key: cord-1002419-ixrak95q
authors: Yu, Bingchen; Li, Shanshan; Tabata, Takako; Wang, Nanxi; Kumar, G. Renuka; Liu, Jun; Ott, Melanie M.; Wang, Lei
title: Accelerating PERx Reaction Enables Covalent Nanobodies for Potent Neutralization of SARS-Cov-2 and Variants
date: 2022-03-14
journal: bioRxiv
DOI: 10.1101/2022.03.11.483867
sha: 2286d28b439690f07bc11dfde6fcc8bfacafb480
doc_id: 1002419
cord_uid: ixrak95q

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.

In December 2019 a novel coronavirus (SARS-CoV-2) caused an outbreak of coronavirus disease-19 (Covid-19) pandemic, 1 which has been ongoing for over 2 years and resulted in an unprecedented burden to public health globally. In addition to vaccines, 2 incessant efforts have been spent on developing drugs to inhibit the virus as prophylactics and treatment. Since cell entry of SARS-CoV-2 depends on the binding of the viral Spike protein to the human cellular angiotensin-converting enzyme 2 (ACE2) receptor, 3, 4 various types of reagents have been developed to block the Spike-ACE2 interaction to neutralize SARS-CoV-2. 5 Biologics such as protein-based drugs exert their function through non-covalent interactions, which are reversible to allow drug dissociation such that the unblocked virus can re-access and infect cells. In addition, rapid evolving of the SARS-CoV-2 RNA genome has led to variants that escape neutralization by human immune system or various noncovalent reagents. 13 14 15 Covalent small molecule drugs have been shown to possess enhanced potency, prolonged duration of action, and ability to mitigate drug resistance. 16 17 We thus envisioned that protein drugs able to bind the virus irreversibly in covalent mode would be highly desirable to attain potent and complete inhibition of viral infection, as well as to minimize viral escape through mutation. However, whether and how covalent protein drugs can increase potency in neutralizing viral infection await exploration.

Natural proteins generally lack the ability to bind with target covalently. 18 To break this natural barrier, we recently reported a Proximity-Enabled Reactive therapeutics (PERx) strategy to generate covalent protein drugs. 19 20 A latent bioreactive unnatural amino acid (Uaa) is incorporated into the protein drug through genetic code expansion, 21 which reacts with a natural residue on the target only upon drug-target binding, realizing specific cross-linking of the drug to the target covalently. We have demonstrated that a covalent PD-1 drug efficiently inhibits tumor growth in mice, with therapeutic efficacy superior to that of an FDA-approved antibody. 19 This initial success was achieved on suppression of tumor growth, which is a relatively long time process taking days to weeks. It remains to be established whether PERx can be generally applicable to other proteins for enhanced efficacy and to acute processes requiring fast and prompt reaction.

Here we developed covalent nanobodies to irreversibly inhibit the infection of both SARS-CoV-2 and its variants via the PERx principle. To cope with acute viral infection, a novel latent bioreactive Uaa, fluorine substituted fluorosulfate-L-tyrosine (FFY), was designed and genetically encoded to accelerate the PERx reaction rate by 2.4 fold over the original FSY, enabling fast crosslinking within 10 min. Mechanistic insight was uncovered on how to translate the increase in antagonizing Spike-ACE2 interaction into more potent virus neutralization. Consequently, FFYbased nanobodies exhibited drastic potency increase in neutralizing SARS-CoV-2 (41-fold) as well as its Alpha (23-fold) and Delta (39-fold) variants. Using PERx strategy, covalent soluble human ACE2 was also generated to bind to the Spike protein irreversibly. This PERx-based covalent nanobody strategy may provide a new route to developing effective therapeutics for viral infections.

