Camelid nanobodies targeting shared epitopes of the SARS-CoV-2 RBD with its receptor ACE2

Abstract

The present application provides a camel-derived nanobody targeting the shared epitope of SARS-CoV-2 RBD and its receptor ACE2, which simultaneously binds to the key sites F486, Q493 and S494 of SARS-CoV-2 RBD. The present application screens the purified nanobody VHH5-05, which not only shows strong binding capacity to phages displaying wild-type SARS-CoV-2 RBD, but also shows certain binding capacity to phages of Beta mutant strain RBD and Delta mutant strain RBD.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibody technology, and particularly relates to a camelid nanobody targeting the shared epitope of SARS-CoV-2 RBD and its receptor ACE2, and the use of the antibody in preventing and / or treating diseases related to SARS-CoV-2 virus infection. BACKGROUND

[0002] SARS-CoV-2 is a single-stranded positive-sense RNA enveloped virus, and its membrane surface expresses a large number of spike proteins (S-protein, S protein for short), thereby forming the crown appearance of SARS-CoV-2. The S protein is actually a homotrimeric protein, and each protein monomer contains an S1 subunit (14-685 aa) and an S2 subunit (686-1273 aa). The S protein can recognize and bind to the angiotensin converting enzyme 2 (ACE2) on the cell membrane surface, and after being cut by the cell surface serine protease TMPRSS2, it mediates the fusion of the viral envelope and the cell membrane, and promotes the entry of the viral RNA genome into the host cell. The receptor-binding domain (RBD) of the S1 subunit is the key region for studying the binding of SARS-CoV-2 to the ACE2 receptor and the recognition of antibodies, and is considered to be the most effective target for anti-SARS-CoV-2 neutralizing antibodies (NAbs) so far.

[0003] Neutralizing antibody therapy is one of the advantageous weapons in the process of human response to major public health crises, and is concerned and expected because it can achieve the dual effect of treatment and prevention. Neutralizing antibodies often work by binding to the RBD of the virus, thereby preventing the virus from adsorbing to the ACE2 receptor and entering the cell to replicate and proliferate. In addition, neutralizing antibodies form immune complexes with viruses, which are easily phagocytosed and cleared by macrophages.

[0004] The single domain antibody (sdAb) of camel immunoglobulin is a kind of antibody naturally lacking light chain, only containing a heavy chain variable region and two conventional CH2 and CH3 regions. The single domain antibody binds to the antigen through the variable region on the heavy chain, which can exist stably in vitro alone and is called the variable domain of heavy chain of heavy-chain antibody (VHH) or nanobody (Nb), with a molecular weight of 12-15 kilodaltons (KDa), which is the smallest fragment known to bind to antigens. Compared with traditional monoclonal antibodies (150 KDa), the single domain antibody has further characteristics, such as more available epitopes, relatively low production cost, higher tissue penetration, easier production in prokaryotic expression system, etc. SUMMARY

[0005] In the present application, phage display technology is used for nanobody screening. In this technology, foreign gene fragments are inserted into the genome of phage by genetic engineering methods, so that the encoded proteins or polypeptides form fusion proteins with phage coat proteins and are displayed on the surface of phage. The displayed proteins can maintain relative spatial structure and biological activity, so that the target protein is used as "bait" to fish out the target protein combined with it, and the phage library is screened.

[0006] Based on the method of phage display, the excellent biological characteristics of nanobody and the basic principle of RBD and ACE2 binding mediating virus entry into host cells, the present application uses the constructed phage nanobody immune library to screen nanobody by using wild type SARS-CoV-2 RBD (denoted as SARS-CoV-2 RBD WT) as target antigen and adopting RBD-ACE2 competitive panning method, and expresses and identifies the screened VHH, so as to obtain high affinity nanobody specifically combined with SARS-CoV-2 RBD recombinant protein.

