ANTI-SARS-CoV-2 ANTIBODY

Abstract

To provide an antibody that binds to the N protein of SARS-CoV-2 and a method for using the same.SOLUTION: Provided is an antibody that binds to SARS-CoV-2 having one or more structural domains comprising CDRs 1 to 3, each consisting of a specific amino acid sequence. The antibody of the present invention can bind universally to the N protein of the mutant strain. Therefore, the antibody enables rapid detection of SARS-CoV-2 including mutant strains, i.e., acute respiratory disease (COVID-19), which is SARS-CoV-2 infection.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antibody that binds to the nucleocapsid protein of SARS-CoV-2.

Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the genus Betacoronavirus, like SARS coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and is a SARS-related coronavirus that causes severe acute respiratory disease (COVID-19). Its occurrence was first confirmed near Wuhan City, Hubei Province, China in 2019, and subsequently, it has caused a global pandemic of COVID-19.

[0003] The viral genome of SARS-CoV-2 is about 29,903 bases and is a single-stranded positive-strand RNA virus. Also, the virus particles (virions) are about 50 to 200 nm in size. Similar to common coronaviruses, it is composed of four proteins known as spike protein, nucleocapsid protein, membrane protein, and envelope protein, and RNA. Among these, the nucleocapsid protein binds to RNA to form a nucleocapsid, and the spike protein, membrane protein, and envelope protein bound to lipids surround it to form an envelope (Non-Patent Documents 2 and 3).

[0004] The nucleocapsid protein of SARS-CoV-2 (hereinafter, N protein) is a protein that binds to the RNA genome of SARS-CoV-2. It has been reported that in addition to being involved in virus particle formation such as folding of the viral genome, assembly of virus proteins, and budding of the virus, it also has a function of suppressing the response of host cells (Non-Patent Document 4).

[0005] The virological diagnosis of COVID-19 primarily relies on the detection of SARS-CoV-2 genes using gene amplification (PCR). PCR testing is considered to be the most sensitive virus test currently available. Other detection methods include antigen tests, which detect fragments of proteins on the surface of the virus (e.g., spike proteins). Antigen test kits based on immunochromatography and ELISA are already commercially available. While generally less sensitive than PCR testing, they are superior in terms of speed and ease of use.

[0006] In viral testing, methods that eliminate false positives and false negatives are required. For false positives, a measurement principle with high specificity for the target molecule is crucial, while for false negatives, the detection sensitivity of the test method is important. Furthermore, even in bodily fluids (such as saliva and pharyngeal mucus) and sewage from SARS-CoV-2 infected individuals, some viral structures are present, albeit at low concentrations. Existing PCR and antigen tests may not be sufficient to detect these viruses. Therefore, while PCR and antigen tests for SARS-CoV-2 have become commonplace technologies, there is a strong need for the development of technologies that can detect SARS-CoV-2 more specifically and with higher sensitivity.

[0007] The N protein of SARS-CoV-2 has been reported to be one of the most abundant proteins in the virus (Non-Patent Literature 4). Because the number of N proteins is far greater than the spike proteins exposed on the surface of SARS-CoV-2, it is a preferred target molecule for SARS-CoV-2 antigen testing, particularly in terms of detection sensitivity. Furthermore, the N protein exists bound to the SARS-CoV-2 RNA genome as a nucleocapsid. Therefore, if an antibody is developed that can selectively and strongly bind to the N protein, for example, and the SARS-CoV-2 N protein-RNA genome complex can be enriched, it will be possible to detect SARS-CoV-2 with higher sensitivity even in PCR testing. Similarly, by combining the process of enriching the N protein with antigen testing, it will also be possible to increase the sensitivity of SARS-CoV-2 antigen testing. Furthermore, the N protein of SARS-CoV-2 is one of the proteins mainly expressed in infected cells and exhibits strong immunogenicity. ELISA methods for the N protein of SARS-CoV, which was prevalent from November 2002, have been reported to have high specificity, enabling the diagnosis of patients in the early stages of infection (Non-Patent Literature 5).

[0008] Conventional antibodies (such as IgG antibodies) bind to antigens using two variable regions: a heavy chain and a light chain. However, heavy chain antibodies found in the serum of camelid animals exhibit binding activity and antigen specificity using only one variable region. This variable region of the antibody is called VHH (Variable domain of Heavy chain of Heavy chain antibody), and it is considered to have the lowest molecular weight (about one-tenth that of conventional antibodies (such as IgG antibodies)) among immunoglobulin fragments that can bind to antigens (Non-patent Literature 6). Functional characteristics of VHH antibodies include: 1) having binding activity equal to or greater than that of conventional antibodies; 2) being able to penetrate regions inaccessible to conventional antibodies; 3) being highly resilient to the formation and regeneration of physiological structures; and 4) having a very simple structure because it is a single-chain peptide. In particular, due to reason 3), VHH antibodies are extremely stable against heat and pressure, and it is possible to mass-produce functional antibodies in prokaryotes (such as Escherichia coli, Bacillus subtilis, and Corynebacterium), which was previously difficult. Furthermore, for the reasons stated in 4), it is easy to modify its function through protein engineering and chemical modification, and it has the advantage of being easy to create antibody-drug conjugates (ADCs).

[0009] Thus, VHH antibodies have many advantages in terms of properties and production compared to conventional antibodies (such as IgG antibodies). Therefore, detection technology using VHH antibodies that target the SARS-CoV-2 N protein is expected to be more advantageous in terms of performance and cost than conventional technology using IgG antibodies. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar;579(7798):270-273. [Non-Patent Document 2] A new coronavirus associated with human respiratory disease in China. Nature. 2020 Mar;579(7798):265-269. [Non-Patent Document 3] The Coronavirus Nucleocapsid Is a Multifunctional Protein. Viruses. Volume 6, Issue 8, August 2014, Pages 2991-3018. [Non-Patent Document 4] The SARS-CoV nucleocapsid protein: A protein with multifarious activities. Infection, Genetics and Evolution. Volume 8, July 2008, Pages 397-405. [Non-Patent Document 5] Diagnosis of Severe Acute Respiratory Syndrome (SARS) by Detection of SARS Coronavirus Nucleocapsid Antibodies in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay. Journal of Clinical Microbiology. Volume 41, No. 12, December 2003, Pages 5781-5782. [Non-Patent Document 6] The Therapeutic Potential of Nanobodies. BioDrugs. Volume 34, November 2019, Pages 11-26. [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention relates to providing an antibody that binds to the N protein of SARS-CoV-2 and a method for utilizing the same. [Means for solving the problem]

[0012] The inventors investigated how to obtain a VHH antibody that binds to the N protein of SARS-CoV-2. As a result, they succeeded in obtaining clones that are highly reactive to the N protein of SARS-CoV-2 from a VHH antibody library containing CDR1-3 constructs with specific amino acid numbers, by screening using cDNA display.