Nanobodies (single-domain antibodies) are generally heat stable, easier to produce in bacteria, have small size (~15 kDa) to increase binding density on virus for efficient blockage, 8 4 and can be humanized to minimize potential immunogenicity. Multiple groups have selected nanobodies binding to the Spike protein with high affinities, showing promising inhibition against SARS-CoV-2 infection of Vero cells or ACE2-expressing HEK-293 cells. 22, 23 7 Our strategy was to genetically incorporate a latent bioreactive unnatural amino acid (Uaa) into the nanobody that specifically binds to the receptor binding domain (RBD) of the SARS-CoV-2 viral Spike protein (Figure 1a) . Upon binding of the nanobody with the Spike RBD, the latent bioreactive Uaa would be brought into close proximity to a target residue of the Spike RBD, which enables the Uaa to react with the target residue specifically, irreversibly cross-linking the nanobody with the Spike RBD. The bound nanobody would prevent the Spike RBD interact with human ACE2 receptor, blocking viral infection. The resultant covalent nanobody would thus function as proximityenabled reactive therapeutics (PERx). Compared with the conventional nanobodies, which bind in non-covalent mode and are in dynamic association and dissociation with Spike RBD, covalent nanobodies would permanently bind to Spike RBD and neutralize the virus with enhanced potency (Figure 1b) . Only upon nanobody binding with  the Spike RBD, the latent bioreactive Uaa reacts with the target natural residue, covalently cross-linking the   nanobody with the Spike RBD. (B) While conventional nanobody can dissociate from the Spike RBD, covalent   nanobody binds with the Spike RBD irreversibly, permanently preventing viral binding with ACE2 receptor and thus blocking infection more effectively.

We initially chose to use the latent bioreactive Uaa FSY for its stability and ability to react with Tyr, His, or Lys via proximity-enabled sulfur fluoride exchange (SuFEx) reaction under biocompatible and cellular conditions (Figure 2A) . 24 On the basis of the crystal structure of human SARS-CoV-2 Spike RBD in complex with nanobody H11-D4, 25 nanobody MR17-K99Y, 23 or nanobody SR4, 23 we decided to incorporate FSY at site R27, S30, E100, W112, D115, or Y116 of nanobody H11-D4 (Figure 2B) , site Y99 or D101 of nanobody MR17-K99Y (Figure 2C) , and site Y37, H54 or S57 of nanobody SR4 (Figure 2D) , respectively. Western blot analysis of lysates of cells expressing these mutant nanobody genes and the tRNA Pyl /FSYRS pair 24 confirmed that FSY was successfully incorporated into the nanobodies in the presence of FSY ( Figure S1 ). WT and FSY-incorporated nanobodies were purified with affinity chromatography. Mass spectrometric analysis of the intact SR4(57FSY) protein confirmed that FSY was incorporated into SR4 at site 57 with high fidelity (Figure 2E ).

To test if FSY-incorporated nanobodies could bind to the Spike RBD covalently in vitro, we 

We next tested the efficacy of SR4 nanobodies to inhibit the binding of the Spike RBD-mFc (a mouse Fc tag appended at the C-terminus of Spike RBD) to 293T-ACE2 cells, a HEK293T cell line stably expressing human ACE2 protein on cell surface. Different concentrations of SR4(WT) or SR4(57FSY) were individually incubated with the Spike RBD-mFc at 37 °C for 12 h to allow cross-linking, followed by incubation with 293T-ACE2 cells for 1 h. After incubation, cells were stained with FITC labeled anti-mFc and analyzed with flow cytometry ( Figure 2K ). As expected, SR4(WT) bound to the Spike RBD reversibly such that the Spike RBD could still bind to ACE2 on the cell surface, resulting in an IC50 of 1980 nM. In contrast, the covalent SR4(57FSY) showed highly efficient blocking of the Spike RBD binding to 293T-ACE2 cells with an IC50 of 15.8 nM ( Figure 2K ). The IC50 of SR4(57FSY) was 125-fold lower than that of SR4(WT), demonstrating the drastic improvement of the covalent nanobody in inhibiting the binding of Spike RBD to cell surface ACE2 receptor.

