Nanobody against novel coronavirus and application thereof

Abstract

The application discloses a nanobody for a novel coronavirus and a preparation method and application thereof. A VHH chain of the nanobody comprises CDR1 of an amino acid sequence as shown in SEQ ID NO:1, CDR2 of an amino acid sequence as shown in SEQ ID NO:2 and CDR3 of an amino acid sequence as shown in SEQ ID NO:3, and specific sequence information is shown in the application. The nanobody has broad-spectrum neutralization activity, can effectively inhibit the novel coronavirus from infecting a host cell, and the neutralization capacity can reach a level below nanomolar.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine; specifically, it relates to a nanobody targeting the novel coronavirus, encoding an isolated nucleic acid thereof, an expression vector containing the nucleic acid, a host cell containing the vector, a pharmaceutical composition containing the antibody, a chimeric antigen receptor, a genetically modified cell, an antibody-drug conjugate, a reagent kit and a kit, and the application of the nanobody in the preparation of drugs for the prevention and treatment of diseases caused by the novel coronavirus. Background Technology

[0002] Coronaviruses belong to the order Nidovirales, family Coronaviridae, and genus Coronavirus. They are enveloped viruses with linear single-stranded positive-sense RNA (ssRNA) as their genetic material and are widely distributed in nature (Weiss et al., 2005). To date, seven coronaviruses that infect humans have been identified: HCoV-229E (α genus, 1965), HCoV-OC43 (β genus, 1967), SARS-CoV-1 (β genus, 2003), HCoV-NL63 (α genus, 2004), HCoV-HKU1 (β genus, 2005), MERS-CoV (β genus, 2012), and SARS-CoV-2 (β genus, 2019). All seven coronaviruses can cause respiratory illnesses in humans. Among them, HKU1, NL63, OC43, and 229E cause mild respiratory symptoms and are the second largest category of viruses associated with the common influenza. SARS-CoV-1, MERS-CoV, and SARS-CoV-2 can cause severe respiratory illnesses (Corman et al., 2018), with SARS-CoV-2 being particularly contagious and more dangerous.

[0003] SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its overall morphology is round or oval, approximately 80-120 nM in diameter, and it is composed of four structural proteins: the envelope protein (E protein), the membrane protein (M protein), the spike protein (S protein), and the nucleocapsid protein (N). The N protein encapsulates the viral nucleic acid material, while the S, E, and M proteins together form the viral capsid. The S protein, expressed on the surface of the SARS-CoV-2 viral particle, is a homotrimer composed of the S1 and S2 subunits linked by non-covalent bonds (Wrapp et al., 2020b). The viral particle binds to host cells expressing ACE-2 through the receptor-binding domain (RBD) of its surface spike protein, and then fuses with the host cell through the functional domain of the S2 subunit to infect the host. After the SARS-CoV-2 virus invades the human body, it triggers an immune response, causing the body to secrete interferon (IFN) and chemokines to inhibit viral proliferation and recruit leukocytes to attack the virus; or to produce specific antibodies to block the binding of the viral S protein to the body's ACE-2 for reinfection. However, the S protein is prone to forming glycoproteins due to the presence of numerous N-glycosylation sites. Extensive glycosylation can alter the protein's spatial structure to some extent, blocking or destroying antigenic epitopes, inhibiting the body's immune response, and thus allowing the virus to evade immunity. In addition, SARS-CoV-2 has undergone numerous mutations, is highly infectious, and can survive for up to 9 days at room temperature after leaving the host, and can remain infectious for 2 hours to 9 days on air or on surfaces of different materials (Kampf et al., 2020).

[0004] Vaccines are one of the effective preventive measures against infectious diseases. However, it often takes up to two months for an individual to develop sufficiently strong immunity after vaccination. For some elderly or adolescent populations with weakened immune systems or who cannot effectively produce antibodies, vaccines may not provide the necessary protection. Broad-spectrum neutralizing antibodies can rapidly respond to sudden large-scale viral infection events or promptly address viral mutations or immune escape phenomena during an epidemic, offering both preventative and therapeutic benefits. They can compensate for the limitations of vaccines and also serve as a reserve of antibody candidate molecules for potential future outbreaks of new mutant strains or other sudden viral infection events.

[0005] Currently, most SARS-CoV-2 neutralizing antibodies achieve their preventative and therapeutic effects primarily by competitively blocking the binding of the viral redox domain (RBD) to host cell ACE-2, thereby inhibiting viral infection. A significant number of these are traditional human IgG neutralizing antibodies targeting RBD or non-RBD binding epitopes isolated from the serum of recovered SARS-CoV-2 patients (Brouwer et al., 2020; Cao et al., 2020; Hansen et al., 2020; Liu et al., 2020; Robbiani et al., 2020; Wec et al., 2020; Wrapp et al., 2020a; Xiang et al., 2020). However, these traditional antibodies are costly to produce and expensive, hindering large-scale deployment; furthermore, the impact of antibody-dependent enhancement (ADE) needs to be assessed. Furthermore, the broad-spectrum neutralizing activity of neutralizing antibodies isolated from recovered patient serum is often limited by viral strain variations. Developing broad-spectrum neutralizing antibodies to prevent immune escape caused by amino acid mutations in the viral S protein is currently a key focus in neutralizing antibody drug development. Based on differences in their mechanisms of action and antigen-binding epitopes, developing antibodies targeting conserved antigen-binding epitopes of the viral S protein, or combinations of two mechanisms of action and different epitopes, holds promise for developing broad-spectrum neutralizing antibodies against the novel coronavirus. This could address immune escape caused by viral mutations and potential new outbreaks in the future.

[0006] Most existing neutralizing antibodies against SARS-CoV-2 are traditional monoclonal antibodies isolated from recovered COVID-19 patients. These traditional antibodies are expensive to produce, hindering their large-scale application in pandemic infectious diseases; furthermore, the impact of antibody-dependent enhancement needs to be assessed. Additionally, the broad-spectrum neutralizing activity of neutralizing antibodies isolated from the serum of recovered patients is often limited by factors such as viral strain mutations. Developing broad-spectrum neutralizing antibodies to prevent immune escape due to amino acid variations in the viral S protein is currently a key focus in neutralizing antibody drug development.

[0007] Nanobodies (Nb) are a novel type of antibody derived from the variable domains of heavy chain antibodies in camelids. High-quality Nb can overcome the risk of Fc-related antibody-dependent enhancement (ADE) and are promising candidates for neutralizing antibody therapy. Nb consists of four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The number of amino acids in their CDR regions is generally longer than that of conventional human or mouse CDR3, allowing them to form convex ring structures (conventional antibodies typically have concave or flat antigen-binding sites). The presence of numerous CDR rings exposed to the solvent enhances the specificity and affinity for antigen binding. Furthermore, due to their small molecular weight, they can more easily bind to some difficult-to-reach antigenic epitopes than conventional antibodies (Vanlandschoot et al., 2011b; Desmyter et al., 2001), which is beneficial for screening broad-spectrum neutralizing antibodies. The four hydrophilic residues in the FR2 region of Nb replace the four hydrophobic residues in the FR2 region of traditional antibodies, resulting in higher water solubility. The internal disulfide bonds enhance its heat and acid / alkali resistance compared to traditional antibodies, reducing its polymerization tendency. It retains biological activity even after prolonged storage under high temperature or strong denaturing conditions, exhibiting higher in vitro stability (Vanlandschoot et al., 2011b; Muyldermans et al., 1994; Hamers-Casterman et al., 1993). It can be amplified and expressed in large quantities using prokaryotic and yeast systems, resulting in relatively lower production costs. The Nb gene sequence is highly homologous to the human VH gene family 3 sequence (VH3), exhibiting relatively low immunogenicity in humans. The presence of the nanobody drug caplacizumab (trade name: Cablivi) on the market demonstrates the druggability and application value of nanobodies (Duggan, 2018), indicating significant development potential. Summary of the Invention

[0008] To address the lack of antibodies targeting the novel coronavirus in existing technologies, this invention provides a nanobody targeting the novel coronavirus and its applications.

