Llama-derived nanobody n112 and applications thereof

Abstract

The present application relates to a llama-derived nanobody and its application, in particular to a llama-derived nanobody N112 or an antigen-binding fragment thereof binding to SARS-CoV-2 NTD and its application, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises the following CDRs: 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. The nanobody N112 of the present application has a great potential application prospect in the clinical treatment, prevention and / or detection of SARS-CoV-2 and its variant strain infection.

Description

[0001] Cross-references This application claims priority to Chinese Patent Application No. 202111363956.4, filed on November 17, 2021, entitled “An Alpaca-Derived Nanobody N112 and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to an alpaca-derived nanobody N112 and its application, and more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 NTD, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition containing the above, and their application in the preparation of drugs for the prevention or treatment of COVID-19. Background Technology

[0003] Neutralizing antibody drugs mainly work by binding to antigens on the surface of pathogenic microorganisms, preventing specific molecules expressed by the pathogenic microorganisms from binding to cell surface receptors, thus achieving a "neutralizing" effect.

[0004] Both SARS-CoV and SARS-CoV-2 viruses possess a glycosylated spike protein (S) on their surface. This S protein interacts with the host cell receptor protein ACE2 and triggers membrane fusion; therefore, blocking the binding of the S protein to ACE2 is an effective approach to treating SARS-CoV-2 infection. The S protein contains two functional subunits, S1 and S2. The S1 subunit, in addition to the receptor-binding domain (RBD), also contains an N-terminal domain (NTD). Antibodies targeting the NTD can also exert their antiviral effects through a neutralization mechanism. However, the RBD region, which binds to ACE2 on host cells, is prone to mutation and immune escape, making it difficult for single-specific antibodies to meet long-term therapeutic needs. Therefore, isolating and identifying neutralizing antibodies targeting the NTD region is equally important for the prevention and treatment of SARS-CoV-2 infection.

[0005] Nanobodies, also known as single-domain antibodies (VHHs), possess several unique advantages compared to traditional mAbs (~150 kDa): smaller molecular weight (~15 kDa), lower immunogenicity, better solubility and stability, and a longer CDR3 region. These properties allow nanobodies to be used as single domains or modular units to construct more complex molecules, such as multivalent antibodies targeting different antigens to broaden their spectrum. Importantly, nanobodies can be easily nebulized and delivered directly to the lungs via inhalers, making them potential drugs for treating respiratory diseases. Therefore, isolating and identifying cross-reactive nanobodies is crucial for building a potential drug reserve to address the current COVID-19 pandemic and future coronavirus infections. Summary of the Invention

[0006] Purpose of the invention The present invention aims to provide an alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 NTD, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a method for its preparation, transformed cells, and a pharmaceutical composition comprising the above, as well as their application in the preparation of drugs for the prevention or treatment of COVID-19. The alpaca-derived nanobody or its antigen-binding fragment of the present invention is a highly neutralizing nanobody with strong binding affinity to the SARS-CoV-2 NTD protein, effectively inhibiting infection by the original SARS-CoV-2 strain and its variants. This nanobody has advantages such as small molecular weight (~15 kDa), low immunogenicity, better solubility and stability, and a longer CDR3 region, allowing for nebulized administration, direct delivery to the lungs, and faster onset of action, providing a potential therapeutic strategy for COVID-19 or other coronavirus infections.

[0007] Solution To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD, wherein the antibody comprises a heavy chain variable region, and the heavy chain variable region comprises the following CDRs: The amino acid sequence is CDR1 as shown in SEQ ID NO: 1 (i.e., GFTFSNFA). The amino acid sequence is CDR2 as shown in SEQ ID NO: 2 (i.e., ITTGSFT). And CDR3 with an amino acid sequence as shown in SEQ ID NO: 3 (i.e., NAYLKVFKTYDY).

[0008] In a specific implementation, the heavy chain variable region further includes four frame regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0009] In a preferred embodiment, the amino acid sequences of FR1-4 are as shown in SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLSCAAS), SEQ ID NO:5 (i.e., MRWYRQASGKERELVAD), SEQ ID NO:6 (i.e., NYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC), and SEQ ID NO:7 (i.e., WGQGTQVTVSS).

