JP2022022974A - DRUG AND INSPECTION KIT USING ANTI-SARS-CoV-2 ANTIBODY - Google Patents

Abstract

To provide a method for detecting SARS-CoV-2 using an antibody binding to SARS-CoV-2, and a drug and an inspection kit using the antibody.SOLUTION: A method for detecting SARS-CoV-2 in a sample comprises bring an antibody that binds to SARS-CoV-2 having one or more structural domains into contact with a test sample, where the structural domain comprises: CDR1 consisting of an amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence; CDR2 consisting of an amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence; and CDR3 consisting of an amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting SARS-CoV-2 using an antibody that binds to SARS-CoV-2, a drug and a test kit using the antibody.

SARS coronavirus-2 (Severe acute respiratory syndrome 2,; SARS-CoV-2) belongs to the genus Beta coronavirus like SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV), and acute respiration. It is a SARS-related coronavirus that causes organ disease (COVID-19). The outbreak was first confirmed in 2019 near Wuhan City, Hubei Province, China, and has since caused a global pandemic of COVID-19.

SARS-CoV-2 has a viral genome of about 29,903 bases and is a single-strand plus-strand RNA virus. The virus particles (virions) have a size of about 50 to 200 nm. Like a common coronavirus, it is composed of four proteins known as spike proteins, nucleoproteins, endogenous membrane proteins, and enveloped proteins, and RNA. Of these, nucleoprotein binds to RNA to form nucleocapsid, and spike protein, endogenous membrane protein, and envelope protein bound to lipid surround it to form an envelope (Non-Patent Documents 1 and 2).

Peplomers are large transmembrane proteins of type I consisting of two subunits, S1 and S2, where S1 is primarily a receptor binding domain (RBD) that recognizes cell surface receptors and S2 is a membrane fusion. Contains the necessary elements. Peplomers have been implicated in inducing neutralizing antibodies and T cell responses, as well as in protective immunity.

Known receptors that bind to S1 include ACE2 (angiotensin converting enzyme 2), DPP4 (dipeptidyl peptidase-4), APN (aminopeptidase N), CEACAM (cancer fetal antigen-related cell adhesion molecule 1), O- There is ac Sia (O-acetylated sialic acid), and SARS-CoV-2 has been reported to infect human respiratory epithelial cells via human ACE2 (Non-Patent Document 3).

For the virological diagnosis of COVIDEO-19, the gene detection of SARS-CoV-2 is mainly performed by the gene amplification method (PCR). The PCR test is considered to be the most accurate virus test available at the moment, but there is a problem that it takes time and effort to perform the test and it is difficult to expand the PCR test to the number really required. .. On the other hand, in the detection of a virus, unlike PCR in which a genetic substance of a virus is detected, there is also an antigen test for detecting a fragment of a protein on the surface of the virus. If the antigen can be detected, it can be diagnosed in a few minutes without expensive machinery, training, or labor. Therefore, if there is a reliable antigen test, the scale of the test can be easily expanded, and COVID-19 can be diagnosed at home or in a clinical setting, and it is required to introduce it to a clinical setting as soon as possible. In addition, a serological diagnostic method using an immunochromatography method for detecting a virus-specific antibody in serum or an enzyme-linked immunosorbent assay (ELISA) is being studied. In particular, COVID-19 is considered to have a long incubation period from infection to onset in many cases. In addition, there have been reports of cases with a long clinical course, such as the rapid deterioration of symptoms about one week after the onset, leading to severe pneumonia. Therefore, it is expected that serological diagnosis will be useful in the diagnosis of COVID-19.

On the other hand, VHH antibody, which is a natural single domain antibody using the variable region of heavy chain antibody found in the serum of camelids, has a molecular weight as small as 1/10 of that of IgG antibody, and has acid resistance and heat resistance. Excellent for. In addition, IgG antibody needs to be produced using cultured cells, but VHH antibody can be produced in Escherichia coli or yeast. Further, since the VHH antibody is a single-stranded peptide, it has a feature that the function can be easily modified by protein engineering or chemical modification, and an antibody drug conjugate (ADC) can be easily produced. On the other hand, there is a problem that it is difficult to obtain a VHH antibody having a high affinity with a substrate.

A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar; 579 (7798): 270-273. A new coronavirus associated with human respiratory disease in China. Nature. 2020 Mar; 579 (7798): 265-269. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Cell. Volume 181, Issue 2, 16 April 2020, Pages 281-292.e6

The present invention relates to a method for detecting SARS-CoV-2 using an antibody that binds to SARS-CoV-2, and to provide a drug and a test kit using the antibody.

As a result of investigating to obtain an antibody that binds to SARS-CoV-2, the present inventors examined SARS-CoV- from a VHH antibody library containing CDR1 to 3 having a specific amino acid number in a construct by screening by the cDNA display method. We succeeded in obtaining a highly reactive clone in 2.

That is, the present invention relates to the following 1) to 3).
1) CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one amino acid sequence represented by SEQ ID NO: 2 or one amino acid sequence. A structural domain containing CDR2 consisting of an amino acid sequence in which an amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A method for detecting SARS-CoV-2 in a sample, which comprises contacting an antibody that binds to SARS-CoV-2 having one or more of the amino acids with a test sample.
2) CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one amino acid sequence represented by SEQ ID NO: 2 or one amino acid sequence in the amino acid sequence. A structural domain containing CDR2 consisting of an amino acid sequence in which an amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A SARS-CoV-2 detection kit containing an antibody that binds to SARS-CoV-2 having one or more of.
3) CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one amino acid sequence represented by SEQ ID NO: 2 or one amino acid sequence. A structural domain containing CDR2 consisting of an amino acid sequence in which an amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A drug containing an antibody that binds to SARS-CoV-2 having one or more of.

According to the present invention, SARS-CoV-2 can be detected, that is, acute respiratory disease (COVID-19), which is a SARS-CoV-2 infection, can be rapidly detected, and the disease can be prevented or treated. Will be.

Distribution of frequency of appearance of anti-SARS-CoV-2 antibody candidates that appeared after each round selection (displayed only in the top 10). Arrows indicate the frequency of appearance of CoVHH1. Alignment diagram of amino acid sequences of CoVHH1 and CoVHH1 mutant selected by selection. * Indicates a conserved amino acid. Compared with the amino acid sequence of CoVHH1, the place where the amino acid is substituted is shown by enclosing it in a square. Western blotting image of Bacillus subtilis culture supernatant incorporating the VHH gene. * Indicates the band of the target protein. Measurement of binding activity of SARS-CoV-2 to S1 protein of CoVHH1 by biolayer interferometry. The gray line shows the coupling divergence curve based on the measured raw data, and the black line shows the coupling divergence curve after fitting. Measurement of binding activity of SARS-CoV-2 to S1 protein of CoVHH1 and CoVHH1 dimers by ELISA method. Measurement of binding activity of SARS-CoV-2 to S1 protein of CoVHH1 by sandwich ELISA method. Evaluation of binding specificity of CoVHH1 to coronavirus S1 protein by ELISA method. Measurement of binding activity of SARS-CoV-2 to S1 protein of CoVHH1 by biolayer interferometry. The black line shows the coupling divergence curve based on the measured raw data, and the gray line shows the coupling divergence curve after fitting. Measurement of binding activity of CoVHH1 to S1 protein at pH 7.4 by surface plasmon resonance method. The black line shows the coupling divergence curve based on the measured raw data, and the gray line shows the coupling divergence curve after fitting. Measurement of binding activity of CoVHH1 to S1 protein at pH 6.5 by surface plasmon resonance method. The black line shows the coupling divergence curve based on the measured raw data, and the gray line shows the coupling divergence curve after fitting. Measurement of binding activity of SARS-CoV-2 to S1 protein of CoVHH1 by sandwich ELISA method.

In the antibody that binds to SARS-CoV-2 of the present invention (hereinafter referred to as "antibody of the present invention"), the amino acid sequence represented by SEQ ID NO: 1 or one amino acid in the amino acid sequence is replaced with another amino acid. CDR1 consisting of the amino acid sequence and the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and the amino acid sequence represented by SEQ ID NO: 3 or the said. An anti-SARS-CoV-2 antibody having one or more structural domains containing CDR3 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence.

SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory disease (COVID-19), and its viral genome is a single-strand plus-strand RNA virus of about 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 S1 subunit of the spike protein of SARS-CoV-2.

The structural domain of the antibody of the present invention has three CDRs, CDR1, CDR2 and CDR3. CDRs (Complementarity Determining Regions) include sequence-variable antigen recognition sites or random sequence regions, and are also referred to as hypervariable regions. In the structural domain of the antibody of the present invention, the three CDRs exist in the order of CDR1, CDR2, and CDR3 from the N-terminal side.

In the structural domain of the antibody of the invention
CDR1 consists of the amino acid sequence shown by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence.
CDR2 consists of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence.
CDR3 consists of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence.
Here, the amino acid sequence shown by SEQ ID NO: 1 is GSTFSDYVMA, the amino acid sequence shown by SEQ ID NO: 2 is TISRNGGTTT, and the amino acid sequence of CDR3 consisting of the amino acid sequence shown by SEQ ID NO: 3 is VGGDGDS.