[0009] This invention utilizes in vitro passive cross-immunization with camel-like animals, combined with phage display technology, to develop broad-spectrum neutralizing nanoantibodies against SARS-CoV-2 and its variants. Furthermore, it has undergone multivalent modification to enhance the antibody's permeability in vivo and prolong its half-life, enabling it to effectively neutralize and eliminate the virus. This provides a reserve of antibody drugs for epidemic prevention and control and for potential new emergencies in the future.

[0010] The technical solution of this invention mainly involves: cross-immunizing alpacas with wild-type and mutant SARS-CoV-2 proteins, and detecting and evaluating to ensure that high-affinity and broad-spectrum serum antibody titers are obtained in the alpacas. After immunization, peripheral blood is collected from alpacas, and plasma and peripheral blood lymphocytes (PBMCs) are separated. Total RNA is extracted from PBMCs and reverse transcribed into cDNA. Alpaca nanobodies (VHHs) are then amplified by multiplex PCR, and a nanobodies phage library is constructed. A multi-antigen cross-panning method is then used to screen for SARS-CoV-2 RBD nanobodies with high affinity and broad-spectrum activity. The nanobodies are then subjected to multivalent modification and bioactivity testing and evaluation, assessing antibody binding affinity, broad-spectrum activity, antigen-binding epitopes, and antiviral neutralizing activity.

[0011] The following is the technical solution provided by the present invention to solve the above-mentioned technical problems.

[0012] The present invention provides a nanobody against the novel coronavirus, wherein the VHH chain of the nanobody comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:3;

[0013] Alternatively, the VHH chain of the nanobody comprises CDR1 with an amino acid sequence as shown in SEQ ID NO:4, CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and CDR3 with an amino acid sequence as shown in SEQ ID NO:6;

[0014] Alternatively, the VHH chain of the nanobody may contain amino acid sequences such as CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9.

[0015] Preferably, the nanobody further includes a framework region FR, the FR comprising: FR1 with an amino acid sequence as shown in SEQ ID NO:10, FR2 with an amino acid sequence as shown in SEQ ID NO:11, FR3 with an amino acid sequence as shown in SEQ ID NO:12, and FR4 with an amino acid sequence as shown in SEQ ID NO:13;

[0016] Alternatively, the nanobody may further include a framework region FR, the FR comprising: FR1 with an amino acid sequence as shown in SEQ ID NO:14, FR2 with an amino acid sequence as shown in SEQ ID NO:15, FR3 with an amino acid sequence as shown in SEQ ID NO:16, and FR4 with an amino acid sequence as shown in SEQ ID NO:17.

[0017] Alternatively, the nanobody may further include a framework region FR, the FR comprising: FR1 with an amino acid sequence as shown in SEQ ID NO:18, FR2 with an amino acid sequence as shown in SEQ ID NO:19, FR3 with an amino acid sequence as shown in SEQ ID NO:20, and FR4 with an amino acid sequence as shown in SEQ ID NO:13.

[0018] In one specific embodiment, the VHH chain comprises an amino acid sequence as shown in SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0019] The present invention also provides an antibody against the novel coronavirus, comprising one or more nanobodies against the novel coronavirus as described above. The antibody is preferably a monomer, a bivalent antibody, or a multivalent antibody.

[0020] The antibody preferably contains the sequence shown in SEQ ID NO:27.

[0021] The antibody may preferably be a bispecific antibody or a multispecific antibody.

[0022] The present invention also provides a chimeric antigen receptor comprising the nanobody or the antibody as described above.

[0023] The present invention also provides an isolated nucleic acid that encodes a nanobody or an antibody as described above.

[0024] The present invention also provides a recombinant expression vector comprising the isolated nucleic acid as described above.

[0025] Preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0026] The present invention also provides a transformant comprising the recombinant expression vector as described above, or having the isolated nucleic acid as described above integrated into its genome.

[0027] Once a recombinant expression vector or DNA sequence for expression has been prepared, the recombinant expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0028] The present invention also provides a method for preparing nanobodies or antibodies against the novel coronavirus, which includes culturing a transformant as described above and obtaining the nanobodies or antibodies from the culture.

[0029] The present invention also provides another transformant, namely a genetically modified cell comprising the nanobodies, antibodies, or chimeric antigen receptors described above. The originating host of the transformant is a cell. Preferably, the cell is a mammalian cell, such as human 293 cells, CHO cells, or T cells.

[0030] Preferably, the cells are mammalian cells such as human 293 cells, CHO cells, or T cells.

[0031] The present invention also provides an antibody-drug conjugate comprising the nanobody or antibody as described above, and a cytotoxic agent.

[0032] Preferably, the cytotoxic agent is MMAF or MMAE.

[0033] The present invention also provides a pharmaceutical composition comprising the nanobody described above, or the antibody, chimeric antigen receptor, or antibody-drug conjugate described above.

[0034] Preferably, the pharmaceutical composition further includes other antibodies against SARS-CoV-2, or small molecule drugs, nucleic acid drugs, or antibodies targeting other viruses for the treatment of SARS-CoV-2.

[0035] In some embodiments, the pharmaceutical compositions or formulations of the present invention comprise suitable pharmaceutically acceptable carriers, such as pharmaceutical excipients, including buffers, as known in the art. As used in this invention, a "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Suitable pharmaceutical carriers for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextran and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For information on the use and applications of excipients, see also “Handbook of Pharmaceutical Excipients”, 5th edition, R.C. Rowe, P.J. Seskey and S.C. Wen, Pharmaceutical Press, London, Chicago. The compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers, if desired. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, saccharin. Pharmaceutical formulations or compositions comprising the invention can be prepared by mixing the antibody or its antigen-binding fragment of the invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of lyophilized formulations or aqueous solutions. The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, said active ingredient being required for the specific indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody or antigen-binding fragment of the present invention, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0036] The present invention also provides a kit for detecting the novel coronavirus, which includes the nanobody or the antibody as described above;

[0037] Preferably, the kit further includes (i) a means for administering the nanobody or antibody; and / or (ii) instructions for use.

[0038] The present invention also provides a medicine box set, which includes medicine box A and medicine box B, wherein:

[0039] The kit A contains the nanobody, the antibody, the pharmaceutical composition, the chimeric antigen receptor, the genetically modified cell, or the antibody-drug conjugate as described above.

[0040] The kit B contains other antibodies against the novel coronavirus or a pharmaceutical composition containing said other antibodies against the novel coronavirus, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, diagnostic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0041] The present invention also provides the use of the nanobodies, antibodies, chimeric antigen receptors, nucleic acids, recombinant expression vectors, transformants, antibody-drug conjugates or pharmaceutical compositions described above in the preparation of medicaments for treating and / or preventing diseases or symptoms caused by the novel coronavirus.

[0042] The tenth aspect of the present invention provides a nanobody, antibody, chimeric antigen receptor, antibody-drug conjugate or pharmaceutical composition as described above for the treatment of diseases related to the novel coronavirus.

[0043] The eleventh aspect of the present invention provides a method for treating diseases related to the novel coronavirus, characterized in that an effective amount of the nanobody, antibody, chimeric antigen receptor, antibody-drug conjugate, or pharmaceutical composition as described above is administered to a subject in need.

[0044] As used herein, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, "therapeutic effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function.

[0045] The method may also be a combination therapy, which includes administering the drugs described above to patients in need, and also administering a second therapeutic agent; the second therapeutic agent preferably comprises other antitumor antibodies or a pharmaceutical composition comprising said other antitumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0046] This invention also provides a method for immunoassay or determination of the novel coronavirus, comprising using the nanobody of this invention, mixing the antibody with the sample to be tested. This method can be used for non-diagnostic purposes or for diagnostic purposes. Non-diagnostic applications include, for example, in scientific research, detecting the presence of the novel coronavirus in samples in a laboratory; or, in screening new drugs, detecting the presence of the novel coronavirus in the components used for drug screening.