[0010] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8: QVQLQESGGGLVQPGGSLRLSCAAS GFTFSNFA MRWYRQASGKERELVAD ITITGSFT NYADSVKGRF TISRDNAKNTVYLQMNNLKPEDTAVYYC NAYLKVFKTYDY WGQGTQVTVSS The underlined parts represent the frame regions FR1-4, and the bolded parts represent the heavy chain variable regions CDR1, CDR2, and CDR3.

[0011] In a second aspect, the present invention provides a polynucleotide encoding an alpaca-derived nanobody or its antigen-binding fragment as described in the first aspect above.

[0012] Furthermore, the polynucleotide is DNA or mRNA.

[0013] Furthermore, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:9: CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTTTGCCATGAGGTGGTACCGCCAGGCTTCAGGGAAGGAGCGCGAGTTGGTCGCAGATATTACGATTACTGGTAGTTTTACTAACTA TGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGGTGTATTTGCAAATGAACAACCTGAAGCCTGAGGACACGGCCGTATATTATTGTAACGCCTATTTGAAGGTTTTCAAGACGTATGACTACTGGGGCCAGGGGGACCCAGGTGACCGTGAGCTCT.

[0014] Thirdly, the present invention provides a nucleic acid construct comprising the polynucleotides described in the second aspect above.

[0015] More preferably, the nucleic acid construct further comprises at least one expression regulatory element operatively linked to the polynucleotide, such as a histidine tag, a stop codon, etc.

[0016] Fourthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0017] Fifthly, the present invention provides a transformed cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0018] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or transformed cells as described in the fifth aspect above, and a pharmaceutically acceptable carrier and / or excipient.

[0019] Preferably, the pharmaceutical composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation.

[0020] More preferably, the nasal spray is selected from aerosols, sprays, and powders.

[0021] More preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments; More preferably, the tablet is a sublingual tablet; More preferably, the granules are fine granules; More preferably, the powder is a granule; More preferably, the pills are small pills.

[0022] More preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; even more preferably, the injectable preparation is a push-in preparation.

[0023] In a seventh aspect, the present invention provides the use of an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or a transformed cell as described in the fifth aspect above, or a pharmaceutical composition as described in the sixth aspect above, in the preparation of a medicament for the prevention, treatment, or detection of COVID-19 infection.

[0024] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0025] More preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0026] Eighthly, the present invention provides a method for preventing or treating COVID-19, comprising: administering to a subject in need a preventive or therapeutically effective amount of an alpaca-derived nanobody or antigen-binding fragment thereof that is conjugated to SARS-CoV-2 NTD as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, or a transformed cell as described in the fifth aspect above, or a pharmaceutical composition as described in the sixth aspect above.

[0027] In a ninth aspect, the present invention provides a method for detecting the novel coronavirus, comprising using an alpaca-derived nanobody or an antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in the first aspect above.

[0028] Preferably, the novel coronavirus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0029] More preferably, the SARS-CoV-2 variant strain is an Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and / or Delta (B.1.617.2) variant strain of SARS-CoV-2.

[0030] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0031] Beneficial effects This invention develops nanobody drugs targeting the novel coronavirus. The process involves immunizing alpacas with SARS-CoV-2 RBD and NTD proteins, constructing an antibody library, and using phage display technology to screen for specific nanobodies. The resulting nanobodies specifically bind to SARS-CoV-2. CoV 2. The nanobody of NTD, named N112 nanobody in this paper. Surface plasmon resonance (SPR) technology confirmed that the N112 nanobody of this invention specifically binds to SARS with high affinity. CoV 2. NTD proteins of the original strain and its variants. Furthermore, virus neutralization assays (including true virus neutralization and sham virus neutralization assays) have confirmed that the nanobody N112 of this invention can neutralize the original SARS-CoV-2 strain and its series of variants with high neutralizing activity.