In the amino acid sequence represented by SEQ ID NOs: 1 to 3, the CDR consisting of an amino acid sequence in which one amino acid is replaced with another amino acid has a mutation introduced into the CDR consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 3. It means CDR. Further, even if a mutation is introduced into CDR1, CDR2, and CDR3, it is included in the CDR of the antibody of the present invention as long as it has a binding ability to SARS-CoV-2.
Here, the position of the amino acid to be substituted is not limited, but preferred positions include the 3, 4, 5, or 10th position in CDR1 and 3, 4, 5, 6, 7, 8, 9 or in CDR2. The tenth is mentioned, and in CDR3, the first, second, third or fourth are mentioned.
The type of amino acid after the substitution is not limited, but a preferable substitution includes substitution with an amino acid having a similar polarity and size. Also, replacement of threonine residues with asparagine residues, isoleucine residues or methionine residues, substitution of phenylalanine residues with isoleucine residues, substitution of serine residues with arginine residues or asparagine residues, alanine residues. Substitution of threonine residue, substitution of arginine residue with serine residue, substitution of asparagine residue with tyrosine residue, substitution of glycine residue with serine residue, phenylalanine residue or isoleucine of valine residue Substitution with residues, substitution of aspartic acid residues with glycine or valine residues are also preferred substitutions.
A preferred combination of the position of the amino acid to be substituted and the type of amino acid after substitution is the substitution of the third threonine residue with the asparagine residue and the substitution of the fourth phenylalanine residue with the isoleucine residue in CDR1. , Substitution of the 5th serine residue to the arginine residue, 10th substitution of the alanine residue to the threonine residue,
In CDR2, the third serine residue is replaced with an asparagine residue, the fourth arginine residue is replaced with a serine residue, the fifth asparagine residue is replaced with a tyrosine residue, and the sixth glycine residue is present. Substitution of the base serine residue, substitution of the 7th glycine residue with the serine residue, substitution of the 8th threonine residue with the methionine residue, substitution of the 9th threonine residue with the isoleucine residue Substitution of the 10th threonine residue with a methionine residue,
In CDR3, the first valine residue is replaced with isoleucine residue, the third glycine residue is replaced with valine residue, and the fourth aspartic acid residue is replaced with glycine residue or valine residue. , Are preferred.

The binding ability to SARS-CoV-2 can be evaluated by a method known to those skilled in the art. Specifically, as shown in Examples described later, it can be evaluated by obtaining the equilibrium deviation constant KD by the biolayer interferometry. Further, for example, it can be evaluated by a method using an ELISA method in which an antigen is immobilized, an immunochromatography method, an isothermal titration calorimetry method, a surface plasmon resonance measurement method, or the like.

The structural domain of the antibody of the present invention may have framework regions at both ends of CDR1, CDR2, and CDR3.
The framework region is a region excluding the complementarity determining region in the variable region of the antibody molecule, and refers to a region with high conservativeness.
That is, the structural domain of the present invention is, in one aspect, 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. Those having regions (FR4) in this order can be mentioned.
The amino acid sequences of the framework region in the structural domain include the following.
FR1: Amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having 80% or more identity with the amino acid sequence FR2: Amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having 80% or more identity with the amino acid sequence. FR3: Amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 80% or more identity with the amino acid sequence FR4: Amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence having 80% or more identity with the amino acid sequence.

The framework region consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NOs: 4 to 7 is preferably 85% or more, more preferably 90% or more, more preferably 95% or more, and more. A framework region consisting of an amino acid sequence having an identity of 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more can be mentioned.

Here, the identity of the amino acid sequence means the ratio (%) of the number of positions where the same amino acid residue exists in both sequences to the total number of amino acid residues when the two amino acid sequences are aligned. Sequence identity can be calculated, for example, by performing analysis using BLAST (Basic Local Alignment Sensor Tool) of National Center for Biotechnology Information (NCBI).

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 by SEQ ID NO: 1, CDR2 is the amino acid sequence shown by SEQ ID NO: 2, and CDR3. Is the amino acid sequence shown by SEQ ID NO: 3, FR1 is the amino acid sequence shown by SEQ ID NO: 4, FR2 is the amino acid sequence shown by SEQ ID NO: 5, FR3 is the amino acid sequence shown by SEQ ID NO: 6, and FR4 is the amino acid sequence shown by SEQ ID NO: 7. The antibody having the structural domain (SEQ ID NO: 9) which is the amino acid sequence shown in 1 is a highly reactive clone (CoVHH1) of SARS-CoV-2 obtained by screening by the cDNA display method in the examples described later. Yes, it is a suitable anti-SARS-CoV-2 antibody.

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 is not limited to a single domain antibody (also called a nanobody) such as a VHH antibody, a single chain antibody, or a heavy chain antibody. It may be a multimer in which an antibody or a variable region fragment is bound with a peptide linker or the like, for example, a dimer, or a multimer in which one or a plurality of variable region fragments having different antigen specificities are linked to the variable region fragment. ..
Further, the antibody of the present invention may be humanized. Since the humanized antibody can be administered to humans, it can be applied as a medicine.

The method for producing the antibody of the present invention is not particularly limited, and the antibody can be easily produced by a technique known in the art. For example, it can be prepared by combining a peptide solid phase synthesis method and a native chemical ligation (NCL) method, or can be genetically engineered, but the nucleic acid encoding the antibody of the present invention is suitable. It is preferable to incorporate it into a vector, introduce it into a host cell, and produce it as a recombinant antibody.

Host cells used in the production of recombinant antibodies include, for example, Escherichia coli, bacillus, mold, animal cells, plant cells, baculovirus / insect cells, yeast cells and the like.
As an expression vector for expressing an antibody, a vector suitable for various host cells can be used. As the expression vector, for example, a vector derived from Escherichia coli such as pBR322, pBR325, pUC12, pUC13; a vector derived from bacteriophage such as pUB110, pTP5, pC194; a shuttle vector that can be shared with E. coli such as pHY300PLK; Yeast-derived vectors 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 thereto can be used.
These expression vectors have replication initiation sites, selectable markers and promoters suitable for each vector, and optionally include enhancers, transcriptional concentrating sequences (terminators), ribosome binding sites, polyadenylation signals and the like. You may have. Further, in order to facilitate the purification of the expressed polypeptide, a base sequence for fusing and expressing a FLAG tag, a His tag, an HA tag, a GST tag, or the like may be inserted into the expression vector.

When the expressed antibody of the present invention is extracted from cultured cells or cells, after culturing, the cells or cultured cells are collected by a known method, suspended in an appropriate buffer, and ultrasonically or lysoteam. And / or after destroying the cells or cells by freezing and thawing, a soluble extract is obtained by centrifugation or filtration. From the obtained extract, the desired antibody can be obtained by appropriately combining known separation / purification methods.
Known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, methods that mainly utilize differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-PAGE, and ions. A method that utilizes the difference in charge such as exchange chromatography, a method that utilizes a specific affinity such as affinity chromatography, a method that utilizes a difference in hydrophobicity such as reverse phase high-speed liquid chromatography, or an isoelectric focusing method. A method that utilizes the difference in isoelectric focusing such as is used.

Since the antibody of the present invention binds to SARS-CoV-2, it is contained in the sample by contacting it with a test sample containing or may contain SARS-CoV-2. -It can be confirmed that CoV-2 exists or does not exist.
Specifically, in the detection of SARS-CoV-2 using the antibody of the present invention, the antibody of the present invention is brought into contact with the test sample, and the antibody of the present invention and SARS-CoV-2 in the test sample are combined. It is provided with a step of forming the conjugate of SARS-CoV-2 and a step of detecting SARS-CoV-2 in the conjugate.
In addition, the antibody of the present invention has a SARS-CoV against an anti-SARS-CoV-2 antibody (for example, a serum antibody) contained in an immobilized test sample (serum) in the detection of a virus-specific antibody in serum. -It can also be used as an antibody for confirming the presence of the SARS-CoV-2 antigen in the bound state by adding a part of the -2 antigen and binding the antibody.

Test samples include tracheal swabs, nasal swabs, pharyngeal swabs, nasal lavage fluids, nasal suction fluids, nasal juices and nasal chews, saliva, sputum, blood, serum, urine, feces, tissues, cells, tissues or crushed cells. In addition to biological samples such as the above, a sample collected from a solid surface to which a virus may be attached, for example, a door knob, a nasal cavity, or the like can be mentioned. The antibody may or may not be immobilized on the solid phase.
The step of detecting SARS-CoV-2 in the above-mentioned conjugate is, for example, 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. It can be done by letting it. Alternatively, a homogenius assay may detect SARS-CoV-2 in the above conjugate in the liquid phase.

In addition, the antibody of the present invention can be a component of the SARS-CoV-2 detection kit. 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.
In addition to the anti-SARS-CoV-2 antibody of the present invention, the detection kit can include reagents and instruments necessary for detection, such as an antibody, a solid phase carrier, a buffer solution, an enzyme reaction terminator, 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 the solid phase include beads, a membrane, side surfaces and bottom surfaces of a reaction vessel, a plate-like substrate such as a slide glass, a well substrate such as an immunoplate, and the like, and the antibody of the present invention is directly or indirectly immobilized. Ru.

