Nanobodies as novel agents for disease diagnosis and therapy

Abstract

The discovery of naturally occurring, heavy-chain only antibodies in Camelidae, and their further development into small recombinant nanobodies, presents attractive alternatives in drug delivery and imaging. Easily expressed in microorganisms and amenable to engineering, nanobody derivatives are soluble, stable, versatile, and have unique refolding capacities, reduced aggregation tendencies, and high-target binding capabilities. This review outlines the current state of the art in nanobodies, focusing on their structural features and properties, production, technology, and the potential for modulating immune functions and for targeting tumors, toxins, and microbes.

Keywords: antibody expression; formatting; heavy chain antibodies; molecular display; nanobodies.

Figure 1

Nb research trends (2004–2012) placing emphasis on original works on Camelidae HCAbs retrieved from the Web of Science® database (Thomson Reuters, Philadelphia, PA, USA). Notes: The clustering into scientific fields is based on the science overlay mapping approach,– which uses the subject categories that the Web of Science® assigns to journals: for a set of publications indexed by the Web of Science®; Nbs are located via the journals in which they appear. The assignment of each paper into a scientific field is determined by the classification of (i) the cited references in the paper and (ii) the citations that the paper received. Abbreviations: Nb, nanobody; HCAbs, heavy-chain antibodies.

Figure 2

The scientific roadmap of university-derived advancements in Camelidae HCAbs. Notes: Since their discovery in 1989, research groups moved fast to integrate Nbs in the well-established scientific frame of Ab-based biomedical tools. Although a clear distinction between the exploration and the exploitation phase cannot be made, and since even the latest papers are, to some extent, concerned with elucidation of mechanisms and physical chemistry, while the applicability domain was clearly defined in the introductory paper of 1993, a turning point can be found around 1998. As the spinoff, Ablynx, was moving to patenting and clinical testing at the early 2000s, the academic research groups were feeding a knowledge push mechanism until 2008, offering relations between properties and potential (until 2004), engineering trends (until 2006) and technology optimizations (until 2008). More researchers were attracted to join and extend the scientific network. Thereafter, the transition to a market pull mechanism becomes apparent, justifying Nbs as viable alternatives to Ab fragments, while a clear therapeutic advantage remains to be clinically proven. Superscripted numbers refer to published papers that document either a Nb asset or a stated intended use. Abbreviations: Ab, antibody; HC, heavy chain; Nb, nanobody; SPR, surface plasmon resonance; PEGylation, polyethylene glycol treatment; H, heavy; sd, single-domain; NMR, nuclear magnetic resonance; ER, endoplasmic reticulum.

Figure 3

Schematic representations of intact IgG1, fragmentation products, engineered fragments and minibodies, IgG2 and IgG3, and the VHH domain or nanobody. Notes: Schematic representations of (A) intact IgG1; (B) fragmentation products; (C) engineered fragments like diabodies, formed by interlinking two scFv molecules via a short peptide, and minibodies with a diabody structure equipped with an additional CH3 domain; (D) IgG2 and IgG3 HCAbs in the sera of camelids; and (E) the VHH domain or nanobody, which can be easily engineered to bispecific formats or conjugated to the VTB, which self assembles into a homopentamer. Abbreviations: VH, variable heavy chain; CH, constant heavy chain; VL, variable light chain; CL, constant light chain; Fab, antigen-binding fragment; Fc, crystallizable fragment; scFV, single-chain variable fragment; VHH, single-variable domain; Ig, immunoglobulin; VTB, B-subunit of Escherichia coli verotoxin; HCAbs, heavy-chain antibodies.