VHH against SARS-cov2 and fusion protein

Abstract

[Problem] To provide a VHH which specifically binds to receptor binding domains of spike proteins of various types of variants of SARS-CoV2. [Solution] A VHH having any one or more of the characteristic properties mentioned below can recognize various types of variants of SARS-CoV2. (a) The VHH binds to Wuhan-Hu-1, delta variant, or micron variant of SARS-CoV2. (b) CDR1, CDR2, and CDR3 include the amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively; and / or (c) the VHH recognizes the amino acid sequence represented by SEQ ID NO: 4 and / or the amino acid sequence represented by SEQ ID NO: 5. This fusion protein which is obtained by fusing the VHH with ACE2 or an Fc form thereof enhances the SARS-CoV2 neutralizing capability and can be used in a pharmaceutical composition for treating or preventing COVID-19.

Description

VHH and fusion protein against SARS-CoV-2

[0001] The present invention relates to VHH against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), and in particular, to a VHH capable of specifically binding to a receptor binding region common to various mutant strains of SARS-CoV-2, a fusion protein of the VHH and ACE2, a detection composition for SARS-CoV-2 containing the VHH, and their uses.

[0002] SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a coronavirus belonging to the SARS-related coronavirus (SARSr-CoV), and is also referred to as the novel coronavirus. SARS-CoV-2 occurred in Wuhan City, Hubei Province, the People's Republic of China in December 2019, and the infection of SARS-CoV-2 caused COVID-19 (coronavirus disease-2019), which spread worldwide and caused a pandemic.

[0003] SARS-CoV-2 is an enveloped single-stranded RNA (+) virus belonging to the Orthocoronavirinae subfamily (coronavirus). SARS-CoV-2 can cause infections in humans and can cause severe pneumonia, similar to other SARS coronaviruses (SARS-CoV) and MERS coronaviruses (MERS-CoV). SARS-CoV-2, like other coronaviruses, is composed of four proteins known as spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein), and RNA. Among these, the N protein binds to RNA to form a nucleocapsid, and the S protein, E protein, and M protein bound to lipids surround the nucleocapsid to form an envelope. The S protein located on the outermost side of the envelope binds to the ACE2 receptor on the cell surface and then initiates entry into human cells.

[0004] Regarding the novel coronavirus (SARS-CoV-2), particularly since around 2020, various mutant strains with mutations, such as the Delta strain and Omicron strain, have successively emerged, leading to explosive outbreaks. These mutant strains have many mutations in the S protein, especially its receptor-binding domain (RBD), and are thought to have enhanced immune evasion, infectivity, and pathogenicity. As a result, it is known that the neutralizing ability of monoclonal antibodies and serum-derived antibodies is lost, and the effectiveness of vaccines is reduced against mutated viruses.

[0005] Several antibodies that bind to SARS-CoV-2 have been obtained. For example, Patent Documents 1 and 2 describe monoclonal antibodies. Patent Documents 3 and 4 describe antibodies or antigen-binding fragments thereof that specifically bind to the coronavirus spike protein.

[0006] Special table No. 2024-528710 Publication Special table No. 2024-518335 Publication Special table No. 2024-527607 Publication Special table No. 2024-518335

[0007] The inventors have completed the present invention by creating VHH that specifically binds to the RBD (receptor-binding domain) of the spike protein (S protein) of various SARS-CoV2 mutants. This enables more accurate detection of SARS-CoV2. Furthermore, a fusion protein of VHH and ACE2 was created by further binding the VHH of the present invention to an ACE2-Fc compound, which is formed by linking the Fc region of an antibody to ACE2, the receptor for the S protein. This fusion protein has a higher neutralizing ability against SARS-CoV2 compared to the ACE2-Fc compound. Utilizing this neutralizing ability, the VHH-ACE2 fusion protein of the present invention can be used as a pharmaceutical composition for the treatment or prevention of COVID-19.

[0008] The present invention relates to the following [1] to

[18] . [1] A VHH that specifically binds to the RBD (receptor-binding domain) of the SARS-CoV2 spike protein and has at least one of the following characteristics: (a) Binds to Wuhan-Hu-1, delta strain, and omicron strain of SARS-CoV2; (b) CDR1, CDR2, and CDR3 each contain an amino acid sequence having at least 90% homology to the amino acid sequences shown in SEQ ID NOs. 1, 2, and 3, and / or; (c) Recognizes the amino acid sequence shown in SEQ ID NOs. 4 and / or the amino acid sequence shown in SEQ ID NOs. 5. [2] The VHH according to item [1], which binds to the RBD (receptor-binding domain) of SARS-CoV2 and CDR1, CDR2, and CDR3 each contain an amino acid sequence having at least 90% homology to the amino acid sequences of SEQ ID NOs. 1, 2, and 3. [3] An antibody or antigen-binding fragment thereof that recognizes the amino acid sequence shown in SEQ ID NO: 4 and / or the amino acid sequence shown in SEQ ID NO: 5, or VHH. [4] VHH comprising an amino acid sequence having at least 90% homology to the amino acid sequence shown in SEQ ID NO: 6, and binding to SARS-CoV2 Wuhan-Hu-1, Delta strain, and Omicron strain.