Further, it is considered that the antibody of the present invention binds to the S1 subunit of SARS-CoV-2 and inhibits the binding of the virus to cells. Therefore, the antibody of the present invention can be administered to mammals and used as a medicine for preventing or treating SARS-CoV-2 infection. Examples of mammals include humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs and the like, but humans are preferred.
When the antibody of the present invention is used as a pharmaceutical, the embodiment may be oral or parenteral, and can be appropriately used in combination with a well-known pharmaceutically acceptable non-toxic carrier or diluent. The parenteral administration is typically an injection, but it can also be administered by inhalation together with a spray or the like.

In such a pharmaceutical, the content of the antibody of the present invention in the composition can be appropriately adjusted.
In addition, such a drug should be administered as an antibody of the present invention in an effective amount at intervals of about 1 to several times a week to a target, for example, a patient who needs prevention or treatment of SARS-CoV-2 infection. Can be applied by. In this case, the administration method preferably includes intravenous injection, infusion and the like.

In the present invention, the following aspects are further disclosed with respect to the above-described embodiment.
<1> CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one in the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence. A structure containing CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A method for detecting SARS-CoV-2 in a sample, which comprises contacting a test sample with an antibody that binds to SARS-CoV-2 having one or more domains.
<2> CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one in the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence. A structure containing CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A SARS-CoV-2 detection kit containing an antibody that binds to SARS-CoV-2 having one or more domains.
<3> CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one in the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence. A structure containing CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A drug containing an antibody that binds to SARS-CoV-2 having one or more domains.
<4> The drug of <3> for the prevention or treatment of SARS-CoV-2 infection.
<5> The method of <1>, the kit of <2>, or the pharmaceutical of <3> or <4>, wherein the antibody is a VHH antibody, a heavy chain antibody, or a VHH antibody multimer.
<6> The method, kit or pharmaceutical of <5>, wherein the VHH antibody multimer is a multimer in which a plurality of the structural domains are linked.
<7> The method, kit or pharmaceutical of <5>, wherein the VHH antibody multimer is a multimer in which one or more of the structural domains and one or more of structural domains having different antigen specificities from the structural domains are linked. ..

<8> CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence for producing a drug, and the amino acid sequence represented by SEQ ID NO: 2. Alternatively, CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and an amino acid sequence represented by SEQ ID NO: 3 or an amino acid in which one amino acid is replaced with another amino acid in the amino acid sequence. Use of an antibody that binds to SARS-CoV-2 having one or more structural domains containing CDR3 consisting of a sequence.
<9> CDR1 consisting of the amino acid sequence shown by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence for use as a medicine, and the amino acid sequence shown by SEQ ID NO: 2. Alternatively, CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and an amino acid sequence represented by SEQ ID NO: 3 or an amino acid in which one amino acid is replaced with another amino acid in the amino acid sequence. An amino acid that binds to SARS-CoV-2 having one or more structural domains containing CDR3 consisting of a sequence.
<10> Use of <8> or antibody of <9>, wherein the drug is a drug for the prevention or treatment of SARS-CoV-2 infection.
<11> CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and one in the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence. A structure containing CDR2 consisting of an amino acid sequence in which one amino acid is replaced with another amino acid, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence. A method for preventing or treating a SARS-CoV-2 infection, which is administered to a subject in need of an antibody that binds to SARS-CoV-2 having one or more domains.
<12> Use of <8> or <10>, the antibody of <9> or <10>, or the method of <11>, wherein the antibody is a VHH antibody, heavy chain antibody or VHH antibody multimer.
<13> The use, antibody or method of <12>, wherein the VHH antibody multimer is a multimer in which a plurality of the structural domains are linked.
<14> Use of <12>, an antibody, or a VHH antibody multimer, which is a multimer in which one or more of the structural domains and one or a plurality of structural domains having different antigen specificities from the structural domains are linked. Method.

<15> In the amino acid sequence shown in SEQ ID NO: 1, the substitution of one amino acid with another amino acid is replaced with the asparagine residue of the third threonine residue to the isoleucine residue of the fourth phenylalanine residue. The method of <1>, the kit of <2>, <3> or the substitution of the 5th serine residue with an arginine residue or the 10th alanine residue with a threonine residue. The drug of <4>, the use of <8>, the antibody of <9>, or the method of <11>.
<16> In the amino acid sequence shown in SEQ ID NO: 2, the substitution of one amino acid with another amino acid is the substitution of the third serine residue with the asparagine residue and the substitution of the fourth serine residue with the serine residue. Substitution, substitution of the 5th asparagine residue with a tyrosine residue, substitution of the 6th glycine residue with a serine residue, substitution of the 7th glycine residue with a serine residue, 8th threonine residue The method of <1>, the kit of <2>, which is the substitution of a methionine residue with a methionine residue, the substitution of a 9th threonine residue with an isoleucine residue, or the substitution of a 10th threonine residue with a methionine residue. , <3> or <4> drug, <8> use, <9> antibody, or <11> method.
<17> In the amino acid sequence shown by SEQ ID NO: 3, the substitution of one amino acid with another amino acid, and in CDR3, the substitution of the first valine residue with the isoleucine residue and the substitution of the third glycine residue. The method of <1>, the kit of <2>, the pharmaceutical of <3> or <4>, which is a substitution with a valine residue or a substitution with a glycine residue or a valine residue of the fourth aspartic acid residue. , <8> use, <9> antibody, or <11> method.
<18> The structural domain includes the first framework region (FR1), CDR1, the second framework region (FR2), CDR2, the third framework region (FR3), CDR3, and the fourth framework region (FR4). The method of <1>, the kit of <2>, the drug of <3> or <4>, the use of <8>, the antibody of <9>, or the method of <11>, which are possessed in order.
<19> The method, kit, drug, use or antibody of <18>, wherein FR1 to FR4 consist of the following amino acid sequences.
FR1: Amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having 80% or more identity with the amino acid sequence FR2: Amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having 80% or more identity with the amino acid sequence. FR3: Amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 80% or more identity with the amino acid sequence FR4: Amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence having 80% or more identity with the amino acid sequence. <20> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown by SEQ ID NO: 1, CDR2 is the amino acid sequence shown by SEQ ID NO: 2, and CDR3 is SEQ ID NO: 3. FR1 is the amino acid sequence shown by SEQ ID NO: 4, FR2 is the amino acid sequence shown by SEQ ID NO: 5, FR3 is the amino acid sequence shown by SEQ ID NO: 6, and FR4 is the amino acid sequence shown by SEQ ID NO: 7. The method, kit, drug, use or antibody of <18>, which is a sequence.

Example 1 Screening for anti-SARS-CoV-2 antibody <Materials and methods>
1. 1. Target molecule for screening
As a target molecule, SARS-CoV-2 (2019-nCoV) Spike Protein (S1 subunit, His Tag) (His-tagged S1 protein, SinoBiological) was used.