[0032] This invention provides potential nanobody drugs for the clinical prevention, treatment and detection of the original strain of the novel coronavirus and its series of variant strains. Attached Figure Description

[0033] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0034] Figure 1 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 WT RBD-his protein described in Example 1 of this invention; Figure 2 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 WT NTD-his protein described in Example 1 of this invention; Figure 3 This is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the nanobody N112 described in Example 4 of the present invention; Figure 4 This is a schematic diagram illustrating the effect of the nanobody N112 on SARS-CoV-2 WT pseudovirus infection as measured in Example 7 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0036] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0037] The present invention will now be described in detail.

[0038] definition "Nanobodies," also known as "single-domain antibodies," contain only one variable domain of heavy chain (VHH) and, unlike other antibodies, naturally lack the light chain.

[0039] Due to their inherent biophysical advantages, nanobodies can be easily atomized and delivered directly to the lungs via inhalers to treat viral respiratory infections, making them a highly promising antibody drug.

[0040] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the nanobody of the present invention to the SARS-CoV-2 NTD protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0041] The reagents, enzymes, culture media, antibiotics, and milk used in the following examples of the present invention are all commercially available products. For example, TRIzol was purchased from Invitrogen, and the Superscript II First-Strand Synthesis System for RT-PCR kit was purchased from Invitrogen.

[0042] Some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. For example, the pCAGGS vector was purchased from MiaoLing Plasmid, and 293F cells and HEK293T cells were purchased from ATCC; electrocompetent cells... E. coli TG1 cells were purchased from Lucigen, VCSM13 helper phage from StrataGene, and plasmid pMES4 from Addgene; Vero cells were purchased from ATCC CCL81.

[0043] Some synthetic biological materials, such as primers and sequences, which require artificial synthesis, are outsourced to synthetic companies. For example, the primers (SED ID NO: 14~19) in this invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0044] Example 1: Expression and purification of SARS-CoV-2 original strain (WT) RBD-his, SARS-CoV-2WTNTD-his and SARS-CoV-2WT and variant strains Alpha, Beta, Gamma, Delta and KappaNTD-hFc proteins The coding sequence of the signal peptide (as shown in SEQ ID NO:11) was linked to the 5' end of the SARS-CoV-2 WT RBD protein coding sequence (as shown in SEQ ID NO:10), and the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were linked to the 3' end. The sequence was constructed into the pCAGGS vector through the restriction endonuclease sites EcoRI and XhoI, and transfected into 293F cells for expression of SARS-CoV-2 WT RBD-his protein. Similarly, the coding sequence of the signal peptide (as shown in SEQ ID NO:13) was linked to the 5' end of the SARS-CoV-2 WTNTD protein coding sequence (as shown in SEQ ID NO:12), and the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA were linked to the 3' end. The pCAGGS vector was constructed using EcoRI and XhoI restriction endonuclease sites and transfected into 293F cells to express the SARS-CoV-2 WT NTD-his protein.

[0045] Cell culture medium containing the target protein was subjected to nickel ion affinity chromatography (HisTrap). TM Excel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification using a 200 Increase 10 / 300 GL column (GE Healthcare), a relatively pure target protein can be obtained. The SDS-PAGE analysis of the SARS-CoV-2 WT RBD-his protein revealed a size of approximately 30 kDa, as shown in the results. Figure 1 The SDS-PAGE analysis of the SARS-CoV-2 WT NTD-his protein revealed a size of approximately 60 kDa, as shown in the results. Figure 2 .

[0046] Furthermore, the coding sequences for signal peptides (as shown in SEQ ID NO:11) and human Fc tag (hFc) codons (as shown in SEQ ID NO:22) were added to the 5' end of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Delta, and Kappa NTD proteins (the coding sequences for the NTD proteins of these viral strains are available in the public database NCBI). The expression supernatant was then constructed into the pCAGGS vector (purchased from Invitrogen) by linking EcoRI and XhoI, and transfected into 293F cells (purchased from Invitrogen) for NTD-hFc protein expression.TM Excel (Cytiva) and Superdex gel filtration chromatography TM After purification and identification, 200Increase 10 / 300 GL column (Cytiva) was used for surface plasmon resonance analysis.