[0047] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0048] The reagents and raw materials used in this invention are all commercially available.

[0049] The positive and progressive effects of this invention are as follows:

[0050] This invention utilizes multi-antigen cross-immunization of alpacas with SARS-CoV-2 and its variants to obtain broad-spectrum and high-affinity immune serum titers. Then, using phage display technology, multi-antigen cross-selection and screening were performed to identify nanoantibodies with broad-spectrum neutralizing activity. Specifically:

[0051] (1) The RBD nanobodies obtained by screening through multi-antigen cross-immunization and panning have broad-spectrum binding activity and can effectively bind to SARS-CoV-2 and several highly infectious and harmful viral variants, such as the Alpha coronavirus mutant strain (B.1.1.7) from the UK, the Beta coronavirus mutant strain (B.1.351) from South Africa, the Gamma coronavirus mutant strain (P.1) from Brazil, and the Delta coronavirus mutant strain (B.1.617.2) from India, which has recently experienced outbreaks in various countries.

[0052] (2) The RBD nanobody of the present invention can be expressed efficiently in pronuclear cells. Compared with the expression of traditional antibodies in eukaryotic mammalian cells, the production cost is lower and it is more conducive to its promotion and application in such infectious diseases.

[0053] (3) The RBD nanobody of the present invention has a binding capacity with SARS-CoV-2 S1 or RBD at the sub-nanomolar or even picomolar level, which is superior to commercial detection antibodies.

[0054] (4) The RBD nanobody and its multivalent modified nanobody of the present invention can effectively inhibit the infection of host cells by the novel coronavirus, and the neutralization capacity reaches the level of nanomolar.

[0055] (5) The RBD nanobody of the present invention has a new and different competitive binding epitope compared with the RBD nanobody of another invention (R-30). It has strong neutralizing activity and also has broad-spectrum activity, making it a new type of RBD nanobody. Attached Figure Description

[0056] Figure 1 The results are from the detection of alpaca plasma titer after immunization.

[0057] Figure 2 The image shows the purified nanobody as detected by SDS-PAGE electrophoresis; where M is the protein marker; 1 is the expression lysis supernatant; 2 is the target protein elution buffer 1; 3 is the target protein elution buffer 2; and 4 is the washing buffer.

[0058] Figure 3A and Figure 3B This demonstrates the ability of the nanobody to bind to the S1 antigen.

[0059] Figure 4 Displays competition-based epitope predictions; ordinate OD450 S1-NC The OD450 values ​​of the nanobody reaction wells and the S1 antigen reaction wells are represented by the OD450 values ​​of the PBS reaction wells.

[0060] Figure 5 The nanobody was shown to inhibit the binding of wild-type and mutant S1 to ACE-2.

[0061] Figure 6 This study demonstrates the inhibitory effect of nanobodies on wild-type and Indian delta mutant pseudovirus infection. The effective concentration of the nanobodies was 15 μg / ml. NC represents wells without nanobodies (opti-MEM medium). PC represents the positive control antibody included in the pseudovirus kit, with a concentration of 40 μg / ml for wild-type detection and 100 μg / ml for Indian delta mutant pseudovirus detection. Blank represents wells without both nanobodies and pseudovirus (opti-MEM medium), indicating the background value of the luciferase assay.

[0062] Figure 7 The experiment demonstrated the detection of bivalent nanobodies under non-reducing conditions via electrophoresis. The purified R-47-FC bivalent nanobodies had a band size of approximately 80 kDa and a purity > 95%. The non-reducing buffer was 5×native loading buffer, and the electrophoresis conditions were 140 V for 50 min.

[0063] Figure 8 The detection of the neutralizing and inhibitory effect of bivalent nanobodies on pseudoviruses was shown.

[0064] Figure 9 This study demonstrates the neutralizing and inhibitory effect of bivalent nanobodies on SARS-CoV-2 and its mutant pseudoviruses. Detailed Implementation

[0065] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0066] Example 1: Multi-antigen cross-immunization

[0067] Initial immunization: 200 μg of wild-type SARS-CoV-2 S1 full-length antigen (sigma) was mixed with an equal volume of Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple sites in the alpaca's neck for the initial immunization. Subsequent cross-immunizations were performed using SARS-CoV-2 spike protein-binding domain (RBD) protein and South African beta mutant S1 protein. Each immunization consisted of 100-200 μg of protein, with an equal volume of incomplete Freund's adjuvant (Sigma), for a total of four immunizations, with a two-week interval between each immunization. On day 10 after the immunization period, 20-30 ml of peripheral blood was collected from the alpaca vein, and plasma and PBMC samples were separated for subsequent immunobank construction.

[0068] Example 2: Plasma titer detection

[0069] 100 ng of RBD and mutant S1 antigen were used to coat an ELISA plate and incubated overnight at 4°C. The plate was washed three times with 0.05% PBST. 200 μl of 2% BSA was added to each well and incubated at room temperature for 2 hours. The plate was then washed three times with PBST. The plasma from Example 1 before and after immunization was diluted 10 μL. 3 10 4 10 5 10 6 and 10 7 The solution was diluted 2500 times and then added to the corresponding ELISA plate. The plate was incubated at room temperature for 1 hour. After washing 5 times with PBST, 100 μl of 2500-fold diluted anti-alpaca H&L IgG HRP was added to each well, and the plate was incubated at room temperature in the dark for 1 hour. After washing 5 times with PBST, 100 μL of TMB chromogenic buffer (abcam) was added, and the plate was incubated for 10 minutes. An equal volume of TMB stop buffer (abcam) was then added to stop the OD450 reading.

[0070] ELISA test results are as follows ( Figure 1The results showed that plasma titers increased significantly after immunization, with wild-type RBD reaching an immunogenic titer of 10. 6 The immunogenicity of mutant strain S1 was 10. 5 Up to 10 6 The presence of these antibodies indicates that, through cross-immunization, the alpaca has acquired abundant RBD or mutant S1 antibodies.

[0071] Example 3: Construction of a phage display library for nanobodies

[0072] (1) PBMC isolation. Blood samples were taken from alpacas after immunization. Lymphocytes in the peripheral blood of alpacas were isolated and purified using lymphocyte separation medium (GE, 17-1440-02) and density gradient centrifugation. The cells were washed with PBS two to three times and then used for RNA extraction.

[0073] (2) Construction of nanobody library.

[0074] Total RNA extraction.

[0075] Take the lymphocytes isolated in (1), add 1 ml of Trizol reagent (INVITROGEN, 15596-018), pipette and mix well to fully lyse the cells. After standing at room temperature for 10 min, add 0.2 ml of chloroform, shake vigorously for 15 s, stand on ice for 5-10 min, place in a refrigerated centrifuge at 4℃, centrifuge at 12,000 rpm for 10 min, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, stand at room temperature for 15 min, wait for nucleic acid precipitation, centrifuge at high speed to remove the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge at high speed to remove the supernatant, drain the water, dissolve the RNA in nuclease-free water, and take 1 μl for concentration and purity determination.

[0076] cDNA synthesis.

[0077] Take 20 μg of RNA and use SuperScript. TM III. First-Strand Synthesis SuperMix (Invitrogen) kit and procedure were used to synthesize cDNA, which was then stored at -20°C.

[0078] PCR amplification.

[0079] Using the reverse transcription product cDNA as a template, the variable region (VHH) of the camel heavy chain antibody was amplified by Nest-PCR. Table 1 shows the names and sequences of the primers used for amplification.

[0080] Table 1. Primer information used for alpaca VHH fragment amplification.