[0013] In other words, the present invention relates to the following 1) to 5). 1) An antibody that binds to SARS-CoV-2 having one or more structural domains containing a CDR as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence indicated by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence indicated by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence indicated by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence indicated by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence indicated by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence indicated by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by SGT. 2) The nucleic acid that codes for the antibody in 1). 3) A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with an antibody that binds to SARS-CoV-2, having one or more structural domains containing CDRs as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by NHRFTGRVY (SEQ ID NO: 12) (e) CDR1 consisting of the amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15) (f) CDR1 consisting of the amino acid sequence represented by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence represented by LALSSDRLGLYAY (SEQ ID NO: 18) (g) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by SGT 4) An SARS-CoV-2 detection kit containing the antibody of 1). 5) A pharmaceutical containing the antibody of 1). [Advantages of the Invention]

[0014] According to the present invention, a highly reactive anti-SARS-CoV-2 N protein antibody can be provided. Since the antibody of the present invention can bind universally to the N protein of mutant strains, by using the antibody, it becomes possible to detect SARS-CoV-2 including mutant strains, that is, to rapidly detect acute respiratory diseases (COVID-19) which are SARS-CoV-2 infections. [Brief Description of the Drawings]

[0015] [Figure 1] Alignment diagram of the amino acid sequences of EN2, EN3 and EN4 obtained by screening and the amino acid sequences of each CDR variant. Amino acids with a gray background indicate mutated amino acids. [Figure 2] Alignment diagrams of the amino acid sequences of EN5, EN6, EN11, and EN12 obtained through screening, and the amino acid sequences of each CDR variant. Amino acids shown in gray background indicate mutated amino acids. [Figure 3] Detection of His-tagged EN2, EN3, EN4, EN5, EN6, EN11, and EN12 using SDS-PAGE. [Figure 4] Evaluation of the binding specificity of coronavirus N proteins EN2, EN3, EN4, EN5, EN6, EN11, and EN12 by ELISA method. [Figure 5] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 variant coronaviruses using ELISA. [Figure 6] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 variant coronaviruses using ELISA. [Figure 7] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 omicron strain coronavirus by ELISA method. [Modes for carrying out the invention]

[0016] The antibody that binds to SARS-CoV-2 according to the present invention (hereinafter referred to as "the antibody of the present invention") is an anti-SARS-CoV-2 antibody having one or more structural domains containing CDR1-3 as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence indicated by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence indicated by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence indicated by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence indicated by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence indicated by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence indicated by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by SGT.

[0017] SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory illness (COVID-19), and its viral genome is a single-stranded positive-sense RNA virus with approximately 29,903 nucleotides. The antibody of the present invention is an antibody that binds to SARS-CoV-2, and more specifically, an antibody that binds to the N protein of SARS-CoV-2.

[0018] The structural domain of the antibody of the present invention has three CDRs: CDR1, CDR2, and CDR3. A CDR (Complementarity Determining Region) is a sequence-variable antigen recognition site or random sequence region, also known as a hypervariable region. In the structural domain of the antibody of the present invention, the three CDRs are located in the order of CDR1, CDR2, and CDR3 from the N-terminus.

[0019] The binding ability to SARS-CoV-2 can be evaluated by methods known to those skilled in the art. Specifically, as shown in the examples described later, it can be evaluated by determining the equilibrium dissociation constant KD by surface plasmon resonance. It can also be evaluated by methods such as ELISA, immunochromatography, isothermal titration calorimetry, and biolayer interferometry.

[0020] The structural domains of the antibody of the present invention may have framework regions at both ends of CDR1, CDR2, and CDR3. The framework region refers to the region of the antibody molecule's variable region excluding the complementarity-determining region, and is a highly conserved region. In other words, one embodiment of the present invention includes a structural domain having a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4) in this order. The amino acid sequences of the framework region in the structural domain include the amino acid sequences shown below or amino acid sequences that have 80% or more identity with said amino acid sequences. FR1: EVQLVESGGGLVQAGGSQKLSCTTS (Sequence ID 19) EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 20) EVQLVESGGGSVQAGGSLRLSCAAS (Sequence ID 21) EVQLVESGGGLVTAGGSLSLSCAAS (Sequence ID 22) EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 23) EVQLVESGGGLVQPGGSLRLTCVAS (Sequence ID 24) FR2: WFRQAPGKEREFVA(Sequence ID 25) WVRQVPGKGLEWVS (Sequence ID 26) WYRQAPGKQRELVA (Sequence ID 27) WVRQAPGKGLEAVS(Sequence ID 28) FR3: YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRH (Sequence ID 29), YADSVKGRFTISRDNSKSTVYLQMNNLTPEDTAVYYCVG (Sequence ID 30), YAESVKGRFTISRDSAKRTVYLQMNSLKPEDTAVYYCNA (Sequence ID 31), YDESVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCYA (Sequence ID 32), YGNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCIA (Sequence ID 33), YGDSVKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCYA(Sequence ID 34) YADSVKGRFHISRDKTKNTVDLFIRSLKPEDSGVYYCRA (Sequence ID 35) FR4: WGQGTQVTVSS (Sequence ID 36) WGQGTQVTVTS (Sequence ID 37) RGQGTQVTVSS (Sequence ID 38) WGPGTQVTVSE (Sequence ID 39)

[0021] Framework regions consisting of amino acid sequences having 80% or more identity with the amino acid sequences shown in SEQ ID NOs. 19-39 are preferably framework regions consisting of amino acid sequences having 85% or more identity, more preferably 90% or more identity, more preferably 95% or more identity, more preferably 96% or more identity, more preferably 97% or more identity, more preferably 98% or more identity, and more preferably 99% or more identity.