[0047] SEQ ID NO:22---Encoding sequence of human Fc tag (hFc) ; Example 2: Construction of an alpaca immune and antibody library 200 µg each of the SARS-CoV-2 WT RBD and NTD proteins with six histidine tags prepared in Example 1 were diluted with PBS to a final volume of 1 mL, emulsified with 1 mL of complete Freund's adjuvant for 5 min, and administered via subcutaneous injection at multiple sites for immunization. Immunization was then performed every two weeks. On day 12 after the fourth immunization, 50-60 mL of blood was collected, and PBMCs (peripheral blood mononuclear cells) were isolated. The isolated PBMCs were added to 1 mL of TRIzol, and total RNA was extracted according to the manufacturer's instructions. Using the extracted total RNA as a template, the Superscript II First-Strand Synthesis System for RT-PCR kit was used with random primers oligo-dT... 12-18 cDNA was synthesized using primers. Using the cDNA as a template, PCR was performed using specific primers CALL001 and CALL002 (primer sequences shown in Table 1). The 700 bp band was excised from the gel and recovered. The purified DNA was then used as a template for nested PCR using nested primers VHH-BACK and PMCF to amplify the nanobody (VHHs) sequence. The purified VHHs sequence, approximately 400 bp in size, was recovered.

[0048] The VHHs fragment was ligated into plasmid pMES4 using a double restriction enzyme digestion method, via restriction sites PstⅠ and BstEⅡ. The purified cloning vector and electrocompetent cells were then... E. coli TG1 cells were mixed, and the cloning vector was transformed into electrocompetent cells using an electroporator (MicroPulser BIO-RAD electroporator). E. coli TG1 cells were spread on selective medium containing ampicillin and cultured overnight at 37°C. All colonies were collected in LB medium, centrifuged and the supernatant was discarded. The cells were resuspended in LB medium to form an antibody library.

[0049] Table 1. Reaction primers

[0050] Example 3: Screening for specific nanobodies using phage display technology Take the recombinant plasmid transfected in Example 2 E. coli TG1 was inoculated with VCSM13 helper phage at a ratio of approximately 20 multiplicity of infection (MOI). After overnight incubation, the mixture was centrifuged at 4000 rpm, and the supernatant was collected. After filtration through a 0.22 µm membrane, PEG6000 / NaCl was added at a volume ratio of 1:4. The mixture was then incubated at 4°C for at least 1 hour, centrifuged at 8000×g for 30 min, and the supernatant was discarded. The precipitate was resuspended in PBS to obtain the collected phage particles, and the phage titer was determined.

[0051] 2×10 11 The collected phages were mixed with an equal volume of 5% (w / v) skim milk and added to a 96-well plate coated with SARS-CoV-2 WT NTD-his antigen. After incubation at room temperature for 1 hour, the specific phages were eluted with 0.2 M glycine and neutralized with Tris-HCl (pH 9.1). The phages were then used to infect... E. coli TG1 cells were used to amplify the phages. A second round of panning was performed on 96-well plates coated with the SARS-CoV-2 WT NTD-his antigen to enrich phages expressing specific nanobodies. A total of three rounds of panning were conducted. After each round, different single colonies were randomly selected from the agar plates containing colonies and cultured in a shaker at 37°C. VCSM13 helper phages were then added for overnight amplification. The next day, the culture medium was centrifuged, and the phage supernatant was used for ELISA experiments (using SARS-CoV-2 WT NTD-his protein as the coating antigen). When the OD... 450nM When the result is >0.2, it is considered a positive reaction. The corresponding clone is then taken, and the plasmid is sequenced using specific primers MP57 and GⅢ (primers are shown in Table 2) to obtain the sequence encoding VHHs in the plasmid. The core coding sequence of N112 is obtained through sequencing.