[0007] In an embodiment, the present application provides a camelid nanobody or antigen binding fragment thereof targeting an epitope shared by SARS-CoV-2 RBD and its receptor ACE2, which simultaneously binds to key sites F486, Q493 and S494 of SARS-CoV-2 RBD, which has at least 95% homology to SEQ ID NO: 1 : ATGGCCCAGTTGCAGCTCGTGGAGTCCGGAGGAGGCGCGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGAACCATCAGTCACTATCGCATGGGCTGGTACCGCCAACGTCCAAGGGGGCCGCGCGAGAAGGTTGCGATCATTACTATTAATGCTTCGACTGACTATGACGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTTTATCTCCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCGACCCCCCGGGACTGTCTCAGAATGACTACTGGGGGCCGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC.

[0008] In an embodiment, the nanobody or antigen binding fragment thereof is SEQ ID NO: 1 : ATGGCCCAGTTGCAGCTCGTGGAGTCCGGAGGAGGCGCGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGAACCATCAGTCACTATCGCATGGGCTGGTACCGCCAACGTCCAAGGGGGCCGCGCGAGAAGGTTGCGATCATTACTATTAATGCTTCGACTGACTATGACGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTTTATCTCCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCGACCCCCCGGGACTGTCTCAGAATGACTACTGGGGGCCGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC.

[0009] In an embodiment, the present application provides an isolated nucleic acid molecule encoding the above-mentioned camelid nanobody or antigen-binding fragment thereof targeting the SARS-CoV-2 RBD shared epitope with its receptor ACE2.

[0010] In an embodiment, the present application provides an antibody conjugate comprising the above-mentioned antibody.

[0011] In an embodiment, the present application provides a composition comprising the above-mentioned camelid nanobody or antigen-binding fragment thereof targeting the SARS-CoV-2 RBD shared epitope with its receptor ACE2.

[0012] In an embodiment, the present application provides the use of the above-mentioned camelid nanobody or antigen-binding fragment thereof targeting the SARS-CoV-2 RBD shared epitope with its receptor ACE2 in the manufacture of a medicament for preventing or treating a disease or condition caused by wild-type SARS-CoV-2 RBD, Beta mutant RBD and Delta mutant RBD virus infection.

[0013] Most of the residues on the epitope of the antibody RBD-VHH5-05 of the present application overlap with the RBD-ACE2 binding interface, and in particular the key sites F486, Q493 and S494 of the SARS-CoV-2 RBD are involved in its binding to VHH5-05, which is due to its small size, enabling the nanobody to bind deeply into the interior of the SARS-CoV-2 RBD and ACE2 binding epitope with excellent targeting. The present application screens and purifies the nanobody VHH5-05, which not only shows strong binding ability to phages displaying wild-type SARS-CoV-2 RBD, but also shows certain binding ability to phages displaying Beta mutant RBD and Delta mutant RBD. The antibody VHH5-05 of the present application can efficiently neutralize WT pseudovirus, with an IC50 of 0.026 ug / ml. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0015] Figure 1 is a schematic diagram of the process of phage nanobody library RBD-ACE2 competitive panning.

[0016] Figure 2 is a Phage-ELISA identification A450 value result graph of phage monoclonal.

[0017] Figure 3 Figure 6 is the result of VHH phage competing with ACE2 to bind SARS-CoV-2 RBD.

[0018] Figure 4 Figure 7 is the VHH 5-05 Figure 8 is the result of SDS-PAGE identification analysis of induced expression and purification of nanobodies, wherein Figure 4 A is the result of protein expression SDS-PAGE identification, M: standard protein Marker; 1: whole bacteria after induction; 2: supernatant after ultrasonic crushing; 3: crushed sediment after ultrasonic. 4B is the result of protein purification SDS-PAGE analysis, M: standard protein Marker; 1: supernatant after crushing; 2: flow-through; 3: elution.

[0019] Figure 5 Figure 9 is the nanobody VHH 5-05 Figure 10 is the result of specificity of binding SARS-CoV-2 RBD WT.

[0020] Figure 6 Figure 11 is the nanobody VHH 5-05 Figure 12 is the result of affinity of binding SARS-CoV-2 RBD WT.

[0021] Figure 7 Figure 13 is the result of ELISA verification of nanobodies competing with ACE2 to bind RBD.

