Novel coronavirus sars-cov-2 broad-spectrum neutralizing nanobodies and uses thereof

Abstract

This invention discloses a broad-spectrum neutralizing nanobody against SARS-CoV-2 and its applications. Specifically, it discloses a nanobody that can effectively inhibit multiple SARS-CoV-2 mutant strains. Utilizing phage antibody library technology, this invention successfully obtained a broad-spectrum neutralizing nanobody A2 that specifically binds to the SARS-CoV-2 spike protein RBD. The nanobody of this invention exhibits high affinity for the RBD region; furthermore, it demonstrates good broad-spectrum neutralizing activity against multiple major circulating strains. The nanobody of this invention can be expressed and purified in prokaryotic Escherichia coli with high purity, which is beneficial for production. Furthermore, the physicochemical properties of the nanobody remain unchanged before and after nebulization, showing promising application prospects and significant importance in the fields of preventive and therapeutic inhaled medications and clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a broad-spectrum neutralizing nanobody against SARS-CoV-2 and its applications. More specifically, it relates to a broad-spectrum neutralizing nanobody that effectively inhibits multiple SARS-CoV-2 mutant strains. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a single-stranded RNA virus, spherical in shape with protrusions on its surface, and has four main structural proteins: spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E). Among these, the S protein is one of the main proteins responsible for the virus's "corona" morphology and is closely related to its infectivity. The S protein comprises S1, S2, and a receptor-binding domain (RBD). The S protein, composed of the S1 and S2 domains, is exposed on the SARS-CoV-2 viral capsid and plays a crucial role in viral attachment, fusion, entry, and transmission. Specifically, the receptor-binding domain (RBD) of S1 binds to the SARS-CoV-2 cellular receptor angiotensin-converting enzyme 2 (ACE2), thereby enabling viral entry into the cell. As time goes by, the novel coronavirus continues to evolve. The continuous mutation of the spike protein site enhances its ability to evade immunity, but the hidden sites inside the spike protein trimer remain largely unchanged. Therefore, research on highly conserved sites in the S protein and RBD of SARS-CoV-2 mutant strains will help design viral vaccines and develop new broad-spectrum neutralizing antibody drugs against coronaviruses.

[0003] In the early 1990s, Hamers-Casterman and her team discovered a pure heavy-chain antibody without a light chain in camels. Its variable heavy chain domain (VHH) retained its complete antigen-binding ability, and this is now called a nanobody. Nanobodies can be expressed using prokaryotic cell expression systems, which can greatly reduce antibody production costs. Nanobodies are the smallest known functional single-domain antibodies that can stably bind to antigens, and their binding to antigens at certain specific targets is superior to that of monoclonal antibodies. Nanobodies also exhibit significant stability. These characteristics give them significant advantages as antibody drugs, such as the ability to choose multiple routes of administration, rapid systemic diffusion after drug entry, and good tissue penetration. The research and development of nanobodies has a very broad prospect and great significance in the fields of drug application and clinical diagnostics.

[0004] Target-specific nanobodies can be obtained by screening nanobody phage libraries. Nanobodies obtained by immunizing alpacas, camels, and llamas not only increase the diversity of the obtained nanobodies, but also allow them to systematically recognize discontinuous amino acid fragments and conserved regions of antigens in native protein conformations.

[0005] SARS-CoV-2, as a single-stranded RNA virus, exhibits higher variability compared to DNA viruses. Mutations at key sites on the S protein can affect its binding to receptors, thereby altering viral characteristics such as infectivity, lethality, and immune evasion. With the emergence of SARS-CoV-2 mutant strains, hundreds or even thousands of sites have appeared, leading to the ineffectiveness or significantly reduced neutralizing capacity of monoclonal antibody drugs. Nanobodies, due to their small size and high specificity, can enter the spike protein trimer interface and bind to latent epitopes, thereby altering the spike protein trimer state and inactivating it, thus exhibiting a certain degree of neutralizing effect on the virus. These nanobodies, capable of binding to latent or highly conserved epitopes, can bind to various SARS-CoV-2 mutant strains, demonstrating a broad-spectrum neutralizing effect.

