Review
Nanobody-based products as research and diagnostic tools

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Highlights

  • New production platforms for VHH and VHH-Fc antibodies were recently explored.

  • VHHs have distinct properties and often outperform conventional antibodies.

  • Several VHHs for protein purification and localisation are commercially available.

Since the serendipitous discovery 20 years ago of bona fide camelid heavy-chain antibodies, their single-domain antigen-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 multiple applications, including fundamental research, diagnostics, 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.

Section snippets

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 humanised 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 antibody fragment, the 28-kDa single-chain variable fragment (scFv) (Figure 1A), has not yet been approved, but several are being evaluated in clinical trials

VHH GFP-binding protein (GBP) sets the tone

A clear breakthrough for VHHs in research was the development 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 intracellular target localisation studies (Figure 2A), avoiding the need for genetic modification of

Implementation of VHHs as sensitive detection probes

Detection probes should ideally meet most of the following characteristics: high probe accessibility, stability and selectivity towards the antigen, even in complex samples, and cost-effective large-scale production [28]. Conversely, probe accessibility is determined by probe size. When small antibody fragments are coated on adsorptive plates, the vicinity of the adsorbing surface might hinder the antigen–probe interaction [29]. However, probe accessibility is also dependent on a uniform,

Production of VHHs and VHH-derived formats

Evidently, large quantities of VHHs are required for some of these applications. To meet these demands, VHHs and VHH-based proteins have been expressed in bacteria, fungi, mammalian cell lines, plants, and insects.

Concluding remarks

The peculiar properties of VHHs have stimulated their introduction in various applications: their high stability allows oral administration and in vivo immunomodulation; their distinct paratope enables recognition of epitopes inaccessible for full-length antibodies; and their small size make them better diagnostic detection probes. However, besides VHHs, non-immunoglobulin protein scaffolds are also considered highly potent binders for diverse applications [78]. Hence, we believe VHHs will

Acknowledgements

The authors thank Annick Bleys for help in preparing the manuscript. T.D.M. is indebted to the Agency for Innovation by Science and Technology (IWT) for a predoctoral fellowship.

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