2. 2. Synthesis of cDNA display (1) Synthesis of initial library 1x10 by PCR amplification using VHH naive DNA library AlipLib L hinge (60 ng / μL; RePHAGE) or AlipLib Shinge (60 ng / μL; RePHAGE) as template DNA. AlipLib Lhinge 1st PCR products or AlipLib Shinge 1st PCR products with ¹⁴ varieties were prepared. Primers page_to_DNA_dis. And LHinge-GGGS-His-Rs were used for PCR amplification of AlipLib Linge. Primers page_to_DNA_dis. And SHinge-GGGS-His-R were used for PCR amplification of AlipLive Hinge.
The composition of the PCR solution was adjusted to a 250 μL reaction scale and dispensed into 5 test tubes. Using 300 ng of VHH naive DNA library as template DNA per test tube, the above primers or primers SHinge-GGGS-His-R, PrimeSTAR DNA polymerase (Takarabio), 5x PrimeSTAR Buffer (Takarabio), 2.5 mM It was assumed to include dNTP mix and _H2O (Ultra Pure DNase / RNase-Free Distilled Water, Thermo Fisher Scientific).
The PCR product was purified using Agencourt AMPure XP (Beckman Coulter). Approximately 2 volumes of beads per 1 μL of PCR product were added, pipetted and mixed, allowed to stand for a few minutes, then the supernatant was removed and the beads were washed with 70% ethanol. After that, the beads were air-dried, an appropriate amount of _H2O was added to suspend the beads, and the supernatant was collected. Then, the DNA concentration was quantified by absorbance measurement (A260 / A280).
Next, the AlipLib L hinge 2nd PCR project or the AlipLib Hinge 2nd PCR was prepared by PCR amplification using the AlipLib L hinge 1st PCR product or the AlipLib Shinge 1st PCR product as the template DNA. T7-Ω and His-C (Ytag cnvK) were used as primers for PCR amplification.
The PCR solution was prepared on a 500 μL reaction scale and dispensed into 10 test tubes. Each test tube contains 3000 ng of template DNA, primers (T7-Ω and His-C), PrimeSTAR DNA polymerase (Takara Bio), 5x PrimeSTAR Buffer (Takara Bio), 2.5 mM dNTP mix, H ₂ O (Ultra Pure). DNase / RNase-Free Distilled Water, Thermo Fisher Scientific) was included.
The PCR product was subjected to agarose gel electrophoresis, a gel containing the VHH library fragment (PCR product) separated at the desired position was cut out, and then purified using NucleoSpin Gel and PCR Clean-up (Takara Bio). AlipLib Linge 2nd PCR project and AlipLib Schinge 2nd PCR product were obtained. The DNA concentration was measured by absorbance measurement (A260 / A280), and the DNA concentration was quantified.
4,500 ng of AlipLib L hinge 2 ^nd PCR project and 4,500 ng of AlipLib Shinge 2nd PCR project (Epsilon Molecular Engineering) were amplified by PCR. The reaction mixture was 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kappa biosystems), 1.5 μL of 10 μM Forward primer, 1.5 μL of 10 μM Reverse index primer, and 300 μL of 10 μM Reverse index primer, 300 NG. It was prepared to be. Primers used were 5'-GATCCCGCGAAAATTAATACGACTCATCATAGGG-3'(SEQ ID NO: 10) and 5'-TTTCCACGCCGCCCCCCGTCCT-3' (SEQ ID NO: 11). PCR was performed at 95 ° C. for 5 minutes, then 98 ° C. for 20 seconds, 65 ° C. for 15 seconds, 72 ° C. for 30 seconds for 5 cycles, and 72 ° C. for 5 minutes. Purification of PCR products is Agencourt AMPure
XP (Beckman Coulter) was used. 1.8 μL of beads per 1 μL of PCR product was added, mixed by pipetting, and allowed to stand for 5 minutes. After allowing to stand on a magnetic plate until it became transparent, the supernatant was removed. After adding 1.4 mL of 70% ethanol and allowing to stand for 30 seconds, the supernatant was removed. This ethanol washing was performed 3 times. After removing the supernatant, the beads were allowed to stand on a magnetic plate for 3 minutes and the beads were air-dried. The tube was removed from the magnetic plate, 100 μL of UNCLEASE FREE WATER was added, and the beads were suspended by pipetting and allowed to stand for 5 minutes. The tube was allowed to stand on a magnetic plate for 2 minutes, and then the supernatant was collected.

(2) In vitro transcription T7 RiboMAX Express Large Scale RNA Production System (Promega) was used. 25 μL of RivoMAX ™ Express T7 2X Buffer, 20 pmol PCR product, 5 μL of Enzyme Mix were mixed and incubated at 37 ° C. for 30 minutes. Subsequently, 5 μL of RQ1 RNase-Free DNase was added and incubated at 37 ° C. for 15 minutes. RNA Clean XP (Beckman Coulter) was used for purification of the transcript. 1.8 μL of beads per 1 μL of transcript was added, mixed by pipetting and allowed to stand for 5 minutes. After allowing to stand on a magnetic plate until it became transparent, the supernatant was removed. 200 μL of 70% ethanol was added, and the mixture was allowed to stand for 30 seconds, and then the supernatant was removed. This ethanol washing was performed 3 times. After removing the supernatant, the beads were allowed to stand on a magnetic plate for 10 minutes and the beads were air-dried. The tube was removed from the magnetic plate, 20 μL of UNCLEASE FREE WATER was added, and the beads were suspended by pipetting and allowed to stand for 5 minutes. The tube was allowed to stand on a magnetic plate for 1 minute, and then the supernatant was collected. After purification, RNA was quantified by NanoPad DS-11FX (DeNovix).

(3) Coupling of mRNA and puromycin linker 4 μL of 0.25M Tris-HCl (pH 7.5), 4 μL of 1M NaCl, 20 pmol of mRNA, and 20 pmol of cnvK riboG linker are mixed to make 20 μL with NUCLASE FREE WATER. A reaction solution was prepared in. Annealing was performed using a ProFlex PCR System (Life technologies) at 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./sec. Subsequently, ultraviolet rays having a wavelength of 365 nm were irradiated for 5 minutes using Handheld UV Lamp, 6W, UVGL-58, 254/365 nm, 100 V (Anallytic jena US, An Endress + Houser Company). The mRNA-linker conjugate was shaded and ice-cooled until use.

(4) Cell-free translation Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega) was used for the synthesis of mRNA-VHH conjugate from mRNA-linker conjugate. 10.5 μL NUCLEASE FREE WATER, 1.5 μL 20x Translation Mix, 52.5 μL Rabbit Reticulocyte Lysate, 1.5 μL RNassin ™ Ribonuclease Inhibitor (40U / μL) Was mixed. After incubating this reaction solution at 37 ° C. for 15 minutes, 36 μL of IVV formation buffer (3M KCl, 1M MgCl ₂ ) mixture was added, and the mixture was further reacted at 37 ° C. for 20 minutes to synthesize an mRNA-VHH conjugate. ..

(5) Immobilization on streptavidin magnetic beads 60 μL of Dynabeads My One Streptavidin C1 (Thermo Fisher Scientific) with 60 μL of 2x Binding buffer (20 mM Tris-HCl, 2M mM NaCl) ) Was added and suspended by pipetting for 1 minute. It was allowed to stand on a magnetic plate for 1 minute, and the supernatant was discarded. This wash was performed twice. It was mixed with 75 μL of mRNA-VHH conjugate, 75 μL of 2xBindering buffer, and washed streptavidin magnetic beads and incubated for 30 minutes at room temperature. After allowing to stand on a magnetic plate for 1 minute, the supernatant was removed. 200 μL of Binding buffer (10 mM Tris-HCl, 1M NaCl, 0.1% Tween 20, 1 mM EDTA, pH 8) was added, pipetting for 1 minute, and then the supernatant was removed. This wash was performed twice.

(6) Reverse transcription reaction 55.5 μL of UNCLEASE FREE WATER, 15 μL of 5x RiverTra Ace Buffer (Toyobo Life Scene), 3 μL of 25 mM dNTP Mix, 1.5 μL of RiverTlaCe (100 μL) did. A cDNA-VHH conjugate was synthesized by adding an mRNA-VHH conjugate immobilized on streptavidin magnetic beads to the above reaction solution and incubating at 42 ° C. for 30 minutes to carry out a reverse transcription reaction. ..

(7) Cut out from beads His-tag wash buffer (20 mM Sodium phosphate, 500 mM NaCl, 5 mM Imidazole, 0.05% Tween 20, for the cDNA-VHH conjugate immobilized on the streptavidin magnetic beads, pH 7.4) was added, and the mixture was suspended by pipetting for 1 minute. It was allowed to stand on a magnetic plate for 1 minute, and the supernatant was discarded. Subsequently, 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 allowed to stand at 37 ° C. for 15 minutes for cDNA-VHH linkage. The body was eluted.

(8) Purification 30 μL of His Mag Sepharose Ni Beads (GE Health Care) was allowed to stand on a magnetic plate for 1 minute, the supernatant was discarded, and then the cells were resuspended in His-tag wash buffer. This cleaning operation was performed twice. The eluate of the cDNA-peptide conjugate was mixed with the His Mag Sepharose Ni Beads suspension, incubated for 30 minutes at room temperature, then allowed to stand 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. It was allowed to stand on a magnetic plate for 1 minute, and the supernatant was discarded. This cleaning operation was performed twice. The cDNA-VHH conjugate is eluted by adding 10 μL of His-tag elution buffer (20 mM Sodium phosphate, 500 mM NaCl, 250 mM Imidazole, 0.05% Tween 20, pH 7.4) and incubating at room temperature for 15 minutes. did.

3. 3. Selection (1) Immobilization and blocking of target molecule SARS-CoV-2 (2019-nCoV) Spike Protein (S1 subunit, His Tag) (SinoBiological) was used as the target molecule. 100 μL of 100 ug / mL (selection round 1, R1), 10 ug / mL (selection round 2, R2), 1 ug / mL (selection round 3, R3) in each well of Nunc-Immuno ™ Plate II (Thermo Fisher Scientific). ) SARS-CoV-2 S1 protein / PBS was added, and after sealing, the mixture was allowed to stand at 4 ° C. overnight. After carefully removing the S1 protein / PBS that was not adsorbed to the wells using a pipette, 200 μL of 3% BSA / PBST (PBS containing 0.02% Tween 20) was added, and the mixture was incubated at room temperature for 2 hours. After carefully removing BSA / PBST using a pipette, add 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween 20, pH 7.2), allow to stand for 5 minutes, and then carefully use a pipette. Removed to. This cleaning operation was performed three times.