[0009] [5] A nucleic acid containing the base sequence of Sequence ID No. 7. [6] A nucleic acid containing the base sequence encoding VHH as described in any of items [1] to [4]. [7] A vector containing the nucleic acid described in item [5] or [6]. [8] A host cell containing the nucleic acid described in item [5] or [6], or the vector described in item [7].

[0010] [9] A detection composition for SARS-CoV2 comprising VHH as described in any of items [1] to [4].

[10] The detection composition according to item [9], wherein VHH is labeled.

[11] The detection composition according to item

[10] or

[11] , wherein the label is selected from fluorescent labeling, enzyme labeling, biotin, magnetic beads, agarose beads, magnetic agarose beads, and gold colloid.

[0011]

[12] A method for detecting SARS-CoV2, comprising the steps of: contacting SARS-CoV2 contained in a sample solution with a detection composition described in any of items [9] to

[11] to bind the SARS-CoV2 to the detection composition; and detecting the presence of SARS-CoV2 by the presence of the detection composition.

[13] The detection method according to item

[12] , wherein the step of binding SARS-CoV2 to the detection composition is performed by contacting the sample solution with a binding molecule that binds to SARS-CoV2 fixed on a solid phase to bind the SARS-CoV2 in the sample solution to the binding molecule, and then further contacting the detection composition with the SARS-CoV2 bound to the binding molecule.

[14] The detection method according to item

[13] , wherein the binding molecule is (a) an antibody that binds to SARS-CoV2, (b) ACE2 or an ACE2 variant that binds to SARS-CoV2, or (c) ACE2-Fc obtained by linking ACE2 or an ACE2 variant that binds to SARS-CoV2 to the Fc region of the antibody.

[0012]

[15] A fusion protein comprising (a) (i) an ACE2 variant having binding affinity to ACE2 or SARS-CoV2, or (ii) ACE2-Fc obtained by linking an ACE2 variant having binding affinity to ACE2 or SARS-CoV2 to the Fc region of an antibody, and (b) a VHH as described in any of items [1] to [4].

[16] The fusion protein according to item

[15] , wherein ACE2 is a mutant ACE2 with higher binding affinity to the S protein of SARS-CoV-2 than the wild type.

[17] A composition for neutralizing SARS-CoV-2, comprising the fusion protein according to item

[15] or

[16] .

[18] The composition according to item

[17] , which is a pharmaceutical composition for the treatment or prevention of COVID-19.

[0013]

[19] A dimer of VHH as described in any of items [1] to [4].

[20] A homodimer of VHH as described in item

[19] .

[21] A heterodimer of VHH as described in item

[19] .

[22] A dimer of an anti-albumin antibody and VHH as described in any of items [1] to [4], as described in item

[19] .

[0014] This graph shows the evaluation of the binding affinity of VM777 to each SARS-CoV-2 S protein RBD mutant. This graph shows the results of competitive inhibition tests of VM777 against ACE2 binding to SARS-CoV-2 S protein RBD mutants. This graph shows the analysis of the binding site of VHH to SARS-CoV-2 RBD. This is a schematic diagram showing the analysis of the binding site of VHH to SARS-CoV-2 RBD. This graph shows the evaluation of the neutralizing activity of the VM777-ACE2 fusion protein using a pseudovirus. This is a schematic diagram of a virus detection test using a fluorescent protein and VM777 fusion, and a photograph of the results.

[0015] "SARS-CoV-2" refers to SARS coronavirus 2, or severe acute respiratory syndrome-associated coronavirus 2, and unless otherwise specified, includes Wuhan-Hu-1, the Wuhan strain first detected in Wuhan, and various variant strains. Wuhan-Hu-1 refers to the Wuhan strain (GenBank_MN908947) first detected in Wuhan. The Delta strain refers to the B. 1.617.2 lineage variant, and the Omicron strain also includes subtype lineages such as B. 1.1.529, BA5, BA2.75, and XBB. In this specification, these variant strains are used in a concept that also includes lower lineages and sublineages. For example, the Omicron strain is a concept that includes BA. 1 lineage, BA. 2 lineage, BA. 3 lineage, etc., which are lower lineages of its lower lineage B. 1.1.529 lineage, as well as several of its sublineages. In this specification, "SARS-CoV-2" is also referred to as the novel coronavirus or SARS coronavirus 2.