[0022] Figure 8 Figure 14 is the result of structural analysis of RBD-VHH5-05 and RBD-hACE2 complex and comparison of their epitopes, wherein Figure 8 a is the crystal structure of hACE2-SARS-CoV-2 RBD complex, RBD and ACE2 are shown in blue and light pink, respectively; Figure 8 b is the superimposition of VHH5-05 and RBD, VHH5-05 is shown in cyan; Figure 8 c is the 2D map of the complex.

[0023] Figure 9 Figure 15 is the result of SARS-CoV-2 pseudovirus neutralization ability of VHH 5-05

[0024] Figure 10 Figure 16 is the result of VHH 5-05 Figure 17 is the analysis of VHH binding to recombinant phage displaying RBD of mutant strains of coronavirus. DETAILED DESCRIPTION

[0025] ​In order for those skilled in the technical field to better understand the technical solutions in the present application, the present application will be further described below in conjunction with embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work should fall within the scope of protection of the present application. Embodiment one RBD-ACE2 competition panning of nanobodies

[0026] As shown in Figure 1 A camel-derived nanobody phage library against wild-type SARS-CoV-2 RBD was panned using solid-phase panning, with RBD coated on a high-adsorption plate as the target protein. After adding the phage library, the successfully bound phage was left over after panning. In the first round of panning, phage that did not bind or weakly bound to RBD was removed. In the second and third rounds, free ACE2 protein was added to compete with the phage for binding to the immobilized target protein RBD.

[0027] 100 uL of 5 ug / mL RBD diluted in coating solution was added to a high-adsorption enzyme-labeled plate well, and was coated at 4°C overnight. The next day, after drying, PBST was repeatedly washed five times, 300 uL of blocking buffer (PBST containing 2% BSA) was added, and 2 h of 37°C blocking was performed. At 1 h, the phage library diluted in blocking buffer was pre-blocked in another enzyme-labeled well at 37°C. After washing the antigen-coated well where blocking was completed, the pre-blocked phage nanobodies were transferred into it, and 2 h of 37°C incubation was performed for binding. PBST was used for washing 10 times, and PBS was used for washing 5 times to strictly wash away the unbound phage. 100 ug / mL of trypsin was added to the well to elute the bound phage. The eluted phage was added to 2 mL of TG1 bacteria liquid in the logarithmic growth phase, mixed well, and 37°C incubated for 1 h. Then, 10 mL of 2xYT liquid medium containing 2% glucose and 100 ug / mL ampicillin (Amp) was added, and the culture was shaken until the logarithmic phase. Then, helper phage was added at a multiplicity of infection (MOI) of 20:1, and 37°C incubated for 1 h. After centrifugation, the bacteria were resuspended in 100 ug / mL Amp and 50 ug / mL kanamycin (Kana) 2xYT liquid medium, and were cultured overnight. The next day, after centrifugation, the supernatant was added to 1 / 5 volume of PEG / NaCl, and the phage was precipitated in an ice bath. The phage library after the first round of panning was collected, the titer was determined, and was used for the next round of panning.

[0028] The second, third and fourth rounds of panning were performed by adding 100 uL of 2 ug / mL (1 ug / mL for the third round) ACE2 diluted in blocking solution to the antigen-coated wells, followed by 1 h of pre-incubation at 37°C. The wells were then washed with PBST for 5 times and PBS for 5 times, and the pre-blocked phage library was transferred into the wells for 2 h of incubation at 37°C. The subsequent steps were the same as described above. The three rounds of panning were completed to enrich the phage expressing nanobodies against SARS-CoV-2 RBD on the surface. The material information and recovery rate of the panning process are summarized in Table 1.

[0029] Table 1 Recovery rate after three rounds of panning

[0030]

[0031] Note: The recovery efficiency of each round was calculated by dividing the amount of phage eluted by the amount of phage input. pfu, plaque-forming unit.

[0032] Example Two Phage-ELISA identification of positive clones

[0033] Twenty-four single colonies were randomly picked from the plates after three rounds of screening, inoculated in 2% glucose, 100 ug / mL Amp 2xYT liquid medium, and cultured at 37°C for 8 h. The bacterial solution was inoculated into new 2% glucose, 100 ug / mL Amp medium and cultured to the logarithmic growth phase. The helper phage was added at a MOI of 20:1 and incubated at 37°C for 1 h. The culture was centrifuged to collect the bacterial cells, which were resuspended in 100 ug / mL Amp, 50 ug / mL Kana 2xYT liquid medium and cultured overnight to produce new nanobody phage.

