Nanobodies to the rescue: The new answer to viral infections

The COVID-19 pandemic was an extraordinary time. The pandemic saw scientific boundaries pushed to their limits, as research teams across the world raced to gather as much knowledge as they could to produce their best attempt at stopping the virus in its tracks. During the pandemic, the main thing you usually heard on the news was about the race to create a vaccine. While this is a very good preventative measure for viruses such as SARS-CoV-2, it wasn’t completely effective. Mutations in the virus led to new variants resulting in vaccines, especially those based on earlier variants, becoming less effective over time. This made effective antiviral treatments more desirable, with therapies involving human monoclonal antibodies seeing major progress.

Human antibodies are made up of two protein chains, the heavy and the light chain. The epitope binding region (paratope) is spread across both the heavy and light chains on their variable domains. In addition to the conventional, camelids can produce a single heavy-chain variant that doesn’t have a light chain. The paratope of the heavy chain only antibody is confined to the 130-residue-variable domain (VHH)(1).

Figure 1. From reference (1), Nanobodies, (a) a conventional antibody, the complementarity determining regions (CDRs) (yellow) are formed by both the heavy (wine) and the light (green) chains. (b) Camelid antibodies only have a heavy chain; thus, the CDRs (yellow tips) are located within one domain (red). The isolated red domain is known as the nanobody.

Typically, nanobodies were derived from immunisation of a llama with a small amount of the desired purified protein injected into the animal, however more recently there has been a rise in research groups using animal-free methods of nanobody generation. This is done through screening a commercial naïve library to identify binders, by looking at low affinity levels (KD in the nM to µM range) and then optimised by methods such as PCR mutagenesis and error prone PCR. Nanobodies can then be engineered in a straightforward manner and heterologously expressed in bacteria, yeast and human cells (1).

The small size and stability of nanobodies gives it an advantage over regular antibodies in terms of greater tissue penetration and has a greater potential as inhaled biotherapeutics. There have been multiple studies in which nanobodies have been used as a therapeutic treatment as well as proactively, both showing significant signs of success (2, 3).

When researching nanobodies to test neutralisation against SARS-CoV-2, researchers go through a series of steps to identify, test and structurally characterise each nanobody that they see as having neutralisation capabilities. The library can be constructed via llama immunisation of a purified protein, which in this case would be the S protein of a SARS-CoV-2 variant, and then analysed using an ELISA to identify binding abilities of SARS-CoV-2 to human cells. Neutralisation of SARS-CoV-2 is achieved by targeting antibodies to the spike (S) protein’s receptor binding domain (RBD), the RBD then engages with the angiotensin-converting enzyme (ACE-2) to facilitate cell entry (2, 4). This is a very important process taken into account during testing for nanobodies. Epitope mapping is then undertaken followed by preclinical evaluation which can be done through in vivo and in vitro studies. The Syrian golden Hamster model is widely used as a model of COVID-19. When the model contracts the disease, it loses weight based on severity of infection, a clear indicator of worsening conditions (2, 4).

References

1)      Tang, Q., Owens, R. J., & Naismith, J. H. (2021). Structural Biology of Nanobodies against the Spike Protein of SARS-CoV-2. Viruses, 13(11), 2214. https://doi.org/10.3390/v13112214

2)      Cornish K et al. 2024 Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2. Open Biol. 14: 230252. https://doi.org/10.1098/rsob.230252

3)      Palomo C, Mas V, Detalle L, Depla E, Cano O, Vázquez M, Stortelers C, Melero JA. Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection. Antimicrob Agents Chemother. 2016 Oct 21;60(11):6498-6509.

doi: 10.1128/AAC.00842-16. PMID: 27550346; PMCID: PMC5075053.

4)      Stefan, Maxwell A., Yooli K. Light, Jennifer L. Schwedler, Peter R. McIlroy, Colleen M. Courtney, Edwin A. Saada, Christine E. Thatcher, et al. 2021. “Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies.” mAbs 13 (1). doi:10.1080/19420862.2021.1958663.