(2) Selection A cDNA display library prepared to 200 pmol in selection round 1 (R1) and 6 pmol in selection round 2 (R2) and selection round 3 (R3) was used. Each cDNA display library was prepared to 100 μL by adding HBST (20 mM HEPES, 500 mM NaCl, 0.02% Tween 20, pH 7.2, 0.5% Bovine serum albumin). The S1 protein was added to the immobilized well and incubated at room temperature for 1 hour. After carefully removing the cDNA display solution using a pipette, 200 μL of HBST was added, and the mixture was incubated at room temperature for 5 minutes and then carefully removed using a pipette. This cleaning operation was performed 10 times. 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) was added, and after careful pipetting, the mixture was allowed to stand at room temperature for 5 minutes. After standing, the supernatant was collected in a new tube.

4. The PCR amplified eluate was subjected to PCR. The reaction mixture was 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5 μL of 10 μM Forward primer, 1.5 μL of 10 μM Reverse index primer, 12.5 μL of 12.5 μL of Kapa Bihot Start Ready Mix (2X). 5
μL of Nuclease free water was mixed and prepared. Primers used were 5'-GATCCCGCGAAAATTAATACGACTCACTATAGGGAAGTTATTTTTACACAAATTACCAACCA-3'(SEQ ID NO: 12) and 5'-TTTCCACGCCGCCCCCCGTCCT-3'(SEQ ID NO: 11). PCR is performed at 95 ° C for 2 minutes, then at 98 ° C for 20 seconds, at 68 ° C for 15 seconds, at 72 ° C for 20 seconds for 22 cycles (R1) or 24 cycles (R2 or later), and at 72 ° C for 5 minutes. went. The PCR product was purified using Agencourt AMPure XP (Beckman Coulter). 1.8 μL of beads per 1 μL of PCR product was added, mixed by pipetting, and allowed to stand for 5 minutes. After allowing to stand on a magnetic plate until it became transparent, the supernatant was removed. 200 μL of 70% ethanol was added, and the mixture was allowed to stand for 30 seconds, and then the supernatant was removed. This ethanol washing was performed twice. After removing the supernatant, the beads were allowed to stand on a magnetic plate for 5 minutes and the beads were air-dried. The tube was removed from the magnetic plate, 45 μL of UNCLEASE FREE WATER was added, and the beads were suspended by pipetting and allowed to stand for 5 minutes. The tube was allowed to stand on a magnetic plate for 1 minute, and then the supernatant was collected.

5. Extraction of VHH antibody candidate sequence (1) Quantification and concentration preparation of PCR product PCR product was quantified using PicoGreen ™ dsDNA reagent kit (Thermo Fisher Scientific). The concentration of each sample was adjusted to 100 ng / mL according to the quantitative value.

(2) Preparation of sequence library 1st ^PCR was performed. Reaction solution is 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kappa biosystems) per sample, 0.5 μL of 10 μM Forward primer, 0.5 μL of 10 μM Reverse primer, 2.5 μL of the preceding paragraph. 9 μL of Nuclease free water was mixed and prepared. Primers were 5'-TCGTCGGGCAGGCGTCAGATGTGTAATAAGAGACAGNNNNNATGCTGAGGTGCAGCTCGTG-3'(SEQ ID NO: 13) and 5'-GTCTCGTGGGCGCTCGGAGATGTGTAGTAAGACAGNACGNNTGACGSATGACTG. PCR was performed at 95 ° C. for 3 minutes, then 98 ° C. for 20 seconds, 62 ° C. for 15 seconds, 72 ° C. for 20 seconds for 16 cycles, and 72 ° C. for 5 minutes.
The PCR product was purified using Agencourt AMPure XP (Beckman Coulter). 1.8 μL of beads per 1 μL of PCR product was added, mixed by pipetting, and allowed to stand for 5 minutes. After allowing to stand on a magnetic plate until it became transparent, the supernatant was removed. 200 μL of 70% ethanol was added, and the mixture was allowed to stand for 30 seconds, and then the supernatant was removed. This ethanol washing was performed twice. After removing the supernatant, the beads were allowed 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 UNCLEASE FREE WATER was added, and the beads were suspended by pipetting and allowed to stand for 5 minutes. The tube was allowed to stand on a magnetic plate for 1 minute, and then the supernatant was collected.
Subsequently, Index PCR was performed. The reaction solution was 12.5 μL of KAPA HiFi HotStart Ready Mix (2X) (Kappa biosystems), 1 μL of 10 μM Forward index primer (Nuclease XT Index Primer), 1 μL IX Index Kit v2, Illumina. Kit v2, Illumina), 2.5 μL of template DNA, and 8 μL of Nuclease free water were mixed and prepared. PCR was performed at 95 ° C. for 3 minutes, then 98 ° C. for 20 seconds, 55 ° C. for 15 seconds, 72 ° C. for 30 seconds for 8 cycles, and 72 ° C. for 5 minutes. The PCR product was 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 values, it was prepared to 4 nM by diluting with nucleoASE FREE WATER.
DNA concentration [ng / μL] / (660 [g / mol] × 550 [bp]) × 10 ⁶ = DNA concentration [nM] ... (1)

(3) Sequence Prepared according to the recommended protocol when using the 4nM library. The final concentration of the denatured and diluted sample library was adjusted to 7 pM. In addition, the PhiX Control in the sample library was adjusted to be 5%. Sequences were performed using MiSeq (Illumina) and MiSeq Reagent Nano Kit v2 500 cycle (Illumina).

(4) Extraction of amino acid sequence of VHH antibody from sequence data and multiple measurement The sequence data demolated using MiSeq Controller was used for analysis. The base sequence region in which the VHH antibody was encoded was extracted from the sequence data and translated into an amino acid sequence. Then, the number of amino acid sequences that completely matched each amino acid sequence was counted.

(5) Selection of anti-SARS-CoV-2 VHH antibody candidate sequences The amino acid sequences of VHH antibodies that commonly appear in the libraries after selection of R2 and R3 and the number of clones of each sequence were calculated. As a result of determining the number of clones by perfect matching of the amino acid sequences of the full length of the VHH antibody, it was found that CoVHH1 was the most dominant in the library (Fig. 1).
The entire base sequence encoding CoVHH1 is as shown in SEQ ID NO: 8, and the amino acid sequence thereof is AEVQLVESGGGQVETGGSLRLSCQASGSTFSDYVMAWFRQRPGKEREFVATISRNGGTTYGSSVKGRFTISRDNAKSTVYLQSRGDYGSVTYLGSTVISSRDNAKSTVYLQMNDSSWVGSTVG The base sequence encoding the amino acid is not limited to the codon shown in SEQ ID NO: 8. In the amino acid sequence, Nos. 1 to 26 are FR1, Nos. 27 to 36 are CDR1, Nos. 37 to 50 are FR2, Nos. 51 to 60 are CDR2, Nos. 61 to 99 are FR3, Nos. 100 to 106 are CDR3, 108 to 117. The number is FR4.

(6) Extraction of CDR Region Amino Acid Sequences of VHH Antibodies CoVHH1 mutants that commonly appeared in the libraries after selection of R2 and R3 and had similar amino acid sequences to CoVHH1 that appeared most frequently were found in the CDR regions. Comparisons were made (Fig. 2). As CoVHH1 variants, the CDR1 region variant has 4 sequences (CoVHH1301 (SEQ ID NO: 34), CoVHH319 (SEQ ID NO: 35), CoVHH39 (SEQ ID NO: 36), CoVHH327 (SEQ ID NO: 37)), and the CDR2 region variant has 8 sequences (SEQ ID NO: 37). CoVHH47 (SEQ ID NO: 38), CoVHH16 (SEQ ID NO: 39), CoVHH49 (SEQ ID NO: 40), CoVHH58 (SEQ ID NO: 41), CoVHH417 (SEQ ID NO: 42), CoVHH118 (SEQ ID NO: 43), CoVHH416 (SEQ ID NO: 44), CoVHH3342. (SEQ ID NO: 45)), the CDR3 region variant has 6 sequences (CoVHH201 (SEQ ID NO: 46), CoVHH20 (SEQ ID NO: 47), CoVHH112 (SEQ ID NO: 48), CoVHH2017 (SEQ ID NO: 49), CoVHH137 (SEQ ID NO: 50), CoVHH96 (SEQ ID NO: 51)) was found.

Example 2 Production of VHH by protease-deficient recombinant bacillus (1) Artificial synthesis of gene The amino acid sequence of CoVHH1 synthesized is a His tag sequence (SEQ ID NO: 25, SEQ ID NO: 25, via a hinge sequence on the C-terminal side of SEQ ID NO: 9. EPKTPKPQSHHHHH) is assigned SEQ ID NO: 26. Therefore, the artificially synthesized gene for CoVHH1 expression is SEQ ID NO: 27 in which a stop codon is added to the 3'end of the base sequence encoding SEQ ID NO: 26. Further, as a sequence for inserting an artificially synthesized gene for CoVHH1 expression into a plasmid vector, GCACTAACTTGCAGCA (SEQ ID NO: 28) is at the 5'end and TCTATTAAACTAGTT (SEQ ID NO: 29) is at the 3'end with respect to SEQ ID NO: 27. The added product was artificially synthesized by Eurofins Scientific and used for the experiment.

