org/10.1016/s0161-5890(01)00057-8

    Article  CAS  PubMed  Google Scholar 

  13. 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. https://doi.org/10.1016/s0161-5890(03)00084-1

    Article  CAS  PubMed  Google Scholar 

  14. 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 naïve nurse sharks. Antib Ther 2:1–11. https://doi.org/10.1093/abt/tby011

    Article  CAS  PubMed  Google Scholar 

  15. 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. https://doi.org/10.3389/fimmu.2017.01121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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. https://doi.org/10.1016/j.molimm.2016.05.009

    Article  PubMed  PubMed Central  Google Scholar 

  17. 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 10:526. https://doi.org/10.3389/fimmu.2019.00526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Seth D. Ludwig, Angela Zhu & Jamie B. Spangler

  2. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Ankith P. Maremanda

  3. Department of Microbiology and Immunology, Institute of Marine and Environmental Technology (IMET), Baltimore, MD, USA

    Helen M. Dooley

  4. University of Maryland School of Medicine, Baltimore, MD, USA

    Helen M. Dooley

  5. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

  6. Translational Tissue Engineering Center Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

  7. Department of Oncology, Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

  8. Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

  9. Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

  10. Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA

    Jamie B. Spangler

Authors
  1. Seth D. Ludwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angela Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ankith P. Maremanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Helen M. Dooley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jamie B. Spangler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jamie B. Spangler .

Editor information

Editors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Millicent O. Sullivan

  2. Department of Molecular Genetics and Microbiology School of Medicine, University of New Mexico, Albuquerque, NM, USA

    Bryce Chackerian

  3. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Wilfred Chen

Rights and permissions

Reprints and permissions

Copyright information

© 2024 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

Ludwig, S.D., Zhu, A., Maremanda, A.P., Dooley, H.M., Spangler, J.B. (2024). Design and Construction of Antibody Fusion Proteins Incorporating Variable New Antigen Receptor (VNAR) Domains. In: Sullivan, M.O., Chackerian, B., Chen, W. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 2720. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3469-1_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3469-1_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3468-4

  • Online ISBN: 978-1-0716-3469-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation