ANTI-SARS-CoV-2 ANTIBODY

Abstract

To provide an antibody that is bound to the nucleocapsid protein (N protein) of SARS-CoV-2.SOLUTION: The present invention provides an antibody that is bound to SARS-CoV-2, comprising one or more structural domains with CDR1-3 comprising a specific amino acid sequence, such as a single domain antibody like VHH, or a multimer comprising one or more structural domains linked to one or more structural domains with different antigen specificity from these structural domains. Also provided is a nucleic acid coding for the antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies that bind to the nucleocapsid protein of SARS-CoV-2. [Background technology]

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a SARS-related coronavirus that causes acute respiratory disease (COVID-19) and belongs to the betacoronavirus genus, just like SARS-CoV and MERS-CoV. It was first identified in 2019 near Wuhan, Hubei Province, China, and has since caused the COVID-19 pandemic.

[0003] SARS-CoV-2 is a single-stranded positive-strand RNA virus with a viral genome of about 29,903 bases. The virus particle (virion) is about 50 to 200 nm in size. Like common coronaviruses, it is composed of four proteins known as spike protein, nucleocapsid protein, integral membrane protein, and envelope protein, and RNA. Of these, the nucleocapsid protein binds to RNA to form a nucleocapsid, and the spike protein, integral membrane protein, and envelope protein bound to lipids surround it to form an envelope (Non-Patent Documents 1 and 2).

[0004] The SARS-CoV-2 nucleocapsid protein (hereinafter referred to as N protein) is a protein that binds to the RNA genome of SARS-CoV-2. It is reported that it is involved in the formation of virus particles, such as the folding of the viral genome, the assembly of viral proteins, and the budding of the virus, as well as suppressing the response of host cells (Non-Patent Document 3). It has been reported that the N protein of SARS-CoV-2 is one of the most abundant proteins that constitute the virus (Non-Patent Document 4). The number of N proteins is far greater than that of the spike proteins exposed on the surface of SARS-CoV-2, making it a preferred target molecule for SARS-CoV-2 antigen testing, especially in terms of detection sensitivity. In addition, the N protein of SARS-CoV-2 is one of the proteins that is mainly expressed in infected cells and shows strong immunogenicity. It has been reported that the ELISA method for the N protein of SARS-CoV, which has been prevalent since November 2002, has high specificity and can diagnose patients who are considered to be in the early stages of infection (Non-Patent Document 5).

[0005] In response to the recent global pandemic of COVID-19 caused by SARS-CoV-2, the need for POCT (Point of Care Testing: simple and rapid testing) such as antigen testing based on immunochromatography has increased more than ever before. In immunochromatography, an antibody bound to gold colloids or latex particles (hereinafter referred to as labeled antibody) and an antigen contained in a sample form a complex and move on a nitrocellulose membrane by capillary action, and an antibody (hereinafter referred to as capture antibody) that has been immobilized on the nitrocellulose membrane in advance binds to the antigen-labeled antibody complex, causing color development. Currently, most commercially available immunochromatography kits use monoclonal or polyclonal IgG antibodies derived from mice, rabbits, etc.

[0006] Meanwhile, heavy chain antibodies found in the serum of camelids exhibit binding activity and antigen specificity with only one variable domain, whereas normal antibodies (such as IgG antibodies) bind to antigens with two variable domains, a heavy chain and a light chain. The variable domain of this antibody is called VHH (Variable domain of Heavy chain of Heavy chain antibody), and is considered to have the lowest molecular weight (about one-tenth that of normal antibodies (such as IgG antibodies)) among immunoglobulin fragments capable of binding to antigens (Non-Patent Document 6). While VHHs exhibit binding activity equivalent to that of IgG antibodies, they have the following characteristics: (1) their molecular weight is about one-tenth that of IgG antibodies, and they are expected to bind to new epitopes that cannot be bound by conventional antibodies, (2) unlike IgG antibodies, they have a highly reversible protein structure and are highly resistant to heat and pressure, and (3) unlike IgG antibodies, they can be produced using microorganisms such as yeast and bacteria. Furthermore, (4) VHHs have a very high affinity for in-vitro antibody selection techniques such as cDNA display and phage display, and can be developed in a shorter period of time than IgG antibodies obtained by immunization of mice, rabbits, etc.

[0007] In addition, VHHs are also clearly superior in the development of test drugs. In particular, the following characteristics of VHHs are considered to be superior to IgG antibodies: (1) VHHs can be densely immobilized on particles or nitrocellulose membranes, allowing more paratopes to be presented on the substrate, (2) VHHs have excellent protein stability, and products can be expected to have better storage stability, (3) VHHs can be mass-produced inexpensively using microorganisms, allowing for further reduction in manufacturing costs, and (4) VHHs do not have an Fc region that can cause nonspecific reactions in immunochromatography.

[0008] However, there are problems with using VHH in antigen testing methods such as immunochromatography. It is known that VHH consisting only of variable regions (1) have a low molecular weight compared to IgG antibodies, and therefore have low physical adsorption performance to substrates, (2) are prone to a decrease in binding activity due to the protein denaturation effect when immobilized on a substrate, and (3) unlike IgG antibodies, VHH do not have an Fc region, making it difficult to control the orientation in random adsorption to a substrate. For these reasons, when VHH are used as capture antibodies, the binding activity is prone to decrease, which is a problem. In other words, it is known that it is difficult to detect antigens with high sensitivity when VHH are used as capture antibodies in immunochromatography.

[0009] To address this issue, Non-Patent Document 7 uses a method in which a conjugation pad is impregnated with two types of antibodies, VHH as a labeled antibody and biotin-labeled VHH, and streptavidin, which exhibits binding activity to biotin, is immobilized on a nitrocellulose membrane. That is, a complex of labeled antibody-antigen-biotinylated VHH is formed on the nitrocellulose membrane, and the complex is captured by streptavidin (Prior Art 1).

[0010] In addition, Non-Patent Document 8 uses VHH as a labeled antibody, and uses a method in which a pre-prepared biotinylated VHH-streptavidin complex is immobilized on a nitrocellulose membrane as a capture antibody. In other words, this method makes the immobilization of VHH on the nitrocellulose membrane dependent on the interaction between streptavidin and the nitrocellulose membrane (prior art 2).

[0011] In addition, Non-Patent Document 9 uses a single-chain variable region fragment in which a variable region composed of VH and VL, the minimum unit for an IgG antibody to recognize an antigen, is linked by a peptide linker as a capture antibody in an immunochromatography method using a cellulose membrane. When a single-chain variable region fragment is used as a capture antibody, there is a problem with detection sensitivity, but this is solved by using a carbohydrate-binding module that binds to the cellulose membrane (Prior Art 3). That is, it is reported that by linking the single-chain variable region fragment to a carbohydrate-binding module, the amount and orientation of the single-chain variable region fragment immobilized on the cellulose membrane can be controlled, and the binding activity of the single-chain variable region fragment used as a capture antibody is improved.

[0012] In the case of conventional techniques 1 and 2, it is necessary to modify VHH with biotin, and in many cases, chemical modification methods such as amine coupling are used. However, it is known that the amine coupling method often leads to a decrease in binding activity because it is difficult to site-specifically modify VHH. In addition, the use of biotin modification or streptavidin is not a preferable method in terms of work steps and costs. In particular, streptavidin also binds to vitamins other than avidin. When such vitamins are contained in the sample, the use of streptavidin as a part of the capture antibody poses the problem that the function of the capture antibody is reduced due to competitive inhibition.

[0013] Regarding Prior Art 3, the method of linking an adsorption module to a substrate to VHH is an excellent method in terms of the amount of immobilization on the substrate, orientation, and avoidance of protein denaturation effects during adsorption. However, it is necessary to search for an adsorption module for each substrate used, and it is difficult to find one with excellent versatility. Another issue is that depending on the type of adsorption module linked to VHH, production using microorganisms may be difficult. In addition, it is described that the carbohydrate binding module described in Non-Patent Document 4 does not exhibit binding activity to nitrocellulose membranes, and Prior Art 3 is an antibody immobilization method limited to cellulose membranes as a substrate for immobilizing capture antibodies.

[0014] Therefore, there has been a demand for the development of a VHH that can detect an antigen with high sensitivity in an immunochromatography method without using the methods used in conventional techniques 1 to 3. [Prior art documents] [Non-patent literature]

[0015] [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. [Non-Patent Document 7] Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus. mSphere. 2016;1:e00219-16. [Non-Patent Document 8] Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections. Sci Rep. 2018;8:9019. [Non-Patent Document 9] Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays. Sci Rep. 2021;11:7880. Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention relates to providing an antibody capable of specifically binding to the N protein of SARS-CoV-2 and detecting the N protein of SARS-CoV-2. [Means for solving the problem]

[0017] The inventors conducted studies to obtain VHHs that bind to the N protein of SARS-CoV-2, and as a result, they succeeded in obtaining clones highly reactive to the N protein of SARS-CoV-2 by screening a VHH library containing CDRs 1 to 3 with a specific number of amino acids in the construct using the cDNA display method, and found that these could become anti-SARS-CoV-2 antibodies suitable for immunochromatography.

[0018] That is, the present invention relates to the following 1) and 2). 1) An antibody that binds to SARS-CoV-2, having one or more structural domains including CDR1 to 3 selected from the following (a), (b), (c), (d), (e) and (f). (a) CDR1 consisting of the amino acid sequence represented by GRTFSDYDMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SITSGGSTK (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by HDYFYVDNWESY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSRYDMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by AISWNGGSTY (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by LDWLQWDWAY (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GFTFSSYAMT (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GQHHQELQHWYAWDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AITWNGGSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by LQDHNSVLADAY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AISWSGDSTH (SEQ ID NO: 13), and CDR3 consisting of the amino acid sequence represented by LGEIDGLEENDY (SEQ ID NO: 14). (f) CDR1 consisting of the amino acid sequence represented by GFTFSDYAMG (SEQ ID NO: 15), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by LGQNKEHVGKRIEDY (SEQ ID NO: 16). 2) A nucleic acid encoding the antibody of 1). Effect of the Invention

[0019] According to the present invention, a highly reactive anti-SARS-CoV-2 N protein antibody can be provided. The antibody is useful as an antibody used in immunochromatography, and is therefore useful for rapid detection of SARS-CoV-2, i.e., for simple and rapid diagnosis of acute respiratory disease (COVID-19), which is a SARS-CoV-2 infection. [Brief description of the drawings]

[0020] [Figure 1] SDS-PAGE results of His-tagged VHH produced in Bacillus subtilis. [Diagram 2] Evaluation of the binding activity of His-tagged PN4, His-tagged PN10, His-tagged PN27, His-tagged PN44, His-tagged PN46, and His-tagged PN63 to the N protein of SARS-CoV-2 using the Sandwich ELISA method. [Diagram 3]Evaluation of the binding specificity of His-tagged PN4, His-tagged PN10, His-tagged PN27, His-tagged PN44, His-tagged PN46, and His-tagged PN63 to the N protein of coronaviruses by direct ELISA. [Figure 4] A test to examine whether it is possible to construct an immunochromatography system using His-tagged VHH as the labeled antibody and capture antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The antibody of the present invention that binds to SARS-CoV-2 (hereinafter referred to as the "antibody of the present invention") is an antibody that binds to SARS-CoV-2 and has one or more structural domains including CDR1 to 3 selected from (a), (b), (c), (d), (e), and (f) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSDYDMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SITSGGSTK (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by HDYFYVDNWESY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSRYDMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by AISWNGGSTY (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by LDWLQWDWAY (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GFTFSSYAMT (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GQHHQELQHWYAWDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AITWNGGSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by LQDHNSVLADAY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AISWSGDSTH (SEQ ID NO: 13), and CDR3 consisting of the amino acid sequence represented by LGEIDGLEENDY (SEQ ID NO: 14). (f) CDR1 consisting of the amino acid sequence represented by GFTFSDYAMG (SEQ ID NO: 15), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by LGQNKEHVGKRIEDY (SEQ ID NO: 16).

[0022] SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory disease (COVID-19), and its viral genome is a single-stranded positive-strand RNA virus with approximately 29,903 bases. 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.

[0023] The structural domain of the antibody of the present invention has three CDRs, namely, CDR1, CDR2, and CDR3. A CDR (Complementarity Determining Region) includes a sequence-variable antigen recognition site or a random sequence region, and is also called a hypervariable region. In the structural domain of the antibody of the present invention, the three CDRs are present in the order of CDR1, CDR2, and CDR3 from the N-terminus.

[0024] The binding ability to SARS-CoV-2 can be evaluated by methods known to those skilled in the art, such as ELISA, immunochromatography, isothermal titration calorimetry, biolayer interference, surface plasmon resonance, and equilibrium dissociation constant KD.

[0025] The structural domain of the antibody of the present invention may have framework regions on both ends of CDR1, CDR2, and CDR3. The framework region is a region in the variable region of an antibody molecule excluding the complementarity determining regions, and refers to a highly conserved region. That is, one embodiment of the structural domain of the present invention includes a first framework region (FR1), CDR1, second framework region (FR2), CDR2, third framework region (FR3), CDR3 and fourth framework region (FR4) in this order. Examples of amino acid sequences of framework regions in structural domains include the amino acid sequences shown below or amino acid sequences having 80% or more identity to said amino acid sequences.

[0026] FR1: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 17) FR2: WYRQAPGKGLEWVA (SEQ ID NO: 18) WFRQAPGKGREFVA (SEQ ID NO: 19) WFRQAPGKGREGVA (SEQ ID NO: 20) FR3: YADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 21) YAESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 22) YADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAV (SEQ ID NO: 23) YAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAA (SEQ ID NO: 24) FR4: WGQGTLVTVSS (SEQ ID NO: 25)

[0027] Examples of framework regions consisting of amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 17 to 25 include framework regions consisting of amino acid sequences having preferably 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.

[0028] Here, the identity of amino acid sequences refers to the percentage (%) of the number of positions at which identical amino acid residues exist in two amino acid sequences when the two sequences are aligned relative to the total number of amino acid residues. The identity of sequences can be calculated, for example, by performing an analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

[0029] Among the antibodies of the present invention, an antibody having a structural domain (SEQ ID NO: 26) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, CDR3 being the amino acid sequence shown in SEQ ID NO: 3, FR1 being the amino acid sequence shown in SEQ ID NO: 17, FR2 being the amino acid sequence shown in SEQ ID NO: 18, FR3 being the amino acid sequence shown in SEQ ID NO: 21, and FR4 being the amino acid sequence shown in SEQ ID NO: 25, and an antibody having a structural domain (SEQ ID NO: 26) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 4 an antibody having a structural domain (SEQ ID NO:27) 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, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:19, FR3 is the amino acid sequence shown in SEQ ID NO:22, and FR4 is the amino acid sequence shown in SEQ ID NO:25; an antibody having a structural domain (SEQ ID NO:28) in which 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:17, FR2 is the amino acid sequence shown in SEQ ID NO:20, FR3 is the amino acid sequence shown in SEQ ID NO:21, and FR4 is the amino acid sequence shown in SEQ ID NO:25; and an antibody having a structural domain (SEQ ID NO:29) in which CDR1 is the amino acid sequence shown in SEQ ID NO:10, CDR2 is the amino acid sequence shown in SEQ ID NO:13, CDR3 is the amino acid sequence shown in SEQ ID NO:14, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:20, FR3 is the amino acid sequence shown in SEQ ID NO:21, and FR4 is the amino acid sequence shown in SEQ ID NO:25; and an antibody having a structural domain (SEQ ID NO:30) in which CDR1 is the amino acid sequence shown in SEQ ID NO:10, CDR2 is the amino acid sequence shown in SEQ ID NO:13, CDR3 is the amino acid sequence shown in SEQ ID NO:14, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:20, FR3 is the amino acid sequence shown in SEQ ID NO:21, and FR4 is the amino acid sequence shown in SEQ ID NO:25;The antibodies having a structural domain (SEQ ID NO: 31) in which CDR2 is the amino acid sequence shown in SEQ ID NO: 8, CDR3 is the amino acid sequence shown in SEQ ID NO: 16, FR1 is the amino acid sequence shown in SEQ ID NO: 17, FR2 is the amino acid sequence shown in SEQ ID NO: 20, FR3 is the amino acid sequence shown in SEQ ID NO: 24, and FR4 is the amino acid sequence shown in SEQ ID NO: 25 are clones highly reactive to SARS-CoV-2 obtained by screening using the cDNA display method in the Examples described below (SEQ ID NO: 26 is PN4, SEQ ID NO: 27 is PN10, SEQ ID NO: 28 is PN27, SEQ ID NO: 29 is PN44, SEQ ID NO: 30 is PN46, and SEQ ID NO: 31 is PN63), and are suitable anti-SARS-CoV-2 antibodies.

[0030] The form of the antibody of the present invention is not limited as long as it has at least one of the above structural domains, and it may be a single domain antibody or a multimer (e.g., a dimer) in which multiple single domain antibodies are linked. Multimers include multimers in which multiple structural domains of the present invention are linked, as well as multimers in which one or more of the structural domains are linked to one or more other structural domains that have different antigen specificity from the structural domains. A single domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region (antigen-binding domain). Single domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy chain single domain antibody) and antibodies whose variable region consists only of the variable region of a light chain (light chain single domain antibody). VHH, which is a heavy chain antibody identified in camelids (e.g., camel, llama, alpaca, etc.), and VNAR, which is a heavy chain antibody derived from cartilaginous fish (e.g., shark), are known as types of single domain antibodies, and VHH is preferred in the present invention. The antibody of the present invention may be humanized. Humanized antibodies can be administered to humans and therefore can be used as medicines.

[0031] The method for producing the antibody of the present invention is not particularly limited, and the antibody can be easily produced by known techniques in the art. For example, the antibody can be produced by combining solid-phase peptide synthesis and native chemical ligation (NCL) or by genetic engineering, but a preferred method is to design an artificial gene optimized for the expression of the antibody of interest in a host cell by subjecting a nucleic acid encoding the antibody of the present invention to a process such as codon optimization, incorporate the artificial gene into an appropriate vector, and introduce the vector into a host cell to produce the antibody as a recombinant antibody.

[0032] Examples of host cells used in the production of recombinant antibodies include Escherichia coli, Bacillus subtilis, mold, animal cells, plant cells, baculovirus / insect cells, and yeast cells. The expression vector for expressing the antibody can be a vector suitable for various host cells. Examples of expression vectors that can be used include vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common between Escherichia coli and Bacillus subtilis such as pHY300PLK; vectors derived from yeast such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified from these. These expression vectors have a replication origin, a selection marker, and a promoter suitable for each vector, and may have an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, in order to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a base sequence for expressing a fused tag such as a FLAG tag, a His tag, an HA tag, or a GST tag.

[0033] When extracting the expressed antibody of the present invention from cultured bacteria or cells, after culturing, the bacteria or cultured cells are collected by a known method, suspended in an appropriate buffer, and the bacteria or cells are disrupted by ultrasonic waves, lysozyme and / or freeze-thawing, etc., and then a soluble extract is obtained by centrifugation or filtration. The target antibody can 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 utilize mainly 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 reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0034] Since the antibody of the present invention binds to the N protein of SARS-CoV-2, by contacting it with a test sample that contains or may contain SARS-CoV-2, it is possible to confirm the presence or absence of SARS-CoV-2 in the sample. Specifically, 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 complex between the antibody of the present invention and SARS-CoV-2 in the test sample, and detecting SARS-CoV-2 in the complex. In addition, the antibody of the present invention can also be used as an antibody for detecting virus-specific antibodies in serum, by adding a portion of an antigen containing the N protein of SARS-CoV-2 to anti-SARS-CoV-2 antibodies (e.g., serum antibodies) contained in an immobilized test sample (serum) to bind to the antibodies, and confirming the presence of SARS-CoV-2 antigen in the bound state.

[0035] Examples of test samples include biological samples such as tracheal swabs, nasal swabs, pharyngeal swabs, nasal washings, nasal aspirates, nasal mucus, nasal mucus, saliva, sputum, blood, serum, urine, feces, tissues, cells, tissue or cell fragments, as well as samples collected from solid surfaces to which the virus may be attached, such as doorknobs and toilets. From the viewpoint of binding to the N protein, it is preferable to dissolve the virus in the test sample in a solution containing a surfactant and bind it to the antibody in the solution. The antibody may or may not be fixed to 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 that of the antibody of the present invention in the conjugate. Alternatively, SARS-CoV-2 in the above-mentioned conjugate may be detected in a liquid phase by a homogeneous assay.

[0036] The antibody of the present invention can also be a component of a kit for detecting SARS-CoV-2. In particular, the antibody of the present invention is useful as a capture antibody or labeled antibody used in immunochromatography (lateral flow assay), and is therefore suitable as a component of a strip in a lateral flow assay. The kit can be used as a diagnostic agent for SARS-CoV-2 infection (COVID-19) and as a tool for developing a therapeutic agent 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 instruments 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 the antibody of the present invention, a solid phase support, a buffer solution, an enzyme reaction stop solution, a microplate reader, etc. In the detection kit, the antibody of the present invention may be immobilized on a solid phase, such as beads, a membrane, the side or bottom surface of a reaction vessel, a plate-like substrate such as a slide glass, a well substrate such as an immunoplate, or a strip in a lateral flow assay, to which the antibody of the present invention is directly or indirectly immobilized.

[0037] In addition, the antibody of the present invention is considered to bind to the N protein of SARS-CoV-2 and inhibit the replication of the virus in the cell by translocating the antibody into the cell, for example by introducing a cell membrane-permeable peptide. Therefore, the antibody of the present invention can be administered to a mammal and used as a medicine for preventing or treating SARS-CoV-2 infection. Mammals include humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc., with humans being preferred. When the antibody of the present invention is used as a pharmaceutical, it may be administered either orally or parenterally, and may be appropriately combined with a known, pharma- ceutically acceptable, non-toxic carrier or diluent. Parenteral administration typically includes injections, but administration by inhalation together with a spray or the like is also possible.

[0038] In such a pharmaceutical composition, the content of the antibody of the present invention in the composition can be appropriately adjusted. Such a medicine can be applied by administering an effective amount of the antibody of the present invention to a patient at intervals of about once to several times a week. In this case, suitable methods of administration include intravenous injection, drip infusion, etc.

[0039] In the present invention, the following aspects are further disclosed in relation to the above-mentioned embodiment. <1> An antibody that binds to SARS-CoV-2, having one or more structural domains including CDR1 to CDR3 selected from (a), (b), (c), (d), (e), and (f) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSDYDMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SITSGGSTK (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by HDYFYVDNWESY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSRYDMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by AISWNGGSTY (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by LDWLQWDWAY (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GFTFSSYAMT (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GQHHQELQHWYAWDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AITWNGGSTY (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by LQDHNSVLADAY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by RSIFSGNAMG (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by AISWSGDSTH (SEQ ID NO: 13), and CDR3 consisting of the amino acid sequence represented by LGEIDGLEENDY (SEQ ID NO: 14). (f) CDR1 consisting of the amino acid sequence represented by GFTFSDYAMG (SEQ ID NO: 15), CDR2 consisting of the amino acid sequence represented by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by LGQNKEHVGKRIEDY (SEQ ID NO: 16). <2> Binds to N-protein <1> antibodies. <3> A single domain antibody or a multimer thereof. <1> or <2> antibodies. <4> the single domain antibody is a VHH, <3> antibodies. <5> A multimer of a single domain antibody is a multimer in which multiple structural domains are linked together. <3> antibodies. <6> The antibody according to claim 3, wherein the single domain 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.

[0040] <7> The structural domain has, in this order, a first framework region (FR1), a CDR1, a second framework region (FR2), a CDR2, a third framework region (FR3), a CDR3 and a fourth framework region (FR4); <1> ~ <6> Any of the antibodies. <8> FR1 to FR4 are composed of the following amino acid sequences: <7> antibodies. FR1: the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having 80% or more identity thereto FR2: an amino acid sequence shown in SEQ ID NO: 18, 19, or 20, or an amino acid sequence having 80% or more identity thereto FR3: an amino acid sequence represented by SEQ ID NO: 21, 22, 23, or 24, or an amino acid sequence having 80% or more identity thereto FR4: the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having 80% or more identity thereto <9> a structural domain (SEQ ID NO:26) in which 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, and CDR3 is the amino acid sequence shown in SEQ ID NO:3, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:18, FR3 is the amino acid sequence shown in SEQ ID NO:21, and FR4 is the amino acid sequence shown in SEQ ID NO:25; <7> antibodies. <10> a structural domain (SEQ ID NO:27) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO:4, CDR2 is the amino acid sequence shown in SEQ ID NO:5, and CDR3 is the amino acid sequence shown in SEQ ID NO:6, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:19, FR3 is the amino acid sequence shown in SEQ ID NO:22, and FR4 is the amino acid sequence shown in SEQ ID NO:25; <7> antibodies. <11> a structural domain (SEQ ID NO:28) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO:7, CDR2 is the amino acid sequence shown in SEQ ID NO:8, and CDR3 is the amino acid sequence shown in SEQ ID NO:9, FR1 is the amino acid sequence shown in SEQ ID NO:17, FR2 is the amino acid sequence shown in SEQ ID NO:19, FR3 is the amino acid sequence shown in SEQ ID NO:23, and FR4 is the amino acid sequence shown in SEQ ID NO:25; <7> antibodies. <12> a structural domain (SEQ ID NO: 29) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 10, CDR2 is the amino acid sequence shown in SEQ ID NO: 11, and CDR3 is the amino acid sequence shown in SEQ ID NO: 12, FR1 is the amino acid sequence shown in SEQ ID NO: 17, FR2 is the amino acid sequence shown in SEQ ID NO: 20, FR3 is the amino acid sequence shown in SEQ ID NO: 21, and FR4 is the amino acid sequence shown in SEQ ID NO: 25; <7> antibodies. <13> a structural domain (SEQ ID NO: 30) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 10, CDR2 is the amino acid sequence shown in SEQ ID NO: 13, and CDR3 is the amino acid sequence shown in SEQ ID NO: 14, FR1 is the amino acid sequence shown in SEQ ID NO: 17, FR2 is the amino acid sequence shown in SEQ ID NO: 20, FR3 is the amino acid sequence shown in SEQ ID NO: 21, and FR4 is the amino acid sequence shown in SEQ ID NO: 25; <7> antibodies. <14> a structural domain (SEQ ID NO: 31) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 15, CDR2 is the amino acid sequence shown in SEQ ID NO: 8, CDR3 is the amino acid sequence shown in SEQ ID NO: 16, FR1 is the amino acid sequence shown in SEQ ID NO: 17, FR2 is the amino acid sequence shown in SEQ ID NO: 20, FR3 is the amino acid sequence shown in SEQ ID NO: 24, and FR4 is the amino acid sequence shown in SEQ ID NO: 25; <7> antibodies.

[0041] <15> <1> ~ <14> A nucleic acid encoding any one of the antibodies. EXAMPLES

[0042] Example 1: Screening of VHHs that bind to the novel coronavirus N protein Materials and Methods 1. Screening target molecule The target molecule used was SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein, Sino Biological).

[0043] 2. Synthesis of Display cDNA (1) Synthesis of initial DNA library PCR amplification was performed using the synthetic VHH library PharmaLogical library (PL library), PL_Upright6-Y, PL_Upright12-Y, PL_Upright6-V, PL_Upright12-V, PL_Roll_12, and PL_Roll_15, as template DNA. The PCR solution was prepared for a 500 μL reaction scale. Each test tube contained 150 μL of each VHH library as template DNA, 7.5 μL of 20 μM Newleft primer (5'-GATCCCGCGAAATTAATACGACTCACTATAGGG-3', sequence number 32), 7.5 μL of 20 μM NewYtag_for_polyA_cnvk-linker primer (5'-TTTCCACGCCGCCCCCCGTCCT-3', sequence number 33), 250 μL 2×PrimeSTAR MAX (Takara Bio), 85 μL of H 2O was added. PCR was performed at 98°C for 1 min, followed by 5 cycles of 98°C for 10 s, 60°C for 5 s, 72°C for 10 s, and 72°C for 1 min. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. An equal amount of beads was added per 1 μL of PCR product, mixed by pipetting, and left to stand for 2 min, after which the supernatant was removed and the beads were washed three times with 400 μL of 70% ethanol. After air drying the beads, 500 μL of Nuclease Free Water (NFW) was added to suspend the beads, and the supernatant was collected. The DNA concentration was then quantified by absorbance measurement (A260 / A280).

