Llama-derived nanobody n36 and applications thereof

Abstract

The present application relates to a llama-derived nanobody N36 and its application, in particular to a llama-derived nanobody N36 or antigen-binding fragment thereof binding to SARS-CoV-2 NTD and its application, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising 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 N36 of the present application can neutralize SARS-CoV-2 pseudovirus, and has a great application prospect in clinical treatment and prevention of SARS-CoV-2 infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a llama-derived nanobody N36 and its application, more particularly to a llama-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2 NTD, polynucleotides encoding the same, nucleic acid constructs comprising the same, expression vectors comprising the nucleic acid constructs, methods of making the same, transformed cells, and pharmaceutical compositions comprising the same, and their use in the preparation of a medicament for the prevention or treatment of COVID-19. BACKGROUND

[0002] Since the end of 2019, the epidemic caused by the novel coronavirus of the family Coronaviridae (also known as SARS-CoV-2 or COVID-19) has been spreading worldwide, and the newly emerging and re-emerging viruses pose a great threat to global public health.

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

[0004] Both SARS-CoV and SARS-CoV-2 viruses have a glycosylated spike protein (S) on their surface, which can interact with the host cell receptor protein ACE2 and trigger membrane fusion. Therefore, blocking the binding of S protein to ACE2 is an effective way to treat COVID-19 infection. The S protein contains two functional subunits, S1 and S2, and in addition to the receptor binding domain RBD, the N-terminal domain NTD is located in the S1 subunit. Antibodies targeting NTD can also exert their antiviral effects through the neutralization mechanism. At the same time, the RBD region that binds to the host cell ACE2 is prone to mutation and immune escape, and a single specific antibody is difficult to meet the long-term treatment needs. Therefore, it is also very important to isolate and identify neutralizing antibodies targeting the NTD region for the prevention and treatment of COVID-19 infection.

[0005] Nanobodies, also known as single-domain antibodies (VHH), have some unique advantages compared to traditional mAbs (~150 kDa): small molecular weight (~15 kDa), low immunogenicity, better solubility and stability, and longer CDR3 regions. With these properties, nanobodies can be used as single domains or modular units to build more complex molecules, such as multivalent antibodies against different antigens to expand the spectrum. Importantly, nanobodies can be easily aerosolized and delivered directly to the lungs by inhalers, making them potential drugs for treating respiratory diseases. Therefore, isolating and identifying nanobodies with cross-reactivity is to provide potential drug reserves for the current COVID-19 pandemic and possible future coronavirus infections. SUMMARY

[0006] Invention objectives

[0007] The present application aims to provide a llama-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD, a polynucleotide encoding the same, a nucleic acid construct comprising the same, an expression vector comprising the same, a method for preparing the same, a transformed cell, and a pharmaceutical composition comprising the same, and their use in the preparation of a drug for preventing or treating COVID-19. The llama-derived nanobody or antigen-binding fragment thereof of the present application is a high-titer nanobody with strong binding ability to SARS-CoV-2 NTD protein and can effectively inhibit the infection of SARS-CoV-2 original strain and a series of variant strains. The nanobody has the advantages of small molecular weight (~15 kDa), low immunogenicity, better solubility and stability, and longer CDR3 region, can be aerosolized for administration, can reach the lungs directly, and has a faster onset, providing a potential treatment strategy for COVID-19 or other coronavirus infections.

[0008] Solution

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] In a first aspect, the present application provides a llama-derived nanobody or antigen-binding fragment thereof that binds to SARS-CoV-2 NTD, wherein the antibody comprises a heavy chain variable region comprising the following CDRs:

[0011] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 (i.e., GSIFGIYV),

[0012] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2 (i.e., ISSGGINY),

[0013] and CDR3 is shown as SEQ ID NO: 3 (i.e., QDGVPPLGYDY).

[0014] In a particular embodiment, the heavy chain variable region further comprises four framework regions FR1-4, which are staggered in order with the CDR1, CDR2 and CDR3.

[0015] In a preferred embodiment, the amino acid sequences of the FR1-4 are shown as SEQ ID NO: 4 (i.e., QVQLQESGGGLVQPGGSLRLSCAAS), SEQ ID NO: 5 (i.e., MGWYCQAPGKQRELVAT), SEQ ID NO: 6 (i.e., ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYV) and SEQ ID NO: 7 (i.e., WGQGTQVTVSS), respectively.