(2) Construction of plasmid for expressing VHH with His tag Using the recombinant plasmid pHY-S237 (Japanese Patent Laid-Open No. 2014-158430) prepared based on pHY300PLK as a template, 5'-GATCCCCGGGGAATTTCCTGTATAAAAAAAAG-3'(SEQ ID NO: 15) and 5 The plasmid sequence was amplified by PCR using the primer set of'-ATGATGTTAAGAAGAAAACAAAGCAG-3'(SEQ ID NO: 16) and PrimeSTAR Max DNA polymerase (TaKaRa). A promoter derived from the spoVG gene was amplified by PCR using the genome of the 168 strain as a template and the primer sets of 5'-GAATTCCGGGGATCTAAGAAAGTGATTCTGGGAGAG-3'(SEQ ID NO: 17) and 5'-CTTTCTTAACTCATCATCATCATCACCACTTTTCCC-3'(SEQ ID NO: 18). .. The obtained promoter DNA was incorporated into a plasmid sequence using an In-Fusion HD Cloning Kit (Takara) to construct a plasmid for VHH expression linked to the spoVG promoter. The plasmid sequence was amplified by PCR using the primer sets of 5'-TGCTGCAAGAGCTGGCCGGAAATAAA-3'(SEQ ID NO: 19) and 5'-TCTATTAAACTAGTTATAGGG-3' (SEQ ID NO: 20) and PrimeSTAR Max DNA polymerase (TaKaRa). A DNA containing the artificially synthesized gene (1) was incorporated into the obtained PCR fragment using an In-Fusion HD Cloning Kit (Takara) to construct a plasmid for expressing VHH containing each of the artificially synthesized VHH genes. This His-tagged CoVHH1 expression plasmid is called CoVHH1-His-pHY.
The CoVHH dimer was constructed as follows. For the CoVHH1 dimer, the artificially synthesized gene for CoVHH1 expression is used as a template, and the primer set of 5'-GCAGCTCTTGCAGCCGCTGAAG-3'(SEQ ID NO: 21) and 5'-CGCGCGCTGGACTGAACTCACGGTTACCTGAG-3'(SEQ ID NO: 22), and VHH1 are used as templates. A fragment obtained by PCR with PrimeSTAR Max DNA polymerase (TaKaRa) using the primer sets of 5'-AGTTCAGCCGCCGCGGAAGTGCAACTGGGTTGGTCG-3'(SEQ ID NO: 23) and 5'-AACTAGTTTAATAGATTAATG-3'(SEQ ID NO: 24). The gene) was integrated into a VHH expression plasmid linked to the spoVG promoter in the same manner as above using the In-Fusion HD Cloning Kit (Takara). This His-tagged CoVHH1 dimer expression plasmid is called CoVHH1 (dimer) -His-pHY.

(3) Construction of FLAG-tagged plasmid for VHH expression Using CoVHH1-His-pHY as a template, 5'-GCAGCTCTTGCAGCAGCTGAAGTAGCAACTGGGTTGAG-3'(SEQ ID NO: 31) and 5'-ACTAGTTTAATAGGATTTTTGTTGTCATGTCATGTGTCATGTGTCATGTGTcat A PCR fragment of FLAG-tagged CoVHH1 was amplified by PCR with PrimeSTAR Max DNA polymerase (TaKaRa). Primer sets of 5'-TGCTGCAAGGCTGCCGGAAATAAA-3'(SEQ ID NO: 19) and 5'-TCATTATAAAACTAGTTATAGGG-3' (SEQ ID NO: 20) and PrimeSTAR Max DNA polymerase (SEQ ID NO: 20) using the plasmid for VHH expression linked to the above-mentioned spoVG promoter as a template. The plasmid sequence was amplified by PCR using TaKaRa). A PCR fragment of FLAG-tagged CoVHH1 was incorporated into the obtained plasmid PCR fragment using an In-Fusion HD Cloning Kit (Takara). This FLAG-tagged CoVHH1 dimer expression plasmid is called CoVHH1-FLAG-pHY.
The constructed plasmid was transferred from Bacillus subtilis 168 strain according to the procedure shown in (4) below, according to the method described in JP-A-2006-174707, and 9 kinds of extracellular protease genes (epr, wprA, mpr, nprB, bpr). , NprE, vpr, aprE and aprX) were all deleted, and the strain was introduced into a strain (Dpr9ΔsigF) obtained by deleting the sigF gene involved in sporulation according to the method described in Japanese Patent No. 4336882.

(4) Preparation of recombinant Bacillus subtilis The plasmid was introduced into the Bacillus subtilis strain by the protoplast method shown below. Bacillus subtilis stocked with glycerol was inoculated into 1 mL of LB liquid medium and cultured at 30 ° C. and 210 rpm with shaking overnight. The next day, 10 μL of this culture solution was inoculated into a new 1 mL LB liquid medium and shake-cultured at 37 ° C. and 210 rpm for about 2 hours. The culture was collected in 1.5 mL tubes, centrifuged at 1,2000 rpm for 5 minutes, the pellet from which the supernatant had been removed was suspended in 500 μL of SMMP containing 4 mg / mL of Lysozyme (SIGMA), and incubated at 37 ° C. for 1 hour. Then, the pellet was centrifuged at 3,500 rpm for 10 minutes, and the pellet from which the supernatant had been removed was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with various plasmids, and 100 μL of 40% PEG was further added for vortexing. 350 μL of SMMP was added to this solution, mixed by inversion, shaken at 30 ° C. and 210 rpm for 1 hour, and then the whole amount was applied to a DM3 agar plate and incubated at 30 ° C. for 2 to 3 days.

(5) VHH production The recombinant Bacillus subtilis prepared in (4) was inoculated into LB medium containing 1 mL of 50 ppm tetracycline and shaken back and forth at 32 ° C. overnight to prepare a preculture solution. Dpr9ΔsigF was inoculated at 1% in 20 mL of 2 × L-mal medium in which the preculture solution was placed in a pleated Erlenmeyer flask, and cultured at 30 ° C. for 72 hours with shaking. At the end of the culture, 1 mL of the culture solution was centrifuged in a microtube at 4 ° C., 15,000 rpm for 5 minutes, and the supernatant was collected. Each VHH was purified using Ni-NTA agarose beads (Fuji Film Wako Pure Chemical Industries, Ltd.) according to the protocol of the kit. PBS (50 mM imidazole) was used as the eluate.
Western blotting was performed to confirm the production of the antibody in the culture supernatant. For SDS-PAGE, use Super Cep Ace, 15-20% (Tricin gel) (Fuji Film Wako Pure Chemical Industries, Ltd.) as the gel, and apply a sample containing 0.5 μL of the culture supernatant to each well, and then apply the sample at 120 V for 3 hours. It was run. An xL ladder (Broad) (Aproscience) was used as the molecular weight marker. Proteins were transferred from SDS-PAGE gels to PVDF membranes using Trans-Blot Turbo Mini PVDF Transfer Packs (BIO-RAD) and Trans-Blot Turbo System (BIO-RAD). The antibody was 6 × -His Tag Monoclonal Antibody (3D5), HRP (Invitrogen) or ANTI-FLAG M2-peroxidase (HRP) conjugate (Sigma-Aldrich), and iBind Western System (Invitrogen) was used for the antibody reaction. The target protein was detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific) (Fig. 3).

As a result, it was confirmed that CoVHH1 and CoVHH1 dimer were synthesized. The amino acid sequence of His-tagged CoVHH1 synthesized by this method is as shown in SEQ ID NO: 26, the His-tagged CoVHH1 dimer is as shown in SEQ ID NO: 30, and the amino acid sequence of FLAG-tagged CoVHH1 is SEQ ID NO: As shown in 33.

Example 3 Measurement of binding activity by biolayer interferometry The binding activity of CoVHH1 immobilized on a Ni-NTA sensor chip to the S1 protein was measured using Blitz bio-layer interferometer (Fortebio). The Advanced kinetics mode was selected, and 250 μL of each measurement solution was added to Black 0.5 mL Tube for measurement. Prior to measurement, the tip was hydrated by immersing the tip of Dip and Read ™ Ni-NTA (NTA) Biosensors (Fortebio) in 100 μL PBST (PBS containing 0.1% Tween 20, pH 7.4) overnight. rice field. The measurement order for each run is as follows; 1) Baseline step: Measurement in PBST for 30 seconds, 2) Loading step: Measurement in 1-10 μg / mL His-tagged CoVHH1 diluted with PBST for 120 seconds, 3) Baseline step: Measurement in PBST for 30 seconds, 4) Association step: 0-693.1 nM SARS-CoV-2 S1 subunit-Fc (Fc-tagged S1 protein, The Native Agent Company) prepared by diluting with PBST. Measurement for 180 seconds, 5) Dilution step: Measurement for 300 seconds in PBST. Global Fitting was performed using BLItz Pro software version (1.2.1.5) (Molecular Devices) to calculate binding activity (FIG. 4).
As a result, the equilibrium deviation constant (KD) of CoVHH1 with respect to the S1 protein was ^7.131x10-9M (ka = 1.604x10 ⁴ (1 / Ms), kd = 1.144x10 ^-4 (1 / s)). ..

