NANOBODIES®: A Review of Diagnostic and Therapeutic Applications

Abstract

NANOBODY® (a registered trademark of Ablynx N.V) molecules (Nbs), also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, nanobodies possess the potential to overcome several of the limitations of conventional monoclonal antibodies. For many years, nanobodies have been of great interest in a wide variety of research fields, particularly in the diagnosis and treatment of diseases. This culminated in the approval of the world's first nanobody based drug (Caplacizumab) in 2018 with others following soon thereafter. This review will provide an overview, with examples, of (i) the structure and advantages of nanobodies compared to conventional monoclonal antibodies, (ii) methods used to generate and produce antigen-specific nanobodies, (iii) applications for diagnostics, and (iv) ongoing clinical trials for nanobody therapeutics as well as promising candidates for clinical development.

Keywords: diagnostics; therapeutics; NANOBODY®; NANOBODY® generation; NANOBODY® production.

Figure 1

(A) Diagrammatic representation of the structure of conventional m(IgG)Ab, HCAb, and VHH (Nb), (B) an outline of the unique characteristics found only in Nbs, and (C) an outline of the advantages of Nbs compared to other mAb formats.

Figure 2

Schematic representation of the conventional method of generating antigen-specific Nbs using a phage display library. For an immune library, the camelid is inoculated with the antigen of interest, but no inoculation occurs for the generation of a naïve library. Blood is then collected, lymphocytes extracted, mRNA extracted, and a Nb sequence library is constructed via two rounds of PCR and a gel electrophoresis to select for the smaller Nb sequences. In the case of a synthetic library, a suitable Nb scaffold is selected, and the CDR are varied through amino acid randomization. The library is inserted into phage vectors and transformed into E. coli cells, for the production of phages containing the Nb nucleotide sequence and displaying the Nb on the outside. Multiple rounds of biopanning are then carried out to isolate the highest affinity binding phages, which are then used to reinfect E. coli cells for the creation of more phages with the high-affinity Nb. These phages are then validated using phage-ELISA; and Nb candidates can then be sequenced, have their affinity measured, and be used to create glycerol stock for future production [7,8,63,65,69,70,71].

Figure 3

Schematic representation of the conventional protocol for the laboratory scale production of Nbs starting from glycerol stock (frozen Nb expressing E. coli cells). LB medium (supplemented with glucose, ampicillin, and MgCl₂) is inoculated with the glycerol stock and incubated overnight. This LB medium is used to inoculate a TB medium (supplemented with glucose, ampicillin, and MgCl₂) and incubated for 3–4 h. Nb expression is induced via the addition of IPTG and incubated overnight. After each incubation, the absorbance is measured to ensure that cell multiplication (absorbance limit 0.6–0.8) and Nb expression (absorbance limit 2.0–3.0) occurred satisfactorily. The cell pellet of the TB medium is then aggregated through multiple rounds of centrifugation at 4000 RPM (using a tabletop centrifuge with TB medium in 50 mL falcon tubes) for 20 min each round. The aggregated cell pellet then undergoes periplasm lysis through the addition of a recommended quantity of TES buffer followed by the addition of TES/4 buffer. The periplasmic extract (supernatant) is then harvested through a round of centrifugation at 4000 RPM for 60 min using a tabletop centrifuge with TB medium in 50 mL falcon tubes) and the pellet is discarded. The periplasmic extract is then purified through IMAC and the Nb is eluted through the addition of imidazole. It is also possible to add a further purification step by size exclusion chromatography. The Nb then undergoes quality control via SDS-PAGE, Coomassie blue staining, western blotting, and differential scanning fluorimetry [71,78].

Figure 4

Flowchart representation of Nb use for diagnostics. Yellow represents the format in which Nbs are applied. Blue represents the method/device in which these Nb formats are used. Green represents the type of target (biomarker/protein/toxin/pathogen/molecule) for each of the assay formats.