[0016] In a preferred embodiment, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 8:

[0017] wherein the underlined parts are framework regions FR1-4, respectively, and the blacked parts are CDR1, CDR2 and CDR3 of the heavy chain variable region, respectively.

[0018] In a second aspect, the present application provides a polynucleotide encoding the llama-derived nanobody or antigen-binding fragment thereof according to the first aspect described above.

[0019] Further, the polynucleotide is DNA or mRNA.

[0020] Further, the polynucleotide has a nucleotide sequence shown as SEQ ID NO: 9:

[0021] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCGGCATCTATGTCATGGGCTGGTACTGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAACCATTAGTAGTGGTGGTATAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTATTGTTATGTTCAAGATGGAGTACCCCCGCTGGGCTATGACTACTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCT.

[0022] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide according to the second aspect as described above.

[0023] Further preferably, the nucleic acid construct further comprises at least one expression regulatory element operably linked to the polynucleotide. For example, a histidine tag, a stop codon, etc.

[0024] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid construct according to the third aspect as described above.

[0025] In a fifth aspect, the present application provides a transformed cell comprising the polynucleotide according to the second aspect as described above, the nucleic acid construct according to the third aspect as described above, or the expression vector according to the fourth aspect as described above.

[0026] In a sixth aspect, the present application provides a pharmaceutical composition comprising the llama-derived nanobody or antigen-binding fragment thereof binding to SARS-CoV-2 NTD according to the first aspect as described above, the polynucleotide according to the second aspect as described above, the nucleic acid construct according to the third aspect as described above, the expression vector according to the fourth aspect as described above, or the transformed cell according to the fifth aspect as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0027] Further preferably, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation.

[0028] Further preferably, the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

[0029] Further preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, granules, granulates, soft / hard capsules, film-coated tablets, pellets, sublingual tablets and pastes.

[0030] Further preferably, the parenteral formulation is a transdermal formulation, an ointment, a plaster, a liquid for external use, an injectable or a bolus formulation.

[0031] In a seventh aspect, the present application provides use of a llama-derived nanobody or an antigen-binding fragment thereof that binds to the NTD of SARS-CoV-2 as defined in the first aspect above, a polynucleotide as defined in the second aspect above, a nucleic acid construct as defined in the third aspect above, an expression vector as defined in the fourth aspect above, or a transformed cell as defined in the fifth aspect above, or a pharmaceutical composition as defined in the sixth aspect above, in the manufacture of a medicament for preventing, treating or detecting a coronavirus infection.

[0032] Preferably, the coronavirus is a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0033] Further preferably, the SARS-CoV-2 variant strain is 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.

[0034] In an eighth aspect, the present application provides a method for preventing or treating a coronavirus, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of a llama-derived nanobody or an antigen-binding fragment thereof that binds to the NTD of SARS-CoV-2 as defined in the first aspect above, a polynucleotide as defined in the second aspect above, a nucleic acid construct as defined in the third aspect above, an expression vector as defined in the fourth aspect above, or a transformed cell as defined in the fifth aspect above, or a pharmaceutical composition as defined in the sixth aspect above.

[0035] In a ninth aspect, the present application provides a method for detecting a coronavirus, comprising using a llama-derived nanobody or an antigen-binding fragment thereof that binds to the NTD of SARS-CoV-2 as defined in the first aspect above.

[0036] Preferably, the coronavirus is a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0037] Further preferably, the SARS-CoV-2 variant strain is 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.

[0038] The amount of the effective component of the pharmaceutical composition of the present application to be administered varies depending on the subject to be administered, the organ of the subject, the symptoms, the method of administration, and the like, and can be determined according to the kind of the dosage form, the method of administration, the age and body weight of the patient, the symptoms of the patient, and the like, based on the judgment of a medical doctor.

[0039] Advantages

[0040] The present application is directed to the development of nanobody drugs against SARS-CoV-2 by immunizing alpacas with SARS-CoV-2 RBD and NTD proteins, constructing antibody libraries, and screening specific nanobodies using phage display technology. The nanobody that specifically binds to SARS-CoV-2 NTD, designated as nanobody N36, was screened. The nanobody N36 of the present application was confirmed to specifically bind to SARS-CoV-2 NTD with high affinity by surface plasmon resonance technology. In addition, the nanobody N36 of the present application was confirmed to neutralize SARS-CoV-2 wild-type strain and its series of variant strains with high neutralization activity by virus neutralization test including authentic virus neutralization test and pseudovirus neutralization test.