[0022] Here, amino acid sequence identity refers to the percentage (%) of the total number of amino acid residues at the positions where two amino acid sequences are identical when aligned. Sequence identity can be calculated, for example, by performing analysis using BLAST (Basic Local Alignment Search Tool) from the National Center for Biotechnology Information (NCBI).

[0023] Among the antibodies of the present invention, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SEQ ID NO: 3, and FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 36, and CDR1 is shown in SEQ ID NO: 4 An antibody having a structural domain (SEQ ID NO: 41) in which CDR2 is the amino acid sequence shown in SEQ ID NO: 5, CDR3 is the amino acid sequence shown in SEQ ID NO: 6, FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 26, FR3 is the amino acid sequence shown in SEQ ID NO: 30, and FR4 is the amino acid sequence shown in SEQ ID NO: 37; and an antibody having a structural domain (SEQ ID NO: 41) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 7, CDR2 is the amino acid sequence shown in SEQ ID NO: 8, CDR3 is the amino acid sequence shown in SEQ ID NO: 9, and FR1 is the amino acid sequence shown in SEQ ID NO: 21 An antibody having a structural domain (SEQ ID NO: 42) in which the amino acid sequence is shown, FR2 is the amino acid sequence shown in SEQ ID NO: 27, FR3 is the amino acid sequence shown in SEQ ID NO: 31, and FR4 is the amino acid sequence shown in SEQ ID NO: 38, and CDR1 is the amino acid sequence shown in SEQ ID NO: 10, CDR2 is the amino acid sequence shown in SEQ ID NO: 11, CDR3 is the amino acid sequence shown in SEQ ID NO: 12, FR1 is the amino acid sequence shown in SEQ ID NO: 22, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 32, and FR4 is the sequence number An antibody having a structural domain (SEQ ID NO: 43) which is the amino acid sequence shown in SEQ ID NO: 36, and an antibody having a structural domain (SEQ ID NO: 44) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 13, CDR2 is the amino acid sequence shown in SEQ ID NO: 14, CDR3 is the amino acid sequence shown in SEQ ID NO: 15, FR1 is the amino acid sequence shown in SEQ ID NO: 23, FR2 is the amino acid sequence shown in SEQ ID NO: 28, FR3 is the amino acid sequence shown in SEQ ID NO: 33, and FR4 is the amino acid sequence shown in SEQ ID NO: 36, and CDR1 is the amino acid sequence shown in SEQ ID NO: 16,An antibody having a structural domain (SEQ ID NO: 45) in which CDR2 is the amino acid sequence shown in SEQ ID NO: 17, CDR3 is the amino acid sequence shown in SEQ ID NO: 18, FR1 is the amino acid sequence shown in SEQ ID NO: 24, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 34, and FR4 is the amino acid sequence shown in SEQ ID NO: 36; and an antibody having a structural domain (SEQ ID NO: 45) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SGT, FR1 is the amino acid sequence shown in SEQ ID NO: 19, and FR2 is the sequence number Antibodies having a structural domain (SEQ ID NO: 46) where the amino acid sequence is shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 35, and FR4 is the amino acid sequence shown in SEQ ID NO: 39 are clones that are highly reactive to SARS-CoV-2 (SEQ ID NO: 40 is EN2, SEQ ID NO: 41 is EN3, SEQ ID NO: 42 is EN4, SEQ ID NO: 43 is EN5, SEQ ID NO: 44 is EN6, SEQ ID NO: 45 is EN11, and SEQ ID NO: 46 is EN12), obtained by screening using the cDNA display method in the examples described later, and are suitable anti-SARS-CoV-2 antibodies.

[0024] The antibody of the present invention is not limited in form as long as it has at least one of the above-described structural domains, and may be a single-domain antibody (also called a nanobody) such as a VHH antibody, a single-chain antibody, a heavy-chain antibody, a multimer formed by linking variable region fragments with a peptide linker, for example a dimer, or even a multimer formed by linking a variable region fragment with one or more variable region fragments having different antigen specificities. Furthermore, the antibodies of the present invention may be humanized. Since humanized antibodies can be administered to humans, they can be applied as pharmaceuticals.

[0025] The method for producing the antibody of the present invention is not particularly limited and can be easily produced by known techniques in the art. For example, it can be produced by combining peptide solid-phase synthesis and native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves designing an artificial gene optimized for the expression of the target antibody in host cells by performing steps such as codon optimization on the nucleic acid encoding the antibody of the present invention, incorporating it into a suitable vector, introducing it into host cells, and producing it as a recombinant antibody.

[0026] Examples of host cells used in the production of recombinant antibodies include Escherichia coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus / insect cells, or yeast cells. Expression vectors suitable for various host cells can be used to express the antibodies. Examples of expression vectors include E. coli-derived vectors such as pBR322, pBR325, pUC12, and pUC13; Bacillus subtilis-derived vectors such as pUB110, pTP5, and pC194; shuttle vectors that can be used in both E. coli and Bacillus subtilis, such as pHY300PLK; yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, and baculoviruses; and modified vectors thereof. These expression vectors have replication start sites, selection markers, and promoters suitable for each vector, and may optionally contain enhancers, transcription aggregation sequences (terminators), ribosome binding sites, and polyadenylation signals. Furthermore, the expression vectors may have nucleotide sequences inserted to facilitate the purification of the expressed polypeptides by fusing them with FLAG tags, His tags, HA tags, and GST tags.

[0027] When extracting the antibody expressed in the present invention from cultured bacterial cells or cultured cells, after culturing, the bacterial cells or cultured cells are collected by a known method, suspended in a suitable buffer, and the bacterial cells or cells are destroyed by sonication, lysozyme, and / or freeze-thaw cycles. A soluble extract is then obtained by centrifugation or filtration. The target antibody can then be obtained from the obtained extract by appropriately combining known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that primarily utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; or methods that utilize differences in isoelectric point, such as isoelectric focusing electrophoresis.

[0028] Since the antibody of the present invention binds to the N protein of SARS-CoV-2, contacting it with a test sample containing or potentially containing SARS-CoV-2 can confirm whether or not SARS-CoV-2 is present in the sample. Specifically, the detection of SARS-CoV-2 using the antibody of the present invention comprises the steps of: contacting the antibody of the present invention with a test sample to form a conjugate of the antibody of the present invention and SARS-CoV-2 in the test sample; and detecting the SARS-CoV-2 in the conjugate. Furthermore, the antibody of the present invention can also be used in the detection of virus-specific antibodies in serum by adding a portion of the antigen containing the SARS-CoV-2 N protein to the anti-SARS-CoV-2 antibody (e.g., serum antibody) contained in an immobilized test sample (serum) and binding it to confirm the presence of the SARS-CoV-2 antigen in the conjugated state.