[0081]

[0082]

[0083] The PCR reaction conditions are as follows:

[0084] Round 1

[0085]

[0086] Reaction conditions: 95℃, 5 min; 94℃, 45 s; 56℃, 45 s; 72℃, 45 s per cycle; 72℃, 5 min; amplification 25 cycles.

[0087] Second round

[0088]

[0089] Reaction conditions: 95℃, 5 min; 94℃, 30 s; 56℃, 45 s; 72℃, 35 s per cycle; 72℃, 10 min;

[0090] Amplification 17 cycles

[0091] After the PCR reaction, the target fragment was recovered using the QIAgen gel purification kit (Qiagen) or PCR purification kit (Qiagen) and their operating procedures. The recovered product was then tested for concentration and purity using Nanodrop 2000 and stored at -20°C.

[0092] (3) Construction of phage display library.

[0093] Enzyme digestion and ligation. The VHH fragment and pMECs obtained in (2) were double-digested with restriction endonucleases NotI and PstI (NEB), respectively.

[0094] Vector digestion system:

[0095]

[0096] Add H2O to a final volume of 300 μl;

[0097] Enzyme digestion overnight at 37℃;

[0098] Fragment digestion system:

[0099]

[0100] Add H2O to 200 μl

[0101] Enzyme digestion overnight at 37°C.

[0102] The enzyme digestion products of the VHH fragment and pMECs vector were recovered by agarose gel electrophoresis and agarose gel recovery kit (QIAGEN, 20051), and then the enzyme digestion products were ligated.

[0103] The connection system is as follows:

[0104]

[0105] Connect overnight at 16°C.

[0106] Transformation. Take 1 μl of the ligation product and mix it with 30 μl of TG1 supercompetent cells. Incubate on ice for 5 min. Transfer the mixture to an electroporation cuvette and electroporate at 1.5 kV. After electroporation, add 1 ml of SOC medium, pipette and mix well, then transfer to a 2 ml centrifuge tube. Incubate at 37°C for 1 h. Serially dilute 10⁻⁶ times. 2 10 3 10 4 The diluted bacterial culture was spread onto plates and incubated overnight at 37°C. The colony count was calculated the following day, reaching approximately 10. 7-8 One clone. The transformed bacterial culture becomes the antibody phage library, which is then added with an equal volume of 50% glycerol and stored at -20°C.

[0107] (4) Detection of the diversity of nanobodies in phage display library.

[0108] 54 clones from (3) were randomly selected and sent to 3730 for sequencing. The antibody VHH region sequence was obtained and compared. It was found that only 3 clones had the same sequence, and 51 clones had different VHH sequences, accounting for 94.4%, indicating that the antibody library has good diversity.

[0109] (5) Phage amplification and rescue.

[0110] The obtained library was amplified, and a phage strain rescuing the nanobodies was added using helper phage. The phage library preserved in (3) was inoculated into 100 ml of culture medium and cultured to the logarithmic growth phase. 20 μl of helper phage (pfu = 2 × 10^12) was added, and the culture was allowed to stand at room temperature for 30 min. After centrifugation at low speed, the precipitate was resuspended in culture medium and inoculated into 300 ml of culture medium for overnight culture. The next day, the phage was centrifuged at 3,000 g for 30 min, the supernatant was collected, PEG solution was added to precipitate the phage, and the culture was allowed to stand on ice for 30 min. After centrifugation at 2,200 g for 30 min, the phage library carrying the nanobodies was precipitated. The precipitate was resuspended in an appropriate amount of PBS to a titer of 2 × 10^12. 12 pfu / ml.

[0111] Example 4: Obtaining high-affinity RBD or mutant S1 nanobodies using phage display technology

[0112] (1) Washing of the affinity RBD and mutant strain S1 nanobody phage library.

[0113] Take 1 μg of RBD, mutant strain S1, and 5% milk to coat ELISA plates, and incubate overnight at 4°C. The next day, add 2 × 10⁶ nanoparticles of the phage obtained in (5) above to the 5% milk wells. 11 / well, incubate at room temperature for 1 hour, aspirate the supernatant and add it to the RBD and S1 coated wells, incubate at room temperature for 1-2 hours; wash 10 times with PBST, add 100 μl of triethylamine elution buffer, incubate at room temperature for 10-30 minutes, and collect the phages, which are the RBD or S1 nanobody phage libraries obtained by affinity washing; take 10 μl of each to infect TG1 cells and spread on plates, and use the remaining phages for further amplification and rescue.

[0114] (2) Amplification and rescue of the phage library after screening.

[0115] The amplification and rescue methods are the same as in Example 3(5). The phage library obtained after the first round of screening is stored at 4°C and the screening is repeated for 2-3 rounds.

[0116] (3) Cross-screening of RBD and mutant S1 high-affinity nanobodies.

[0117] As above, 100 ng of RBD and mutant strain S1 antigen were used to coat the ELISA plate as the experimental group. At the same time, an equal number of wells were left uncoated as negative controls. The plates were incubated overnight at 4°C. From the plates selected in step 3 above, single clones were randomly picked and placed in 1 ml of culture medium. The plates were cultured at 37°C until the logarithmic phase was reached. 1 mM IPTG was added for induction overnight. The next day, the bacterial sediment was collected by centrifugation. After disruption, the plates were centrifuged at 5,000 g for 15 min and the supernatant was collected. At the same time, 2% BSA was added to the ELISA plate and the plates were blocked at room temperature for 1 h. The supernatant of the single clone was added to each well of the experimental group and the negative control group and incubated at room temperature for 2 h. The plates were washed 10 times with PBST and anti-HA-HRP-tagged antibody (abcam) was added and the plates were incubated at room temperature for 1 h. The plates were washed 5 times with PBST and the substrate chromogenic agent was added. The reaction was carried out for 10-20 min and the stop agent was added. The absorbance was read on the microplate reader. When the ratio of the absorbance to the control well was greater than 2.1, the plate was considered a positive clone. In this study, three nanobodies with specificity and affinity were screened. The identification numbers and amino acid sequence information of the three nanobodies are shown in Tables 2 and 3, respectively.

[0118] Table 2. Amino acid sequence numbers of the three nanobodies

[0119] serial number CDR1 CDR2 CDR3 FR1 FR2 FR3 FR4 VHH R-47 1 2 3 10 11 12 13 21 S1-96 4 5 6 14 15 16 17 22 R-45 7 8 9 18 19 20 13 23

[0120] 1) R-47

[0121] The amino acid sequence is:

[0122] QVQLQESGGGLVQPGGSLRLSCAVSGMTLDYYAIAWFRQAPGKEREGVSRISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAASPLTYYSGTYYFPGEYDYWGQGTQVTVSS(SEQ ID NO:21)

[0123] The nucleotide sequence is:

[0124] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTC TGAGGCTCTCCTGTGCAGTCTCTGGAATGACTTTGGATTATTATGCCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCACGTATTAGTAGTAGCGATG GTAGCACATCCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGGCG TTTATTACTGTGCAGCCTCCCCCCTTACATATTATAGTGGTACTTACTACTTTCCCGGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO: 24)

[0125] 2) S1-96

[0126] The amino acid sequence is:

[0127] QVQLQESGGGLVQPGGSLRLSCTASGSIFSIDNMSWYRQAPGKPREWVAAATSG GAANYADFVKGRFTISRDNAKNTVYLQMNNLKPDDTAVYYCYVVDATMDYWGEGT QVTVSS(SEQ ID NO:22)

[0128] The nucleotide sequence is:

[0129] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTC TGAGACTCTCCTGTACAGCCTCTGGAAGTATTTTCAGTATCGATAACATGAGCTGGTACCGCCAGGCTCCAGGGAAGCCTCGCGAGTGGGTCGCAGCTGCAACTAGTGGTGG AGCCGCAAACTATGCAGACTTCGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAACCTGAAGCCTGACGACACGGCCGT CTATTACTGTTATGTAGTGGACGCGACCATGGACTACTGGGGCGAAGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO: 25)