[0041] The present application provides potential nanobody new drugs for the clinical prevention, treatment and detection of SARS-CoV-2 wild-type strain and its series of variant strains. BRIEF DESCRIPTION OF DRAWINGS

[0042] One or more embodiments are illustrated by way of example in the figures that constitute a part of this patent document. This exemplary illustration is not intended to limit the embodiments to the illustrated embodiments or examples. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0043] Figure 1 is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 RBD-his protein described in Example 1 of the present application;

[0044] Figure 2 is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the SARS-CoV-2 NTD-his protein described in Example 1 of the present application;

[0045] Figure 3Figure 2 is a schematic diagram of the molecular sieve chromatography and SDS-PAGE identification results of the nanobody N36 described in Example 4 of the present application. DETAILED DESCRIPTION

[0046] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application. Unless otherwise explicitly indicated, in the entire specification and claims, the term “comprise” or its variants such as “contain” or “include” and the like are understood to include the stated element or component without excluding other elements or components.

[0047] In addition, in order to better illustrate the present application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0048] Hereinafter, the present application will be described in detail.

[0049] Definitions

[0050] “Nanobody”, i.e. “heavy-chain single-domain antibody”, this kind of antibody only contains one heavy-chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody), compared with other antibodies, the light chain is naturally missing.

[0051] Due to the biophysical advantages of nanobodies themselves, they can be easily atomized and directly delivered to the lungs through inhalers, thereby treating respiratory viral infections, and are considered to be very potential antibody drugs.

[0052] When referring to ligand / receptor, antibody / antigen or other binding pairs, “specific” binding refers to the determination of the presence or absence of a binding reaction between the protein, e.g. the nanobody of the present application, and the SARS-CoV-2 RBD protein in a heterogeneous population of proteins and / or other biological reagents. Therefore, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in a significant amount.

[0053] The chemical materials used in the following examples of the present application, such as reagents, enzymes, culture medium, antibiotics and milk, are commercially available products. For example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

[0054] Some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available products. For example, pCAGGS vector is purchased from MiaoLing Plasmid, 293F cells, HEK293T cells, etc. are purchased from ATCC; electrocompetent E. coli TG1 cells are purchased from Lucigen, VCSM13 helper phage is purchased from StrataGene, and plasmid pMES4 is purchased from Addgene; protein A chip is purchased from GE Healthcare; Vero cells are purchased from ATCC CCL81.

[0055] Some synthetic biological materials, such as primers, sequences, etc., which need to be artificially synthesized, are all entrusted to synthesis companies to complete. For example, the primers (SED ID NO: 14-19) in the present application are synthesized by Beijing Chengke Biological Technology Co., Ltd.

[0056] Example 1: Expression and purification of SARS-CoV-2 RBD-his and SARS-CoV-2 NTD-his proteins

[0057] A signal peptide (as shown in SEQ ID NO: 11) is connected to the 5' end of the SARS-CoV-2 RBD protein coding sequence (as shown in SEQ ID NO: 10), and a coding sequence of a hexa-His-tag and a translation termination codon TGA are connected to the 3' end of the SARS-CoV-2 RBD protein coding sequence. Through the restriction enzyme sites EcoRI and XhoI, it is constructed into the pCAGGS vector, transfected into 293F cells, and the expression of SARS-CoV-2 RBD-his protein is carried out. Similarly, a signal peptide (as shown in SEQ ID NO: 13) is connected to the 5' end of the SARS-CoV-2 NTD protein coding sequence (as shown in SEQ ID NO: 12), and a coding sequence of a hexa-His-tag and a translation termination codon TGA are connected to the 3' end of the SARS-CoV-2 NTD protein coding sequence. Through the EcoRI and XhoI restriction enzyme sites, it is constructed into the pCAGGS vector, transfected into 293F cells, and the expression of SARS-CoV-2 NTD-his protein is carried out.

[0058] The cell culture solution containing the target protein is subjected to nickel ion affinity chromatography (HisTrap TMexcel (GE Healthcare) and gel filtration chromatography (Superdex TM 200Increase 10 / 300 GL column (GE Healthcare) after purification, the relatively pure target protein can be obtained.

