Scientists around the world are working to understand the various aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from its infection mechanisms to its molecular engagements. In-depth knowledge of the virological dynamics of SARS-CoV-2 will be crucial in developing effective vaccines and therapeutics to combat the ongoing coronavirus disease (COVID-19) pandemic in the long term.

The first step by which SARS-CoV-2 infects its host is by establishing an interaction between the receptor-binding domain (RBD), present on the Spike (S) protein of the virus, and the human receptor, namely, angiotensin-converting enzyme 2 (ACE2). Several neutralizing antibodies target this event, such that the RBD cannot engage with ACE2. However, mutations that have occurred at these sites may threaten the current vaccines and therapeutics that have taken aim at these antigenic sites.

At present, the SARS-CoV-2 Delta strain is the dominant strain circulating globally, possessing both the mutations seen in the UK and South African variants. The extent to which this variant has challenged the available vaccines is not yet clear. The substantial time lost between an outbreak caused by new mutants and the development of vaccines/monoclonal antibodies (mAbs) against these mutants shows the importance of identifying new antibodies with broad reactivity.

Study: Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism. Image Credit: Huen Structure Bio / Shutterstock

What are nanobodies?

Typically, antibodies contain two heavy chains and two light chains that are joined to form a “Y” shaped structure. The variable region of the heavy-chain antibodies is known as nanobodies due to their small size, i.e., ~14 kDa. These small, single-chained structures can target antigens, such as the SARS-CoV-2 S protein, with comparable selectivity and affinity to conventional antibodies. Owing to their small size, nanobodies are extremely stable and can be easily synthesized in large quantities using microbes, cost-effectively.

As they are amenable to protein engineering, for example, fusion in various forms, the newly modified structure may possess much improved binding affinity and high neutralizing activity. Protein engineers can easily fuse nanobodies that recognize non-competing epitopes to produce biparatopic nanobodies that have shown high tolerance to escape mutant strains. Another interesting property of nobodies is that they are heat stable.

This character increases their possibility of being used as inhaling drugs for respiratory diseases. Further, nanobodies possess other useful advantages, i.e., they offer hassle-free storage and transportation. Recently, several neutralizing nanobodies against SARS-CoV-2 have been reported.

There are many similarities and differences between antibodies and nanobodies. Besides the difference in their sizes, they have different modes of action. For example, the minute size of the nanobodies enables more efficient binding to the receptor surfaces, which might not be accessible for conventional antibodies. Additionally, similar to several conventional antibodies, nanobodies can disrupt S trimer post binding.

Isolation and characterization of nanobodies against SARS-CoV-2 variants

A new study has been published on the bioRxiv* preprint server, which has identified and characterized two RBD-targeting neutralizing nanobodies, namely, DL4 and DL28. These nanobodies were isolated from immunized alpaca, a South American camelid mammal. This study has revealed that RBM-antibodies tend to escape mutants and has discovered nanobodies that can be used to develop potent therapeutics against SARS-CoV-2 variants.

A biolayer interferometry assay revealed that both DL4 and DL28 bind to the S protein firmly at the receptor-binding motif (RBM) of the RBD with sub-picomolar affinities. This indicated that nanobodies could bind with antigens with high affinity, which is comparable with Fab (almost four times larger in size). DL4 was found to neutralizes the Alpha strain. However, such activity was not seen against the Beta variant. DL28 could neutralize both the Alpha and Beta variants.

The neutralizing mechanisms of DL4 and DL28 are different. In the case of DL28, it identifies RBD at a region adjacent to RBM, distorts the RBM, and reduces the ACE2-binding. The study of the mechanism of DL28 has shown that the affinity of nanobodies is dependent on their shape, i.e., high affinity is observed when the shape of the nanobody is complementary to the antigen.

Even though, typically, avidity is known to increase potency by enhancing the binding affinity, in the case of DL4 and DL28 low avidity does not limit the binding affinity. This is because of the fusion with Fc, which might have initiated an additional steric hindrance to block RBD-ACE2 binding. Thereby, excellent binding kinetics of these nanobodies has been observed.

Further, an in vivo experiment from previous research revealed that Fc fusion increases the potency of nanobodies by extending the serum half-life from several minutes to several days. Despite having comparable binding kinetics, DL28 had shown less neutralizing activity (~5 fold) than DL4.

Development of biparatopic nanobodies

The study authors further explained the production of biparatopic nanobodies. The main mechanism behind biparatopic nanobodies is that DL28’s epitope slightly overlaps with the RBM such that DL28 can bind to the RBD in the presence of other RBM-targeting nanobodies and human monoclonal antibodies.

Hence, such pairing offers the development of biparatopic nanobodies, which have superior tolerance to escape mutants. The present research has opened new avenues for the development of therapeutic nanobodies against SARS-CoV-2.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.