[0016] The SARS-CoV-2 spike protein (S protein) is a protein present on the outer shell of the viral particle. By attaching to a receptor called angiotensin-converting enzyme 2 (ACE2) on respiratory cells, it mediates the entry of the virus into host cells. Therefore, the spike protein can be a target for therapeutic purposes such as vaccine development, antiviral antibodies, and entry inhibitors, but the amino acids that make up the protein are prone to change due to viral mutations. The spike protein contains two subunits, S1 and S2, and the receptor-binding domain (RBD) contained in S1 is responsible for recognizing receptors on the cell surface.

[0017] The VHH of the present invention specifically binds to the RBD (receptor-binding domain) of the SARS-CoV-2 spike protein. "Specifically binding" means that VHH does not cause an antigen-antibody reaction with proteins other than the RBD of the spike protein, or if an antigen-antibody reaction occurs, it is not detected, or even if an antigen-antibody reaction with proteins other than the RBD of the spike protein is detected, it causes a reaction that is significantly weaker than that with other proteins.

[0018] VHH (Variable domain of heavy chain of heavy chain antibody) refers to the variable domain of heavy chain-only antibodies that consist only of heavy chains found in the blood of camelid animals, or antibodies derived from antibodies consisting only of heavy chains of camelid animals. The molecular weight of VHH is 15 kDa, which is about one-tenth that of IgG antibodies. VHH can be produced in E. coli and yeast and has excellent acid and heat resistance. Furthermore, because VHH does not contain light chains like ordinary antibodies and consists of only one polypeptide chain, it is easy to improve affinity and modify its function. From a formulation design perspective, in addition to injection, administration via the lung and nasal channels is being considered for VHH, and recently, VHH that reaches the colonic mucus and suppresses inflammation when administered orally has been reported. Humanized VHH is preferred as the VHH of this invention.

[0019] Compared to a typical antibody having six complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3 in the heavy and light chains respectively, VHH has three CDRs (CDR1, CDR2, and CDR3). The positions of the CDRs in the amino acid sequence can be determined based on numbering such as Kabat, Chothia, or IMGT. The amino acid sequences of CDR1-3 of the VHH of the present invention may include substitutions, insertions, or deletions, as long as they specifically bind to the RBD (receptor-binding domain) of the spike protein of SARS-CoV2 Wuhan-Hu-1, Delta, and Omicron strains. That is, the sequences of CDR1-3 of the VHH of the present invention include amino acid sequences that are at least 90% homologous to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. For example, as long as the same epitope is recognized as that of VHH containing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 as CDR1-3, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 may contain a total of 1 to 10 amino acid substitutions, insertions, and / or deletions. Conservative substitutions, which are substitutions between amino acids with the same properties, are preferred. Furthermore, the amino acid sequences of CDR1-3 of VHH may have approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. VHHs containing amino acid sequences as CDR1-3 that have at least 90% homology to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or VHHs containing amino acid sequences as CDR1-3 that include a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and that recognize the same epitopes as VHHs containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 as CDR1-3, can be screened by competitive testing or affinity maturation.

[0020] The amino acid sequence of VHH may include substitutions, insertions, or deletions, as long as it specifically binds to the receptor-binding region common to the spike proteins of SARS-CoV2 Wuhan-Hu-1, Delta strain, and Omicron strain. For example, SEQ ID NO: 6 may contain a total of 1 to 10 amino acid substitutions, insertions, and / or deletions. Substitutions, insertions, or deletions may be contained in the CDR, in the framework region, or in both the CDR and the framework region, as long as they recognize the same epitope as VHH containing the amino acid sequence shown in SEQ ID NO: 6. Conservative substitutions, which are substitutions between amino acids of the same properties, are preferred. Furthermore, the amino acid sequence of VHH may have approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6. Proteins having an amino acid sequence containing 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 6, and recognizing the same epitope as VHH containing the amino acid sequence shown in SEQ ID NO: 6, can be screened by competitive testing or affinity maturation.