[0059] Example 2: Immunization of Llama and Construction of Antibody Library

[0060] 200μg of SARS-CoV-2 RBD and NTD proteins with 6 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 immunized by subcutaneous multiple point injection. After that, immunization was performed once every two weeks. On the 12th day after the fourth immunization, 50-60 mL of blood was collected, and PBMCs (peripheral blood mononuclear cells) were separated. The separated PBMCs were added to 1 mL of TRIzol, and the total RNA was extracted according to the steps of the instruction manual. Using the extracted total RNA as a template, the Superscript II First-Strand Synthesis System for RT-PCR kit was used to synthesize cDNA with random primer oligo-dT 12-18 Primers were synthesized to synthesize cDNA. Using specific primers CALL001 and CALL002 (primer sequences are shown in Table 1), PCR was performed using cDNA as a template, and a 700 bp band was cut and recovered. The purified DNA was used as a template, and nest PCR was performed using nest primers VHH-BACK and PMCF to amplify the nanobody (VHH) sequence, and the VHH sequence with a size of about 400 bp was recovered and purified.

[0061] Using double enzyme digestion method, the VHH fragment was connected to the plasmid pMES4 through the restriction enzyme cutting sites Pst I and BstE II. The purified cloning vector and electro-competent E. coli TG1 cells were mixed, and the cloning vector was transformed into the electro-competent E. coli TG1 cells using an electroporator (BIO-RAD electroporator MicroPulser). All were coated on a selective medium containing ampicillin, and after overnight culture at 37°C, all colonies were collected in LB medium, centrifuged and the supernatant was discarded, and the cells were resuspended in LB. This is the antibody library.

[0062] Table 1. Reaction primers

[0063]

[0064]

[0065] Example 3: Screening of specific nanobodies by phage display technology

[0066] The E. coli TG1 transfected with the recombinant plasmid of Example 2 was taken, and VCSM13 helper phage was added at a multiplicity of infection (MOI) of about 20. After overnight culture, centrifugation was performed at 4000 rpm, and the supernatant was taken and filtered through a 0.22 μm membrane. PEG6000 / NaCl was added at a volume ratio of 1:4, and after mixing, the mixture was placed at 4°C for at least 1 hour. Centrifugation was performed at 8000 x g for 30 min, the supernatant was discarded, and the precipitate was resuspended with PBS. The collected phage particles were obtained, and the phage titer was determined.

[0067] 2 x 10 11 The above collected phage was mixed with an equal volume of 5% (w / v) skimmed milk, and added to a 96-well plate coated with SARS-CoV-2 NTD-his antigen. After incubation at room temperature for 1 h, specific phage was eluted with 0.2 M glycine, and the eluted phage was neutralized with Tris-HCl (pH 9.1). Then, the phage was used to infect E. coli TG1 cells, and the phage was amplified. A 96-well plate coated with SARS-CoV-2 NTD-his antigen was prepared again, and the second round of panning was performed to enrich phage expressing specific nanobodies. A total of 3 rounds of panning were performed. After each round of panning, different single colonies were randomly picked from the agar plate with colonies, and cultured in a 37°C shaker. Then, VCSM13 helper phage was added for overnight expansion. The next day, the culture solution was centrifuged, and the phage supernatant was taken for ELISA experiment (using SARS-CoV-2 NTD-his protein as coating antigen). When OD 450nM >0.2, it was determined as a positive reaction, and the corresponding clone was taken, and specific primers MP57 and GIII were used for sequencing of the plasmid (primers are shown in Table 2), and the sequence encoding VHHs in the plasmid was obtained. Through sequence determination, the core coding sequence of N36 was obtained.

