Shaping Nanobodies and Intrabodies against Proteoforms.

Proteoforms expand genomic diversity and direct developmental processes. While high-resolution mass spectrometry has accelerated characterization of proteoforms, molecular techniques working to bind and disrupt the function of specific proteoforms have lagged behind. In this study, we worked to develop intrabodies capable of binding specific proteoforms. We employed a synthetic camelid nanobody library expressed in yeast to identify nanobody binders of different SARS-CoV-2 receptor binding domain (RBD) proteoforms. Importantly, employment of the positive and negative selection mechanisms inherent to the synthetic system allowed for amplification of nanobody-expressing yeast that bind to the original (Wuhan strain RBD) but not the E484 K (Beta variant) mutation. Nanobodies raised against specific RBD proteoforms were validated by yeast-2-hybrid analysis and sequence comparisons. These results provide a framework for development of nanobodies and intrabodies that target proteoforms.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor.

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

In Silico Optimization of SARS-CoV-2 Spike Specific Nanobodies.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, caused a global pandemic, and killed millions of people. The spike protein embedded in the viral membrane is essential for recognizing human receptors and invading host cells. Many nanobodies have been designed to block the interaction between spike and other proteins. However, the constantly emerging viral variants limit the effectiveness of these therapeutic nanobodies. Therefore, it is necessary to find a prospective antibody designing and optimization approach to deal with existing or future viral variants. METHODS: We attempted to optimize nanobody sequences based on the understanding of molecular details by using computational approaches. First, we employed a coarse-grained (CG) model to learn the energetic mechanism of the spike protein activation. Next, we analyzed the binding modes of several representative nanobodies with the spike protein and identified the key residues on their interfaces. Then, we performed saturated mutagenesis of these key residue sites and employed the CG model to calculate the binding energies. RESULTS: Based on analysis of the folding energy of the angiotensin-converting enzyme 2 (ACE2) -spike complex, we constructed a detailed free energy profile of the activation process of the spike protein which provided a clear mechanistic explanation. In addition, by analyzing the results of binding free energy changes following mutations, we determined how the mutations can improve the complementarity with the nanobodies on spike protein. Then we chose 7KSG nanobody as a template for further optimization and designed four potent nanobodies. Finally, based on the results of the single-site saturated mutagenesis in complementarity determining regions (CDRs), combinations of mutations were performed. We designed four novel, potent nanobodies, all exhibiting higher binding affinity to the spike protein than the original ones. CONCLUSIONS: These results provide a molecular basis for the interactions between spike protein and antibodies and promote the development of new specific neutralizing nanobodies.

Development of functional anti-Gn nanobodies specific for SFTSV based on next-generation sequencing and proteomics.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next-generation sequencing (NGS) with proteomics technology based on affinity purification-mass spectrometry (AP-MS) and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, of which six sequences were selected for recombinant protein expression and purification. Among these six anti-Gn nanobodies, nanobody 57,493 was validated to be highly specific for Gn. The innovative high-throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS-CoV-2.

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.

SARS-CoV-2 Delta Variant Decreases Nanobody Binding and ACE2 Blocking Effectivity.

The Delta variant spreads more rapidly than previous variants of SARS-CoV-2. This variant comprises several mutations on the receptor-binding domain (RBDDelta) of its spike glycoprotein, which binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. The RBD-PD interaction has been targeted by antibodies and nanobodies to prevent viral infection, but their effectiveness against the Delta variant remains unclear. Here, we investigated RBDDelta-PD interactions in the presence and absence of nanobodies H11-H4, H11-D4, and Ty1 by performing 21.8 µs of all-atom molecular dynamics simulations. Unbiased simulations revealed that Delta variant mutations strengthen RBD binding to ACE2 by increasing the hydrophobic interactions and salt bridge formation, but weaken interactions with H11-H4, H11-D4, and Ty1. Among these nanobodies H11-H4 and H11-D4 bind RBD without overlapping ACE2. They were unable to dislocate ACE2 from RBDDelta when bound side by side with ACE2 on RBD. Steered molecular dynamics simulations at comparable loading rates to high-speed atomic force microscopy (AFM) experiments estimated lower rupture forces of the nanobodies from RBDDelta compared to ACE2. Our results suggest that existing nanobodies are less effective to inhibit RBDDelta-PD interactions and a new generation of nanobodies is needed to neutralize the Delta variant.

Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor-binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.Abbreviations: ACE2 - angiotensin converting enzyme 2BSA - buried surface areaCDR - complementary determining regionRBD - receptor binding domainRBM - receptor-binding motifSARS-CoV-2 - severe acute respiratory syndrome coronavirus 2.

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.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

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.

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo.

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Šoverall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Šresolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19.

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

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.

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.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

Rapid generation of potent antibodies by autonomous hypermutation in yeast.

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.