[0130] 3) R-45

[0131] The amino acid sequence is:

[0132] QVQLQESGGGLVQPGGSLTLSCAASGDIFSIYAMGWYRQAPGRQREAVATISTSGT TSYARSGKGRFTIFRDNAKNTAYLQMNSLEPEDTAVYYCHAVNSRSGGDYWGQGTQVTVSSS(SEQ ID NO: 23)

[0133] The nucleotide sequence is:

[0134] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTC TGACGCTCTCCTGTGCAGCCTCTGGAGACATCTTCAGTATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAGGCAGCGCGAGGCGGTCGCAACTATTAGTACTAGTGGT ACCACAAGTTATGCACGCTCCGGGAAGGGCCGATTCACCATCTTCAGAGACAACGC CAAGAACACGGCCTATCTGCAAATGAACAGTCTGGAACCTGAGGACACGGCCGTCTATTACTGCCATGCCGTCAACAGTCGGTCCGGTGGGGACTACTGGGGCCAGGGGGACC CAGGTCACCGTCTCCTCA (SEQ ID NO: 26)

[0135] Table 3. Sequences and corresponding numbers of each nanobody.

[0136]

[0137]

[0138] Example 5: Induction, Expression, and Purification of Nanobodies

[0139] (1) Induced expression of nanobodies.

[0140] Five nanobody monoclonal strains from Example 4 were selected and inoculated into 10 ml of ampicillin-containing medium, and cultured overnight at 37°C and 220 rpm. The next day, 2 ml of the overnight culture was inoculated into 200 ml of ampicillin-containing medium, and cultured at 37°C and 220 rpm until the logarithmic growth phase (OD200). 600 The concentration of the bacterial culture was 0.6-0.8 g / mL. IPTG was added overnight to induce nanobody expression. The next day, the bacterial cell pellet was collected, and the cells were lysed using a hypotonic method. The supernatant was then collected by high-speed centrifugation for subsequent protein purification.

[0141] (2) Purification of nanobodies.

[0142] Purified nanobodies were obtained using His-nickel packing material (Ni packing material, BioRad). The Ni packing material was packed into a column, washed first with ultrapure water, then with equilibration buffer (PBS). The supernatant was added to the purification column at a flow rate of 1 ml / min. Impurities were washed away with an appropriate volume of PBS until the OD280 was below 0.0001. The target protein was then eluted with 10 volumes of elution buffer (150 mM imidazole). An equal volume of the purified target protein was analyzed using 12% SDS-PAGE to detect the expression and purification status of the nanobodies. Figure 2 As shown in the figure, the nanobody bands are around 15KD in size and have a purity of >95%, and all three nanobody strains were successfully and correctly expressed.

[0143] Example 6: ELISA analysis of the binding ability of nanobodies to wild-type and mutant antigens.

[0144] Coat an ELISA plate with 100 ng of RBD antigen or mutant S1 antigen and incubate overnight at 4°C. Wash three times with PBST (0.05%). Add 200 μL of 2% BSA to each well and incubate at room temperature for 2 hours. Wash three times with PBST. Serially dilute the nanobody purified in Example 5 and the irrelevant negative control VHH antibody (NC). The starting concentration is 3 μg / ml (~200 nM), and each well is serially diluted 3-fold for a total of 8 concentration gradients. Add 100 μL to each well, repeating each concentration gradient three times, and incubate at room temperature for 1 hour. Wash five times with PBST, add 100 μL / well of 2000-3000 times diluted anti-HA, HRP (abcam), and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 100 μl of TMB chromogenic solution (abcam), develop for 10 min, add an equal volume of TMB stop buffer (abcam) to stop the chromogenic process, and analyze the binding ability of the nanobody to wild-type RBD and mutant S1 antigen.

[0145] The ELISA results (Figure 3) show that R-47 and S1-96 have high binding affinity and specificity to both wild-type and South African beta mutant S1. R-47 showed the best performance, with EC50 values ​​of 1.728 nM and 5.128 nM for wild-type and beta mutant S1 antigens, respectively. R-45 showed high binding ability to wild-type S1 (EC50 of 1.525 nM), but this antibody could not bind to the South African beta mutant S1 antigen.

[0146] Example 7: Determination of the affinity constants of nanobodies to wild-type and mutant antigens using the SPR method.

[0147] The CM5 chip was sequentially coupled with the SARS-CoV-2 S1 antigen. The nanobody protein solution was then diluted with HBS-P buffer to create suitable concentration gradients. In this example, the starting concentrations were approximately 0.96 μg / ml (64 nM) or 0.24 μg / ml (16 nM), with subsequent 2-fold serial dilutions, resulting in six concentration gradients. Each injection lasted 120 s, followed by 180 s of dissociation at a flow rate of 30 L / min. Regeneration was then performed with 10 mM pH 2.0 Gly-HCl, and the cycle was repeated until all concentration gradients were injected. After the program ran, the built-in analysis program of the Biacore T200 (GE) instrument was used for fitting analysis to obtain the nanobody affinity constants (Table 4).

[0148] Table 3 Affinity constants of RBD nanobodies

[0149]

[0150] Note: " / " indicates that the antibody has no binding affinity and cannot be detected.

[0151] The results showed that in this case, both R-47 and S1-96 could bind to the S1 antigen of wild-type, South African Beta, and Indian Delta mutant strains; R-45 only had a high affinity for wild-type S1 and could not bind to the other two mutant strains. R-47 had a high affinity for wild-type S1, reaching 0.531 nM, while its affinity for South African Beta and Indian Delta mutant S1 strains was slightly weaker, at 13 nM and 1 nM, respectively. S1-96 had the smallest dissociation constant with wild-type S1 and was the least likely to dissociate, with a binding affinity of 5.98 pM; however, its affinity for the other two mutant S1 strains was weak, at 20.4 and 12.6 nM, respectively.

[0152] Example 8: Predicting Nanobody Binding Competitive Epitopes Using ELISA

[0153] The purified R-47-FC from Example 11 was coated onto an ELISA plate; washed three times with PBST, blocked with 2% BSA, and incubated at room temperature for 2 hours. After washing three times with PBST, 50 μl of 1.5 μg / ml S1 antigen (sigma) was added to one half of the wells, and PBS was added to the other half as a negative control well (no antigen reaction), incubated at room temperature for 1 hour or overnight at 4°C. After washing five times with PBST, 50 μl of the three monovalent nanobodies purified in Example 5 and nanobodies R-30, S1-28, S1-51, and S1-24 were added to each well, incubated at room temperature for 2 hours. After washing five times with PBST, 50 μl of 7000-fold diluted anti-HA HRP (abcam) detection reagent was added, incubated at room temperature in the dark for 1 hour. After washing five times with PBST, 50 μl of TMB solution was added for color development in the dark for 20 minutes, and then 50 μl of TMB stop buffer (abcam) was added to stop the color development. Finally, the ELISA plate was placed in a BioTek microplate reader to read the values. The difference between the OD450 values ​​of the nanobodies with S1 antigen added and the OD450 values ​​of the negative control wells with PBS added was analyzed. Nanobodies with a difference similar to that of R-47 were considered to have similar or identical competitive epitopes to R-47; nanobodies with a difference significantly different from that of R-47 were considered to have different competitive binding epitopes.

[0154] The results are as follows Figure 4 As shown, in this embodiment, three nanobodies have similar competitive binding epitopes and can no longer bind to the S1 antigen bound to R-47-FC, indicating that their binding epitopes are the same or similar to those of R-47. Four nanobodies, R-30, S1-28, S1-51, and S1-24, can still bind to the S1 antigen bound to R-47-FC, indicating that these four nanobodies have different competitive binding epitopes compared to R-47. These types of nanobodies can enhance the neutralizing effect of viral strains and prevent immune escape by being used in combination or constructed into bispecific / multispecific nanobodies.