[0068] Table 2. Reaction primers

[0069]

[0070] Example 4: Expression and purification of nanobody N36

[0071] To make the heavy chain variable region of N36 more complete, the coding sequence of QVQLQ (CAGGTGCAGCTGCAG) was connected to the 5' end of the core coding sequence of N36 obtained in Example 3, and the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT) was connected to the 3' end, to obtain a nucleotide sequence as SEQ ID NO: 9, which is the coding sequence of the N36 nanobody of the application. Then a signal peptide (SEQ ID NO: 20) was connected in front of it, and the coding sequence of a hexa-His-tag and a translation termination codon TGA were connected behind it. Through restriction enzyme cutting sites EcoRI and XhoI, it was constructed into a pCAGGS vector, transfected into 293F cells, and cultured for 5 days. After centrifugation at 5000 rpm for 30 min, the supernatant was filtered through a 0.22 μm filter, and the target protein was purified by nickel ion affinity chromatography (HisTrap TM excel((GE Healthcare)) and gel filtration chromatography (Superdex TM 75Increase 10 / 300 GL column (GE Healthcare). After purification, the target protein was obtained in a relatively pure state. The target peak was determined by SDS-PAGE, and the results are shown in Figure 3 , to obtain the purified nanobody N36.

[0072] It was confirmed by surface plasmon resonance technology that the nanobody N36 of the application can specifically bind to SARS-CoV-2 NTD with high affinity (data not shown).

[0073] And it was confirmed by antibody neutralization test (including true virus and false virus neutralization assay) that the nanobody N36 of the application can neutralize SARS-CoV-2 original strain and its series of variant strains with high neutralization activity (data not shown).