We next tested the neutralization activity of SR4(WT) or SR4(57FSY) nanobody against SARS-CoV-2 spike pseudotyped lentivirus using a widely adopted protocol. 26 27 SARS-CoV-2 reporter virus particles display antigenically correct Spike protein on a heterologous virus core and carry a modified genome that expresses a convenient GFP reporter gene, which is integrated and expressed upon successful viral entry into cells harboring the ACE2 receptor. Briefly, the pseudoviruses were incubated with various concentrations of SR4(WT) or SR4(57FSY) at 37 °C for 1 h, and subsequently used to infect 293T-ACE2 cells for 48 h. Cells were then analyzed by flow cytometry for GFP signal to determine the percentage of infected cells. The IC50 for SR4(WT) was measured to be 602.8 nM (Figure 2L) , close to the literature reported 390 nM, 23 whereas the IC50 for SR4(57FSY) was measured to be 151.6 nM. This 4-fold decrease of IC50 demonstrated that the covalent SR4(57FSY) was more potent in inhibiting pseudovirus infection of ACE2expressing human cells than SR4(WT), but the enhancement was moderate.

We further tested the neutralization ability of SR4(WT) or SR4(57FSY) nanobody against authentic SARS-Cov-2 infection of ACE2 expressing human cells. Surprisingly, no significant enhancement of SR4(57FSY) over SR4(WT) was measured (Data not shown). The lack of enhancement in inhibiting authentic virus infection seemed to be discrepant with the fact that SR4(57FSY) drastically enhanced inhibition of the Spike RBD binding with cell surface ACE2 receptor (125 fold). We reasoned that the discrepancy could be accounted for by how SR4 accesses and binds to the Spike RBD on the live SARS-CoV-2 virus, which may be different from the purified Spike RBD in isolation. In fact, the Spike RBD in SARS-Cov-2 virus exchanges between the active up state and the inactive down state. 28, 29 To achieve potent inhibition of viral infection, binding and locking the Spike RBD in the inactive down state could be more effective. 7 However, the crystal structure of SR4 in complex with RBD indicates that SR4 binds with the up state of RBD instead. 23 We therefore sought to covalently engineering a nanobody that could bind the Spike RBD in the inactive down state. Generate covalent nanobodies from mNb6 that binds the down-state of RBD Nanobody mNb6 was isolated through screening a library against the Spike ectodomain stabilized in the prefusion conformation, and thus bound the Spike RBD in the down state. 7 To identify which sites of mNb6 would allow covalent crosslink of the Spike RBD, we incorporated FSY individually at 30 different sites located at the three complementarity-determining regions (CDR) of mNb6 ( Figure 3A) . Although the crystal structure of mNb6 in complex with RBD is available, 7 we performed this comprehensive site screening without inspecting the structure to show that for proteins with well-defined regions, such as the nanobody, one could readily determine the appropriate sites for FSY cross-linking without sophisticated high-throughput screening or detailed structures. We incubated 2 µM mNb6(WT) and its FSY mutant proteins with 0.5 µM Spike RBD in PBS (pH 7.4) at 37 °C for 12 h, followed by Western blot analysis ( Figure   3B -D). When FSY was incorporated at site 27 in CDR1, site 55 in CDR2, or sites 102-108 in CDR3, a covalent complex of mNb6 with the Spike RBD was detected, indicating that multiple sites in mNb6 allowed covalent cross-linking with the Spike RBD, which may not be obvious by only inspecting the crystal structures. We then performed kinetic study on the four more efficient sites on CDR3 (Figure 3E) , and found that mNb6(108FSY) showed the fastest cross-linking rate.

We thus chose site 108 to incorporate Uaa for subsequent experiments.