[0155] The amino acid sequence of R-30 is:

[0156] QVQLQESGGGLVQSGGSLRLSCTASGGIIRLNSMGWYRQAPGKQREPVATIVSDV GTNYADSVKGRFTISRDNAKNTIYLQMNSLKFEDTAVYYCVADRAFVLRGEYEYWG QGTQVTVSS(SEQ ID NO:28)

[0157] The amino acid sequence of S1-28 is as follows:

[0158] QVQLQESGGGLVQPGGSLRLSCTASGGIIRLNSMGWYRQAPGKQREPVATIVSDV GTNYADSVKGRFTISRDNAKNTIYLQMNSPKFEDTAVYYCVADRAFVLRGEYEYWGQGTQVTVSS(SEQ ID NO:29)

[0159] The amino acid sequence of S1-51 is as follows:

[0160] QVQLQESGGGLVQSGGSLRLSCAASGGVSRLNSMGWYRQAPGKQRELVATIISD VGTNYADSVKGRFTISRDNAANTVYLQMNSLKFEDTAVYYCVADRAFVLRGEYEYWGQGTQVTVSS(SEQ ID NO:30)

[0161] The amino acid sequence of S1-24 is as follows:

[0162] QVQLQESGGGLVQSGGSLRLSCAASGGVSRLNSMGWYRQAQGKQRELVATIVN DVGTNYADSVKGRFTISRDNAANTVYLLMNSLKFEDTAVYNCVADRAFVLRGEYEYWGQGTQVTVSS(SEQ ID NO:31)

[0163] Example 9: Detection of the blocking effect of nanobodies on wild-type and mutant S1 antigen / ACE-2 using the SPR method.

[0164] Take the protein A chip and capture 2.5 μg / ml ACE-2-FC (Beijing Baipusaisi) protein solution to 160 RU. Dilute three nanobodies and two control nanobodies S1-28 and S1-24 from another patent to 30 μg / ml. Replace the negative control (NC) with PBS. Separately, dilute wild-type and mutant S1 antigen (South African beta) to 15 μg / ml. Take an equal volume of nanobody dilution and NC sample, mix with S1 antigen dilution, and load the sample at 30 μl / min for 60 s, followed by 120 s of dissociation. After each round of injection, regenerate using 10 mM pH 1.5 Gly-HCl, and repeat the above steps until all samples are injected. After the program runs, analyze the data using the built-in analysis program of the Biacore T200 (GE) instrument, export all experimental detection data, and analyze the blocking inhibition rate. The calculation formula is: (NC - test sample) / NC × 100%.

[0165] Depend on Figure 5The results showed that, at the same effective concentration, R-47 could strongly inhibit the binding of wild-type and mutant S1 to ACE-2, with inhibition rates of 91.8% and 96.9%, respectively; superior to S1-96 (51.2% and 54.7%) and R-45 (19.6% and 28.6%), and also superior to two nanobodies in another patent, S1-28 (64.7% and 30.3%) and S1-24 (63.2% and 35.1%).

[0166] Example 10: Detection of the neutralizing effect of nanobodies using wild-type and Indian delta mutant pseudoviruses.

[0167] Three RBD nanobodies were diluted to 30 μg / ml using Opti-MEM. Positive control antibodies (from a pseudovirus detection kit, GenScript, SC2087A and SC2087V) were diluted to 40 μg / ml (wild-type detection concentration) and 200 μg / ml (Indian delta mutant detection concentration) using Opti-MEM. In white-walled 96-well cell culture microplates (neutralization plate), 25 μl of sample was added to each well; 25 μl of positive antibody (both from GenScript pseudovirus kits) was added to each positive control well; and 25 μl of Opti-MEM was added to each negative control and blank control well, with two replicates per group. The pseudoviruses were removed from -196°C and thawed by rapid, gentle shaking in a 37°C water bath. The thawed pseudoviruses were then added to a 15 ml tube containing 1500 μl of Opti-MEM and mixed thoroughly. 25 μl of pseudovirus solution was added to each well of the 96-well plate for the sample, positive, and negative controls. 25 μl of Opti-MEM was added to the blank control group. After this addition, each well contained 50 μl of solution. The sample and pseudovirus mixture were mixed thoroughly and incubated at room temperature for 1 h. After neutralization of the sample and pseudovirus, Opti-HEK293 / ACE2 cells were immediately revived and diluted to a concentration of 6 × 10⁻⁶. 5 / ml, and place in a cell culture incubator for later use. After the sample and pseudovirus incubation is complete, remove the cell suspension from the cell culture incubator. After thoroughly mixing the cell suspension, add 50μl of cell suspension to each well. After 24 hours, add 50μl of pre-warmed fresh DMEM complete medium to each well and continue incubating in the cell culture incubator for another 24 hours. Carefully aspirate the medium from the 96-well plate using a pipette, discarding it along with the pipette tip into a waste container pre-filled with 10-fold diluted 84 disinfectant. Add 50μl of freshly prepared luciferase chromogenic solution (GenScript) and incubate at room temperature for 3-5 minutes. Place the 96-well plate in a microplate reader (BioTek) to read the chemiluminescence signal of each well. Analyze the inhibition efficiency of the nanobody based on the detection results: Inhibition rate = 1 - (average detection value - average value of blank wells) / (average negative control value - average value of blank wells)

[0168] Table 4 and Figure 6 The results showed that all three nanobodies effectively inhibited wild-type pseudovirus infection of ACE-2 cells, with inhibition efficiencies reaching 98%-100%. Meanwhile, R-47, at a concentration of 15 μg / ml, effectively inhibited the infection of receptor cells by the Indian delta mutant pseudovirus, with an efficacy similar to that of high-concentration positive antibodies (100 μg / ml), at 100% and 98%, respectively. R-45 and S1-96 showed weaker inhibitory effects against delta mutant pseudovirus infection, achieving only about 30% inhibition. Therefore, R-47 exhibits stronger neutralizing and inhibitory effects and can effectively inhibit infection by the Indian delta mutant virus.

[0169] Table 4. Inhibitory efficiency of monovalent nanobodies against wild-type and Indian delta mutant SARS-CoV-2.

[0170]

[0171] Example 11: Bivalent modification of monovalent nanobodies, eukaryotic expression and purification

[0172] Based on the results of pseudovirus neutralization, R-47 nanobodies were selected for bivalent structural modification. First, the R-47 nanobodies were fused with a human IgG1 Fc amino acid sequence for gene synthesis (amino acid sequence see notes). The synthesized gene sequence was then processed using seamless cloning PCR technology (specific procedures refer to Hieff). (Plus One Step Cloning Kit instructions) Subcloned into expression vector pCDNA4 (Invitrogen, Cat V86220). The recombinant single-domain antibody VHH-FC fusion protein particle was transfected into HEK293T cells for expression. The recombinant expression plasmid was diluted with PBS and PEI (polyethylenimine) solution for transformation was added. After mixing, the mixture was added to the HEK293T cell suspension and incubated at 37°C, 8% CO2, and ≥80% relative humidity at 150 rpm. After 5-6 days of culture, the transient expression culture supernatant was collected. The supernatant was used for subsequent protein purification, which was performed by protein A affinity chromatography to obtain the bivalent nanobody (…). Figure 7 ).

[0173] The results showed that the purified bivalent nanobody bands were 80 kDa in size, which was consistent with the theoretical size, with a purity greater than 95% and a transient expression yield of about 100 mg / L.

[0174] The amino acid sequence of R-47-FC is as follows:

[0175] QVQLQESGGGLVQPGGSLRLSCAVSGMTLDYYAIAWFRQAPGKEREGVSRISSSD GSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAASPLTYYSGTYYFPGEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 27).

[0176] Example 12: ELISA analysis of the binding ability (broad-spectrum binding activity) of bivalent nanobodies to wild-type and mutant antigens.