[0029] Test samples include biological samples such as tracheal swabs, nasal swabs, pharyngeal swabs, nasal lavage solutions, nasal aspirates, nasal discharge, saliva, sputum, blood, serum, urine, feces, tissues, cells, and tissue or cell lysates, as well as samples collected from solid surfaces that may have virus contamination, such as doorknobs and toilet bowls. From the viewpoint of binding to the N protein, it is preferable to dissolve the virus in the test sample with a solution containing a surfactant and then bind it with the antibody in that solution. The antibody may or may not be immobilized on a solid phase. The step of detecting SARS-CoV-2 in the above-mentioned conjugate can be carried out, for example, by reacting the above-mentioned conjugate with an anti-SARS-CoV-2 antibody that recognizes an epitope different from the antibody of the present invention in the conjugate. Alternatively, SARS-CoV-2 in the above-mentioned conjugate may be detected in the liquid phase by a homogeneous assay.

[0030] Furthermore, the antibody of the present invention can be a component of a SARS-CoV-2 detection kit. This kit can be used as a diagnostic agent for SARS-CoV-2 infection (COVID-19) and as a tool for the development of therapeutic agents for SARS-CoV-2 infection. The detection kit may include, in addition to the anti-SARS-CoV-2 antibody of the present invention, reagents and equipment necessary for detection, such as an antibody that recognizes the antibody of the present invention or an N protein antibody that recognizes an epitope different from that of the antibody of the present invention, a solid support, a buffer, an enzyme reaction stop solution, a microplate reader, and the like. In the detection kit, the antibody of the present invention may be immobilized on a solid phase. Examples of solid phases include beads, membranes, the sides or bottom of a reaction vessel, plate-shaped substrates such as glass slides, and well substrates such as immunoplates, on which the antibody of the present invention is immobilized directly or indirectly.

[0031] Furthermore, it is conceivable that the antibody of the present invention, when introduced into cells by means of introducing a cell membrane-permeable peptide, binds to the N protein of SARS-CoV-2 and inhibits viral replication within cells. Therefore, the antibody of the present invention can also be administered to mammals and used as a pharmaceutical for the prevention or treatment of SARS-CoV-2 infection. Examples of mammals include humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs, but humans are preferred. When the antibody of the present invention is used as a pharmaceutical, it may be administered orally or parenterally, and can be used in combination with a well-known, pharmaceutically acceptable, and non-toxic carrier or diluent as appropriate. Parenteral administration typically involves injection, but administration by inhalation with a spray or the like is also possible.

[0032] In such pharmaceuticals, the content of the antibody of the present invention in the composition can be adjusted as appropriate. Furthermore, such pharmaceuticals can be administered to patients by administering an effective amount of the antibody of the present invention at intervals of one to several times per week. Preferred methods of administration in this case include intravenous injection and intravenous drip.

[0033] In relation to the embodiments described above, the following embodiments are further disclosed in the present invention. <1> An antibody that binds to SARS-CoV-2 having one or more structural domains containing CDRs as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence indicated by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence indicated by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence indicated by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence indicated by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence indicated by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence indicated by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by SGT. <2> Binding to N-protein, <1> Antibodies of [the substance]. <3> It is a VHH antibody, a heavy chain antibody, or a VHH antibody polymer. <1> or <2> Antibodies of [the substance]. <4> The VHH antibody polymer is a polymer formed by linking multiple of the aforementioned structural domains. <3> Antibodies of [the substance]. <5> The VHH antibody polymer is a dimer formed by linking two of the aforementioned structural domains. <3> Antibodies of [the substance]. <6> The VHH antibody polymer is a polymer formed by linking one or more of the aforementioned structural domains with one or more structural domains that have different antigen specificity from the aforementioned structural domains. <3> Antibodies of [the substance].

[0034] <7> The structural domain has the following regions in this order: 1st framework region (FR1), CDR1, 2nd framework region (FR2), CDR2, 3rd framework region (FR3), CDR3, and 4th framework region (FR4). <1> ~ <9> One of the following antibodies. <8> FR1~FR4 consists of the following amino acid sequence: <7> Antibodies of [the substance]. FR1: The amino acid sequence shown in SEQ ID NOs. 19, 20, 21, 22, 23, or 24, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR2: The amino acid sequence shown in Sequence ID No. 25, 26, 27, or 28, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR3: The amino acid sequence shown in SEQ ID NOs. 29, 30, 31, 32, 33, 34, or 35, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR4: The amino acid sequence shown in SEQ ID NOs. 36, 37, 38, or 39, or an amino acid sequence having 80% or more identity with said amino acid sequence. <9> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, and CDR3 being the amino acid sequence shown in SEQ ID NO: 3. It also has a structural domain (SEQ ID NO: 40) where FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <7> Antibodies of [the substance]. <10> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 4, CDR2 being the amino acid sequence shown in SEQ ID NO: 5, and CDR3 being the amino acid sequence shown in SEQ ID NO: 6. It has a structural domain (SEQ ID NO: 41) where FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 26, FR3 is the amino acid sequence shown in SEQ ID NO: 30, and FR4 is the amino acid sequence shown in SEQ ID NO: 37. <7> Antibodies of [the substance]. <11> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 7, CDR2 being the amino acid sequence shown in SEQ ID NO: 8, and CDR3 being the amino acid sequence shown in SEQ ID NO: 9. It has a structural domain (SEQ ID NO: 42) where FR1 is the amino acid sequence shown in SEQ ID NO: 21, FR2 is the amino acid sequence shown in SEQ ID NO: 27, FR3 is the amino acid sequence shown in SEQ ID NO: 31, and FR4 is the amino acid sequence shown in SEQ ID NO: 38. <7> Antibodies of [the substance]. <12> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 10, CDR2 being the amino acid sequence shown in SEQ ID NO: 11, CDR3 being the amino acid sequence shown in SEQ ID NO: 12, and FR1 being the amino acid sequence shown in SEQ ID NO: 22, FR2 being the amino acid sequence shown in SEQ ID NO: 25, FR3 being the amino acid sequence shown in SEQ ID NO: 32, and FR4 being the amino acid sequence shown in SEQ ID NO: 36, forming a structural domain (SEQ ID NO: 43). <7> Antibodies of [the substance]. <13> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 13, CDR2 being the amino acid sequence shown in SEQ ID NO: 14, and CDR3 being the amino acid sequence shown in SEQ ID NO: 15. It also has a structural domain (SEQ ID NO: 44) where FR1 is the amino acid sequence shown in SEQ ID NO: 23, FR2 is the amino acid sequence shown in SEQ ID NO: 28, FR3 is the amino acid sequence shown in SEQ ID NO: 33, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <7> Antibodies of [the substance]. <14> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 16, CDR2 being the amino acid sequence shown in SEQ ID NO: 17, and CDR3 being the amino acid sequence shown in SEQ ID NO: 18. It has a structural domain (SEQ ID NO: 45) where FR1 is the amino acid sequence shown in SEQ ID NO: 24, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 34, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <7> Antibodies of [the substance]. <15> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, and CDR3 being the amino acid sequence shown in SGT. It has a structural domain (SEQ ID NO: 46) where FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 35, and FR4 is the amino acid sequence shown in SEQ ID NO: 39. <7> Antibodies of [the substance]. <16> <1> The nucleic acid that codes for antibodies. <17> A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with an antibody that binds to SARS-CoV-2, having one or more structural domains containing CDRs as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence indicated by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence indicated by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence indicated by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence indicated by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence indicated by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence indicated by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence indicated by SGT. <18> <1> ~ <15> A SARS-CoV-2 detection kit containing one of the following antibodies. <19> <1> ~ <15> A pharmaceutical product containing one of the following antibodies. <20> For the prevention or treatment of SARS-CoV-2 infection <1> ~ <15> A pharmaceutical product containing one of the following antibodies. [Examples]