The potency of covalent protein drug would rely on the rate and extent of latent bioreactive Uaa to form a covalent bond with the target residue of the target protein. As protein interactions are dynamic, a fast reaction rate would be essential to ensure covalent bond formation before protein dissociation. Given certain contact time, the reaction extent increases with faster reaction rate, which can be critical to achieve complete inhibition of viral infection. Introducing electronwithdrawing groups on the aromatic ring has been reported to increase the SuFEx rates. 30 We thus envisioned that adding electron-withdrawing substituents to FSY would accelerate its proximityenabled reaction rate when used in PERx. As a result, we designed and evaluated a fluorine substituted fluorosulfate-L-tyrosine (FFY, Figure 4A ). FFY was synthesized using [4-(acetylamino) phenyl]imidodisulfuryl difluoride (AISF), 31 followed with the deprotection of the Boc protecting group using hydrogen chloride. As FFY and FSY have similar structures, we reasoned that FSYRS, a pyrrolysyl-tRNA synthetase (PylRS) mutant we previously evolved to incorporate FSY, 24 should be able to incorporate FFY into proteins as well. To test this idea, the enhanced green fluorescent protein (EGFP) gene containing a TAG codon at permissive site 182 was co-expressed with genes for tRNA Pyl /FSYRS in E. coli. In the absence of FFY, no obvious fluorescence was detected; in the presence of FFY, fluorescence intensity was measured to increase with FFY concentration (Figure 4B) , suggesting FFY incorporation into EGFP. We also co-expressed mNb6(108TAG) with tRNA Pyl /FSYRS in E.

coli, and observed that full-length mNb6 was produced in the presence of 2 mM FFY or 1 mM FSY ( Figure 4C ). mNb6(WT), mNb6 (108FSY), and mNb6(108FFY) proteins were purified with Ni 2+ affinity chromatography. Mass spectrometric analysis of the intact protein confirmed that FFY was incorporated into mNb6 at site 108 in high fidelity. A major peak observed at 13721 Da corresponds to intact mNb6(108FFY) (Figure 4D , expected 13720.7 Da). A minor peak observed at 13702 Da corresponding to mNb6(108FFY) lacking F, suggesting a slight F elimination during mass spectrometric measurement. 24 We also verified mNb6(WT) and mNb6(108FSY) via mass spectrometric analysis of the intact proteins ( Figure 4E and Figure S2) . Figure S4 ). Thus, FFY increased the PERx reaction rate over FSY in the mNb6 system to 240%, so we used mNb6(108FFY) for subsequent viral inhibition tests. Figure 5E , the IC50 of mNb6(WT) in inhibiting authentic SARS-CoV-2 was 69.9 nM whereas the IC50 measured for mNb6(108FFY) was 1.7 nM, indicating a drastic 41-fold improvement in potency. 

In addition to artificial nanobodies specific for the Spike RBD, we also sought to engineer the native receptor, human ACE2, into a covalent binder to block the interaction of SARS-CoV-2 Spike with human ACE2. Using a soluble ACE2 receptor binding to the viral Spike protein, thereby neutralizing SARS-CoV-2, is an attractive strategy. The Spike protein of SARS-CoV-2 binds to the ACE2 receptor with a KD of 4.7 nM, 38 comparable to affinities of mAbs. In addition, ACE2 administration could additionally treat pneumonia caused by SARS-CoV-2. Coronavirus binding leads to ACE2 protein shedding and downregulation, which induces pulmonary edema and acute respiratory distress syndrome (ARDS). Administration of recombinant human ACE2 improves acute lung injury and reduces ARDS in preclinical studies. 39, 40 Moreover, recombinant human ACE2 is safe and well tolerated by patients in phase II trial, 41, 42 and small levels of soluble ACE2 are secreted and circulate in human body. 43 More importantly, the soluble ACE2 therapy is expected to have broad coverage as SARS-CoV-2 cannot escape ACE2 neutralization due to its dependence on the same protein for cell entry. Any mutation of SARS-CoV-2 reducing its affinity for the ACE2-based therapeutics will render the virus less pathogenic.