[0044] (2) In vitro transcription T7 RiboMAX Express Large Scale RNA Production System (Promega) was used. 25 μL of RiboMAX (trademark) Express T7 2×Buffer, 10 pmol of PCR product, and 5 μL of Enzyme Mix were mixed and incubated at 37°C for 30 minutes. 5 μL of RQ1 RNase-Free DNase was then added and incubated at 37°C for 15 minutes. RNAClean XP (Beckman Coulter) was used to purify the transcription product. 1.8 μL of beads were added per 1 μL of transcription product, mixed by pipetting, and left to stand for 5 minutes. After standing on a magnetic plate until it became transparent, the supernatant was removed. 200 μL of 70% ethanol was added, left to stand for 30 seconds, and the supernatant was removed. This ethanol washing was performed three times. After removing the supernatant, the beads were left to stand on a magnetic plate for 10 minutes and air-dried. The tube was removed from the magnetic plate, 300 μL of NFW was added, the beads were suspended by pipetting, and the tube was left to stand for 5 minutes. The tube was left to stand on the magnetic plate for 1 minute, and the supernatant was collected. After purification, RNA was quantified using NanoPad DS-11FX (DeNovix).

[0045] (3) Ligation of mRNA with puromycin linker 20 μL of 0.25 M Tris-HCl (pH 7.5), 20 μL of 1 M NaCl, 100 pmol of the mRNA prepared in (2), and 100 pmol of cnvK riboG linker were mixed to prepare a reaction solution of 100 μL with NFW. Annealing was performed using the ProFlex PCR System (Life technologies) under the conditions of 90°C for 2 minutes, 70°C for 1 minute, 25°C for 30 seconds, and incubation at 4°C. The ramp rate was set to 0.1°C / second. Next, the sample was irradiated with ultraviolet light at a wavelength of 365 nm for 5 minutes using a Handheld UV Lamp, 6W, UVGL-58, 254 / 365 nm, 100V (Analytik jena US, An Endress+Hauser Company). The mRNA-linker conjugate was protected from light and cooled on ice until use.

[0046] (4) Cell-free translation PUREfrex (registered trademark) 1.0 (Gene Frontier) was used to synthesize an mRNA-VHH conjugate from the mRNA-linker conjugate. 12 μL of NUCLEASE FREE WATER, 37.5 μL of Purefrex Solution 1 (Gene Frontier), 3.75 μL of Purefrex Solution 2 (Gene Frontier), 3.75 μL of Purefrex Solution 3 (Gene Frontier), and 18 μL of the mRNA-linker conjugate were mixed. This reaction solution was incubated at 37° C. for 15 minutes, and then 36 μL of IVV formation buffer (3 M KCl, 1 M MgCl 2 ) mixture was added and further reacted at 37°C for 20 minutes to synthesize mRNA-VHH conjugates.

[0047] (5) Immobilization of streptavidin onto magnetic beads 60 μL of 2×Binding buffer (20 mM Tris-HCl, 2 M NaCl, 0.2% Tween 20, 2 mM EDTA, pH 8) was added to 60 μL of Dynabeads My One Streptavidin C1 (Thermo Fisher Scientific), and suspended by pipetting for 1 minute. The mixture was left on a magnetic plate for 1 minute, and the supernatant was discarded. This washing was performed twice. 75 μL of mRNA-VHH conjugate, 75 μL of 2×Binding buffer, and washed streptavidin magnetic beads were mixed and incubated at room temperature for 30 minutes. The mixture was left on a magnetic plate for 1 minute, and the supernatant was removed. 200 μL of Binding buffer (10 mM Tris-HCl, 1 M NaCl, 0.1% Tween 20, 1 mM EDTA, pH 8) was added, and the mixture was pipetted for 1 minute, and the supernatant was removed. This washing was performed twice.

[0048] (6) Reverse transcription reaction 55.5 μL of Nuclease Free Water, 15 μL of 5×RT Buffer (Nippon Gene), and 1.5 μL of GeneAce Reverse Transcriptase (200 U / μL) (Nippon Gene) were mixed. The mRNA-VHH conjugate immobilized on streptavidin magnetic beads was added to the reaction solution, and the mixture was incubated at 42° C. for 30 minutes to carry out reverse transcription, thereby synthesizing a cDNA-VHH conjugate.

[0049] (7) Cutting out beads His-tag wash buffer (20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, 0.05% Tween 20, pH 7.4) was added to the cDNA-VHH conjugates immobilized on streptavidin magnetic beads, and suspended by pipetting for 1 minute. The mixture was left to stand on a magnetic plate for 1 minute, and the supernatant was discarded. Next, 30 μL of His-tag wash buffer containing 10 U RNase T1 (Thermo Fisher Scientific) was added, suspended by pipetting for 1 minute, and then left to stand at 37°C for 15 minutes to elute the cDNA-VHH conjugates.

[0050] (8) Refining 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed on a magnetic plate for 1 minute, the supernatant was discarded, and the beads were resuspended in His-tag wash buffer. This washing procedure was performed twice. The cDNA-VHH conjugate eluate and the His Mag Sepharose Ni Beads suspension were mixed and incubated at room temperature for 30 minutes, then placed on a magnetic plate for 1 minute, and the supernatant was discarded. 200 μL of His-tag wash buffer was added, and suspended by pipetting for 1 minute. The beads were placed on a magnetic plate for 1 minute, and the supernatant was discarded. This washing procedure was performed twice. 10 μL of His-tag elution buffer (20 mM Sodium phosphate, 500 mM NaCl, 250 mM Imidazole, 0.05% Tween 20, pH 7.4) was added, and the beads were incubated at room temperature for 15 minutes to elute the cDNA-VHH conjugate.

[0051] 3. Selection (1) Immobilization of target molecules and blocking In this study, SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein, Sino Biological) was used as the target molecule. The target molecule, which had been adjusted in concentration with PBS, was immobilized in each well of a Nunc-ImmunoTM Plate II (Thermo Fisher Scientific). Specifically, 100 μL of the target factor, which had been adjusted to 50 μg / mL in selection round 1 (R1), 10 μg / mL in R2 (R2), and 1 μg / mL in rounds R3 and after, was added and incubated overnight at 4 °C. After removing the target factor using a pipette, 5% skim milk / PBST was added and incubated at room temperature for 1 hour. Then, the skim milk / PBST was carefully removed. Finally, the operation of adding and removing 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween20) (washing) was repeated three times.

[0052] (2) Screening procedures for selection round 1 (R1) In R1, 100 μL of library solution was used, which was prepared by adding 35 μL of HBT (20 mM HEPES, 0.02% Tween20) and 40 μL of HBST to 200 pmol of the synthesized cDNA-VHH linked library. The library solution was added to the wells where the immobilization of the target molecule was completed, and incubated at room temperature for 1 hour. The library solution was then carefully removed. Next, the operation of adding and removing 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween20) (washing) was repeated six times. Finally, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) was added, carefully pipetted 10 times, and incubated at room temperature for 10 minutes. After that, the wells were carefully pipetted 10 times again, and the eluate was collected in a new tube.

[0053] (3) Screening procedures for R2 and later The synthesized cDNA-VHH linked library was adjusted to 100 μL by adding 35 μL of HBT (20 mM HEPES, 0.02% Tween 20) and 40 μL of HBST. 9 pmol of library solution was used in R2, and 18 pmol in R3 and later. In R2 and later, 100 μL of 5% skim milk / PBST was added to each well in advance, incubated at room temperature for 1 hour, and then washed three times with 200 μL of HBST to block the wells. The library solution was added to the blocked wells and incubated at room temperature for 30 minutes. This operation removed VHH clones with affinity to the blocking agent. After that, the library solution was added to the wells where the target molecule had been solid-phased, and incubated at room temperature for 1 hour. The library solution was then carefully removed. Next, 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween 20) was added and removed (washing) six times. Finally, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) was added and carefully pipetted 10 times, and then incubated at room temperature for 10 minutes. After that, the mixture was carefully pipetted again 10 times, and the eluate was collected in a new tube.