Example 4 ELISA
100 μL 0-10 μg / mL SARS-CoV-2 S1 subunit-Fc (Fc-tagged S1 protein, The Native Engine) diluted with PBS and adjusted to each well of Nunc-Immuno ™ Plate II (Thermo Fisher Scientific). ) Was added, and after sealing, the mixture was allowed to stand at 4 ° C. overnight. After carefully removing the S1 protein dispersion that was not adsorbed to the wells using a pipette, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added, and the mixture was incubated at room temperature for 1 hour. After carefully removing skim milk using a pipette, 200 μL of PBST was added, and the mixture was allowed to stand for 5 minutes and then carefully removed using a pipette. This cleaning operation was performed three times. 100 μL of 2 μg / mL His-tagged CoVHH1 or His-tagged CoVHH1 dimer / PBST was added to each well on which the S1 protein was immobilized and incubated for 1 hour at room temperature. After carefully removing the VHH antibody using a pipette, 200 μL of PBST was added, and the mixture was allowed to stand for 5 minutes and then carefully removed using a pipette. This cleaning operation was performed three times. Anti-His-tag mAb-Biotin (Monoclonal, OGHis) (MEDICAL & BIOLOGICAL LABORATORIES) was used as the primary antibody. The primary antibody was diluted to 1 / 5,000 with PBST. 100 μL of primary antibody was added to each well and incubated for 1 hour at room temperature. After carefully removing the primary antibody using a pipette, 200 μL of PBST was added, and the mixture was allowed to stand for 5 minutes and then carefully removed using a pipette. This cleaning operation was performed three times. Streptavidin HRP Conjugate (Tokyo Chemical Industry) was used for the detection of the primary antibody. Streptavidin HRP Conjugate solution was diluted to 1 / 5,000 with PBST, 100 μL was added to each well, and the mixture was incubated at room temperature for 1 hour. The Streptavidin HRP Conjugate solution was carefully removed using a pipette, 200 μL of PBST was added, and the mixture was allowed to stand for 5 minutes, and then carefully removed using a pipette. This cleaning operation was performed three times. The color-developing substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in a Table Peroxide Substrate Buffer (Thermo Fisher Scientific). After adding 100 μL of a color-developing substrate to each well and incubating for 20 minutes under shading, the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN). As a result, both CoVHH1 and CoVHH1 dimers were found to have binding activity to S1 protein. Further, it was found that the binding activity was improved by dimerizing CoVHH1 (FIG. 5).

Example 5 Neutralizing activity test The neutralizing activity of the His-tagged CoVHH1 and His-tagged CoVHH1 dimers obtained by the above method against SARS-CoV-2 was measured. CoVHH1 solution and CoVHH1 dimer solution are prepared to have concentrations of 323.4 μg / mL and 122.5 μg / mL, respectively, by mixing with 2% Fetal Bovine Serum (FBS) / Dulbecco Modified Eagle's Medium (DMEM). did. A 2-fold dilution is prepared by mixing 100 μL of each mixture with 100 μL of 2% FBS / DMEM, and similarly, a 2-fold dilution series solution of the antibody is prepared using 2% FBS / DMEM up to 128-fold dilution. (Table 1).

Add 100 μL of each 2-fold diluted series solution of His-tagged CoVHH1 or His-tagged CoVHH1 dimer to a 96-well plate, and further add 2.5 × 10 ⁵ pfu / mL SARS-CoV-2 solution (National Institute of Infectious Diseases). (Obtained from the same place) was added in an amount of 100 μL each. Then, after incubating at 37 ° C. for 2 hours, the cells were incubated overnight at 4 ° C. to contact the antibody with the virus. To wells inoculated with Vero-E6 / TMPRSS2 cells (purchased from JCRB cell bank), 100 μL of a solution in which an antibody and a virus were contacted was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ environment. As a control, PBS (containing 50 mM imidazole) used for dissolving the CoVHH1 and CoVHH1 dimers was used. Three days after the start of culture, the cytopathic effect (CPE) was measured with a microscope to evaluate the neutralizing activity. The results are shown in Table 2.

As a result, when imidazole was added, cytopathic effect was observed in all wells, whereas CoVHH1 showed neutralizing activity at 4.043 μg / well, and CoVHH1 dimer showed medium at 0.8750 μg / well. It was clarified that it showed a sum-active ability. That is, it was shown that the neutralization activity of CoVHH1 is enhanced by increasing the number of multimers.

Example 6 Detection of S1 Protein of SARS-CoV-2 by Sandwich ELISA Add 100 μL of 5 μg / mL His-tagged CoVH1 to each well of Nickel Coated Plates, Clear, 96-Well (Thermo Fisher Scientific). Then, it was allowed to stand at 4 ° C. overnight. After carefully removing the VHH antibody that was not adsorbed to the wells using a pipette, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added, and the mixture was incubated at room temperature for 1 hour. After the blocking agent was carefully removed using a pipette, 200 μL of PBST was added and the blocking agent was carefully removed using a pipette. This cleaning operation was performed three times. 100 μL of 0 to 1 μg / mL SARS-CoV-2 S1 subunit-Fc (Fc-tagged S1 protein, The Native Antigen Company) / PBST was added to each well on which the VHH antibody was immobilized, and the mixture was added at room temperature for 50 minutes. Incubated. After carefully removing the Fc-tagged S1 protein using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This cleaning operation was performed three times. Rabbit Anti-Sheep IgG Fc (Abcam) was used as the primary antibody. The primary antibody was diluted to 1 / 5,000 with PBST. 100 μL of primary antibody was added to each well and incubated for 50 minutes at room temperature. After carefully removing the primary antibody using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This cleaning operation was performed three times. Goat Anti-Rabbit IgG H & L (HRP) (abcam) was used as the secondary antibody. The secondary antibody was diluted to 1 / 5,000 with PBST. 100 μL of secondary antibody was added to each well and incubated for 50 minutes at room temperature. After carefully removing the secondary antibody using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This cleaning operation was performed three times. The color-developing substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in a Table Peroxide Substrate Buffer (Thermo Fisher Scientific). After adding 100 μL of a color-developing substrate to each well and incubating for 30 minutes under shading, the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN).

As a result, it was clarified that the S1 protein of SARS-CoV-2 can be detected by immobilizing CoVHH1 on a plate (FIG. 6).

Example 7 Evaluation of CoVHH1 Binding Specificity by ELISA Add 100 μL of 0-10 μg / mL coronavirus His-tagged S1 protein / PBS to each well of Nunc-Immuno ™ Plate II (Thermo Fisher Scientific) after sealing. It was allowed to stand at 4 ° C. overnight. Examples of the coronavirus S1 protein include Human coronavirus HKU1 (isolate N1) (HCoV-HKU1) Spike / S1 Protein (S1 Subunit, His Tag), Human coronavirus (HCoV-NiS), and Human coronavirus (HCoV-NL63). , Human coronavirus (HCoV-229E) Spike / S1 Protein (S1 Subunit, His Tag), Human coronavirus (HCoV-OC43) Spike S1 Protein (HisTag) -SSA , His Tag), MERS-CoV Spike / S1 Protein (S1 Subunit, aa 1-725, His Tag) (all Sino Biological), SARS Coronavirus Spike / Engine (S1) ) Was used. After carefully removing the S1 protein dispersion that was not adsorbed to the wells using a pipette, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added, and the mixture was incubated at room temperature for 1 hour. After the skim milk was carefully removed using a pipette, 200 μL of PBST was added and the skim milk was carefully removed using a pipette. This cleaning operation was performed three times. 100 μL of 20 μg / mL FLAG-tagged CoVHH1 / PBST was added to each well on which the S1 protein was immobilized, and the mixture was incubated at room temperature for 1 hour. After carefully removing the VHH antibody using a pipette, 200 μL of PBST was added, and the VHH antibody was carefully removed using a pipette. This cleaning operation was performed three times. Monoclonal anti-FLAG® M2 antibody-peroxidase (HRP) -labeled mouse host antibody (Merck) was used as the detection antibody. The detected antibody was diluted to 1/5000 with PBST. 100 μL of detection antibody was added to each well and incubated for 1 hour at room temperature. After carefully removing the detected antibody using a pipette, 200 μL of PBST was added, and the detection antibody was carefully removed using a pipette. This cleaning operation was performed three times. The color-developing substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in a Table Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of a chromogenic substrate was added to each well, incubated for 30 minutes under shading, and immediately measured for an absorbance of 450 nm using a Microplate Reader Infinite M1000 PRO (TECAN).

As a result, CoVHH1 showed binding to the S1 protein of SARS-CoV-2, but no binding to the S1 protein of other coronaviruses was observed (FIG. 7).