[0035] (Example 1) cDNA preparation for VHH-presented cDNA display adjustment Construction of a DNA library encoding the single variable domain of the heavy chain of a heavy-chain antibody (VHH) from full-length alpaca-derived antibodies. Using two alpaca-derived naive VHH library genes, S-hinge and L-hinge, provided by RePHAGEN, as templates, the S-hinge VHH-specific primer pair (SEQ ID NOs. 47, 48) and L-hinge VHH-specific primer pair (SEQ ID NOs. 47, 49) were amplified by PCR. Then, to prepare a DNA fragment consisting of a T7 promoter, omega (ω) enhancer, Kozak consensus sequence, VHH gene, His tag, and linker hybridization region (Y tag), the fragments were extended by extension PCR using overlap PCR primers (SEQ ID NOs. 50, 51) to create a full-length VHH-coding DNA library.

[0036] (Example 2) Fabrication of a cDNA display Each cDNA display was prepared according to the following steps (1) to (5). The buffers used are listed in Table 1.

[0037] [Table 1]

[0038] (1) Transcription The full-length VHH-coding DNA library prepared in Example 1 was transcribed using a T7 RiboMAX Express Large Scale RNA Production System (Promega) according to the attached manual. The amount of DNA used was 6.6 μg. The amount of DNA used for each subsequent selection was 0.1 to 1 μg from the 2nd round onward. The obtained transcripts were purified using RNAClean XP (Beckman Coulter) according to the attached manual. The purified products were quantified by concentration using NanoPad DS-11FX (DeNovix).

[0039] (2) Ligation Purified 20 pmol mRNA and a 20 pmol puromycin linker, cnvK riboG linker (Epsilon Molecular Engineering), were mixed with NaCl (final concentration 0.2 M) and Tris-HCl (pH 7.5, final concentration 0.05 M) and incubated at 90°C for 1 minute. The mixture was then cooled to 70°C at a rate of 0.1°C / second and incubated at 70°C for 1 minute. Next, the mixture was cooled to 25°C at a rate of 0.1°C / second, and then to 10°C at a rate of 2°C / second to hybridize the cnvK linker to the 3' end of the mRNA. Subsequently, using a UVP CrossLinker (CL-3000), 365nm, 100-115V (Analytik Jena), 365nm UV was applied at 4060 mJ / cm². 2 Irradiation was performed to photocrosslink the cnvK linker and mRNA, obtaining an mRNA-linker complex.

[0040] (3) Preparation of mRNA display Six pmol of mRNA-linker complexes were incubated at 30°C for 30 minutes using a 50 μL cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), Promega). MgCL2 and KCL were then added to final concentrations of 75 mM and 900 mM, respectively, and incubated at 37°C for 1 hour to display the mRNA-corresponding peptide on the puromycin on the mRNA-linker. Next, EDTA (pH 8.0) was added to a final concentration of 70 mM, and incubated at 4°C for 5 minutes to prepare the mRNA display.

[0041] (4) Preparation of cDNA display 60 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific) was placed in a Protein Lobind Tube, washed with 200 μL of binding buffer, and then the mRNA display prepared by the methods of (1)-(4) above was added and stirred at 25°C for 30 minutes. After washing with 200 μL of binding buffer, a reverse transcription reaction was carried out by incubation at 42°C for 30 minutes in a reaction mixture with the composition shown in Table 2 to prepare mRNA / cDNA-VHH conjugates. After washing with 200 μL of binding buffer, 39 μL of His tag binding / wash buffer and 1 μL of 1,000 U / μL RNase T1 were added and stirred at 37°C for 15 minutes to elute the mRNA / cDNA-VHH conjugates (cDNA display) from Dynabeads Myone streptavidin C1.

[0042] [Table 2]

[0043] (5) Purification of cDNA display 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed in an Eppendorf tube and washed with 200 μL of His tag binding / wash buffer. Then, the cDNA display prepared by the methods described in (1)-(5) above was added and the mixture was stirred at 25°C for 30 minutes. After washing with 200 μL of His tag binding / wash buffer, the cDNA display was eluted by adding 30 μL of His tag elution buffer and stirring at 37°C for 15 minutes.

[0044] (Example 3) Selection of VHH (1) In-vitro selection experiment Selection was performed using cDNA displays prepared from the libraries obtained in Example 2. The synthesis scale of the VHH-presenting cDNA display libraries used in each selection round is shown in Table 3.