[0054] (4) PCR amplification The entire amount of the eluate after selection was used for PCR in R1, half the amount for R2, and 1 / 4 the amount for R3 and after. PCR was prepared by mixing 25 μL of KAPA Hifi Hotstart Ready Mix (×2) (KAPA biosystems), 1.5 μL of 10 μM forward primer (5'-GATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC-3', sequence number 34), 1.5 μL of 10 μM reverse primer (5'-TTTCCACGCCGCCCCCCGTCCT-3', sequence number 35), 12.5 μL of cDNA-VHH conjugate, and 9.5 μL of NFW per PCR tube. Using a ProFlex PCR system (Life technologies), PCR was performed under the following conditions: 2 minutes at 95°C, 22 cycles (R1) or 28 cycles (R2 and later) of [20 seconds at 98°C, 15 seconds at 68°C, 20 seconds at 72°C], and 5 minutes at 72°C. Next, purification of the PCR product was performed using Agencourt AMPue XP (Beckman Coulter). 1.8 μL of beads were added per 1 μL of PCR product, and the mixture was left to stand for 5 minutes after mixing by pipetting. After standing on a magnetic plate until it became transparent, the supernatant was removed. Next, 1 mL of 70% ethanol was added, the mixture was left to stand for 30 seconds, and the supernatant was removed. This operation was repeated twice. After removing the supernatant, the mixture was left to stand on a magnetic plate for 3 minutes, and the beads were air-dried. The tube was removed from the magnetic plate, thoroughly suspended in 45 μL of NFW, and then left to stand for 5 minutes. Finally, the mixture was left to stand on a magnetic plate for 1 minute, and the supernatant was collected. When proceeding to the next round, purified DNA was used for in vitro transcription, followed by synthesis, selection and manipulation of cDNA display.

[0055] 4. Extraction of VHH candidate sequences (1) Quantification and concentration adjustment of PCR products The PCR products were quantified using a PicoGreen™ dsDNA reagent kit (Thermo Fisher Scientific). The concentration of each sample was adjusted to 100 ng / mL according to the quantification value.

[0056] (2) Preparation of sequencing library 1 st PCR was performed. The reaction mixture was prepared by mixing 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 2.5 μL of the PCR product described in the previous paragraph, and 9 μL of NFW per sample. The primers used were 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNATGGAAGTACAATTAGTTGAATCTGGTGGTGGGCTTG-3' (SEQ ID NO: 36) and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNTGAAGAGACTGTCACCAACGTGCCTTG-3' (SEQ ID NO: 37). PCR was performed at 95°C for 3 minutes, followed by 16 cycles of 98°C for 20 seconds, 62°C for 15 seconds, and 72°C for 20 seconds, and then 72°C for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. 1.8 μL of beads were added per 1 μL of PCR product, mixed by pipetting, and left to stand for 5 minutes. The tube was left to stand on a magnetic plate until it became clear, and then the supernatant was removed. 200 μL of 70% ethanol was added, left to stand for 30 seconds, and then the supernatant was removed. This ethanol washing was performed twice. After removing the supernatant, the tube was left to stand on a magnetic plate for 5 minutes, and the beads were air-dried. The tube was removed from the magnetic plate, 20 μL of NFW was added, and the beads were suspended by pipetting and left to stand for 5 minutes. The tube was left to stand on a magnetic plate for 1 minute, and the supernatant was collected. Next, index PCR was performed. The reaction mixture was prepared by mixing 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1 μL of 10 μM forward index primer (Nextera XT Index Kit v2, Illumina), 1 μL of 10 μM reverse index primer (Nextera XT Index Kit v2, Illumina), 2.5 μL of template DNA, and 8 μL of NFW per sample. PCR was performed at 95°C for 3 minutes, followed by 8 cycles of 98°C for 20 seconds, 55°C for 15 seconds, and 72°C for 30 seconds, and then 72°C for 5 minutes. PCR products were purified using Agencourt AMPure XP (Beckman Coulter). After confirming the size of the PCR product by polyacrylamide gel electrophoresis, the PCR product was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and the molar concentration was calculated according to the following formula (1). Based on the obtained quantitative value, the product was diluted with NFW to a concentration of 4 nM. DNA concentration [ng / μL] / (660[g / mol]×550[bp])×10 6 =DNA concentration [nM] (1)

[0057] (3) Sequence The library was prepared according to the recommended protocol for use with 4 nM libraries. The final concentration of the denatured and diluted sample library was adjusted to 7 pM. The PhiX control in the sample library was adjusted to 5%. Sequencing was performed using MiSeq (Illumina) and MiSeq Reagent Nano Kit v2 500 cycle (Illumina).

[0058] (4) Extraction of VHH amino acid sequences from sequence data and measurement of overlaps The sequence data demultiplexed using the MiSeq Controller was subjected to analysis. The base sequence region encoding VHH was extracted from the sequence data and translated into amino acid sequence. The number of amino acid sequences that perfectly matched each amino acid sequence was then counted. As a result, PN4 (SEQ ID NO: 26), PN10 (SEQ ID NO: 27), PN27 (SEQ ID NO: 28), PN44 (SEQ ID NO: 29), PN46 (SEQ ID NO: 30), and PN63 (SEQ ID NO: 31) appeared frequently, and were selected as antibodies with high affinity for N protein.

[0059] In the amino acid sequence of PN4 (sequence number 26), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 1), positions 36 to 49 are FR2 (sequence number 18), positions 50 to 58 are CDR2 (sequence number 2), positions 59 to 97 are FR3 (sequence number 21), positions 98 to 109 are CDR3 (sequence number 3), and positions 110 to 120 are FR4 (sequence number 25).

[0060] In the amino acid sequence of PN10 (sequence number 27), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 4), positions 36 to 49 are FR2 (sequence number 19), positions 50 to 59 are CDR2 (sequence number 5), positions 60 to 98 are FR3 (sequence number 22), positions 99 to 108 are CDR3 (sequence number 6), and positions 109 to 119 are FR4 (sequence number 25).

[0061] In the amino acid sequence of PN27 (sequence number 28), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 7), positions 36 to 49 are FR2 (sequence number 19), positions 50 to 59 are CDR2 (sequence number 8), positions 60 to 98 are FR3 (sequence number 23), positions 99 to 113 are CDR3 (sequence number 9), and positions 109 to 124 are FR4 (sequence number 25).

[0062] In the amino acid sequence of PN44 (sequence number 29), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 10), positions 36 to 49 are FR2 (sequence number 20), positions 50 to 59 are CDR2 (sequence number 11), positions 60 to 98 are FR3 (sequence number 21), positions 99 to 110 are CDR3 (sequence number 12), and positions 109 to 121 are FR4 (sequence number 25).

[0063] In the amino acid sequence of PN46 (sequence number 30), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 10), positions 36 to 49 are FR2 (sequence number 20), positions 50 to 59 are CDR2 (sequence number 13), positions 60 to 98 are FR3 (sequence number 21), positions 99 to 110 are CDR3 (sequence number 14), and positions 109 to 121 are FR4 (sequence number 25).

[0064] In the amino acid sequence of PN63 (sequence number 31), positions 1 to 25 are FR1 (sequence number 17), positions 26 to 35 are CDR1 (sequence number 15), positions 36 to 49 are FR2 (sequence number 20), positions 50 to 59 are CDR2 (sequence number 8), positions 60 to 98 are FR3 (sequence number 24), positions 99 to 113 are CDR3 (sequence number 16), and positions 109 to 124 are FR4 (sequence number 25).