[0177] Coat an ELISA plate with 100 ng of RBD antigen or mutant S1 antigen and incubate overnight at 4°C. Wash three times with PBST (0.05%). Add 200 μL of 2% BSA to each well and incubate at room temperature for 2 h. Wash three times with PBST. Serially dilute the bivalent nanobody R-47-FC purified in Example 11. The starting concentration is 2.4 μg / ml (~30 nM), and each well is serially diluted 5-fold for a total of 8 concentration gradients. Add 100 μL to each well, repeating each concentration gradient in 3 wells, and incubate at room temperature for 1 h. Wash the positive control (Bepsys, AM180) five times with PBST, add 100 μL / well of 8000-fold diluted anti-human IgG, HRP (BETHYL, A80-304P), and incubate at room temperature for 1 h. Wash 5 times with PBST, add 100 μl of TMB chromogenic solution (abcam), develop for 5-10 min, add an equal volume of TMB stop buffer (abcam) to stop the chromogenic process, and analyze the OD450 reading to determine the binding ability of the nanobody to wild-type RBD and mutant S1 antigen.

[0178] ELISA test results are shown in Table 5. Figure 8The results showed that R-47-FC exhibited excellent binding ability to both wild-type and mutant S1 antigens, with a maximum effective concentration (EC50) in the sub-nanomolar range (below 0.1 nM), representing a 20-50 fold increase compared to the monovalent nanobody in Example 6. Compared to the PC positive control (Beijing Baipusaisi, AM180), R-47-FC demonstrated better binding ability and broader-spectrum activity.

[0179] Table 5. Binding ability of bivalent nanobodies to wild-type and mutant S1 antigens

[0180]

[0181] Note: NA indicates that the antibody has no binding affinity, and the test result is not available (NA, cannot be detected).

[0182] Example 13: Determination of the affinity constants of bivalent nanobodies to wild-type and mutant antigens using the SPR method.

[0183] The CM5 chip was used, and wild-type and mutant S1 antigens were sequentially coupled. The nanobody protein solution and positive control antibody (ARCO) were then diluted with HBS-P buffer to create suitable concentration gradients. In this example, the gradients started at approximately 0.8 μg / ml (10 nM) or 0.2 μg / ml (2.5 nM), with sequential 2-fold dilutions, resulting in six concentration gradients. Each injection lasted 120 s, followed by 180 s of dissociation at a flow rate of 30 μg / min. Regeneration was then performed with 10 mM pH 2.0 Gly-HCl, and the cycle was repeated until all concentration gradients were injected. After the program ran, the built-in analysis program of the Biacore T200 (GE) instrument was used for fitting analysis to obtain the nanobody affinity constant results (Table 6). The results showed that after the nanobody was modified into a bivalent nanobody, the affinity constant was within 10... -13 The M level (exceeding the instrument detection limit) was nearly 60 times higher than that of the monovalent nanobody (Example 7, Table 3), and also much higher than that of the positive control sample.

[0184] Table 6. Affinity constants of bivalent RBD nanobodies

[0185]

[0186] Note: " / " indicates that the antibody has no binding ability to the ligand antigen.

[0187] Example 14: Analysis of the neutralizing activity of bivalent nanobodies using a pseudovirus neutralization experiment.