[0006] Nanobodies possess characteristics such as high stability, strong affinity, high specificity, low production cost, and simple humanization, making them an emerging force in antibody therapy and diagnostics. Furthermore, clinical specimen analysis shows that SARS-CoV-2 viral copy numbers are highest in multiple sites of the respiratory tract, while viral loads are lower in the blood. Therefore, direct delivery of biological therapeutic drugs to the site of infection via the respiratory route is an attractive alternative systemic drug delivery method. Due to their small size and excellent physicochemical properties, nanobodies can be developed into nebulized inhalation formulations, theoretically suitable for the treatment of respiratory diseases. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a broad-spectrum neutralizing nanobody that can effectively inhibit the activity of multiple novel coronavirus SARS-CoV-2 mutant strains and its application. The purpose is to solve the described technical subject matter. Those skilled in the art can clearly understand the main technical subject matter of this article through the following description.

[0008] To address the aforementioned technical problems, this invention first provides a nanobody that specifically binds to the SARS-CoV-2 spike protein. The nanobody includes a heavy chain variable region (VHH), which comprises a complementarity-determining region (CDR1), a complementarity-determining region (CDR2), and a complementarity-determining region (CDR3), with the following amino acid sequences:

[0009] Complementary Determinant Region CDR1: AASGYTTT;

[0010] Complementary Determinant Region CDR2: IYTDGTST;

[0011] Complementary determinant region CDR3: AADLAYVGSWYNPASFDY.

[0012] This includes complementarity-determining regions CDR1 (positions 23-30 as shown in SEQ ID No. 1), CDR2 (positions 48-55 as shown in SEQ ID No. 1), and CDR3 (positions 94-111 as shown in SEQ ID No. 1), respectively.

[0013] The nanobody is a broad-spectrum neutralizing nanobody that specifically binds to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein). The nanobody is composed of a heavy chain variable region and is also known as a VHH antibody.

[0014] Furthermore, the nanobody described in this invention also includes a framework region.

[0015] The amino acid sequence of the framework region FR1 of the nanobody A2 is shown in positions 1-22 of SEQ ID No. 1; the amino acid sequence of FR2 is shown in positions 31-47 of SEQ ID No. 1; the amino acid sequence of FR3 is shown in positions 56-93 of SEQ ID No. 1; and the amino acid sequence of FR4 is shown in positions 112-122 of SEQ ID No. 1.

[0016] The amino acid sequence of the heavy chain variable region of the above-mentioned nanobody is shown in SEQ ID No. 1.

[0017] The present invention also provides biomaterials related to the nanobody, wherein the biomaterial is any one of the following:

[0018] (1) A nucleic acid molecule encoding the variable region of the heavy chain of the nanobody;

[0019] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0020] (3) A recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2);

[0021] (4) A recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3);

[0022] (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

[0023] In the above-mentioned biological materials, the nucleic acid molecule is a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 2.

[0024] The DNA molecule shown in SEQ ID No. 2 encodes the single-domain antibody A2 shown in SEQ ID No. 1.

[0025] Variants of the single-domain antibodies described in this invention, exhibiting improved affinity and / or titer, can be obtained using methods known in the art and are included within the scope of this invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve translation efficiency in expression systems used to generate antibodies. Furthermore, polynucleotides comprising sequences whose antibody specificity or neutralizing activity is optimized by applying directed evolution to any nucleic acid sequence of this invention are also within the scope of this invention.

[0026] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC), etc.), viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (such as herpes simplex virus). In one embodiment of the present invention, the vector may specifically be pMECS.

[0027] The microorganisms described herein may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*. In one embodiment of the present invention, the microorganism may specifically be *Escherichia coli* wk6.

[0028] The cell (host cell) refers to a cell that can be used to introduce the vector, including but not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells), plant cells, or prokaryotic cells.

[0029] In one embodiment of the present invention, the recombinant vector may specifically be pMECS-A2.

[0030] The recombinant vector pMECS-A2 is obtained by replacing the DNA molecule between the PstI and NotI restriction sites of the expression vector pMECS with the DNA molecule of A2, resulting in the recombinant prokaryotic expression vector pMECS-A2 expressing the histidine-fused nanobody A2. The DNA molecule of A2 has PstI and NotI restriction sites added to the 5' end (position 1 of SEQ ID No. 2) and 3' end (position 364 of SEQ ID No. 2), respectively.

[0031] In an embodiment of the present invention, the recombinant microorganism (recombinant bacteria) is specifically WK6 / pMECS-A2.

[0032] The recombinant microorganism WK6 / pMECS-A2 contains the DNA molecule shown in SEQ ID No. 2 and expresses a nanobody A2 with a histidine tagging amino acid sequence. The recombinant microorganism WK6 / pMECS-A2 is a recombinant bacterium obtained by electroporating the recombinant vector pMECS-A2 into Escherichia coli WK6.