[0052] Table 2. Reaction Primers

[0053] Example 4: Expression and purification of nanobody N112 To make the heavy chain variable region of N112 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG, SED ID NO:23) was added to the 5' end of the core coding sequence of N112 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT, SED ID NO:24) was added to the 3' end, resulting in the nucleotide sequence as shown in SEQ ID NO:9, which is the coding sequence of the N112 nanobody of this application. Then, a signal peptide (SED ID NO:20) was added before it, followed by the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation stop codon TGA. The sequence was constructed into the pCAGGS vector through the restriction enzyme sites EcoRI and XhoI, transfected into 293F cells, and cultured for 5 days. The supernatant was collected, centrifuged at 5000 rpm for 30 min, filtered through a 0.22 μm filter membrane, and then subjected to nickel ion affinity chromatography (HisTrap). TMExcel (GE Healthcare) and gel filtration chromatography (Superdex) TM After purification (using 75 Increase 10 / 300 GL column (GE Healthcare)), a relatively pure target protein was obtained. The target peak was determined by SDS-PAGE, and the results are as follows: Figure 3 The purified nanobody N112 was obtained.

[0054] Example 5: Detection of antibody binding ability to NTD protein of original SARS-CoV-2 strain and its variants using surface plasmon resonance technology. Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). The specific steps were as follows: A protein A chip (purchased from Cytiva) was used. Based on the affinity between the protein A chip and hFc, the NTD-hFc proteins of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Delta, and Kappa obtained in Example 1 were immobilized on the chip, with an immobilization volume of approximately 100 RU. The N112 antibody protein was serially diluted with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween), loading the samples one by one from low to high concentration. Kinetic curves of the N112 nanobody binding to the NTD proteins of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Delta, and Kappa were obtained. The binding constants of nanobody N112 to the NTD proteins of the original SARS-CoV-2 strain and its variants Alpha, Beta, Gamma, Delta, and Kappa. k a) Dissociation constant ( k d) and equilibrium dissociation constant ( K D As shown in Table 3, these parameters were calculated using BIAevaluation software 8K (Biacore, Inc.).

[0055] Table 3. Nanobody N112 and SARS CoV 2. Affinity results of NTD of the original strain and its variant strains

[0056] Table 3 shows that the nanobody N112 of the present invention can bind to SARS with high affinity and specificity. CoV 2. NTD proteins of the original strain and its variant strains Alpha, Beta, Gamma, Delta and Kappa.

[0057] Example 6: Packaging of pseudoviruses from the original SARS-CoV-2 strain 1) The gene encoding the last 18 amino acids of the S protein of the original SARS-CoV-2 strain (WT) was removed, and the remaining sequence of the S protein was synthesized (synthesis service provided by Suzhou Genewiz), to obtain the nucleotide sequence of the SARS-CoV-2-WT-S-del18 gene, as shown in SEQ ID NO:21.

[0058] 2) The protein gene obtained in 1) was cloned into the pCAGGS vector to obtain the expression plasmid pCAGGS-SARS-CoV-2-WT-S-del18.

[0059] The packaging steps for the original SARS-CoV-2 strain and its variant pseudoviruses are as follows: a. Cell preparation: Seed HEK293T cells in 10 cm cell culture dishes and allow the cell confluence to reach approximately 80% by the second day. The culture medium is DMEM containing 10% FBS.

[0060] b. Transfection: Take the expression plasmid of S protein from step 2) above, and transfect 30 μg plasmid / 10 cm cell culture dish with PEI. Mix the target plasmid and PEI at a ratio of 1:3 before transfection. Change the culture medium (DMEM medium containing 10% FBS) after 4-6 hours and incubate at 37℃ for 24 hours.

[0061] c. Adding to the virus: Packaging the G backbone virus with a fake virus. VSV-delG (purchased from Wuhan Shumi Brain Science Technology Co., Ltd.) was added to the above-transfected HEK293T cells, incubated at 37°C for 2 h, the culture medium was changed (DMEM medium containing 10% FBS), and VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank) was added. The cells were then cultured in an incubator for another 30 h.

[0062] d. Collection of the virus: Collect the supernatant, centrifuge at 3000 rpm for 10 min, filter through a 0.45 μm sterile filter in a laminar flow hood to remove cell debris, aliquot, and freeze at -80℃.