To this end, we explored if the soluble human ACE2 could be engineered into a covalent binder for SARS-CoV-2 Spike protein. On the basis of the crystal structure of the SARS-CoV-2

Spike RBD in complex with human ACE2 (Figure 6A ), 38 we decided to incorporate FSY at site D30, H34, E37, D38, Q42 or Y83 to target the proximal nucleophilic amino acid residues on the Spike RBD. These FSY-incorporated ACE2 mutant proteins were expressed and purified from HEK293 cells and individually incubated with the Spike RBD followed by Western blot analysis ( Figure 6B) . Cross-linking of ACE2(FSY) with the Spike RBD was detected when FSY was incorporated at sites 34, 37, or 42. The ACE2 (34FSY) mutant exhibited the highest cross-linking efficiency (28.3%). The cross-linking kinetic experiments of ACE2(FSY) with the Spike RBD showed that cross-linking was detectable at 1 h and increased with incubation time (Figure 6C) .

Furthermore, we analyzed the cross-linking product of ACE2(34FSY) with the Spike RBD using high-resolution mass spectrometry, which clearly indicated that FSY of ACE2(34FSY) specifically reacted with K417 of the Spike RBD (Figure S7) , as designed based on the crystal structure. Together, these results showed that ACE2(34FSY) covalently bound to the Spike RBD. 

In summary, through genetic incorporation of the latent bioreactive Uaas FSY and FFY into nanobodies, we generated multiple covalent nanobodies that bind with the Spike RBD of SARS-CoV-2 irreversibly via the PERx mechanism. The newly developed FFY increased the PERx reaction rate by 2.4 fold over the original FSY, and the resultant covalent nanobody mNb6(108FFY) efficiently neutralized both SARS-CoV-2 pseudovirus and authentic SARS-CoV-2, increasing the potency by a drastic 36-fold and 41-fold over the noncovalent WT mNb6, respectively. In addition, the covalent mNb6(108FFY) nanobody also potently inhibited the Alpha and Delta variant of SARS-CoV-2 pseudovirus, with a respective 23-and 39-fold increase in potency than the WT mNb6. Moreover, using a similar strategy, the soluble human ACE2 receptor was also engineered into a covalent binder that irreversibly cross-linked with the Spike RBD via PERx.

Our results demonstrate that nanobodies can be readily engineered into covalent binders through incorporating a latent bioreactive Uaa, which reacts with a natural residue of the target protein only upon nanobody-target binding, expanding the scope of PERx that we previously developed and applied to the immune-checkpoint PD-1/PD-L1. 19 20 Sites in nanobody appropriate for FSY or FFY incorporation and cross-linking were identified either by inspecting the structure of nanobody-target complex 44 or simply screening all sites in nanobody's three CDRs. Recent breakthrough in accurate prediction of protein structure and interactions have made structural information available for a broad range of proteins, 45 46 which will greatly facilitate structureguided site identification. On the other hand, there are about 30 total sites in nanobody's three CDRs, screening of which using cross-linking and Western blot analysis can be completed in less than 2 weeks. We have always identified multiple sites in diverse nanobodies with successful cross-linking owing to FSY and FFY's exceptional ability to react with multiple residues including Tyr, His, and Lys, 24 which are often found at protein-protein interface. To react with residues at further distances, we have also developed FSK that has a longer and more flexible side chain with similar reactivity. 44 These site identification strategies are straightforward and obviate sophisticated instrumentation such as mass spectrometry, and should be generally applicable to engineering various proteins such as antibodies, Fab, single-chain variable fragment (ScFv), affibodies, DARPins and so on, into covalent binders.

Compared with conventional noncovalent drugs, drugs working in covalent mode offer desirable features such as increased potency, prolonged duration, and possibility to prevent drug resistance, as demonstrated with covalent small molecule drugs. 16 Here we demonstrate that the covalent nanobodies, representing covalent protein drugs, possessed similar valuable properties as well. In particular, the covalent mNb6(108FFY) drastically increased the potency over WT mNb6 to 36-fold in inhibiting SARS-CoV-2 pseudovirus and to 41-fold in inhibiting authentic SARS-CoV-2. Aside from the WT SARS-Cov-2, mNb6(108FFY) was also able to covalently crosslink and neutralize the Alpha and Delta variants of SARS-CoV-2, with potency increase of 23-and 39fold over the WT mNb6. These potency increases were measured by incubating the nanobody with the virus for 1 h. Because the extent of covalent cross-linking increases with time, the potency of covalent nanobodies should be greater with longer incubation time.