[0045] [Table 3]

[0046] (1-1) Screening procedure for Selection Cycle 1 (R (Round) 1) SARS-CoV2 (2019-nCoV) Nucleocapsid-His Recombinant Protein, BiotinyLated (SinoBioLogicaL) (hereinafter referred to as biotinylated N protein) was added to Dynabeads Myone streptavidin C1 and mixed by inversion at 4°C for 30 minutes to prepare SARS-CoV2 Nucleocapsid-coated beads. The cDNA display prepared in the same manner as in Example 2 on the synthesis scale shown in Table 3 was diluted to 200 μL with PBS-T containing 5 mg / mL heparin sodium, and 100 μL was added to the beads cohesive with 50 pmol of biotinylated N protein and mixed by inversion at 4°C for 30 minutes. The remaining 100 μL of cDNA display solution was mixed with 50 pmol of biotinylated N protein and mixed by inversion at 4°C for 30 minutes. The mixture was then added to Dynabeads Myone streptavidin C1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated N protein containing the cDNA display-N protein complex onto the beads. All beads were then combined and washed four times with PBS-T. The bound cDNA display was then collected and purified using AMpure XP (Beckman Coulter) according to the provided instructions. The purified solution was subjected to PCR using cnvK NewYtag for poly A (SEQ ID NO: 52) and T7 omeganew (SEQ ID NO: 53) as primers, under conditions of annealing temperature 65°C and extension reaction time 10 seconds using PrimeSTAR Max (Takara Bio). The PCR products were then analyzed by 4% denatured PAGE. The PCR products were purified using AMpure XP according to the accompanying manual, and the resulting purified R1 library was subjected to the second round of selection.

[0047] (1-2) Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted with 50 μL of PBS-T containing 5 mg / mL heparin sodium, added to 20 μL of Dynabeads Myone streptavidin T1 (Thermo Fisher Scientific), and mixed by inversion at 4°C for 30 minutes. The supernatant was mixed with 10 pmol of biotinylated N protein and mixed by inversion at room temperature for 60 minutes. The mixture was added to 20 μL of Dynabeads Myone streptavidin T1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated N protein containing the cDNA display-N protein complex onto beads. After washing the beads four times with 200 μL of PBS-T, the bound cDNA display was collected and purified using AMpure XP (Beckman Coulter) according to the accompanying manual. The purified solution was subjected to a PCR reaction under the same conditions as in Example 3 (1-1), and then purified using AMpure XP according to the attached manual to obtain the R2 library.

[0048] (1-3) Screening procedure for the selection cycle (R3) A cDNA display library was prepared from the R2 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted with 50 μL of PBS-T containing 5 mg / mL of heparin sodium, and added to 20 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific). The mixture was inverted and mixed at 4°C for 30 minutes. Half of the supernatant was mixed with 10 pmol of biotinylated N protein and inverted and mixed at room temperature for 60 minutes. The mixture was added to 20 μL of Dynabeads Myone streptavidin T1, and the biotinylated N protein containing the cDNA display-N protein complex was immobilized onto beads by inverted and mixed at 4°C for 30 minutes. The beads were washed three times with 200 μL of PBS-T, then washed again by inverted and mixed with 20 μL of PBST at 25°C for 30 minutes. Subsequently, the bound cDNA display was recovered and purified using AMpure XP (Beckman Coulter) according to the attached manual. The purified solution was subjected to a PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain the R3 library.

[0049] (1-4) Screening procedure for Selection Cycle 4 (R4) A cDNA display library was prepared from the R3 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted in 50 μL of PBS-T containing 5 mg / mL heparin sodium, added to 20 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific), and mixed by inversion at 4°C for 30 minutes. Half of the supernatant was mixed with 5 pmol of biotinylated N protein to form a Positine solution, and the remaining half was left as a Negative solution. Both solutions were mixed by inversion at room temperature for 30 minutes. Each mixture was added to 20 μL of Dynabeads Myone streptavidin T1, and the biotinylated N protein containing the cDNA display-N protein complex was immobilized onto beads by inversion at 4°C for 30 minutes. Each bead was washed three times with 200 μL of PBST containing 1 μM biotin, then 20 μL of PBST containing 1 μM biotin and 12.5 pmol biotinylated N protein was added and washed by inversion mixing at 25°C for 5 minutes. Subsequently, 20 μL of PBST containing 12.5 pmol biotinylated N protein was added and competitive elution was performed by inversion mixing at 25°C for 60 minutes. The bound cDNA display was then collected, and each solution was purified using AMpure XP (Beckman Coulter) according to the attached manual. Each purified solution was subjected to PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain the R4 library.

[0050] (2) Next-generation sequencer analysis To verify the convergence of the DNA library through in vitro selection cycles in detail, sequencing analysis was performed using a next-generation sequencer (NGS). The preparation of the sequencing samples was carried out following the 2-step PCR Amplicon Library Preparation method provided by Illumina. First, using the PCR products after each selection as templates, Amplicon PCR was performed using NGS Fw 1st PCR primer (SEQ ID NO: 54) and NGS Rv 1st PCR primer (SEQ ID NO: 55) as primers. The PCR conditions were 98°C for 1 minute, followed by 98°C for 10 seconds, 62°C for 5 seconds, 72°C for 35 seconds (15 cycles), and then 72°C for 1 minute. After purifying the obtained PCR products according to the instructions for the Agentcourt AMPure XP, Index PCR was performed according to the instructions for Illumina. The obtained PCR products were purified according to the instructions for Agencourt AMPure XP, then their concentrations were quantified using NanoPad DS-11FX (DeNovix), and sequence analysis was performed using MiSeq (Illumina) and MiSeq Reagent Nano kit v2 (500 cycles) according to the instructions for Illumina.

[0051] The obtained DNA sequences were translated into VHH amino acid sequences and ranked by frequency of occurrence. As a result, Table 4 was selected as representative VHHs with high affinity to the N protein. Furthermore, analysis of the library after the R4 selection process revealed that VHHs containing amino acid sequence mutations (including substitutions, insertions, and deletions) were selected in the amino acid sequences of CDR1, CDR2, and CDR3 of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 (these VHHs are called CDR variants). SEQ ID NOs. 77-98 were selected as CDR variants of EN2 (SEQ ID NO: 40), SEQ ID NOs. 99-106 as CDR variants of EN3 (SEQ ID NO: 41), SEQ ID NOs. 107-119 as CDR variants of EN4 (SEQ ID NO: 42), SEQ ID NOs. 120-126 as CDR variants of EN5 (SEQ ID NO: 43), SEQ ID NOs. 127-134 as CDR variants of EN6 (SEQ ID NO: 44), SEQ ID NOs. 135 as CDR variant of EN11 (SEQ ID NO: 45), and SEQ ID NOs. 136-150 as CDR variants of EN12 (SEQ ID NO: 46) (Figures 1 and 2). Since these CDR variants are VHHs selected through the selection process, they are considered highly likely to exhibit binding activity to the SARS-CoV-2 N protein.