[0033] After IPTG induction for 17 hours, antibody protein was collected using a freeze-thaw method, and the antibody was purified by nickel column chromatography. The purified nanobody of this invention has a purity greater than 90%. Figure 3 As shown.

[0034] In Example 3 of the present invention, the nanoantibody of the present invention was analyzed by bio-layer interference (BLI) technology to detect biomolecular interactions, and the antibody showed high affinity for multiple SARS-CoV-2 viruses.

[0035] In embodiments of the present invention, the nanoantibody of the present invention can neutralize SARS-CoV-2 pseudovirus and its various mutant strains.

[0036] The neutralized nanobodies can be used as drugs to improve, prevent or treat diseases caused by SARS-CoV-2 infection, or to inhibit SARS-CoV-2 infection.

[0037] The present invention also provides a pharmaceutical composition comprising the aforementioned nanobody and a pharmaceutically acceptable carrier.

[0038] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant.

[0039] The pharmaceutical composition has a neutralizing antiviral effect, inhibiting or neutralizing SARS-CoV-2 activity. The pharmaceutical composition is used to improve, prevent, or treat illness caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

[0040] Furthermore, the pharmaceutical composition of the present invention comprises a first antibody and a second antibody or an antigen-binding fragment thereof, wherein the first antibody is a single-domain antibody of the present invention, and the second antibody is any antibody or antigen-binding fragment thereof that neutralizes SARS-CoV-2 virus infection.

[0041] In the embodiments of the present invention, the physicochemical properties of the nanoantibody did not change before and after being administered via an atomization drug delivery device (Yuyan Instruments, YAN30012).

[0042] The present invention also provides the use of the nanobodies and / or the biomaterials described herein in the preparation of drugs that inhibit or neutralize SARS-CoV-2 activity.

[0043] In the above applications, the drugs that inhibit or neutralize SARS-CoV-2 activity are used to improve, prevent or treat diseases caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

[0044] The present invention also provides the use of the nanobodies and / or the biomaterials described herein in the preparation of products for detecting SARS-CoV-2 and / or the spike protein of SARS-CoV-2.

[0045] The use of the nanobody of the present invention to monitor the quality of an anti-SARS-CoV-2 vaccine by detecting whether the antigen contains a specific epitope with the correct conformation is also contemplated within the scope of the present invention.

[0046] The products used to detect SARS-CoV-2 levels and / or the spike protein of SARS-CoV-2 include those that detect antigen-antibody binding using methods such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination, or immunoturbidimetry.

[0047] The present invention also provides the use of the nanobody and / or the biomaterial described herein in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by SARS-CoV-2 infection.

[0048] In the above applications, the disease caused by SARS-CoV-2 infection is a respiratory infection. This respiratory infection can be a respiratory tract infection and / or a lung infection.

[0049] In the above applications, the product may be a reagent or a reagent kit.

[0050] The reagents or kits contain any of the single-domain antibodies or combinations thereof described herein. The kits may be chemiluminescent immunoassay kits, enzyme-linked immunosorbent assay kits, colloidal gold immunoassay kits, or fluorescent immunoassay kits, but are not limited thereto.

[0051] In this article, the terms "single-domain antibody that specifically binds to the SARS-CoV-2 spike protein" and "anti-RBD single-domain antibody" have the same meaning and can be used interchangeably.

[0052] In this article, the term "neutralizing antibody" refers to an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of pathogens to initiate and / or maintain infection within the host. As described herein, these antibodies may be used alone or in combination, after appropriate formulation, as prophylactic or therapeutic agents, in conjunction with live vaccines, as diagnostic tools, or as manufacturing tools.

[0053] This invention utilizes phage display technology to enrich phages specific to the RBD protein antigen through multiple rounds of screening, identify positive clones and obtain the corresponding coding sequences, express them in *E. coli* WK6, and purify them using affinity chromatography. This successfully yielded a broad-spectrum neutralizing nanobody A2 that specifically binds to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The nanobody of this invention exhibits high affinity for the RBD region and demonstrates good broad-spectrum neutralizing activity against various major prevalent strains (WT strain, Alpha mutant strain, Beta mutant strain, Gamma mutant strain, Delta mutant strain, and Omicron mutant strain). Furthermore, the nanobody can be expressed and purified in prokaryotic *E. coli* cells with high purity, which is beneficial for production. Additionally, the physicochemical properties of the nanobody remain unchanged before and after nebulization, making it promising and significant for applications as a preventative and therapeutic inhaled drug, as well as in clinical diagnosis. Attached Figure Description

[0054] Figure 1 This is a diagram showing the distribution of the A2 framework region and complementarity-determining region of the nanobody.