A potential caveat is that the cross-linking rate can be negatively impacted when the binding affinity between protein drug and target decreases drastically due to target mutation, such as mNb6(108FFY) and Beta RBD. In rare cases, mutation can even occur at the cross-linking residue, which would abolish cross-linking at that site. However, as we demonstrated here and previously, 19 44 PERx is a general strategy that can be applied on diverse nanobodies and each nanobody can have FFY incorporated at multiple sites to target different natural residues on Spike RBD to achieve crosslink (Figure 3) . Therefore, other nanobodies able to bind mutated Spike RBD could be similarly engineered, and diverse covalent nanobodies could be used in combination to ensure the cross-linking and inhibition of future potential variants. When in use, the irreversible crosslinking ability together with multi-targeting of these covalent nanobodies should achieve complete viral inhibition and mechanistically prevent drug resistance. Nonetheless, animal tests and clinical trial are warranted to confirm these potential benefits of covalent protein drugs.

Fast reaction kinetics would be critical for effective inhibition of viral infection, as it enables covalent crosslinks to promptly form between the protein drug and the target within a shorter contact time, and to reach higher extent for more effective neutralization. By introducing electron withdrawing fluorine substitution we designed and genetically encoded a novel latent bioreactive Uaa FFY, which afforded a marked 2.4-fold increase in reaction rate in the cross-linking of mNb6 with the Spike RBD over the original FSY. In a recent communication, 47 Han et al. exploited the FSY and PERx mechanism developed by us 19, 24 and incorporated FSY into the Spike-specific We also uncovered that potency increase by covalent protein drug in neutralizing SARS-CoV-2 was dependent on the Spike RBD state that the protein drug binds. A puzzling observation was that a striking increase in antagonizing the interaction of Spike RBD with ACE2 in vitro by a covalent binder does not necessarily translate into more potent neutralization of viral infection.

Compared with the WT nanobody SR4, the covalent nanobody SR4(57FSY) inhibited the Spike RBD binding to cell surface ACE2 with an enhanced potency of 125-fold, but it showed no enhancing effect in neutralizing SARS-CoV-2. We discovered that the underlying reason was that SR4 binds the Spike RBD in the latter's active up state. 23 In contrast, mNb6 binds the Spike RBD in the latter's inactive down state, 7 and the covalent nanobody mNb6(108FFY) then drastically increased the potency in neutralizing the WT, Alpha, and Delta variant of SARS-CoV-2. Therefore, to achieve potent neutralization of virus, the covalent binder needs to directly block the viral infection process aside from binding with the viral protein. This mechanistic insight will be valuable in guiding the development of covalent inhibitors for other viral infections.

Covalent nanobodies may lead to new protein drugs that can be readily produced in large scale via bacterial expression, easy to store and distribute due to nanobody's high stability, and aerosolized for self-administered inhalation to nasal and lung epithelia. 7 Further development may afford a medication for COVID-19 patients to prevent significant morbidities and death, and provide a potential prophylactic to give passive immunity to clinical providers at the front line. In addition, the PERx-capable ACE2 drugs can serve as a therapeutic stockpile for future outbreaks of SARS-CoV, SARS-CoV-2, and any new coronavirus or its variants that use the ACE2 receptor for entry. Moreover, through irreversible binding covalent protein drugs can potentially achieve complete viral inhibition and mechanistically prevent viral resistance. Lastly, the principle of generating a covalent binder or a covalent soluble receptor inhibitor via PERx can be generally applied for developing covalent protein drugs to effectively treat various other infectious diseases such as influenza, hepatitis, AIDS, anthrax, and so on.