[0065] Example 2: Production of VHHs using protease-deficient recombinant Bacillus subtilis (1) Construction of plasmid for VHH production PN4 (SEQ ID NO: 26), PN10 (SEQ ID NO: 27), PN27 (SEQ ID NO: 28), PN44 (SEQ ID NO: 29), PN46 (SEQ ID NO: 30), and PN63 (SEQ ID NO: 31) obtained in Example 1 were produced. The amino acid sequence of each VHH synthesized in this Example was given a His tag sequence (SEQ ID NO: 39) via a linker sequence (SEQ ID NO: 38) on the C-terminal side. The VHHs synthesized in this manner are called His-tagged PN4 (SEQ ID NO: 40), His-tagged PN10 (SEQ ID NO: 41), His-tagged PN27 (SEQ ID NO: 42), His-tagged PN44 (SEQ ID NO: 43), His-tagged PN46 (SEQ ID NO: 44), and His-tagged PN63 (SEQ ID NO: 45). These VHHs represented by SEQ ID NOs: 40 to 45 are collectively referred to as His-tagged VHHs. To synthesize these His-tagged VHHs, first, the recombinant plasmid pHY-S237 (JP Patent Publication No. 2014-158430) constructed based on pHY300PLK was used as a template, and the plasmid sequence was amplified by PCR using the primer set 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 46) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 47) and PrimeSTAR Max DNA polymerase (TaKaRa). The genome of Bacillus subtilis 168 strain was used as a template, and the promoter DNA derived from the spoVG gene was amplified by PCR using the primer set 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 48) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 49). The obtained promoter DNA was integrated into a plasmid sequence using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter. A His-tagged VHH expression plasmid was synthesized by integrating the sequence of the His-tagged VHH between SEQ ID NO:50 and SEQ ID NO:51 of the previously constructed VHH expression plasmid using GenPlus cloning by Genescript.The constructed plasmid was introduced into a strain (Dpr8ΔsigF) obtained by deleting eight extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE) from Bacillus subtilis 874 strain according to the method described in JP 2006-174707 A, and further deleting the sigF gene involved in sporulation according to the method described in Japanese Patent No. 4336082, according to the procedure shown in (2) below.

[0066] (2) Construction of recombinant Bacillus subtilis The above-mentioned plasmid was introduced into the Bacillus subtilis strain by the protoplast method described below. Bacillus subtilis was cultured in a 96-well deep well plate. Bacillus subtilis stocked in a solution containing glycerol was inoculated into a well containing 0.8 mL of LB liquid medium, and cultured overnight with shaking at 30°C and 1,500 r / min. Next, 8 μL of the culture solution was inoculated into fresh 0.8 mL of LB liquid medium, and cultured with shaking at 30°C and 1,500 r / min for about 2 hours. After the culture was completed, the culture solution was collected in a 2 mL tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. 500 μL of SMMP containing 4 mg / mL lysozyme (SIGMA) was added to suspend the obtained pellet, and the tube was incubated at 37°C for 1 hour.

[0067] After the incubation, the tube was centrifuged at 3,500 rpm for 10 minutes, and the supernatant was removed. 400 μL of SMMP was added to the tube, and the resulting pellet was suspended to obtain a suspension. 33 μL of the resulting suspension was added to another tube, and each of the plasmids prepared as described above was added and mixed thereto, and 100 μL of 40% PEG was further added and vortexed to obtain a mixture. 350 μL of SMMP was added to this mixture, which was then mixed by inversion and incubated at 30°C for 2 hours. Then, 200 μL of the culture solution after incubation was applied onto the agar medium of a plate containing DM3 agar medium that had been prepared in advance, and incubated at 30°C for 2 to 3 days to obtain recombinant Bacillus subtilis.

[0068] (3) Production of VHH The recombinant Bacillus subtilis prepared in (2) was inoculated into 1 mL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C with shaking to obtain a preculture solution. The preculture solution was inoculated at 1% into 1 mL of 2×L-mal medium placed in each well of a 96-well deep-well plate and cultured at 30°C with shaking for 72 hours. At the end of the culture, all the cultures were collected in 15 mL tubes and centrifuged at 4°C and 7,500 rpm for 5 minutes to collect the supernatant. Each VHH contained in the collected supernatant was purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries) according to the protocol attached to the kit. PBS containing 30 mM imidazole was used as the eluent during purification.

[0069] (4) Confirmation by SDS-PAGE 2 μL of each His-tagged VHH in water (both at 1 mg / mL), 2.5 μL of NuPAGE™ LDS Sample Buffer (4×) (Thermo Fisher Scientific), 1 μL of NuPAGE™ Sample Reducing Agent (10×) (Thermo Fisher Scientific), 4.5 μL of H 2The mixture was mixed with 0 and heated at 100°C. Then, 10 μL of the sample solution was applied to the wells of the gel. As a molecular weight marker, 5 μL of Novex™ Sharp Pre-stained Protein Standard (Thermo Fisher Scientific) was also applied to the wells of the gel. The gel used was NuPAGE™ 10%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well (Thermo Fisher Scientific). The electrophoresis buffer was prepared by diluting NuPAGE™ MES SDS Running Buffer (20×) (Thermo Fisher Scientific) 20 times with water. The XCell SureLock™ Mini Cell Electrophoresis System (Thermo Fisher Scientific) was used as the electrophoresis tank, and electrophoresis was performed for 40 minutes at a voltage of 200 V by connecting it to a PowerEase™ 90W Power Supply (Thermo Fisher Scientific). The gel was then stained with GelCode™ Blue Stain Reagent (Thermo Fisher Scientific) to confirm the presence or absence of a band for the target protein. The results are shown in Figure 1. Bands were confirmed at around 15 kDa for all samples, confirming that each His-tagged VHH was produced.

[0070] Example 3: Evaluation of binding activity of VHH by Sandwich ELISA method Nunc-Immuno(trademark) 100 μL of each His-tagged VHH, prepared at 20 μg / mL in PBS, was added to each well of Plate II (Thermo Fisher Scientific) and incubated at room temperature for 1 hour for immobilization. After that, the VHH solution was carefully removed using a pipette, and then 200 μL of PBST was added and carefully removed using a pipette (washing) three times. Next, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added to each well, and the wells were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and then the washing operation was repeated three times. SARS-CoV-2 Nucleocapsid-Fc fusion protein (Fc-tagged N protein, InvivoGen) was prepared at 0, 10, 100, and 1000ng / mL in dilution buffer (20mM Tris-HCl, 500mM NaCl, 0.05% Tween20). 100μL of prepared N protein was added to each well and incubated at room temperature for 1 hour. After that, the target protein solution was carefully removed using a pipette, and the washing operation was repeated three times. Goat pAb to Hu IgG(HRP)(Abcam) was diluted 1 / 5,000 with PBST and added to each well at a concentration of 100μL as a detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the washing operation was repeated three times. As a luminescent substrate, OPD tablets (Thermo Fisher Scientific) were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific) and added to each well at 100 μL. After incubation at room temperature for 30 minutes in the dark, the absorbance at 450 nm was measured using the GloMax® Explorer System (Promega).

