Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

Single-Domain Antibodies as Therapeutics for Respiratory RNA Virus Infections.

Over the years, infectious diseases with high morbidity and mortality disrupted human healthcare systems and devastated economies globally. Respiratory viruses, especially emerging or re-emerging RNA viruses, including influenza and human coronavirus, are the main pathogens of acute respiratory diseases that cause epidemics or even global pandemics. Importantly, due to the rapid mutation of viruses, there are few effective drugs and vaccines for the treatment and prevention of these RNA virus infections. Of note, a class of antibodies derived from camelid and shark, named nanobody or single-domain antibody (sdAb), was characterized by smaller size, lower production costs, more accessible binding epitopes, and inhalable properties, which have advantages in the treatment of respiratory diseases compared to conventional antibodies. Currently, a number of sdAbs have been developed against various respiratory RNA viruses and demonstrated potent therapeutic efficacy in mouse models. Here, we review the current status of the development of antiviral sdAb and discuss their potential as therapeutics for respiratory RNA viral diseases.

Nanobodies: from structure to applications in non-injectable and bispecific biotherapeutic development.

The increasing demand for convenient, miniaturized and multifunctional antibodies necessitates the development of novel antigen-recognition molecules for biological and medical studies. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, as a new tool, complement the conventional antibodies and are in the stage of rapid development. The outstanding advantages of nanobodies include a stable structure, easy production, excellent water solubility, high affinity toward antigens and low immunogenicity. With promising application potential, nanobodies are now increasingly applied to various studies, including protein structure analysis, microscopic imaging, medical diagnosis, and drug development. The approval of the first nanobody drug Caplacizumab by the FDA disclosed the therapeutic potential of nanobodies. The outbreak of COVID-19 accelerated the development of nanobody drugs in non-injectable and bispecific biotherapeutic applications. Herein, we reviewed recent studies on the nanobody structure, screening and their applications in protein structure analysis and nanobody drugs, especially on non-injectable nanobody and bispecific nanobody development.

Structural basis of nanobodies neutralizing SARS-CoV-2 variants.

Because of the evolutionary variants of SARS-CoV-2, development of broad-spectrum neutralizing antibodies resilient to virus escape is urgently needed. We identified a group of high-affinity nanobodies from camels immunized with receptor-binding domain (RBD) of SARS-CoV-2 spike protein and resolved the structures of two non-competing nanobodies (NB1A7 and NB1B11) in complex with RBD using X-ray crystallography. The structures show that NB1A7 targets the highly conserved cryptic epitope shared by SARS-CoV-2 variants and some other coronaviruses and blocks ACE2 receptor attachment of the spike protein, and NB1B11 epitope overlaps with the contacting surface of ACE2 and is different from the binding site of NB1A7. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, which significantly improved the avidity and neutralization potency and may further inhibit viral escape. The results contribute to the structure-guided design of antibodies against future variants of SARS-CoV-2 virus to combat coronavirus epidemics and pandemics.

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering.

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

A Hybrid Soluble gp130/Spike-Nanobody Fusion Protein Simultaneously Blocks Interleukin-6 trans-Signaling and Cellular Infection with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can induce mild to life-threatening symptoms. Especially individuals over 60 years of age or with underlying comorbidities, including heart or lung disease and diabetes, or immunocompromised patients are at a higher risk. Fatal multiorgan damage in coronavirus disease 2019 (COVID-19) patients can be attributed to an interleukin-6 (IL-6)-dominated cytokine storm. Consequently, IL-6 receptor (IL-6R) monoclonal antibody treatment for severe COVID-19 cases has been approved for therapy. High concentrations of soluble IL-6R (sIL-6R) were found in COVID-19 intensive care unit patients, suggesting the involvement of IL-6 trans-signaling in disease pathology. Here, in analogy to bispecific antibodies (bsAbs), we developed the first bispecific IL-6 trans-signaling inhibitor, c19s130Fc, which blocks viral infection and IL-6 trans-signaling. c19s130Fc is a designer protein of the IL-6 trans-signaling inhibitor cs130 fused to a single-domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. c19s130Fc binds with high affinity to IL-6:sIL-6R complexes as well as the spike protein of SARS-CoV-2, as shown by surface plasmon resonance. Using cell-based assays, we demonstrate that c19s130Fc blocks IL-6 trans-signaling-induced proliferation and STAT3 phosphorylation in Ba/F3-gp130 cells as well as SARS-CoV-2 infection and STAT3 phosphorylation in Vero cells. Taken together, c19s130Fc represents a new class of bispecific inhibitors consisting of a soluble cytokine receptor fused to antiviral nanobodies and principally demonstrates the multifunctionalization of trans-signaling inhibitors. IMPORTANCE The availability of effective SARS-CoV-2 vaccines is a large step forward in managing the pandemic situation. In addition, therapeutic options, e.g., monoclonal antibodies to prevent viral cell entry and anti-inflammatory therapies, including glucocorticoid treatment, are currently developed or in clinical use to treat already infected patients. Here, we report a novel dual-specificity inhibitor to simultaneously target SARS-CoV-2 infection and virus-induced hyperinflammation. This was achieved by fusing an inhibitor of viral cell entry with a molecule blocking IL-6, a key mediator of SARS-CoV-2-induced hyperinflammation. Through this dual action, this molecule may have the potential to efficiently ameliorate symptoms of COVID-19 in infected individuals.

Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant.

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against the SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with single nanobodies Nb6, VHH E, and complex with VHH E/VHH V nanobody combination. We combined coarse-grained and all-atom molecular simulations and collective dynamics analysis with binding free energy scanning, perturbation-response scanning, and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein might exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH E/VHH V nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under "basal" circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.

Llamanade: An open-source computational pipeline for robust nanobody humanization.

Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimize solutions to humanize Nbs. Finally, we used Llamanade to successfully humanize a cohort of structurally diverse and potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a server to support the development of therapeutic Nbs into safe and effective trials.

Structural Biology of Nanobodies against the Spike Protein of SARS-CoV-2.

Nanobodies are 130 amino acid single-domain antibodies (VHH) derived from the unique heavy-chain-only subclass of Camelid immunogloblins. Their small molecular size, facile expression, high affinity and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. The first nanobody agent has now entered the clinic as a treatment against a blood disorder. The spread of the SARS-CoV-2 virus has seen the global scientific endeavour work to accelerate the development of technologies to try to defeat a pandemic that has now killed over four million people. In a remarkably short period of time, multiple studies have reported nanobodies directed against the viral Spike protein. Several agents have been tested in culture and demonstrate potent neutralisation of the virus or pseudovirus. A few agents have completed animal trials with very encouraging results showing their potential for treating infection. Here, we discuss the structural features that guide the nanobody recognition of the receptor binding domain of the Spike protein of SARS-CoV-2.

Crystal structures of the SARS-CoV-2 nucleocapsid protein C-terminal domain and development of nucleocapsid-targeting nanobodies.

The ongoing outbreak of COVID-19 caused by SARS-CoV-2 has resulted in a serious public health threat globally. Nucleocapsid protein is a major structural protein of SARS-CoV-2 that plays important roles in the viral RNA packing, replication, assembly, and infection. Here, we report two crystal structures of nucleocapsid protein C-terminal domain (CTD) at resolutions of 2.0 Å and 3.1 Å, respectively. These two structures, crystallized under different conditions, contain 2 and 12 CTDs in asymmetric unit, respectively. Interestingly, despite different crystal packing, both structures show a similar dimeric form as the smallest unit, consistent with its solution form measured by the size-exclusion chromatography, suggesting an important role of CTD in the dimerization of nucleocapsid proteins. By analyzing the surface charge distribution, we identified a stretch of positively charged residues between Lys257 and Arg262 that are involved in RNA-binding. Through screening a single-domain antibodies (sdAbs) library, we identified four sdAbs targeting different regions of nucleocapsid protein with high affinities that have future potential to be used in viral detection and therapeutic purposes.

The role of single-domain antibodies (or nanobodies) in SARS-CoV-2 neutralization.

The severe acute respiratory syndrome (SARS-CoV-2), a newly emerging of coronavirus, continues to infect humans in the absence of a viable treatment. Neutralizing antibodies that disrupt the interaction of RBD and ACE2 has been under the spotlight as a way of developing the COVID-19 treatment. Some animals, such as llamas, manufacture heavy-chain antibodies that have a single variable domain (VHH) instead of two variable domains (VH/VL) as opposed to typical antibodies. Nanobodies are antigen-specific, single-domain, changeable segments of camelid heavy chain-only antibodies that are recombinantly produced. These types of antibodies exhibit a wide range of strong physical and chemical properties, like high solubility, and stability. The VHH's high-affinity attachment to the receptor-binding domain (RBD) allowed the neutralization of SARS-CoV-2. To tackle COVID-19, some nanobodies are being developed against SARS-CoV-2, some of which have been recently included in clinical trials. Nanobody therapy may be useful in managing the COVID-19 pandemic as a potent and low-cost treatment. This paper describes the application of nanobodies as a new class of recombinant antibodies in COVID-19 treatment.

A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies.

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.

Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis.

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs.

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates.

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting diverse and conserved epitopes.

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.