[0034] The enzyme-labeled plate was coated with 0.5 ug / mL SARS-CoV-2 RBD antigen as sample wells, and the blank wells were coated with coating buffer and incubated at 4°C overnight. The next day, the plate was washed with PBST, and 100 uL of blocking buffer was added to each well and incubated at 37°C for 1.5 h. The monoclonal bacterial solution cultured overnight was centrifuged to collect the supernatant, which was diluted ten-fold in a centrifuge tube with blocking buffer and pre-blocked at 37°C for 1 h. After washing the plate, the sample wells and blank wells were added with the phage supernatant after pre-blocking, and incubated at 37°C for 1 h. The plate was washed again, and a HRP-labeled anti-M13 phage secondary antibody (1:8000) was added for incubation. The color was developed, and the absorbance (A 450 ) value at 450 nm was read in a microplate reader. The results are shown in Table 2 and Figure 2 The higher the A450 value, the stronger the binding ability of the phage to SARS-CoV-2 RBD under the same experimental conditions.

[0035] Table 2 Phage-ELISA identification of positive clones assay results

[0036] Monoclonal No. OD450 value Monoclonal No. OD450 value 1 1.322 13 3.132 2 3.037 14 2.795 3 2.154 15 2.940 4 3.070 16 0.342 5 1.372 17 / 6 3.049 18 3.067 7 2.912 19 / 8 2.327 20 / 9 3.029 21 3.116 10 2.576 22 2.914 11 3.061 23 2.954 12 3.152 24 3.072

[0037] Example III Positive clones phage-ACE2 competition ELISA

[0038] RBD-Fc (0.2 ug / well) was coated in microplates in 100 pL coating buffer (pH 9.6) at 4°C overnight. The next day, blocking was performed with 100 pL / well 2% BSA-PBST for 1.5 h. After 3 washes, serially diluted phage solution was added to RBD-Fc and incubated at 37°C for 1 h. HRP-labeled anti-M13 phage secondary antibody (1 :8000) and TMB were used for signal amplification and color development, respectively. After stopping the reaction with 1.0 M HC1, absorbance was measured at 450 nm. Subsequently, a subsaturating (80% of maximal effect) concentration of phage solution was used for the competition ELISA.

[0039] RBD-Fc (0.2 pg / well) was coated for the competition phage ELISA. After blocking for 1.5 h and 3 washes, a subsaturating concentration of VHH phage solution was mixed with 4 pg / ml ACE2-His. The mixture of phage and ACE2-His was added to the RBD-Fc coated wells and incubated for 1 h, after which the steps were as described before.

[0040] Out of the 10 monoclonals selected by phage ELISA, 4 showed the ability to block RBD binding to ACE2, as shown in Figure 3 Table 3 and Table 4, and in combination with the experimental procedure, either phage displaying VHH directly bound to RBD, or phage displaying VHH was mixed with ACE2 before binding to RBD, it was very clear in the results that due to the competition of ACE2 with VHH 5-05 phage for binding to RBD 5-05 phage, the final A450 value decreased compared to RBD binding to VHH 5-05 phage. As shown in Figure 3 Table 3-4, we selected the A5 clone with the strongest blocking ability for further prokaryotic expression and characterization, called VHH 5-05 .

[0041] Table 3 Positive clones phage (RBD + phage)-ACE2 competition ELISA results

[0042]

[0043] Table 4 Phage (RBD+ACE2+phage)-ACE2 competition ELISA results for positive clones

[0044]

[0045] After sequencing, the sequence of the Nanobody VHH5-05 VHH 5-05 (378bp) is: ATGGCCCAGTTGCAGCTCGTGGAGTCCGGAGGAGGCGCGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGAACCATCAGTCACTATCGCATGGGCTGGTACCGCCAACGTCCAAGGGGGCCGCGCGAGAAGGTTGCGATCATTACTATTAATGCTTCGACTGACTATGACGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTTTATCTCCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCGACCCCCCGGGACTGTCTCAGAATGACTACTGGGGGCCGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC

[0046] The corresponding amino acid sequence of the Nanobody VHH5-05 (126 AA) is:

[0047] MAQLQLVESGGGAVQPGGSLTLSCAASGTISHYRMGWYRQRPRGPREKVAII

[0048] TINASTDYDGSVKGRFTISRDNAKNSVYLQMNNLKPEDTAVYYCNTDPPGLS

[0049] QNDYWGPGTQVTVSSAHHSEDP

[0050] Example Four VHH 5-05 Prokaryotic expression and purification of the Nanobody

[0051] Ligation of the VHH using homologous recombination 5-05The fragments were obtained with pET-22b(+) vector fragments to obtain recombinant plasmids, and the BL21(DE3) strain was transformed for prokaryotic expression. Single colonies on the transformed plate were picked and expanded for overnight activation, and the next day the culture was transferred to the logarithmic phase, and IPTG was added to the culture to induce the expression of the fusion protein. The remaining bacterial solution was centrifuged to leave the precipitate, PBS was added to resuspend the bacterial cells, and an ultrasonic cell crusher was used for crushing. After adding 5x SDS loading buffer to the induced whole bacteria, the precipitate and supernatant samples after crushing, and then performing SDS-PAGE band analysis. The soluble protein (His tag) in the supernatant obtained by centrifugation after ultrasonic crushing was subjected to Ni column affinity purification, and the purified product was subjected to SDS-PAGE analysis of protein size and purity. The results are shown in Figs. Figure 4 (4A and 4B), showing that the target protein eluted has high purity and a molecular weight of about 13KD, which is consistent with the theoretical molecular weight.

[0052] Example Five VHH 5-05 Verification of the binding activity of nanobodies

[0053] 1. Nanobody binding specificity and affinity determination for SARS-CoV-2 RBD WT

[0054] As shown in Figs. Figure 5 , the purified VHH 5-05 The binding activity and specificity of the nanobody to SARS-CoV-2 RBD WT antigen were verified by ELISA, and the results showed that the nanobody obtained by panning could specifically bind to SARS-CoV-2 RBD WT as shown in Figs. Figure 5 and Table 5, BCMA is a recombinant protein, B cell maturation protein, as a negative control for SARS-CoV-2 RBD protein, indicating that the nanobody selected can specifically bind to SARS-CoV-2 RBD WT. The binding affinity of the obtained nanobody to SARS-CoV-2 RBD WT was determined by ELISA saturation concentration method, and the EC 5-05 of the nanobody VHH 50 was 0.42 ng / mL, as shown in Figs. Figure 6 and Table 6.

[0055] Table 5 Nanobody binding specificity determination for SARS-CoV-2 RBD WT

[0056]

[0057] Table 6 Nanobody affinity determination for SARS-CoV-2 RBD WT

[0058]

[0059]

[0060] 2. VHH 5-05 Nanobody competes with recombinant ACE2 protein for binding to SARS-CoV-2 RBD WT

[0061] The purified nanobody was mixed with SARS-CoV-2 RBD and simultaneously competed for binding to immobilized ACE2, as described in Figure 7 and Table 7, the results show that VHH 5-05 the conditions under which the RBD was removed from the plate when it was washed, i.e. VHH occupied part of the binding site of RBD to ACE2, making the RBD that could not continue to bind. This further confirms the feasibility of the competitive panning method of this study, according to which the VHH can block the in vitro binding of RBD to ACE2 protein, it is speculated that the binding site of the nanobody to ACE2-RBD is quite coincident, and the results of this step verification can preliminarily infer that the binding site of the nanobody to ACE2-RBD is quite coincident.

