Abstract
The shark-derived autonomous variable antibody domains known as VNARs are attractive tools for therapeutic and diagnostic applications due to their favorable properties like small size (approximately 12 kDa), high thermal and chemical stability, and good tissue penetration. Currently, different techniques have been reported to generate VNAR domains against targets of therapeutic interest. Here, we describe methods for the preparation of an immune VNAR library based on bacteriophage display, and for the preparation of a synthetic library of VNAR domains using a modified protocol based on Kunkel mutagenesis. Finally, we describe procedures for in silico maturation of a VNAR using a bioinformatic approach to obtain higher affinity binders.
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References
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
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
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
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
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
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
Camacho-Villegas T, Mata-Gonzalez T, Paniagua-Solis J et al (2013) Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark. MAbs 5:80–85
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
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
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 17:1–15
Burgess SG, Oleksy A, Cavazza T et al (2016) Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain. Open Biol 6:160089
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
Dooley H (2003) Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 40:25–33
Shao CY, Secombes CJ, Porter AJ (2007) Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library. Mol Immunol 44:656–665
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
Könning D, Rhiel L, Empting M et al (2017) Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders. Sci Rep 7:9676
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
Millán-Gómez D, Dueñas S, Muñoz PLA et al (2018) In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization. Oncotarget 9:28016–28029
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492
Barbas C, Burton D, Scott J et al (eds) (2001) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Webb B, Sali A (2014) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinform 47:5.6.1–5.6.32
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38
Phillips JC, Braun R, Wang W et al (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26:1781–1802
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
Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7:e52031
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This chapter is dedicated to the memory of Dr. Jorge Gavilondo.
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Gasperin-Bulbarela, J., Cabanillas-Bernal, O., Dueñas, S., Licea-Navarro, A.F. (2022). Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders. 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_4
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DOI: https://doi.org/10.1007/978-1-0716-2075-5_4
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