[0188] Take an appropriate amount of R-47-FC and dilute it twice with Opti-MEM to an initial concentration of 200 nM (16 μg / ml), then perform a 5-fold dilution with Opti-MEM, resulting in a total of 7 concentration gradients. Add 25 μl of sample to each well of a 96-well cell culture microplate (neutralization plate) with a white opaque wall. Wild-type (GenScript, SC2087A), South African beta (GenScript, SC2087L), and Indian delta mutant pseudovirus (GenScript, SC2087V) were removed from -196℃ and thawed by rapid, gentle shaking in a 37℃ water bath. The thawed pseudovirus was then added to a 15 ml tube containing 1500 μl of Opti-MEM and mixed thoroughly. Add 25 μl of pseudovirus solution to each sample well and negative control well of the 96-well plate. After this, each experimental well contains 50 μl of solution. Mix the sample and pseudovirus mixture thoroughly and incubate at room temperature for 1 h. After neutralizing the sample and pseudovirus, the Opti-HEK293 / ACE2 cells in the kit were immediately revived and diluted to a concentration of 6 × 10⁻⁶. 5 / ml, and place in a cell culture incubator for later use. After the sample and pseudovirus incubation is complete, remove the cell suspension from the cell culture incubator. After thoroughly mixing the cell suspension, add 50μl of cell suspension to each well. After 24 hours, add 50μl of pre-warmed fresh DMEM complete medium to each well and continue culturing in the cell culture incubator for another 24 hours. Carefully aspirate the culture medium from the 96-well plate using a pipette, discarding it along with the pipette tip into a waste container pre-filled with 1:10 diluted 84 disinfectant. Immediately add 50μl of freshly prepared luciferase chromogenic solution (GenScript) and incubate at room temperature for 3-5 minutes. Place the 96-well plate in a microplate reader (BioTek) to read the chemiluminescence signal of each well. Analyze the data based on the detection results. Figure 9 The results showed that the R47-FC bivalent nanobody could effectively inhibit the infection of ACE-2 cells by wild-type, South African beta, and Indian delta mutant pseudoviruses (i.e., variant strains), with IC50 values ​​of 0.3013, 0.6227, and 0.1346 nM at the sub-nanomolar level, respectively. The neutralizing and inhibitory effects were significant and significantly better than those of the control antibody WNb10-FC against South African beta and Indian delta mutant pseudoviruses (IC50 values ​​of 3.526 and 1.274 nM, respectively). SEQUENCE LISTING <110> Shenzhen BGI Life Science Research Institute <120> A nanobody targeting the novel coronavirus and its application <130> P210110007C <160> 39 <170> PatentIn version 3.3 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> R-47 CDR1 <400> 1 Gly Met Thr Leu Asp Tyr Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> R-47 CDR2 <400> 2 Ile Ser Ser Ser Asp Gly Ser Thr 1 5 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> R-47 CDR3 <400> 3 Ala Ala Ser Pro Leu Thr Tyr Tyr Ser Gly Thr Tyr Tyr Phe Pro Gly 1 5 10 15 Glu Tyr Asp Tyr 20 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> S1-96 CDR1 <400> 4 Gly Ser Ile Phe Ser Ile Asp Asn 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> S1-96 CDR2 <400> 5 Ala Thr Ser Gly Gly Ala Ala 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> S1-96 CDR3 <400> 6 Tyr Val Val Asp Ala Thr Met Asp Tyr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> R-45 CDR1 <400> 7 Gly Asp Ile Phe Ser Ile Tyr Ala 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> R-45 CDR2 <400> 8 Ile Ser Thr Ser Gly Thr Thr 1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> R-45 CDR3 <400> 9 His Ala Val Asn Ser Arg Ser Gly Gly Asp Tyr 1 5 10 <210> 10 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> R-47 FR1 <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser 20 25 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> R-47 FR2 <400> 11 Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 15 Arg <210> 12 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> R-47 FR3 <400> 12 Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Gly Val Tyr Tyr Cys 35 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> R-47 / R-45 FR4 <400> 13 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 14 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> S1-96 FR1 <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> S1-96 FR2 <400> 15 Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Pro Arg Glu Trp Val Ala 1 5 10 15 Ala <210> 16 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> S1-96 FR3 <400> 16 Asn Tyr Ala Asp Phe Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Asp Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> S1-96 FR4 <400> 17 Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 18 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> R-45 FR1 <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser 20 25 <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> R-45 FR2 <400> 19 Met Gly Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Ala Val Ala 1 5 10 15 Thr <210> 20 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> R-45 FR3 <400> 20 Ser Tyr Ala Arg Ser Gly Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 21 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> R-47 VHH <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Met Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Arg Ile Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Pro Leu Thr Tyr Tyr Ser Gly Thr Tyr Tyr Phe Pro Gly 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 22 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> S1-96 VHH <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Phe Ser Ile Asp 20 25 30 Asn Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Pro Arg Glu Trp Val 35 40 45 Ala Ala Ala Thr Ser Gly Gly Ala Ala Asn Tyr Ala Asp Phe Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Val Val Asp Ala Thr Met Asp Tyr Trp Gly Glu Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> R-45 VHH <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Asp Ile Phe Ser Ile Tyr 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Ala Val 35 40 45 Ala Thr Ile Ser Thr Ser Gly Thr Thr Tyr Ala Arg Ser Gly Lys 50 55 60 Gly Arg Phe Thr Ile Phe Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Ala Val Asn Ser Arg Ser Gly Gly Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ser 115 <210> 24 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> R-47 VHH nucleotide sequence <400> 24 caggtgcagc tgcaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgaggctc 60 tcctgtgcag tctctggaat gactttggat tattatgcca tagcctggtt ccgccaggcc 120 ccagggaagg agcgtgaggg ggtctcacgt attagtagta gcgatggtag cacatcctat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac acaggcgttt attactgtgc agcctccccc 300 cttacatatt atagtggtac ttactacttt cccggtgagt atgactactg gggccagggg 360 acccaggtca ccgtctcctc a 381 <210> 25 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> S1-96 VHH nucleotide sequence <400> 25 caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtacag cctctggaag tattttcagt atcgataaca tgagctggta ccgccaggct 120 ccagggaagc ctcgcgagtg ggtcgcagct gcaactagtg gtggagccgc aaactatgca 180 gacttcgtaa agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240 caaatgaaca acctgaagcc tgacgacacg gccgtctatt actgttatgt agtggacgcg 300 accatggact actggggcga agggacccag gtcaccgtct cctca 345 <210> 26 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> R-45 VHH Switchgear <400> 26 60. caggtgcagc tgcaggagtc tggaggaggc ctggtgcagc ctggggggtc tctgacgctc tcctgtgcag cctctggaga catcttcagt atctatgcca tgggctggta ccgccaggct ccagggaggc agcgcgaggc ggtcgcaact attagtacta gtggtaccac aagttatgca 180 cgctccgggga agggccggatt caccatcttc agagacaacg ccaagaacac ggcctatctg 300. sightseeing gtctggaacc tgaggacacg gccgtctatt actgccatgc cgtcaacagt cggtccggtg gggactactg gggccagggg acccaggtca ccgtctcctc a 351 <210> 27 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> R-47-FC Switchboard <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Met Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Arg Ile Ser Ser Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Pro Leu Thr Tyr Tyr Ser Gly Thr Tyr Tyr Phe Pro Gly 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu 115 120 125 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185 190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 225 230 235 240 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 260 265 270 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 305 310 315 320 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of R-30 <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gly Ile Ile Arg Leu Asn 20 25 30 Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val 35 40 45 Ala Thr Ile Val Ser Asp Val Gly Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Ala Asp Arg Ala Phe Val Leu Arg Gly Glu Tyr Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 29 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of S1-28 <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Gly Ile Ile Arg Leu Asn 20 25 30 Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Pro Val 35 40 45 Ala Thr Ile Val Ser Asp Val Gly Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 Gln Met Asn Ser Pro Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Ala Asp Arg Ala Phe Val Leu Arg Gly Glu Tyr Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 30 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of S1-51 <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Val Ser Arg Leu Asn 20 25 30 Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Ile Ser Asp Val Gly Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Ala Asp Arg Ala Phe Val Leu Arg Gly Glu Tyr Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 31 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of S1-24 <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Val Ser Arg Leu Asn 20 25 30 Ser Met Gly Trp Tyr Arg Gln Ala Gln Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Val Asn Asp Val Gly Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Val Tyr Leu 65 70 75 80 Leu Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Asn Cys Val 85 90 95 Ala Asp Arg Ala Phe Val Leu Arg Gly Glu Tyr Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CALL001 <400> 32 gtcctggctg ctcttctaca agg 23 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CALL002 <400> 33 ggtacgtgct gttgaactgt tcc 23 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CALL001-2 <400> 34 gtcctggctg ctctwytaca agg 23 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CALL001-3 <400> 35 cctggykgca ggtchcmagg tg 22 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> VHH-Back <400> 36 gatgtgcagc tgcaggagtc tggrggagg 29 <210> 37 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> VHH-For <400> 37 ctagtgcggc cgctgaggag acggtgacct gggt 34 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> VHH-Back-2 <400> 38 gatgtgcagc tgcargagyc wggrggagg 29 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> VHH-Back-3 <400> 39 gatgtgcagc tgcaggagtc gggcccagg 29

Claims

1. A nanobody targeting the novel coronavirus SARS-CoV-2, characterized in that, The VHH chain of the nanobody contains amino acid sequences as shown in SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3).

2. The nanobody as described in claim 1, characterized in that, The nanobody further includes a framework region FR, which comprises: FR1 with the amino acid sequence shown in SEQ ID NO: 10, FR2 with the amino acid sequence shown in SEQ ID NO: 11, FR3 with the amino acid sequence shown in SEQ ID NO: 12, and FR4 with the amino acid sequence shown in SEQ ID NO:

13.

3. The nanobody as described in claim 1 or 2, characterized in that, The VHH chain contains an amino acid sequence as shown in SEQ ID NO:

21.

4. A bivalent nanobody targeting the novel coronavirus SARS-CoV-2, characterized in that, The bivalent nanobody comprises two nanobodies as described in any one of claims 1 to 3.

5. The bivalent nanobody as described in claim 4, characterized in that, The bivalent nanobody contains the sequence shown in SEQ ID NO:

27.

6. An isolated nucleic acid encoding a nanobody as described in any one of claims 1 to 3 or a bivalent nanobody as described in claim 4 or 5.

7. The nucleic acid as described in claim 6, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

24.

8. A recombinant expression vector, characterized in that, It contains the isolated nucleic acid as described in claim 6 or 7.

9. A transformant comprising a nanobody as described in any one of claims 1 to 3 or a bivalent nanobody as described in claim 4 or 5; wherein the originating host of the transformant is a bacterium, fungus, or mammalian cell.

10. The transformant as described in claim 9, characterized in that, The mammalian cells are human 293 cells, CHO cells, or T cells.

11. A pharmaceutical composition comprising a nanobody as described in any one of claims 1 to 3 or a bivalent nanobody as described in claim 4 or 5.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition also includes other antibodies against the novel coronavirus, or small molecule drugs or nucleic acid drugs for treating the novel coronavirus, or antibodies targeting other viruses.

13. A kit for detecting the novel coronavirus SARS-CoV-2, comprising a nanobody as described in any one of claims 1 to 3 or a bivalent nanobody as described in claim 4 or 5.

14. The kit according to claim 13, characterized in that, The kit also includes (i) an apparatus for administering the nanobody or bivalent nanobody; and / or (ii) instructions for use.

15. A pillbox set comprising pillbox A and pillbox B, wherein: The kit A contains a nanobody as described in any one of claims 1 to 3, a bivalent nanobody as described in claim 4 or 5, a transformant as described in claim 9 or 10, or a pharmaceutical composition as described in claim 11 or 12. The kit B contains other antibodies against the novel coronavirus or a pharmaceutical composition containing said other antibodies against the novel coronavirus, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, diagnostic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

16. The use of the nanobody according to any one of claims 1 to 3, the bivalent nanobody according to claim 4 or 5, the transformant according to claim 9 or 10, or the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for treating and / or preventing diseases or symptoms caused by the novel coronavirus.

17. A method for immunoassay or determination of the novel coronavirus for non-diagnostic purposes, comprising mixing a sample to be tested with a nanobody as described in any one of claims 1 to 3 or a bivalent nanobody as described in claim 4 or 5.

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