[0074] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the application, and not to limit them; although the application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> A Llama-derived Nanobody N36 and Applications Thereof <130> 1087-210318F <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR1 of the heavy chain variable region of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(8) <400> 1 Gly Ser Ile Phe Gly Ile Tyr Val 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR2 of the heavy chain variable region of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(8) <400> 2 Ile Ser Ser Gly Gly Ile Asn Tyr 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CDR3 of the heavy chain variable region of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(11) <400> 3 Gln Asp Gly Val Pro Pro Leu Gly Tyr Asp Tyr 1 5 10 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR1 of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(25) <400> 4 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 Ala Ser 20 25 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR2 of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(17) <400> 5 Met Gly Trp Tyr Cys Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 15 Thr <210> 6 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR3 of the nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(38) <400> 6 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 1 5 10 15 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Tyr Val 35 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of framework region FR4 of the Nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(11) <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of the Nanobody N36 of the invention <220> <221> DOMAIN <222> (1)..(118) <400> 8 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 Ala Ser Gly Ser Ile Phe Gly Ile Tyr 20 25 30 Val Met Gly Trp Tyr Cys Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ile Asn Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Val 85 90 95 Gln Asp Gly Val Pro Pro Leu Gly Tyr Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 9 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> The nucleotide sequence of the nanobody N36 of this invention <220> <221> misc_feature <222> (1)..(354) <400> 9 caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggaag catcttcggc atctatgtca tgggctggta ctgccaggct 120 ccagggaagc agcgcgagtt ggtcgcaacc attagtagtg gtggtataaa ctatgcagac 180 tccgtgaagg gccgattcac catctccaga gacaacgcca agaacacggt gtatctgcaa 240 atgaacagcc tgaaacctga ggacacggcc gtctattatt gttatgttca agatggagta 300 cccccgctgg gctatgacta ctggggccag gggacccagg tgaccgtgag ctct 354 <210> 10 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 RBD protein coding sequence <220> <221> misc_feature <222> (1)..(669) <400> 10 agagtgcaac ctacagaatc aatcgtgaga tttcctaaca tcacaaacct ttgccctttc 60 ggcgaggtgt ttaacgcaac aagatttgca tcagtgtacg catggaacag aaagcgtata 120 tcaaactgcg tggcagatta ctcagtgctt tacaactcag catcattcag tacgtttaaa 180 tgctacggag tgtcacctac aaagctaaat gatctttgct ttacaaacgt gtacgcagat 240 tcatttgtga tcagaggaga tgaagtgaga caaatcgcac ctggacaaac aggaaagatt 300 gccgattaca actacaaact tcctgatgat ttcaccggct gcgtgatcgc atggaactca 360 aacaaccttg attcaaaggt aggtggtaat tataattatt tgtataggct ctttcgtaag 420 agcaacttaa agccatttga gcgagatatc tcaacagaaa tctaccaagc aggatcaaca 480 ccttgcaacg gagtggaagg atttaactgc tactttcctc ttcaatcata cggatttcaa 540 cctacaaacg gagtgggata ccaaccttac agagtggtgg tgctttcatt tgaacttctt 600 cacgcacctg caacagtgtg cggacctaag aagagcacga accttgtgaa gaataagtgc 660 gtgaacttt 669 <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide sequence for expression of SARS-CoV-2 RBD protein <220> <221> misc_signal <222> (1)..(51) <400> 11 gccaccatgt ttgtgtttct tgtgcttctt cctcttgtgt catcacaatg c 51 <210> 12 <211> 858 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 NTD protein coding sequence <220> <221> misc_feature <222> (1)..(858) <400> 12 acacggaccc agctccctcc cgcctacaca aactctttca cccggggcgt gtactacccc 60 gacaaggtgt tccggtctag cgtgctccac tctacacagg acctgttcct ccctttcttc 120 agcaacgtga catggttcca cgccatccac gtgtctggca caaacggcac aaagcggttc 180 gacaaccccg tgctcccttt caacgacggc gtgtacttcg ccagcaccga gaagtctaac 240 attatccggg gctggatttt cggcaccaca ctcgactcta agacacagtc cctcctgatt 300 gtgaacaacg ccacaaacgt ggtgattaag gtgtgcgagt tccagttctg caacgaccct 360 ttcctgggcg tgtactacca caagaacaac aagtcttgga tggagtctga gttcagagtg 420 tactctagcg ccaacaactg caccttcgag tacgtgtccc agcctttcct catggacctg 480 gagggcaagc agggcaactt caagaacctg agagagttcg tgttcaagaa cattgacggc 540 tacttcaaga tttactctaa gcacacccca attaacctcg tgagggacct ccctcagggc 600 ttctccgcct tagaaccact ggtggacctc cctattggca ttaacatcac acgcttccag 660 acactgctcg ccctccaccg gtcttacctg accccaggcg actctagctc tggctggaca 720 gccggcgccg ccgcctacta cgtgggctac ctgcagccta ggaccttcct cctgaagtac 780 aacgagaacg gcacaattac cgacgccgtg gactgcgccc tggacccact gtccgagaca 840 aagtgcacac tgaagtcc 858 <210> 13 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide sequence for expression of SARS-CoV-2 NTD protein <220> <221> misc_signal <222> (1)..(63) <400> 13 gccaccatgt tcgtgttcct cgtgctcctg cctctggtgt ctagccagtg cgtgaacctg 60 acc 63 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer CALL001 <220> <221> misc_feature <222> (1)..(23) <400> 14 gtcctggctg ctcttctaca agg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer CALL002 <220> <221> misc_feature <222> (1)..(23) <400> 15 ggtacgtgct gttgaactgt tcc 23 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer VHH‑BA <220> <221> misc_feature <222> (1)..(29) <400> 16 gatgtgcagc tgcaggagtc tggrggagg 29 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220>n <223> Primer PMCF <220> <221> misc_feature <222> (1)..(34) <400> 17 ctagtgcggc cgctgaggag acggtgacct gggt 34 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer MP57 <220> <221> misc_feature <222> (1)..(20) <400> 18 ttatgcttcc ggctcgtatg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer G III <220> <221> misc_feature <222> (1)..(19) <400> 19 ccacagacag ccctcatag 19 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide sequence for expression of Nanobody N36 of the application <220> <221> misc_signal <222> (1)..(60) <400> 20 gccaccatgc acagcagcgc cctgctgtgc tgcctggttc tgctgaccgg agtgagggcc 60

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 FR1-4 are shown as 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 6, characterized in that, The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

9.

8. A nucleic acid construct comprising the polynucleotide as described in 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, suppository, transdermal preparation, ointment, plaster, topical liquid, or injectable preparation.

14. The pharmaceutical composition according to claim 13, characterized in that, The nasal spray is selected from aerosols, sprays, and powders; The oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, and film-coated formulations; The injectable formulation is a push-in formulation.

15. The pharmaceutical composition according to claim 14, characterized in that, The tablets are sublingual tablets; The powder is a granule; The pills are small pills; The granules are fine granules.

16. 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 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, a transformed cell as described in claim 11, or a pharmaceutical composition as described in any one of claims 12 to 15 in the preparation of a medicament for detecting infection with the original strain of SARS-CoV-2.

17. A method for detecting a raw SARS-CoV-2 strain for non-diagnostic purposes, comprising using an alpaca-derived nanobody or antigen-binding fragment thereof that binds to a SARS-CoV-2 NTD as described in any one of claims 1 to 4.

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