Example 8 Measurement of binding activity by biolayer interferometry In order to measure the binding activity of CoVHH1 to S1 protein with higher accuracy, the S1 protein of CoVHH1 immobilized on the HIS1K sensor chip using the Octet RED 384 system (Fortebio). The binding activity to was measured. PBST (PBS containing 0.05% Tween 20) was used as a buffer, and the reaction temperature was set to 25 ° C. Each 50 μL reaction was measured in each well of a 384-well black plate (Fortebio). The measurement conditions are as follows. 1) Baseline
step: Measurement in PBST for 30 seconds, 2) Loading step: Measurement with His-tagged CoVHH1 diluted with PBST so that binding is 0.3 nm, 3) Baseline step: Measurement in PBST for 30 seconds, 4 ) Association step: Measurement for 180 seconds with 0-245.8 nM SARS-CoV-2 (2019-nCoV) Spike S1-Fc Recombinant Protein diluted with PBST, 5) Measurement step: Measurement for 240 seconds in PBST. 1: 1 Fitting was performed using ForestBio Octet analysis software (Date Analysis HT Version 11.1.2.48) to calculate the binding activity (FIG. 8). As a result, the equilibrium deviation constant (KD) of CoVHH1 with respect to the S1 protein was ^1.40x10-9M (ka = 6.72x10 ⁴ (1 / Ms), kd = 9.42x10 ^-4 (1 / s)). ..

Example 9 Measurement of binding activity by surface plasmon resonance (SPR) resonance method Binding of His-tagged CoVHH1 to S1 protein immobilized on Series S Sensor Chip NTA (Cytiva) using Biacore ™ T200 (Cytiva). The activity was measured. Measurements were made in Wizard, Kinetics / Efficiency modes. The temperature was set to 25 ° C. Running buffers include 10 mM HEPES, 150 mM NaCl, 50 μM EDTA, 0.005% (v / v) Tween 20 (pH 7.4) or 50 mM MES, 150 mM NaCl, 50 μM EDTA, 0.005% (v / v) Tween 20. (PH 6.5) was used. The measurement order for each run is as follows; 1) VHH solidification: The His tag was set to a flow rate of 10 μL / mL, a 0.5 mM NiCl2 aqueous solution was added for 60 seconds, and then diluted with a running buffer. 200 RU immobilization was performed by adding the attached VHH solution for 60 seconds. 2) Measurement of binding activity: Associated SARS-CoV-2 (2019-nCoV) Spike S1-FcRecombinant Protein (S1 protein with Fc tag, SinoBiological) diluted with running buffer at a flow rate of 30 μL / mL. The interaction was set to Time 180 seconds and Dilution Time 400 seconds. 3) Regeneration of sensor chip surface: The flow rate was set to 30 μL / mL, and 350 mM EDTA solution (pH 8.0) was added for 60 seconds to elute the immobilized His-tagged VHH. Analysis by a 1: 1 binding model was performed using Biacore ™ T200 Assessment (software version 2.0) (Cytiva) to calculate binding activity (FIGS. 9 and 10). As a result, the equilibrium deviation constant (KD) of the His-tagged CoVHH1 with respect to the S1 protein was ^3.77x10-9M (ka = 8.87x10 ⁴ (1 / Ms)) at pH 7.4, and kd = 3.35x10 ^-4 (1). / S)), ^4.32x10-12 M (ka = 6.97x10 ⁴ (1 / Ms), kd = ^3.01x10-7 (1 / s)) at pH 6.5.

Example 10 Synthesis of CoVHH1 and CoVHH1 variants CoVHH1 (SEQ ID NO: 9) and CoVHH1 variants (CoVHH1301 (SEQ ID NO: 34), CoVHH319 (SEQ ID NO: 35), CoVHH39 (SEQ ID NO: 36), CoVHH327 (SEQ ID NO: 37), CoVHH47 (SEQ ID NO: 38), CoVHH16 (SEQ ID NO: 39), CoVHH49 (SEQ ID NO: 40), CoVHH58 (SEQ ID NO: 41), CoVHH417 (SEQ ID NO: 42), CoVHH118 (SEQ ID NO: 43), CoVHH416 (SEQ ID NO: 44), CoVHH3342 (SEQ ID NO: 38). SEQ ID NO: 45), CoVHH201 (SEQ ID NO: 46), CoVHH20 (SEQ ID NO: 47), CoVHH112 (SEQ ID NO: 48), CoVHH2017 (SEQ ID NO: 49), CoVHH137 (SEQ ID NO: 50), CoVHH96 (SEQ ID NO: 51). GGGSHHHHH (SEQ ID NO: 52) was added to the side for ELISA and synthesized according to the method of Example 2. The sequences of artificially synthesized genes for synthesizing each VHH are as shown in Table 3. Based on these artificially synthesized genes, CoVHH1 and CoVHH1 mutants were synthesized according to the procedure from (2) of Example 2. As a result, it was confirmed that all CoVHH1 and CoVHH1 mutants except CoVHH118 were synthesized.

Example 11 Evaluation of binding activity of CoVHH1 mutant by sandwich ELISA 100 μL of 5 μg / mL His synthesized in Example 10 in each well of Piece (trademark) Nickel Coated Plates, Clear, 96-Well (Thermo Fisher Scientific). Tagged CoVHH1 or each CoVHH1 variant was added and allowed to stand overnight at 4 ° C. After carefully removing the VHH antibody that was not adsorbed to the wells using a pipette, 200 μL of 5% skim milk / PBST (PBS containing 0.05% Tween 20) was added, and the mixture was incubated at room temperature for 1 hour. After the blocking agent was carefully removed using a pipette, 200 μL of PBST was added and the blocking agent was carefully removed using a pipette. This cleaning operation was performed three times. 100 μL of 0-0.1 μg / mL SARS-CoV-2 (2019-nCoV) Spike S1-Fc Recombinant Protein (S1 protein with Fc tag, SinoBiological) / PBST for each well on which the VHH antibody was immobilized. It was added and incubated for 50 minutes at room temperature. After carefully removing the Fc-tagged S1 protein using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This cleaning operation was performed three times. Goat Anti-Human IgG Fc (HRP) (abcam) was used as the detection antibody. The primary antibody was diluted to 1 / 5,000 with PBST. 100 μL of detection antibody was added to each well and incubated at room temperature for 50 minutes. After carefully removing the detected antibody using a pipette, 200 μL of PBST was added, and the detection antibody was carefully removed using a pipette. This cleaning operation was performed three times. The color-developing substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in a Table Peroxide Substrate Buffer (Thermo Fisher Scientific). After adding 100 μL of a color-developing substrate to each well and incubating for 20 minutes under shading, the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN).

As a result, among the CoVHH1 variants obtained by screening, CoVHH1301 (SEQ ID NO: 34), CoVHH319 (SEQ ID NO: 35), CoVHH39 (SEQ ID NO: 36), CoVHH327 (SEQ ID NO: 37), CoVHH47 (SEQ ID NO: 38), CoVHH16 (SEQ ID NO: 39), CoVHH49 (SEQ ID NO: 40), CoVHH58 (SEQ ID NO: 41), CoVHH417 (SEQ ID NO: 42), CoVHH416 (SEQ ID NO: 44), CoVHH3342 (SEQ ID NO: 45), CoVHH20 (SEQ ID NO: 47), CoVHH112 (SEQ ID NO: 39). It was confirmed that SEQ ID NO: 48), CoVHH2017 (SEQ ID NO: 49), and CoVHH137 (SEQ ID NO: 50) have the same binding activity of SARS-CoV-2 to the S1 protein as CoVHH1 (FIG. 11). From this, the CoVHH1 mutant (VHH antibody having one amino acid mutation in any of the CDR regions of CoVHH1) obtained through screening by the cDNA display method has a binding activity to the S1 protein of SARS-CoV-2. It has been shown.

Claims (7)

CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and the amino acid sequence represented by SEQ ID NO: 2 or one amino acid in the amino acid sequence. 1 is a structural domain containing CDR2 consisting of an amino acid sequence substituted with another amino acid and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is substituted with another amino acid in the amino acid sequence. A method for detecting SARS-CoV-2 in a sample, which comprises contacting a test sample with one or more antibodies that bind to SARS-CoV-2. CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and the amino acid sequence represented by SEQ ID NO: 2 or one amino acid in the amino acid sequence. 1 is a structural domain containing CDR2 consisting of an amino acid sequence substituted with another amino acid and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is substituted with another amino acid in the amino acid sequence. A SARS-CoV-2 detection kit containing an antibody that binds to SARS-CoV-2 having one or more. CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence in which one amino acid is replaced with another amino acid in the amino acid sequence, and the amino acid sequence represented by SEQ ID NO: 2 or one amino acid in the amino acid sequence. 1 is a structural domain containing CDR2 consisting of an amino acid sequence substituted with another amino acid and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence in which one amino acid is substituted with another amino acid in the amino acid sequence. A drug containing an antibody that binds to SARS-CoV-2 having one or more. The medicine according to claim 3 for the prevention or treatment of SARS-CoV-2 infection. The method according to claim 1, the kit according to claim 2, or the pharmaceutical agent according to claim 3 or 4, wherein the antibody is a VHH antibody, a heavy chain antibody, or a VHH antibody multimer. The method, kit or pharmaceutical according to claim 5, wherein the VHH antibody multimer is a multimer in which a plurality of the structural domains are linked. The method, kit or pharmaceutical according to claim 5, wherein the VHH antibody multimer is a multimer in which one or more of the structural domains and one or a plurality of structural domains having different antigen specificities from the structural domains are linked.

Images