The COVID-19 pandemic, caused by SARS-CoV-2, has had devastating global consequences. While vaccines and drugs have reduced transmission, the virus's rapid mutation rate poses ongoing challenges. SARS-CoV-2 infection is initiated by the spike (S) protein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor via its RBD. Blocking this interaction is a key strategy for antiviral therapy. This study aimed to rapidly engineer a potent, broadly neutralizing nanobody against SARS-CoV-2, leveraging the existing framework of a clinically approved nanobody to expedite the process and reduce development risks. Nanobodies, or VHHs, are single-domain antibody fragments with advantages including small size, ease of production, thermostability, solubility, permeability, and low immunogenicity. The existing approved nanobody caplacizumab served as a template for generating a library of mutants screened for RBD-binding and neutralizing activity. This approach offers a significantly faster alternative to traditional antibody discovery methods, which involve animal immunization, are time-consuming, and pose risks to researchers.

Extensive research has focused on understanding SARS-CoV-2's structure and mechanisms of entry into host cells. The spike protein's RBD and its interaction with ACE2 have been identified as crucial targets for antiviral development. Studies have characterized numerous SARS-CoV-2 variants with mutations in the spike protein, leading to increased transmissibility and escape from neutralizing antibodies. While monoclonal antibodies have shown promise, the emergence of variants necessitates the development of broadly neutralizing agents. Nanobodies have emerged as a promising therapeutic class due to their unique properties, including small size and high stability, making them suitable for various therapeutic approaches including inhalation. Several studies have explored the use of nanobodies against SARS-CoV-2, demonstrating their potential for effective neutralization.

The researchers used caplacizumab, a commercially available nanobody, as a template to generate a library of mutants. They engineered the complementarity-determining regions (CDRs) of caplacizumab to target the SARS-CoV-2 RBD. The library was displayed on the phage surface, subjected to three rounds of selection against the RBD, and positive clones were identified via ELISA. The selected nanobodies were then expressed in ExpiCHO cells, and a second round of ELISA-based screening identified nanobodies capable of blocking the interaction between the RBD and human ACE2. The binding affinity of the selected nanobodies was further characterized using ELISA and surface plasmon resonance (SPR). The neutralizing activity was evaluated using pseudovirus and live virus assays in vitro, and in vivo efficacy was tested in K18-hACE2 transgenic mice challenged with ancestral SARS-CoV-2. The epitope of the most promising nanobody (VHH60) was further analyzed using cryo-electron microscopy (cryo-EM) and X-ray crystallography. Multimeric forms of VHH60 were also investigated.

From the engineered library, VHH60 emerged as a potent nanobody exhibiting single-digit nanomolar affinity for the SARS-CoV-2 RBD (2.56 nM). VHH60 effectively competed with ACE2 for RBD binding. In vitro pseudovirus neutralization assays showed that VHH60 effectively neutralized the ancestral strain and several variants including Omicron subvariants (BA.1, BA.2, and BA.3), exhibiting nanomolar IC50 values. Live virus assays using Vero-E6 cells confirmed the potent neutralizing activity of VHH60 against the ancestral SARS-CoV-2 strain (IC50 = 1.87 ± 0.35 nM), demonstrating better efficacy compared to VHH72, a previously reported nanobody. In vivo studies in K18-hACE2 transgenic mice showed that VHH60 significantly suppressed viral load in the lungs and prolonged survival compared to controls, significantly reducing lung damage as evidenced by H&E and Immunofluorescence staining. Cryo-EM and X-ray crystallography revealed the binding epitope of VHH60 on the RBD, involving residues largely conserved across variants, explaining its broad neutralization activity. Additionally, the creation of a trimeric VHH60 construct showed a significant increase in potency, highlighting the potential for further optimization through multimerization. The study demonstrated that VHH60's epitope overlaps significantly with the ACE2 binding site.

This study successfully demonstrated a rapid and effective method for generating broadly neutralizing nanobodies against SARS-CoV-2. The use of a commercially available nanobody as a template significantly reduced development time compared to traditional antibody discovery methods. The high affinity and broad neutralizing activity of VHH60 against multiple variants make it a promising candidate for therapeutic development. The in vivo efficacy data strongly supports its potential for clinical applications. The detailed structural analysis further illuminates the mechanism of action and suggests potential strategies for further optimization, such as multimerization to improve potency. The methodology presented here is readily adaptable for rapidly responding to future viral outbreaks.

VHH60 represents a promising therapeutic nanobody candidate for COVID-19 due to its high affinity, broad neutralizing capacity, and in vivo efficacy. The rapid engineering strategy employed in this study highlights a valuable approach for developing antiviral therapies in response to emerging viral threats. Future research could focus on optimizing VHH60 for clinical development, exploring different formulations and delivery methods, and evaluating its potential in combination with other therapeutics.

The study used a specific mouse model (K18-hACE2 transgenic mice), which may not perfectly mimic human infection. Further studies are needed to validate VHH60's efficacy in other animal models and ultimately in humans. While VHH60 showed broad neutralization against several variants, the emergence of new variants necessitates ongoing monitoring and potential adaptations. The current study focused on neutralizing activity; further investigation into other potential mechanisms of action or effects of VHH60 is warranted.