Nanobody-based products as research and diagnostic tools Thomas De Meyer 1, 2 , Serge Muyldermans 3, 4 , and Ann Depicker 1, 2 1 Department of Plant Systems Biology, VIB, 9052 Gent, Belgium 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium 3 Structural Biology Research Center, VIB, 1050 Brussel, Belgium 4 Research Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussel, Belgium Since the serendipitous discovery 20 years ago of bona fide camelid heavy-chain antibodies, their single-domain anti- gen-binding fragments, known as VHHs or nanobodies, have received a progressively growing interest. As a result of the beneficial properties of these stable recombinant entities, they are currently highly valued proteins for mul- tiple applications, including fundamental research, diag- nostics, and therapeutics. Today, with the original patents expiring, even more academic and industrial groups are expected to explore innovative VHH applications. Here, we provide a thorough overview of novel implementations of VHHs as research and diagnostic tools, and of the recently evaluated production platforms for several VHHs and VHH- derived antibody formats. From conventional antibodies to antibody fragments To date, the European Medicines Agency and US FDA have approved 35 monoclonal antibodies (mAbs) for therapeutic applications. Most of these antibodies are chimeric or huma- nised full-length antibodies, whereas only a few are derived, next-generation antibody fragments, such as the 55-kDa fragment antigen-binding (Fab) (Figure 1A). Another anti- body fragment, the 28-kDa single-chain variable fragment (scFv) (Figure 1A), has not yet been approved, but several are being evaluated in clinical trials [1]. This trend towards smaller antigen-binding antibody formats applies to both therapeutic and diagnostic antibodies, because antibody fragments are more amenable to faster engineering and cheaper production, and show enhanced tissue penetration and lower immunogenicity [2]. A distinct type of antibody fragment, termed VHH or nanobody, is derived from heavy-chain-only antibodies that circulate in sera of camelids, such as llamas, drome- daries, and camels (Figure 1B). This small-sized (15 kDa), autonomous VHH domain is readily produced as a highly soluble and robust entity. Its single-domain nature and strict monomeric behaviour support easy cloning, fast selection from immune or naı¨ve VHH libraries (Box 1), and straightforward design into multivalent and pluripo- tent antigen-binding formats. Moreover, because VHHs prefer to associate with concave-shaped epitopes (e.g., catalytic sites of enzymes), they are able to recognise sites that are inaccessible or cryptic for conventional antibodies [3,4]. Although several VHHs have been developed as new magic bullets for therapy and are currently evaluated in phase I and II clinical trials by Ablynx (http://www.ablynx.- com), VHHs have also paved the way for novel, highly valuable applications in diagnostics, protein or cell re- search, and even agriculture (Box 2). VHHs in research VHH GFP-binding protein (GBP) sets the tone A clear breakthrough for VHHs in research was the devel- opment of chromobodies. These molecules comprise a VHH fused to a fluorescent protein and, due to the stability of the VHH, fold into functional antigen-binding entities, often even in the reducing environment of the cytoplasm within living cells. After expression and binding their specific antigen, chromobodies serve as tracers for in vivo intracel- lular target localisation studies (Figure 2A), avoiding the need for genetic modification of target proteins with fluores- cent tags. As a proof-of-concept, an anti-GFP VHH, termed GBP, was fused to monomeric RFP and the resulting GFP– chromobody could specifically label cytoplasmic or nuclear localised GFP fusion proteins [5]. Other chromobodies were developed for the direct visualisation of native, endogenous proteins [6] or HIV virions [7] in living cells, and several of these nanobody-based tracers are made available by Chro- moTek (http://www.chromotek.com). The GBP is also applied in super-resolution microscopy for the visualisation of GFP fusion proteins. When full- length antibodies coupled to organic dyes are used as pri- mary antibodies, linkage errors arise because of the distance between the organic dye and the actual localisation of the protein. Due to the smaller size of VHHs, coupling the dye to the GBP results in improved labelling with minimal linkage error [8]. Similarly, gold nanoparticle-coupled GBP is used for single-molecule tracking of GFP-tagged membrane pro- teins and is even internalised by electroporation to track intracellular proteins in living cells [9]. Recently, a GBP-based fluorescent-three-hybrid ap- proach has been developed to study in vivo protein–protein interactions: GBP is first fixed at a particular subcellular Review 0167-7799/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tib- tech.2014.03.001 Corresponding author: Depicker, A. (anpic@psb.vib-ugent.be). Keywords: nanobody; recombinant protein production; antibody fragment; GFP- binding protein; plant production platform; yeast production platform. Trends in Biotechnology, May 2014, Vol. 32, No. 5 263