Plasmid pBAD-H11D4, pBAD-MR17K99Y, or pBAD-SR was transformed into E. coli Tris-HCl, 200 mM NaCI, pH 7.5). To yield pure protein, TALON® metal affinity resin was further applied. The purification procedure was same as described above. The eluted proteins were analyzed by running 12% Tris-glycine SDS−PAGE gel.

The Spike RBD was incubated with wildtype nanobody or nanobody mutants at indicated concentrations in PBS (pH 7.4) at 37 °C for 12 h, after which 5 µL sample was mixed with 10 µL

Laemmli loading dye supplied with 100 mM DTT. The mixture was boiled at 95 °C for 10 min and subjected to Western blot analysis. The bands were detected using an antibody against mouse Fc appended at the C-terminus of the Spike RBD. 

The SARS-CoV-2 WT or variant Spike RBD (Hisx6 tagged, 0.5 µM) was incubated with 5 µM mNb6(108FSY) or mNb6(108FFY) in PBS (pH 7.4 ) at 37 °C. At different time points, 5 µL reaction mixture was extracted and mixed with 5 µL Laemmli loading buffer. The mixture was heated to 95 °C for 10 min and the protein cross-linking was examined by Western blot. The protein band was detected with HRP-conjugated anti-Hisx6 antibody (Proteintech, #HRP-66005).

The Spike RBD band intensity in the Western blot was quantified with Bio-rad imaging software.

The linear plot of natural logarithm (ln) of the Spike RBD band intensity versus time (h) gives kobs.

Binding constant (KD) between Spike protein RBD and nanobody was measured with biolayer interferometry (BLI) using Octet Red384 systems (ForteBio). Biotinylated Spike RBD was firstly loaded to streptavidin (SA) sensor (ForteBio #18-5019) by incubating SA sensor in 100 nM biotinylated Spike RBD in Kinetic Buffer ( 0.005 % (v/v) Tween 20 and 0.1 % BSA in PBS, pH = 7.4 ) at 25 °C. The sensor was equilibrated (baseline step) in Kinetic Buffer for 120 s, after which the sensor was incubated with varying concentrations of nanobody (association step) for 50 s, followed with dissociation step in Kinetic Buffer for 450 s. Data was fitted for a 1:1 stoichiometry and KD was calculated using the built in software.

The SARS-CoV-2 GFP Reporter Virus Particles (RVPs) are SARS-CoV-2 pseudotyped lentivirus and were purchased from Integral Molecular. Catalog numbers for strains are the following: wild-type strain: RVP-701; Alpha: RVP-706; Beta: RVP-714; Delta: RVP-763. One day before transduction, 4 × 10 4 293T-ACE2 cells were plated in each well of a 48-well plate.

Serially diluted nanobodies were incubated with pseudovirus in DMEM at 37 °C for 1 h. The mixture was subsequently transferred to each well of the 48-well plate. The cells were cultured at 37 °C for additional 48 h, after which the cells were harvested for flow cytometric analysis to measure the proportion of GFP positive cells.

SARS-CoV-2 Nanoluciferase (USA/WA1-2020) (SARS-CoV2 nLuc) was a kind gift from Dr. Pei-Yong Shi. The virus stocks were prepared in Vero E6 (ATCC) and titers were determined by plaque assays on Vero-E6 cells. Neutralizing assays were performed in 293T-ACE2 cells 

One day before transfection, 2 × 10 6 Expi293F TM cells were seeded with pre-warmed Freestyle 293 media. The cells were transfected with 25 µg pcDNA-ACE2-34TAG and 25 µg pMP-FSYRS plasmids according to manufacturer's protocol. The cells were incubated for 30 min and then 2 mM FSY was added into the culture media dropwise. The supernatant was collected after 4 days post-transfection. Imidazole (20 mM) and pre-equilibrated Ni-NTA resin were added into the supernatant, followed by incubation 4 °C on the rotator for 1 h. The resin was washed and

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eluted with elution buffer (20 mM Tris-HCl, 300 mM imidazole, 200 mM NaCI, pH 7.5). The eluted protein was concentrated and exchanged with PBS, pH 7.5.