[0071] The ELISA results are shown in Figure 2. It was confirmed that His-tagged PN4, His-tagged PN10, His-tagged PN27, His-tagged PN44, His-tagged PN46, and His-tagged PN63 all showed clear binding activity to the N protein of SARS-CoV-2.

[0072] Example 4: Evaluation of binding specificity of VHH by Direct ELISA method Nunc-Immuno(trademark) 50 μL of N protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, prepared at 10 μg / mL in PBS, was added to each well of Plate II (Thermo Fisher Scientific), and incubated at 4 °C overnight for immobilization. The N proteins used are shown in Table 1. After that, the N protein solution was carefully removed using a pipette, and then 200 μL of PBST was added and the operation of carefully removing the N protein using a pipette (washing) was repeated three times. Next, 200 μL of 5% skim milk / PBST was added to each well, and the wells were incubated at room temperature for 1 hour for blocking. The skim milk / PBST was carefully removed using a pipette, and the washing operation was repeated three times. Each His-tagged VHH was prepared at 1 μg / mL in PBST, and 100 μL was added to each well, followed by incubation at room temperature for 1 hour. After that, the target protein solution was carefully removed using a pipette, and the washing operation was repeated three times. Peroxidase AffiniPure Goat Anti-Alpaca IgG, VHH domain (Jackson ImmunoResearch) was diluted 1 / 5,000 with PBST and added to each well at 100 μL as a detection antibody. After incubation at room temperature for 1 hour, the antibody was carefully removed using a pipette, and the washing operation was repeated three times. As a luminescence substrate, OPD tablets were dissolved in Stable Peroxide Substrate buffer (Thermo Fisher Scientific) and added at 100 μL to each well. After that, the plate was incubated at room temperature and in the dark for 30 minutes, and the absorbance at 450 nm was measured using the GloMax (registered trademark) Explorer System.

[0073] [Table 1]

[0074] The results of the Direct ELISA are shown in Figure 3. It was revealed that His-tagged PN4, His-tagged PN10, and His-tagged PN27 specifically bind to the N protein of SARS-CoV-2. In addition, His-tagged PN44, His-tagged PN46, and His-tagged PN63 showed slight binding activity to the N protein of SARS-CoV, but were confirmed to show specific and clear binding activity to the N protein of SARS-CoV-2.

[0075] Example 5 Lateral flow assay (1) Sensitization of labeled antibodies to pigment particles Colored cellulose particles NanoAct (chemically bonded, Asahi Kasei) were used as the dye particles. Sensitization of the labeled antibody was performed according to the attached protocol. The outline is as follows. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, 540 μL of 100 mM MES (pH 6), 7.5 μL of 4 wt% EDC, and 15 μL of 4 wt% NHS were added thereto, and the tube was left to stand at room temperature for 15 minutes to esterify the carboxyl groups. The tube was centrifuged at 13,000 × g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. 60 μg of EN2 and EN12 described in Patent Application No. 2022-065934, CoVHH-N2 described in International Publication No. 2022 / 181550, or PN4, PN10, PN27, PN46, or PN63 of the present invention was added as a labeled antibody, then vortexed and incubated at 37 ° C. for 120 minutes. Next, 7.2 mL of blocking buffer (1 wt% Casein, 100 mM Boric Acid, pH 8.5) was added, then vortexed and incubated at 37 ° C. for 60 minutes. The mixture was centrifuged at 13,000 × g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. The mixture was centrifuged again at 13,000×g for 15 minutes to remove the supernatant, and 1.3 mL of storage solution (33 mM boric acid, 0.2 wt% casein, 15 wt% sucrose, pH 9.2) was added, sonicated, and stored at 4° C. until use.

[0076] (2) Assembling the Half Strip A nitrocellulose membrane was prepared by cutting Hi-Flow Plus HF120 (Merck) to 5 mm × 40 mm. A 5 mm × 20 mm cut CM5 (Cytiva) was used as an absorbent pad. The nitrocellulose membrane and absorbent pad were laminated together with a 10 mm overlap and attached with a backing sheet (GL-57888, Lohmann) to assemble a half strip.

[0077] (3) Immobilization of capture antibodies onto nitrocellulose membrane The capture antibodies His-tagged EN2, His-tagged EN12, His-tagged CoVHH-N2, ​​His-tagged PN4, His-tagged PN10, and His-tagged PN27 were prepared in advance at 1 mg / mL in a PBS solution containing 30 mM imidazole. 1 μL of the antibodies were spotted with a pipette at a point 2 cm from the bottom end of the strip assembled in (2) above. The antibodies were then immobilized by drying at 37°C for 20 minutes. In the following, the area where the capture antibodies were immobilized is referred to as the test line.

[0078] (4) Preparation of developing solution First, 20 mM HEPES, pH 7 was prepared. NaCl and Tween 20 were added to give final concentrations of 500 mM and 0.5% to prepare an HBST solution, which was used as a developing solution.

[0079] (5) Examination of the feasibility of constructing immunochromatography Fc-tagged N protein was added to the developing solution to be evaluated, and a positive sample was prepared by diluting it to 10 μg / mL. HBST not containing N protein was used as a negative sample. 50 μL of the positive sample solution and 20 μL of the dye particles prepared in (1) were added to one well of a 96-well plate, and mixed well by pipetting. Then, the capture antibody-immobilized membrane prepared in (3) was immersed in the well and left to stand until the liquid had completely flowed out. If coloration was observed only when the positive sample was flowed, the immunochromatography was judged to be "constructible," and if no coloration was observed, it was judged to be "not constructible."

[0080] The feasibility of constructing immunochromatography using each VHH is shown in FIG. 4. Immunochromatography could not be constructed using the three VHHs EN2, EN12, and CoVHH-N2. By using PN4, PN10, PN27, PN44, PN46, and PN63 of the present invention, it became possible to construct immunochromatography with 10 combinations (labeled antibody / capture antibody: EN12 / PN4, CoVHH-N2 / PN27, PN10 / PN4, PN10 / PN27, PN27 / PN4, PN44 / PN4, PN44 / PN27, PN46 / PN4, PN46 / PN27, PN63 / PN27). In other words, it was confirmed that by adopting the VHH of the present invention, it became possible to construct immunochromatography using VHH for both the labeled antibody and the capture antibody.

Claims

1. An immunochromatographic antibody that binds to SARS-CoV-2, having one or more structural domains containing CDR1-3 selected from (c), (f), and (b) below. (b) CDR1 consisting of the amino acid sequence shown by GTFFSRYDMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown by AISWNGGSTY (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown by LDWLQWDWAY (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown by GFTFSSYAMT (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by GQHHQELQHWYAWDY (SEQ ID NO: 9). (f) CDR1 consisting of the amino acid sequence shown by GFTFSDYAMG (SEQ ID NO: 15), CDR2 consisting of the amino acid sequence shown by AISRNGGSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown by LGQNKEHVGKRIEDY (SEQ ID NO: 16).

2. The antibody according to claim 1, which binds to an N-protein.

3. The antibody according to claim 1, which is a single-domain antibody or a polymer thereof.

4. The antibody according to claim 3, wherein the single-domain antibody is VHH.

5. The antibody according to claim 3, wherein the polymer of a single-domain antibody is a polymer formed by linking multiple structural domains.

6. The antibody according to claim 3, wherein the single-domain antibody polymer is a polymer formed by linking one or more of the structural domains with one or more structural domains having different antigen specificity from the said structural domains.

7. A nucleic acid encoding an antibody according to any one of claims 1 to 6.

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