Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

Abstract

Owing to peculiar properties of nanobody, including nanoscale size, robust structure, stable and soluble behaviors in aqueous solution, reversible refolding, high affinity and specificity for only one cognate target, superior cryptic cleft accessibility, and deep tissue penetration, as well as a sustainable source, it has been an ideal research tool for the development of sophisticated nanobiotechnologies. Currently, the nanobody has been evolved into versatile research and application tool kits for diverse biomedical and biotechnology applications. Various nanobody-derived formats, including the nanobody itself, the radionuclide or fluorescent-labeled nanobodies, nanobody homo- or heteromultimers, nanobody-coated nanoparticles, and nanobody-displayed bacteriophages, have been successfully demonstrated as powerful nanobiotechnological tool kits for basic biomedical research, targeting drug delivery and therapy, disease diagnosis, bioimaging, and agricultural and plant protection. These applications indicate a special advantage of these nanobody-derived technologies, already surpassing the "me-too" products of other equivalent binders, such as the full-length antibodies, single-chain variable fragments, antigen-binding fragments, targeting peptides, and DNA-based aptamers. In this review, we summarize the current state of the art in nanobody research, focusing on the nanobody structural features, nanobody production approach, nanobody-derived nanobiotechnology tool kits, and the potentially diverse applications in biomedicine and biotechnology. The future trends, challenges, and limitations of the nanobody-derived nanobiotechnology tool kits are also discussed.

Keywords: HCAbs; IgNAR; V-NAR; VHH; nanobiotechnology; nanobody.

Figure 1

Schematic representations of intact antibodies, including canonical antibodies (IgG1) and heavy-chain antibodies in camels (HCAbs) and sharks (IgNARs), and intact antibody-derived fragments. Notes: (A) Intact canonical antibodies (IgG1) comprising two light chains (VL and CL domains) and two heavy chains (comprising VH, CH1, hinge, and CH2 and CH3 domains). (B) Canonical antibody-derived fragments: Fabs, scFvs, and VH domains. (C) The structure and packing of VH domain from canonical antibodies IgG1. (D) Intact HCAbs in camelids, comprising homodimeric heavy chains (containing VHH and CH2 and CH3 domains), devoid of light chains in intact antibodies and lack of CH1 domains in heavy chains. (E) Camel HCAb-derived single-domain antibodies: VHH. (F) The structure and packing of VHH from camel HCAbs. (G) Intact IgNARs in sharks, comprising homodimeric heavy chains (containing V-NAR and C1–C5 domains), devoid of light chains in antibodies. (H) Shark IgNAR-derived single-domain antibodies: V-NAR. (I) The structure and packing of V-NAR from shark IgNARs. Abbreviations: CDRs, complementarity-determining regions; CH, constant heavy chain; CL, constant light chain; Fabs, antigen-binding fragments; HCAbs, heavy-chain-only antibodies; HV, hypervariable; Ig, immunoglobulin; IgNARs, Ig new antigen receptors; scFvs, single-chain variable fragments; VH, variable heavy chain; VHH, variable domain of HCAbs in camelids; VL, variable light chain; V-NAR, variable domain of IgNARs in sharks.

Figure 2

Schematic of versatile nanobody-derived nanobiotechnological tool kits containing nanobody itself (A), radionuclide-labeled (B) or fluorescent dye-labeled nanobodies (C), fluorescent protein fusion nanobodies (D), chromogenic enzyme fusion nanobodies (E), bivalent nanobodies (F), self-assembly motif-mediated nanobody homo- or heteromultimers (G), nanobody-coated nanoparticles (H), and nanobody-displayed phages (I and J). Note: These nanobiotools have been successfully applied to a variety of biomedical applications.–,,,,– Abbreviations: NPs, nanoparticles; VHH, variable domain of heavy-chain-only antibodies in camelids; V-NAR, variable domain of immunoglobulin new antigen receptors in sharks.

Figure 3

Some challenging structures elucidated using nanobodies as crystallization chaperones. Notes: (A) Nanobody Nb80 (red), as a structural mimic of GαS, stabilizes the active-state conformation of β2-adrenoreceptor (green)., (B) Ribbon representation of the full-length human prion protein (HuPrP, green) in complex with nanobody Nb484 (red). (C) A periplasmic N-terminal domain of GspD (green or cyan) from the type 2 secretion system secretin in complex with nanobody Nb7 (red), forming a compact GspD:Nb7 heterotetramer., (D) Nanobody Nb11 (red)-aided structure determination of EpsI (cyan):EpsJ (green) pseudopilin heterodimer, a component of the bacterial type 2 secretion system in Vibrio vulnificus. Protein Data Bank accession numbers are given in parentheses.