[0062] Table 7. VHH 5-05 nanobody competes with recombinant ACE2 protein for binding to SARS-CoV-2 RBD WT

[0063]

[0064] 3. Prediction of VHH 5-05 binding site to RBD using docking simulation

[0065] The accession number of the S protein sequence of SARS-CoV-2 is YP_009724390.1, and the receptor binding motif (RBM) of SARS-CoV-2 is 437-508 aa. The key amino acids in hACE2 that interact with RBM are K31, E35, D38, M82 and K353. See Figure 8 , those corresponding to SARS-CoV-2 are L455, F486, Q493, S494, N501 and Y505, all molecules are shown in cartoon form as shown in Figure 8 a. According to the analysis of the predicted complex from the RBD-VHH 5-05 docking ( Figure 8 b), the molecular docking score is -78.1905, indicating that VHH 5-05 can bind to RBD very well. The 2D plot of protein-molecule interaction displayed by Ligplot+ ( Figure 8 c) intuitively shows the interaction forces, including hydrogen bonds and hydrophobic interactions, indicating that the simulated conformation binds very strongly. The modeling analysis results show that RBD-VHH 5-05Most of the residues on the epitope overlap with the RBD-ACE2 binding interface, especially the key sites F486, Q493 and S494 of SARS-CoV-2 RBD are involved in its binding to VHH 5-05 , benefiting from its small size, enabling the nanobody to bind deeply into the interior of the SARS-CoV-2 RBD and ACE2 binding epitope with excellent targeting, which confirms why it can effectively block the binding of RBD and ACE2.

[0066] 4. Evaluate the in vitro neutralization ability of VHH 5-05 (Pseudovirus neutralization test)

[0067] Prepare 8 3-fold dilutions of VHH 5-05 in DMEM medium, add pseudovirus 1:1 to 110 μL of each antibody dilution. Pseudovirus: incubate the VHH mixture at 37°C for 1 hour, then mix with 293 / hACE2 cells for 48 hours. After lysing the cells with luciferase substrate and collecting RLU values, calculate IC 50 values by fitting RLU to a sigmoidal dose-response curve. As shown in Figure 9 and Table 8, the results show that VHH 5-05 can efficiently neutralize WT pseudovirus, with an IC 50 of 0.026 ug / ml.

[0068] Table 8 Pseudovirus neutralization experiment of antibodies (determination of IC50)

[0069]

[0070]

[0071] 5. Binding of nanobody VHH 5-05 to recombinant phage displaying SARS-CoV-2 mutant RBD

[0072] Prepare recombinant modified M13KO7 phage containing SARS-CoV-2 mutant RBD fragments, use high adsorption enzyme-labeled plates to coat the purified nanobody, add each mutant RBD recombinant phage to bind the antibody, and use qPCR to amplify the gene fragments of the phage bound to the plate to indirectly reflect the specific binding of the nanobody to the recombinant phage. See Table 9 for details.

[0073] Table 9 Binding of antibodies to mutant RBD recombinant phage

[0074]

[0075] The results show that the nanobody VHH5-05 Not only the phage displaying wild type SARS-CoV-2 RBD showed strong binding ability, but also the phage displaying Beta mutant RBD and Delta mutant RBD showed certain binding, such as Figure 10 and Table 9; the copies / uL value can indicate the binding ability of the coated protein to the RBD phage, the higher the value, the stronger the binding ability.

[0076] Those skilled in the art will further appreciate that the application described herein is amenable to more than one equivalent, without more than routine experimentation. Such equivalents are included within the appended claims.

Claims

1. A camelid nanobody or antigen binding fragment thereof targeting a SARS-CoV-2 RBD shared epitope with its receptor ACE2, characterized in that, The nanobody or antigen-binding fragment thereof binds simultaneously to key sites F486, Q493 and S494 of SARS-CoV-2 RBD, the nanobody or antigen-binding fragment thereof being SEQ ID NO: 1 : ATGGCCCAGTTGCAGCTCGTGGAGTCCGGAGGAGGCGCGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGAACCATCAGTCACTATCGCATGGGCTGGTACCGCCAACGTCCAAGGGGGCCGCGCGAGAAGGTTGCGATCATTACTATTAATGCTTCGACTGACTATGACGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCGGTTTATCTCCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCGACCCCCCGGGACTGTCTCAGAATGACTACTGGGGGCCGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC.

2. An isolated nucleic acid molecule encoding the camelid nanobody or antigen-binding fragment thereof of claim 1 that targets a SARS-CoV-2 RBD shared epitope with its receptor ACE2.

3. A composition comprising the camelid nanobody or antigen-binding fragment thereof of claim 1 that targets a SARS-CoV-2 RBD shared epitope with its receptor ACE2.

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