[0052] [Table 4]

[0053] (Example 4) Production of VHH by protease-deficient recombinant Bacillus subtilis (1) Artificial synthesis of genes The amino acid sequences of the synthesized VHH were modified by adding a His tag sequence (SEQ ID NO: 56) via a linker sequence to the C-terminus of each VHH amino acid sequence listed in Table 4. The resulting synthesized VHHs are called His-tagged EN2 (SEQ ID NO: 57), His-tagged EN3 (SEQ ID NO: 58), His-tagged EN4 (SEQ ID NO: 59), His-tagged EN5 (SEQ ID NO: 60), His-tagged EN6 (SEQ ID NO: 61), His-tagged EN11 (SEQ ID NO: 62), and His-tagged EN12 (SEQ ID NO: 63). These VHHs represented by SEQ ID NOs. 56 to 63 are collectively referred to as His-tagged VHHs. To synthesize these His-tagged VHH cells, we synthesized the following artificial synthesis genes: His-tagged EN2 artificial synthesis gene (SEQ ID NO: 64), His-tagged EN3 artificial synthesis gene (SEQ ID NO: 65), His-tagged EN4 artificial synthesis gene (SEQ ID NO: 66), His-tagged EN5 artificial synthesis gene (SEQ ID NO: 67), His-tagged EN6 artificial synthesis gene (SEQ ID NO: 68), His-tagged EN11 artificial synthesis gene (SEQ ID NO: 69), and His-tagged EN12 artificial synthesis gene (SEQ ID NO: 70), and used them in experiments.

[0054] (2) Construction of a plasmid for VHH expression Recombinant plasmid pHY-S237 (JP 2014-158430 A), prepared based on pHY300PLK, was used as a template, and the plasmid sequence was amplified by PCR using the primer sets 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 71) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 72) and PrimeSTAR Max DNA polymerase (TaKaRa). Promoter DNA derived from the spoVG gene was amplified by PCR using the primer sets 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 73) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 74) with the genomes of 168 strains as templates. The obtained promoter DNA was incorporated into the plasmid sequence using the In-Fusion HD Cloning Kit (Takara), and a VHH expression plasmid linked to the spoVG promoter was constructed. Plasmid sequences were amplified by PCR using the primer sets 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 75) and 5'-TCTATTAAACTAGTTATAGGG-3' (SEQ ID NO: 76) and PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing the synthetically synthesized gene was incorporated into the resulting PCR fragment using the In-Fusion HD Cloning Kit (Takara) to construct VHH expression plasmids containing each of the synthetically synthesized VHH genes.

[0055] (3) Preparation of recombinant Bacillus subtilis From 168 strains of Bacillus subtilis (hereinafter referred to as "168 strains"), knockout strains of extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, aprX) were prepared according to the method described in Japanese Patent No. 4485341. Furthermore, according to the method described in Japanese Patent No. 4336082, the sigF gene, which is involved in spore formation, was deleted from Bacillus subtilis strain Dpr9, which lacked all nine of the above extracellular protease genes. The resulting extracellular protease multiple knockout strain will be described as Dpr9ΔsigF. Plasmid introduction into the Dpr9ΔsigF strain was performed using the protoplast method described below. Bacillus subtilis was inoculated into 1 mL of LB liquid medium from a glycerol stock and incubated overnight at 30°C and 210 rpm with shaking. The following day, 10 μL of this culture was inoculated into a fresh 1 mL of LB liquid medium and incubated at 37°C and 210 rpm with shaking for approximately 2 hours. This culture was collected in a 1.5 mL tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. The resulting pellet was suspended in 500 μL of SMMP (0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic medium 3 (Difco)) containing 4 mg / mL of Lysozyme (SIGMA), and incubated at 37°C for 1 hour. Next, the pellet was centrifuged at 3,500 rpm for 10 minutes, and the supernatant was removed. The pellet was then suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with the various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this mixture and mixed by inversion. After shaking at 30°C and 210 rpm for 1 hour, the entire volume was spread onto a DM3 agar plate and incubated at 30°C for 2-3 days.

[0056] (4) VHH production The recombinant Bacillus subtilis prepared in (3) was inoculated into LB medium containing 1 mL of 50 ppm tetracycline and shaken back and forth overnight at 30°C to prepare the preculture medium. The preculture medium was inoculated at 1% into 20 mL of 2 × L-mal medium in a ribbed Erlenmeyer flask and cultured with shaking at 30°C for 72 hours. At the end of the culture, 1 mL of the culture medium was centrifuged in a microtube at 4°C, 15,000 rpm for 5 minutes, and the supernatant was collected. His-tagged VHH was purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries) according to the kit protocol. The purified protein was dissolved in PBS containing 30 mM imidazole.

[0057] (5) Verification by SDS-PAGE The sample collected in (4) was mixed in equal volumes with Laemmli Sample Buffer (BIO-RAD), and then heat-treated at 99°C for 5 minutes to prepare the sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) gel was used. 5 μL of sample was applied to each well and electrophoresis was performed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used as the molecular weight marker. Protein bands were detected using the ChemiDoc MP Imaging System. The detected proteins were quantified using a calibration curve created based on a lysozyme standard (Sigma-Aldrich). As a result, it was confirmed that the target His-tagged VHH was synthesized (Figure 3).

[0058] (Example 5) Evaluation of binding specificity by ELISA method Each well of the F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific) contains 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein), Human coronavirus (HCoV-229E) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-NL63) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-HKU1) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-OC43) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein) (all from SinoBiological), SARS Nucleocapsid protein, His Tag, MERS nucleocapsid protein, and His Tag (His-tagged N protein) (both from AcroBioSystems) were added, sealed, and allowed to stand overnight at 4°C. After removing the N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well on which N protein was immobilized, and the mixture was incubated at room temperature for 1 hour.After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. Peroxidase-AffiniPure Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) (Jackson Immuno Research Laboratories) was used as the detection antibody. The detection antibody was diluted to 1 / 5,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated for 30 minutes under light shielding, and the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figure 4). As a result, it was confirmed that all of the His-tagged VHH used in this example bound most strongly to the N protein of SARS-CoV-2.