[0063] A pseudovirus was obtained from the original SARS-CoV-2 strain (SARS-CoV-2 WT).

[0064] Example 7: Detection of SARS-CoV-2 pseudovirus infection by nanobody N112 The purified nanobody N112 obtained in Example 4 was serially diluted 10-fold from 50 μg / mL to the 9th gradient (2.56 pg / mL). This diluted nanobody N112 was then mixed with 1.6 x 10⁻⁶ ppm of the solution. 4 TCID 50 The SARS-CoV-2 original strain pseudovirus obtained in Example 6 was mixed and incubated at 37°C for 1 hour, then added to a 96-well plate pre-seeded with Vero cells (purchased from ATCC CCL81). After incubation for 18-20 hours, the results were detected using a CQ1 Confocal Quantitative Image Cytometer (Yokogawa). The neutralizing capacity of the antibody against the SARS-CoV-2 original strain pseudovirus was calculated based on the number of cells exhibiting GFP fluorescence. The results showed an IC50 value. 50 0.68 μg / ml (e.g.) Figure 4 (As shown).

[0065] In summary, nanobody N112 can serve as a high- and high-activity alpaca-derived nanobody against the original strain and variant strains of the novel coronavirus (SARS-CoV-2).

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD, comprising a heavy chain variable region. The heavy chain variable region comprises the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO: 1, CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and CDR3 with the amino acid sequence shown in SEQ ID NO:

3.

2. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 NTD according to claim 1, characterized in that, The heavy chain variable region also includes four frame regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

3. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 NTD according to claim 2, characterized in that, The amino acid sequences of FR1-4 are shown in SEQ ID NO:4, 5, 6, and 7, respectively.

4. The alpaca-derived nanobody or its antigen-binding fragment that binds to SARS-CoV-2 NTD according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

8.

5. A polynucleotide encoding an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in any one of claims 1 to 4.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide is DNA or mRNA.

7. The polynucleotide according to claim 5, characterized in that, The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

9.

8. A nucleic acid construct comprising the polynucleotide of any one of claims 5 to 7.

9. The nucleic acid construct according to claim 8, characterized in that, It also includes at least one expression regulatory element operatively linked to the polynucleotide.

10. An expression vector comprising the nucleic acid construct of claim 8 or 9.

11. A transformed cell comprising the polynucleotide of any one of claims 5 to 7, the nucleic acid construct of claim 8 or 9, or the expression vector of claim 10.

12. A pharmaceutical composition comprising an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in any one of claims 1 to 4, a polynucleotide as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10 or a transformed cell as described in claim 11, and a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is in the form of a nasal spray, oral preparation, or suppository.

14. The pharmaceutical composition according to claim 13, characterized in that, The nasal spray is selected from aerosols, sprays, and powders.

15. The pharmaceutical composition according to claim 13, characterized in that, The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated formulations, and ointments.

16. The pharmaceutical composition according to claim 15, characterized in that, The tablets mentioned are sublingual tablets.

17. The pharmaceutical composition according to claim 15, characterized in that, The granules are fine granules.

18. The pharmaceutical composition according to claim 15, characterized in that, The powder is a granule.

19. The pharmaceutical composition according to claim 15, characterized in that, The pills mentioned are small pills.

20. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition is in the form of a parenteral preparation.

21. The pharmaceutical composition according to claim 20, characterized in that, The parenteral preparations include transdermal preparations, ointments, plasters, topical liquids, and injectable preparations.

22. The pharmaceutical composition according to claim 21, characterized in that, The injectable formulation is a push-in formulation.

23. The use of an alpaca-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD as described in any one of claims 1 to 4, a nucleotide sequence as described in any one of claims 5 to 7, a nucleic acid construct as described in claim 8 or 9, an expression vector as described in claim 10, a transformed cell as described in claim 11, or a pharmaceutical composition as described in any one of claims 12 to 22 in the preparation of a medicament for the prevention, treatment, or detection of SARS-CoV-2 infection, wherein the SARS-CoV-2 is the original SARS-CoV-2 strain.

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