Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity

The COVID-19 pandemic, caused by SARS-CoV-2, highlights the need for diverse therapeutic modalities due to the virus's propensity for evolution and immune evasion. Betacoronaviruses, like SARS-CoV-2, pose a significant global health threat because of their high mortality rates and potential for zoonotic spillover. While effective vaccines have been developed, the emergence of SARS-CoV-2 variants of concern (VoCs) necessitates broadly effective monoclonal antibodies. Camelids and cartilaginous fish possess unique antibodies lacking light chains. These antibodies, particularly the nurse shark's IgNAR antibodies, generate small, stable nanobodies (VNARs) with diverse binding capabilities due to their CDR3 recombination process. This research aimed to identify and characterize novel shark-derived nanobodies with potent neutralizing activity against SARS-CoV-2 and related sarbecoviruses.

A significant body of work exists on the development of therapeutics against SARS-CoV-2, including vaccines and monoclonal antibodies. The literature highlights the challenges posed by the emergence of new variants, which often evade neutralization by existing antibodies. Research has focused on identifying broadly neutralizing antibodies that target highly conserved epitopes on the viral spike protein. The use of alternative antibody formats, such as nanobodies from camelids and sharks, has gained traction due to their unique properties, including small size and high stability. Previous studies have demonstrated the potential of these nanobodies to neutralize various viruses.

Nurse sharks were immunized with three different SARS-CoV-2-based immunogens: recombinant receptor-binding domain (RBD), RBD-ferritin (RFN), and spike protein ferritin nanoparticle (SpFN). The immunizations used complete and incomplete Freund's adjuvant. Blood samples were collected, and IgNAR-specific ELISAs were performed to assess antibody responses. Phage display libraries were constructed from the shark peripheral blood lymphocytes (PBLs), and biopanning was used to isolate VNARs with high affinity to SARS-CoV-2 RBD or SpFN. Unique antigen-positive VNARs were expressed as human IgG1 Fc-fusion chimeras (ShAbs). Binding affinity was assessed using biolayer interferometry (BLI). Epitope mapping was conducted using competition BLI assays. Neutralization assays were performed using SARS-CoV-2 and SARS-CoV-1 pseudoviruses. In vivo protection studies were carried out using K18-hACE2 transgenic mice. The crystal structure of the SARS-CoV-2 RBD in complex with ShAb01 and ShAb02 was determined using X-ray crystallography. Alanine scanning mutagenesis was used to identify critical amino acids for binding. Multi-specific molecules were designed and produced using different strategies to combine ShAb01 and ShAb02. Effector functions, including antibody-dependent cellular phagocytosis (ADCP) and NK cell degranulation, were evaluated in cell culture assays. Finally, the breadth of reactivity of the ShAbs was assessed against a panel of sarbecovirus RBDs from clades 1a, 1b, 2, and 3.

Immunization of nurse sharks yielded high-affinity anti-SARS-CoV-2 nanobodies. Two distinct antigenic groups of ShAbs were identified based on their RBD-binding profiles. ShAb01 and ShAb02, representing each group, showed robust binding to SARS-CoV-2 RBD and S-2P with low nanomolar affinity to various SARS-CoV-2 VoCs, including Alpha, Beta, Gamma, and Delta. While ShAb01 had low affinity to Omicron BA.1, BA.2, and BA.2.12.1, ShAb02 maintained high affinity to these variants. ShAb01 potently neutralized SARS-CoV-2 WA-1, Alpha, Beta, Delta, and SARS-CoV-1, whereas ShAb02 neutralized SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1, and BA.4/5. Both ShAbs provided significant protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model, with ShAb01 offering superior protection. Crystal structure analysis revealed non-overlapping epitopes for ShAb01 and ShAb02, with minimal overlap with the ACE2 binding site. ShAb01's epitope resembled Class IV antibodies, while ShAb02's was similar to Class III antibodies. Multi-specific molecules combining ShAb01 and ShAb02 showed significantly enhanced neutralization capacity and picomolar affinity to various SARS-CoV-2 VoCs and sarbecoviruses. These multi-specific molecules maintained robust ACE2-blocking activity and Fc-mediated effector functions (ADCP and NK cell degranulation). Analysis of sarbecovirus RBDs showed that ShAb01 exhibited strong binding to clades 1a and 1b, while ShAb02 primarily bound to clade 1b. The multi-specific antibodies displayed a similar binding pattern to ShAb01, with BiShAb0201 generally exhibiting higher binding than ShAb01H02K.

This study demonstrates the successful generation of highly potent and broadly neutralizing shark-derived nanobodies against SARS-CoV-2 and related sarbecoviruses. The unique binding properties of ShAb01 and ShAb02, targeting non-overlapping epitopes with minimal interference with the ACE2 binding site, contribute to their broad neutralization capacity. The development of multi-specific molecules further enhanced their efficacy, highlighting the potential of structure-based design for optimizing antibody activity. The in vivo protection studies confirm the therapeutic potential of these nanobodies. The combination of potent neutralization, ACE2 blocking activity, and Fc-mediated effector functions makes these molecules promising candidates for the development of next-generation SARS-CoV-2 therapeutics. The broad reactivity against sarbecoviruses suggests their potential utility in preparing for future pandemics.

This research successfully elicited potent, broadly neutralizing shark nanobodies against SARS-CoV-2 and related sarbecoviruses. The design of multi-specific molecules further improved their efficacy. These findings demonstrate the potential of shark-derived nanobodies as promising therapeutics for current and future sarbecovirus outbreaks. Future research could explore the optimization of these nanobodies for various delivery methods and further investigation into their long-term efficacy and safety.

The study primarily focused on preclinical evaluations. Further research is needed to assess the long-term efficacy, safety, and pharmacokinetics of these nanobodies in humans. The K18-hACE2 mouse model, while useful, may not fully recapitulate human disease. The limited number of sarbecovirus RBDs tested might not encompass the full diversity of these viruses.