[0055] Figure 2 The image shows the pMECS-A2 nanobody.

[0056] Figure 3 This is an immunoblot image of purified nanobody A2.

[0057] Figure 4The statistical results show the affinity of nanobody A2 with pseudoviruses WT, Alpha, Beta, Gamma, Delta, Omicron (B1.1.529), Omicron (BA.5), and Omicron (BQ.1.1).

[0058] Figure 5 The results show the statistical results of the neutralizing activity of nanobody A2 against WT, Alpha, Beta, Gamma, Delta, Omicron (B1.1.529), Omicron (BA.5), and Omicron (BQ.1.1) pseudoviruses.

[0059] Figure 6 This is a Western blot experiment to verify the A2 nanobody before and after atomization. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Example 1: Animal Immunization and Phage Library Screening

[0062] We purchased the following proteins from Beijing Yiqiao Shenzhou Technology Co., Ltd.: SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein (Catalog No.: 40591-V08H), SARS-CoV-2 Spike S1+S2 (T19R, G142D, E156G, HR157-158 deletion, L452R, T478K, D614G, P681R, D950N) Protein (ECD, His Tag) (Catalog No.: 40589-V08B16), and SARS-CoV-2 Spike S1+S2 (G75V, T76I, R246N, (247, 253) deletion, L452Q, F490S, D614G, T859N) Protein (ECD, His Tag). Tag (Catalog No.: 40589-V08B23) contains three protein antigens; camels were immunized using Freund's complete adjuvant + 0.15g of each antigen, with each immunization spaced two weeks apart, for a total of 5 immunizations. OD was measured after immunization using serum diluted 5000-fold. 450 Value > 1, and greater than the OD value of negative serum 450A value of 2 times was used to determine normal immunity. Lymphocytes were then isolated and total RNA extracted. Two rounds of nested PCR were performed to amplify VHH. The pHEN phage vector was digested and ligated with the digested VHH fragment. The ligation product was electroporated into TG1 competent cells, and immediately revive in 2YT medium, yielding a total of 100ml of revival product. The cells were revised at 37℃ for 60min for library construction. Twenty-four single colonies were randomly selected from the plate for culture PCR. Agarose gel electrophoresis showed more than 23 target bands of approximately 300bp, with an insertion rate greater than 95%, indicating a satisfactory antibody library positivity rate. Library screening was then performed, and phages containing antibody genes were enriched after two rounds of screening. Multiple clones from each library were selected for PE-ELISA identification. Positive clones were sequenced, ultimately yielding more than 200 nanobodies with different sequences. Nanobodies with blocking activity were initially screened from the supernatant and expressed in *E. coli* for affinity and neutralizing activity testing. Nanobody A2, exhibiting high affinity and good neutralizing activity, was selected. It is composed sequentially of the frame region FR1, complementarity-determining region CDR1, frame region FR2, complementarity-determining region CDR2, frame region FR3, complementarity-determining region CDR3, and frame region FR4. Specific information is as follows: Figure 1 As shown, its amino acid sequence is shown in SEQ ID No. 1, and the nucleic acid molecule encoding nanobody A2 is shown in SEQ ID No. 2.

[0063] Example 2

[0064] 1. Construction of a prokaryotic expression vector for expressing the single-domain antibody B11

[0065] The DNA molecule between the PstI and NotI restriction sites of the expression vector pMECS was replaced with the DNA molecule of A2 to obtain the recombinant prokaryotic expression vector pMECS-A2 expressing the histidine-fused nanobody A2. (See diagram below.) Figure 2 As shown, the DNA molecule of A2 has PstI and NotI restriction sites added to the 5' end (position 1 of SEQ ID No. 2) and the 3' end (position 364 of SEQ ID No. 2), respectively.

