A recent review describes the role of nanobodies as a new class of recombinant antibodies that are used in the treatment of the coronavirus disease 2019 (COVID-19).

Study: The role of single-domain antibodies (or nanobodies) in SARS-CoV-2 neutralization. Image Credit: Huen Structure Bio / Shutterstock.com

Introduction

To date, there are limited effective treatments available for COVID-19, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of October 20, 2021, SARS-CoV-2 has infected over 242 million and caused over 4.9 million deaths. While vaccination efforts and non-pharmaceutical interventions are helping to control the spread of this virus, emerging SARS-CoV-2 variants are threatening the efficacy of these measures.

Antibody-based therapies are useful to treat mild COVID-19. However, despite their advantages, some of the challenges associated with the use of monoclonal antibodies for treatment include large doses requirements that are typically administered a few grams at a time intravenously.

The production of antibodies is also a lengthy process that is not as economical as alternative treatments. Furthermore, monoclonal antibodies are often unable to target several epitopes simultaneously.

Nanobodies, or camelid antibodies with a single variable domain, present a practical alternative to monoclonal antibody therapy. These antibodies, typically between 13 and 15 kilodaltons (kDa) in size, have also demonstrated high efficiency in neutralizing SARS-CoV-2.

What are nanobodies?

Nanobodies are a relatively new type of recombinant antibody that originate from Camelidae members like dromedaries and camels, as well as llamas and alpacas. As compared to conventional antibodies that mammals naturally produce, nanobodies are only comprised of two heavy chains and a single variable domain (VHH) that acts as the antigen-binding region of the protein.

Some of the advantageous features of nanobodies include higher solubility, small size, greater resistance to denaturation, stability in high temperatures, high/low pH, cost-effective production, high specificity, low immunogenicity, ease of manipulation, and identification of variable epitopes.

Of great therapeutic value are the better tissue penetration and extravasation ability of nanobodies as compared to classical monoclonal antibodies. In particular, the antiviral effects of nanobodies against the SARS-CoV-2 have demonstrated promising results in recent studies.

Nanobodies against SARS-CoV-2

SARS-CoV-2 binds to the host cell's angiotensin-converting enzyme (ACE-2) receptor through its structural spike (s) glycoprotein to enter the cell.

The S protein consists of two subunits, including the S1 and S2 subunits. Whereas the S1 contains the receptor-binding domain (RBD), the S2 subunit induces membrane fusion. Thus, the S protein is a primary target of antibody-mediated neutralization, as antibodies can either block the interaction of S1 and ACE2 or alter the RBD’s structural conformation.

SARS-CoV-2 can be neutralized by the ability of VHH to compete for high affinity with the RBD. Several techniques to identify anti-SARS-CoV-2 nanobodies include llama immunization, phage display of a naive llama nanobody library or humanized synthetic nanobody library, as well as the yeast surface display of synthetic nanobodies.

Several studies have developed nanobodies that are capable of effectively neutralizing both SARS-CoV-2 pseudovirus and the live virus. Because most of the mutations in emerging SARS-CoV-2 variants are also in either of the two S subunits, the affinity for the S protein and neutralization efficacy inhibits the escape of viral mutants. In addition, the nanobodies block the virus via non-specific binding, target conserved epitopes resistant to existing variants of concern (VOCs), or bind to formerly unidentified high-affinity epitopes with strong affinities, which is likely inaccessible to conventional antibodies.

Synthetic nanobodies

While traditional nanobodies are extracted from immunized camelids, highly selective nanobodies, otherwise known as synthetic nanobodies or sybodies, are synthesized quickly. As a result, large libraries of small and stable sybodies are created and can be accessed according to their neutralizing efficiency.

A panel of nanobodies that binds to various epitopes on the SARS-CoV-2 S protein forms a synthetic nanobodies library. This panel includes Class I nanobodies that bind directly to the RBD and compete with the ACE2 receptor on human cell surfaces, as well as Class II nanobodies which are identified as a different binding site, alter the RBD and thus prevent it from recognizing the ACE2 receptor.

Likewise, chimeric nanobodies-Fc are also effective, where the variable area of the nanobody is bonded to the Fc region of the human immunoglobulin.

These synthetic nanobodies are heat stable and can be easily aerosolized. Thus, they are a feasible option for COVID-19 prevention and therapy. Some examples of COVID-19 sybodies that are currently being investigated include SR31 and sybody 23 (Sb23).

With stronger binding affinities and neutralizing activities, these sybodies can also be coupled with monoclonal antibodies or other antibody fragments to reinforce affinity and efficacy.

One major limitation of sybodies is the lack of high binding affinity that is required for therapeutic use. This can be overcome with the use of multivalent or multi-paratopic nanobodies that exploit avidity to improve affinity and effectiveness.

Multivalent nanobodies

Using an in-silico approach, wherein VHHs are fused to Fc domains, multi-specific antibodies with elevated avidity and affinity as well as enhanced S/ACE2 blocking are created. These are multivalent nanobodies or variable domains of heavy-chain Abs developed by knowledge of precise architectures of SARS-CoV-2 epitopes, binding modalities to the S protein of the virus, and fusing the virus to the cell membrane through the S protein.

These multivalent nanobodies counter the rapid mutations of SARS-CoV-2 variants by amplified avidity for the binding domain of ACE2.

The reviewers of the current study presented a range of examples from published studies that showed effective neutralization of both the wild-type SARS-CoV-2 strain and variants. A few examples include the heterodimer nanobody Nb91-Nb3-hFc, several bivalent nanobody cocktails, including WNbFc 2, WNbFc7, WNbFc 15, and WNbFc 36, tri-specific VHH-Fc antibodies, hexavalent VHH-72 nanobody, and three new bispecific nanobodies including Nb15-Fc, Nb22-Fc, and Nb31-Fc.

Nanobodies and inflammation

Inflammation plays an important role in COVID-19 immunopathogenesis and immunological dysregulation. Nanobodies are novel tools that can be adjusted to modulate the inflammatory response in COVID-19 patients.

Nanobodies are better immune-modulators than cytokine-blocking antibodies, better boosters of immune responses from antigen-presenting cells (APCs). They can act as ion channel blocking agents that trigger a pro-inflammatory cascade.

Conclusion

This review concludes that the tiny, stable, and easy-to-make nanobodies have a high therapeutic potential for COVID-19. Most of the developed nanobodies are against the S protein, particularly the RBDs of SARS-CoV-2, thus enabling effective neutralization of the virus. It is evident from numerous studies that nanobodies can neutralize SARS-CoV-2 variants, even if new mutations continue to develop.

Customized nanobodies can also be used to modify the inflammatory responses conducive to the COVID-19 patients’ health. Nanobodies can also be utilized as an inhaler for pulmonary administration to inhibit the infection of the lungs by the virus.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.