Nanobodies: a promising approach to treatment of viral diseases

Abstract

Since their discovery in the 1990s, heavy chain antibodies have garnered significant interest in the scientific community. These antibodies, found in camelids such as llamas and alpacas, exhibit distinct characteristics from conventional antibodies due to the absence of a light chain in their structure. Furthermore, they possess a single antigen-binding domain known as VHH or Nanobody (Nb). With a small size of approximately 15 kDa, these Nbs demonstrate improved characteristics compared to conventional antibodies, including greater physicochemical stability and enhanced biodistribution, enabling them to bind inaccessible epitopes more effectively. As a result, Nbs have found numerous applications in various medical and veterinary fields, particularly in diagnostics and therapeutics. Advances in biotechnology have made the production of recombinant antibodies feasible and compatible with large-scale manufacturing. Through the construction of immune phage libraries that display VHHs and subsequent selection through biopanning, it has become possible to isolate specific Nbs targeting pharmaceutical targets of interest, such as viruses. This review describes the processes involved in nanobody production, from hyperimmunization to purification, with the aim of their application in the pharmaceutical industry.

Keywords: VHH; camelids; heavy chain antibodies; immune library; neutralizing antibodies; phage display; single domain antibodies.

Figure 1

Structure of IgG on the left, with representation of the light and heavy chains, as well as the variable and constant domains, with a focus on the CDRs. HCAb structure on the right, with representation of the heavy chain and variable and constant domains. Below, the representation of the Nbs. Source: by the author, 2023. Created with BioRender.com.

Figure 2

Schematic representation of phage display and biopanning processes. (A) Production of recombinant phages displaying antibodies using a phagemid vector and infection with helper phages. (B) Phages with genome modifications infecting cells to produce phages displaying the antibodies. Source: by the author, 2023. Created with BioRender.com.

Figure 3

Representative scheme of the production steps of nanobodies using phage display technology. Source: by the author, 2023. Created with BioRender.com.

Figure 4

Outline of the methodology for writing the bibliographic review on: “Construction of nanobodies library from the immunization of camelids aiming to obtaining biopharmaceuticals”. Source: by the author, 2023.

Figure 5

Keywords most used in selected articles.

Figure 6

Analyzes related to publications of the nanobodies for the treatment of viral diseases. (A) List of publications selected by year. (B) On the left, list of publications between the SARS-CoV-2 virus and the various selected viruses. (C) list of publications between the years 2018 and 2022. (D) List of applications of nanobodies in selected articles. (E) Relationship of applications of nanobodies in different areas. (F) List of antigenic forms used in the camelid hyperimmunization process. (G) Proportion of administration routes used for antigen application. (H) Proportion of animals used in the production process of nanobodies, from the hyperimmunization of camelids.

Figure 7

Analyzes related to publications of the nanobodies methodologies. (A) Proportion of amplification reactions of genetic material used in the production process of nanobodies. (B) List of cloning processes used in the production of Nbs. (C) List of helper phages used in the cloning process with a phagemid vector. (D) List of supports used for displaying antigens in the biopanning process. (E) List of antigen immobilization methods for biopanning. (F) Ligand detection methods used to select Nbs. (G) Cells used in the nanobodies expression process. (H) Most frequent modifications of Nbs in the selected literature.