[0066] 2. Expression and purification of anti-RBD nanobodies

[0067] The constructed pMECS-A2 vector (such as...) Figure 2 (As shown), the bacteria were electroporated into WK6 competent cells to obtain recombinant strain WK6 / pMECS-A2. The recombinant strain WK6 / pMECS-A2 was inoculated into 5 ml of LB liquid medium and cultured at 37°C and 220 rpm until OD500. 600=0.6, and then the bacterial culture was added to 500ml of 2YT medium containing MgCl2, glucose and AMP+ (ampicillin), and IPTG was added to a final concentration of 0.3mM. The culture was carried out at 28℃ and 220rpm for expression. After 17h, the bacterial cells were collected by centrifugation and stored at -80℃ for later use.

[0068] The obtained bacterial cells were subjected to a freeze-thaw cycle to obtain the supernatant. The supernatant was then passed through an NI-NTA agarose column, followed by washing with 10 column volumes of PBS buffer to remove unbound protein. Finally, the protein was eluted with 15 ml of washing buffer (500 mM imidazole buffer (PBS)). One tube was collected every 5 ml. The purified anti-RBD nanobody A2 was identified by Western blotting (incubation with HIS antibody). Figure 3 (As shown).

[0069] The purified anti-RBD nanobody was placed in a 7KD dialysis bag and dialyzed overnight with PBS to obtain nanobody in PBS buffer.

[0070] Example 3: Affinity determination of broad-spectrum anti-RBD neutralizing A2 nanobodies

[0071] Antibody to be tested: A2 nanobody purified in Example 2.

[0072] Bio-Layer Interferometry (BLI) was used to detect the affinity of A2 nanobodies for antigens and antibodies in the RBD regions of various mutant strains. This label-free technique is based on the principle of optical interference. It offers advantages such as simple operation, short detection time, low sample consumption, and direct use in analysis and detection without the need for manual labels. Through real-time monitoring of the optical interference signal, BLI technology can be widely applied to the analysis and rapid detection of biomolecular interactions.

[0073] When determining the interaction between SARS-CoV-2 RBD protein and A2 nanobody, the antigen RBD protein was first coated onto a chip sensor, and the nanobody A2 was used as the mobile phase to determine the affinity constant.

[0074] Interaction experiment: The same molar amount of biotinylation reagent was added to the RBD antigen; the RBD antigen containing the biotinylation reagent was completely added to the desalting column; 200 μl of 1×PBS was added for flow-through to remove excess biotin, and the flow-through solution containing the biotinylated RBD antigen was collected; the flow-through solution containing the biotinylated RBD antigen was diluted and subjected to affinity testing. The biotinylated RBD antigen was specifically captured using an SA chip, and after the signal reached 4.5 nM, it bound to A2 nanobodies at different concentrations (200 nm, 100 nm, 50 nm, 25 nm, 12.5 nm).

[0075] The program settings are shown in Table 1; and the binding constant (ka), dissociation constant (kd), and affinity constant (KD) are calculated using the software fortebio data analysis 12.0.

[0076] Table 1

[0077] 1 Baseline Baseline 60 2 Association Association 100 3 Dissociation Dissociation 200 4 Custom Custom 3

[0078] The results are as follows Figure 4 As shown in Table 2, a lower KD value indicates a stronger affinity between the antigen and antibody. The A2 nanobody in this study exhibits affinity constants between 0.001 nmol / L and 58.6 nmol / L for various RBD proteins detected. Overall, the A2 nanobody of this invention demonstrates ideal affinity for the RBDs of various SARS-CoV-2 mutant strains.

[0079] Table 2

[0080]

[0081]

[0082] Example 4: Determination of the neutralizing activity of SARS-CoV-2 pseudoviruses against the broad-spectrum A2 nanobody against RBD

[0083] Antibody to be tested: A2 nanobody purified in Example 2.

[0084] SARS-CoV-2 pseudoviruses are novel viral particles assembled from the replication core elements of retroviruses and the envelope spike glycoprotein (S protein) of the SARS-CoV-2 virus. Compared to true viruses, pseudoviruses can only infect cells once, have a broad host range, high titers, and are not easily inactivated by serum complement, making them a viable alternative for neutralization detection. The ability of pseudoviruses to infect cells depends on the type and characteristics of their encapsulating glycoproteins, making them an ideal tool for studying the efficiency of neutralizing antibody inhibition, receptor utilization, and invasion and infection mechanisms of SARS-CoV-2.

[0085] The pseudoviruses of the SARS-CoV-2 mutant strain were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., including SARS-CoV-2 (2019-nCoV) (B.1.617.2) Spike Pseudovirus (Catalog No.: PSV011), SARS-CoV-2 (B.1.1.529) Spike Pseudovirus (Catalog No.: PSV016), and SARS-CoV-2 B.1.1.529 sublineage BA.4 / BA.5 / BA.5.2 (Omicron) Spike Pseudovirus (Catalog No.: PSV022).

