Abstract
The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
Roux KH, Greenberg AS, Greene L, Strelets L, Avila D, McKinney EC, Flajnik MF (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci USA 95:11804–11809
Dooley H, Flajnik MF (2006) Antibody repertoire development in cartilaginous fish. Dev Comp Immunol 30:43–56
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
Barelle C, Gill DS, Charlton K (2009) Shark novel antigen receptors―the next generation of biologic therapeutics? Adv Exp Med Biol 655:49–62
Nuttall SD, Krishnan UV, Hattarki M, De Gori R, Irving RA, Hudson PJ (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
Nuttall SD, Krishnan UV, Doughty L, Nathanielsz A, Ally N, Pike RN, Hudson PJ, Kortt AA, Irving RA (2002) A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis. FEBS Lett 516:80–86
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
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
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
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
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 USA 103:1846–1851
Smith LE, Crouch K, Cao W, Müller MR, Wu L, Steven J, Lee M, Liang M, Flajnik MF, Shih HH, Barelle CJ, Paulsen J, Gill DS, Dooley H (2012) Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias). Dev Comp Immunol 36:665–679
Finlay WJ, Cunningham O, Lambert MA, Darmanin-Sheehan A, Liu X, Fennell BJ, Mahon CM, Cummins E, Wade JM, O’Sullivan CM, Tan XY, Piche N, Pittman DD, Paulsen J, Tchistiakova L, Kodangattil S, Gill D, Hufton SE (2009) Affinity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions. J Mol Biol 388:541–558
Acknowledgments
The authors would like to thank Martin Flajnik for access to mouse monoclonal anti-Nurse shark IgNAR antibodies and both Allan Jensen and Davinder Gill for their support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Müller, M.R., O’Dwyer, R., Kovaleva, M., Rudkin, F., Dooley, H., Barelle, C.J. (2012). Generation and Isolation of Target-Specific Single-Domain Antibodies from Shark Immune Repertoires. In: Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 907. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-974-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-61779-974-7_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-973-0
Online ISBN: 978-1-61779-974-7
eBook Packages: Springer Protocols