[0059] (Example 6) Evaluation of binding activity to mutant N protein by ELISA method Add 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein), SARS-CoV-2 (2019-nCoV) Nucleocapsid (R203K, G204R)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (P13L)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (S194L)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (I292T)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (D3L, R203K, G204R, S235F)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (D3L, S235F)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (T205I) Protein (His Tag), SARS-CoV-2 (2019-nCoV) Nucleocapsid (P80R) Protein (His Tag), SARS-CoV-2 (2019-nCoV) Nucleocapsid (D377Y) Protein (His Tag), SARS-CoV-2 Nucleocapsid (A12G, T205I) Protein (His Tag), SARS-CoV-2 Nucleocapsid (R203M, D377Y) Protein (His Tag), SARS-CoV-2 Nucleocapsid (A119S, R203K, G204R, M234I) Protein (His Tag) (all from SinoBiological) to each well of a F96 Cert.Maxisorp Nunc-Immuno (trademark) Plate (Thermo Fisher Scientific). After sealing, leave it standing at 4 °C overnight. After removing the N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well with immobilized N protein and incubated at room temperature for 1 hour. After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. Peroxidase-AffiniPure Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) (Jackson Immuno Research Laboratories) was used as the detection antibody. The detection antibody was diluted to 1 / 5,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated under light shielding for 30 minutes, and immediately afterward, the absorbance at 450 nm was measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figures 5 and 6). As a result, it was confirmed that all His-tagged VHH used in this example universally bind to the N protein of SARS-CoV-2 mutant strains.

[0060] (Example 7) Measurement of binding activity by biolayer interferometry The concentrations of various His-tagged VHHs were quantified using the BCA method with the Pierce BCA Protein Assay Kit (Thermo Fisher), with bovine serum albumin (Fujifilm Wako Pure Chemical Industries, Ltd.) as the standard protein. The binding activity of various His-tagged VHHs to biotinylated N protein immobilized on a Streptavidin (SA) Biosensor chip (Sartorius) was measured using Octet® RED384 (Sartorius). Each measurement was performed by adding 60 μL of each measurement solution to a 384-well plate. Before measurement, the tip of the SA Biosensor chip was hydrated by immersing it in 200 μL of PBST for 10 minutes. 1) Baseline step: Measurement in PBST for 30 seconds 2) Association step: Measurement for 120 seconds with various His-tagged VHH solutions of 250 nM, 125 nM, 62.6 nM, 31.3 nM, 15.6 nM, and 0 nM, prepared by diluting with PBST. 3) Dissociation step: Measurement in PBS-T for 240 seconds 4) Regeneration step: Measurement is performed by repeating the process three times: 5 seconds in Glycin-HCl (pH 2.2) followed by 5 seconds in PBS-T. The adsorption of various His-tagged VHHs to the SA Biosensor chip was measured in steps 1) to 4) above. Subsequently, the biotinylated N protein was immobilized on the SA Biosensor chip by measuring with PBS-T containing biotinylated N protein for 600 seconds until a response of approximately 6.5 nm was obtained. Steps 1) to 4) above were performed using the SA Biosensor chip with the biotinylated N protein immobilized. Using Octet software version (1.2.1.5) (Molecular Devices), measurements of various His-tagged VHHs at a concentration of 0 nM and the results before immobilization of biotinylated N protein were taken as double references, and binding and dissociation rates were calculated by performing global fitting (Table 5).

[0061] [Table 5]

[0062] (Example 8) Evaluation of binding activity to Omicron strain N protein by ELISA method 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein) and SARS-CoV-2 B.1.529 (Omicron) Nucleocapsid Protein (His Tag) (both SinoBiological) were added to each well of an F96 Cert. Maxisorp Nunc-Immuno® Plate (Thermo Fisher Scientific). After sealing, the plates were incubated overnight at 4°C. After removing any N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well immobilized with N protein, and incubated at room temperature for 1 hour. After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The detection antibody used was Peroxidase-AffiniPure Goat Anti-Alpaca IgG(H+L)(min X Bov,Hu,Ms,Rb,Rat Sr Prot)(Jackson Immuno Research Laboratories) (1.0 mg / mL). The detection antibody was diluted to 1 / 5,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed.This cleaning procedure was performed three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated for 30 minutes under light shielding, and the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figure 7). As a result, it was confirmed that all of the His-tagged VHH used in this example also bind to the N protein of the SARS-CoV-2 Omicron strain.

Claims

1. An antibody that binds to SARS-CoV-2, having one or more structural domains containing CDRs shown in (a), (b), (c), (d), (e), (f) or (g) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence represented by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence represented by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by SGT

2. The antibody of claim 1 which binds to the N-protein.

3. The antibody according to claim 1 or 2, which is a VHH antibody, a heavy chain antibody or a VHH antibody multimer.

4. The antibody according to claim 3 , wherein the VHH antibody multimer is a multimer in which multiple structural domains are linked together.

5. The antibody according to claim 3, wherein the VHH antibody multimer is a multimer in which one or more of the structural domains are linked to one or more structural domains having antigen specificity different from that of the structural domains.

6. A nucleic acid encoding the antibody according to claim 1 or 2.

7. A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with an antibody that binds to SARS-CoV-2, the antibody having one or more structural domains containing CDRs represented by (a), (b), (c), (d), (e), (f) or (g) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by PWGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHYFMH (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by TIGSSSTF (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by RSISRFNVWA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by SITKSGDTN (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by AVFRGITLYTY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GRAFSSYMMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AIGWSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by VINPDGSSTH (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15). (f) CDR1 consisting of the amino acid sequence represented by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by GIRWNNVSTY (SEQ ID NO: 17), and CDR3 consisting of the amino acid sequence represented by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by SGT

8. A SARS-CoV-2 detection kit comprising the antibody according to claim 1 or 2.

9. A pharmaceutical comprising the antibody according to claim 1 or 2.

10. The pharmaceutical composition according to claim 9, for preventing or treating SARS-CoV-2 infection.

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