[0086] Purchased SARS-CoV-2 pseudoviruses were mixed with nanobodies at different dilutions (starting concentration 100 μg / ml, 10 serial dilutions, 2-fold), and then added to 96-well plates pre-inoculated with 293T-ACE2 cells, and incubated for 48 hours. SARS-CoV-2 pseudoviruses contain a luciferase reporter gene; the infectivity and level of the pseudoviruses can be determined by detecting the luciferase reporter gene. Using the Promega Bright-Glo™ luciferase assay kit (catalog number: pro-E2610), cells were lysed according to the product instructions, and the reporter gene activity in the cell lysates was measured. Raw luciferase readings were converted to percentage data for plotting.

[0087] The results are as follows Figure 5 As shown, the results indicate that the nanobody A2 obtained in this invention has significant inhibitory effects on the main circulating strains of SARS-CoV-2 (Alpha mutant, Beta mutant, Gamma mutant, Delta mutant, and Omicron mutant), and exhibits strong inhibitory and neutralizing activity against SARS-CoV-2 pseudoviruses. This demonstrates that the nanobody of this invention has the ability to broadly inhibit infection by multiple mutant strains of SARS-CoV-2 pseudoviruses and is a neutralizing antibody with broad-spectrum activity. Specifically, the half-maximal inhibitory concentration (IC50) is... 50 See Table 3.

[0088] Table 3

[0089] SARS-CoV-2WT 14.98 Alpha 13.82 Beta 2.59 Gamma 3.88 Delta 4.3 Omicron (B1.1.529) 3.34 Omicron (BA.5) 23.64 Omicron(BQ.1.1) 46.19

[0090] Example 5: Performance evaluation of A2 broad-spectrum neutralizing nanobody after atomization

[0091] The nanobody administered via nebulization (Yuyan Instruments) was collected in 150 μl in a sterile 1.5 ml EP tube. Western blotting was performed on the nanobody samples before and after nebulization. 10 μl of the sample was added and incubated with the same concentration of anti-His antibody. The results are as follows: Figure 6 As shown, the nanobody bands after atomization are consistent with those before atomization, and no degradation is observed.

[0092] The remaining 100 μl of nebulized nanobody from the above steps was compared with the same volume of unnebulized nanobody for ELISA affinity assay. PBS buffer was used as a negative control. Table 4 shows A, B, and C as parallel experiments. The results are shown in Table 4.

[0093] Table 4

[0094] A2 antibody before nebulization 0.438 0.535 0.625 After A2 antibody nebulization 0.444 0.538 0.649 negative control 0.17

[0095] The data in Table 4 show that the affinity of the nanobodies after atomization is consistent with that of the nanobodies before atomization, and their performance remains unchanged.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody that specifically binds to the SARS-CoV-2 spike protein, characterized in that, The nanobody includes a heavy chain variable region, which comprises a complementarity-determining region (CDR1), a complementarity-determining region (CDR2), and a complementarity-determining region (CDR3), with the following amino acid sequences: Complementary Determinant Region CDR1: AASGYTTT; Complementary Determinant Region CDR2: IYTDGTST; Complementary determinant region CDR3: AADLAYVGSWYNPASFDY.

2. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

1.

3. A biomaterial related to the nanobody of claim 1 or 2, characterized in that, The biomaterial is any one of the following: (1) A nucleic acid molecule encoding the variable region of the heavy chain of the nanobody as described in claim 1 or 2; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) Recombinant microorganisms containing the nucleic acid molecule described in (1), or recombinant microorganisms containing the expression cassette described in (2), or recombinant microorganisms containing the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

4. The biomaterial as described in claim 3, characterized in that, The nucleic acid molecule is a DNA molecule with a nucleotide sequence as shown in SEQ ID No.

2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the nanobody as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

6. The use of the nanobody of claim 1 or 2 and / or the biomaterial of claim 3 or 4 in the preparation of a drug that inhibits or neutralizes SARS-CoV-2 activity; The drugs that inhibit or neutralize SARS-CoV-2 activity are used to improve, prevent or treat diseases caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

7. The use of the nanobody of claim 1 or 2 and / or the biomaterial of claim 3 or 4 in the preparation of products for detecting the spike protein of SARS-CoV-2.

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