Abstract
Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.
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References
Hamers-Casterman C, Atarhouch T, Muyldermans S et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448
Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26:230–235
Ghahroudi AM, Desmyter A, Wyns L et al (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526
Conrath KE, Lauwereys M, Galleni M et al (2001) Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother 45:2807–2812
Hmila I, Ben Abderrazek R, Saerens D et al (2008) VHH, bivalent domains and chimeric heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’. Mol Immunol 45:3847–3856
Saerens D, Hassanzadeh Gh, Muyldermans S (2008) Single domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 8:600–608
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Saerens D, Stijlemans B, Baral TN et al (2008) Parallel selection of multiple anti-infectome nanobodies without access to purified antigens. J Immunol Methods 329:138–150
Koch-Nolte F, Reyelt J, Schossow B et al (2007) Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J 21:3490–3498
Lauwereys M, Ghahroudi MA, Desmyter A et al (1998) Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17:3512–3520
Hmila I, Saerens D, Ben-Abderrazek R et al (2010) Effective neutralisation of whole scorpion venom by a bispecific nanobody. FASEB J 24:3479–3489
Cortez-Retamozo V, Backmann N, Senter PD et al (2004) Efficient cancer therapy with a nanobody-based conjugate. Cancer Res 64:2853–2857
Baral TN, Magez S, Stijlemans B et al (2006) Experimental therapy of African trypanosomiases with a nanobody-conjugated human trypanolytic factor. Nat Med 12:580–584
Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing of antigens in living cells. Nat Methods 3:887–889
Abbady AQ, Al-Marir A, Zarkawi M et al (2011) Evaluation of nanobody phage display library constructed from Brucella-immunized camel preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. Vet Immunol Immunopathol 142:49–52
Acknowledgements
This work was supported by VIB, the OZR funding of Vrije Universiteit Brussel and AFFINOMICS, the 7th framework programme of the EC-contract 241481.
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Vincke, C., Gutiérrez, C., Wernery, U., Devoogdt, N., Hassanzadeh-Ghassabeh, G., Muyldermans, S. (2012). Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs. 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_8
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DOI: https://doi.org/10.1007/978-1-61779-974-7_8
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