[0021] In one aspect of the present invention, the present invention relates to a VHH or heavy chain antibody, a double-chain antibody consisting of a light chain and a heavy chain that recognizes a specific epitope, or an antigen-binding fragment thereof, specifically a VHH or heavy chain antibody, a double-chain antibody consisting of a light chain and a heavy chain that recognizes an amino acid sequence thought to be shared by SARS-CoV-2 Wuhan-Hu-1 and various mutant strains, or an antigen-binding fragment thereof. Such a VHH or heavy chain antibody, or a double-chain antibody consisting of a light chain and a heavy chain, can be used to create hybridomas from animals immunized with a sequence containing a specific epitope, for example, the amino acid sequence shown in SEQ ID NO: 4 and / or the amino acid sequence shown in SEQ ID NO: 5. To enhance immunogenicity, the amino acid sequence shown in SEQ ID NO: 4 and / or the amino acid sequence shown in SEQ ID NO: 5 can be used, conjugated with a carrier molecule as appropriate. From the obtained hybridomas, the desired VHH or heavy chain antibody, or a double-chain antibody consisting of a light chain and a heavy chain, can be selected by ELISA or the like.

[0022] Furthermore, the amino acid sequences shown in SEQ ID NO: 4 and / or SEQ ID NO: 5 are conserved sequences common to existing variants, and it is highly probable that various SARS-CoV2 variants that may arise in the future will also possess these sequences. Therefore, VHH or heavy chain antibodies, double-chain antibodies consisting of light and heavy chains, or their antigen-binding fragments that recognize the amino acid sequences shown in SEQ ID NO: 4 and / or SEQ ID NO: 5 are highly probable to also recognize the receptor-binding region common to the spike proteins of various SARS-CoV2 variants that may arise in the future.

[0023] Alternatively, various display methods such as phage display, yeast display, mRNA display, and cDNA display can be used to screen display libraries using panning operations or flow cytometry (FACS) to obtain VHH or antigen-binding fragments that recognize specific epitopes. For example, when screening using the cDNA display method, the PharmaLogical® library (manufactured by Epsilon Molecular Engineering Co., Ltd.), a humanized VHH antibody library, can be used.

[0024] The VHH of the present invention can be used as both a monomer and a dimer. The dimerized VHH may be a homodimer or a heterodimer combined with other antibodies, etc. As an example of a heterodimer, for example, it can be a dimer with an anti-albumin antibody to extend its half-life.

[0025] Since the VHH of the present invention can recognize Wuhan-Hu-1 and various mutant strains, it can be used as a detection composition for SARS-CoV2. If necessary, it is desirable to label the VHH with fluorescent labels, enzyme labels, biotin, magnetic beads / agarose beads / magnetic agarose beads, gold colloid, etc.

[0026] Methods for detecting SARS-CoV-2 can be those known in the art. For example, the method involves contacting a detection composition containing VHH of the present invention with a sample solution in which the presence of SARS-CoV-2 is suspected or for which confirmation of the presence or absence of SARS-CoV-2 is required. If SARS-CoV-2 is present in the sample solution, its presence can be detected by the binding of the detection composition of the present invention to SARS-CoV-2.

[0027] For example, a sample solution is brought into contact with a binding molecule that binds to SARS-CoV2, which is pre-immobilized on a solid phase. If SARS-CoV2 is present in the sample solution, SARS-CoV2 will bind to the binding molecule. Furthermore, by bringing the detection composition of the present invention into contact with the SARS-CoV2 bound to the binding molecule, the detection composition will bind to SARS-CoV2, and its presence can be detected. In this case, an antibody that binds to SARS-CoV2, or ACE2, or an ACE2 variant that binds to SARS-CoV2 can be used as the binding molecule.

[0028] Specifically, for example, ELISA, immunochromatography, immunofluorescence assay (IFA), and Western blotting can be used, and are not particularly limited.

[0029] Examples of sample solutions include biological specimens and experimental samples. In particular, nasopharyngeal swabs, nasal cavity specimens, pharyngeal swabs, saliva, and sputum can be used as biological specimens.

[0030] ACE2 is the infection receptor for SARS-CoV-2, and its use as a decoy to neutralize the virus is expected to have applications in infection control and treatment. Furthermore, decoys with enhanced binding affinity to the SARS-CoV-2 S protein have been developed by introducing mutations into wild-type ACE2. See, for example, WO2024 / 030962. When the Fc region of an antibody is conjugated to wild-type ACE2 or an ACE2 mutant to form ACE2-Fc, the pharmacokinetics in the body are stabilized, and dimerization improves neutralizing activity. Therefore, ACE2-Fc is considered desirable as a decoy for neutralizing SARS-CoV-2.

[0031] By using a fusion protein in which the VHH of the present invention is further fused to such an ACE2-Fc compound, the neutralizing ability of SARS-CoV-2 can be further enhanced. Although not bound by any particular theory, it is thought that the VHH of the present invention enhances the neutralizing ability of the decoy by binding to the RBD of the S protein of SARS-CoV-2 without interfering with the binding of SARS-CoV-2 to ACE2, and without allowing SARS-CoV-2 to escape.

[0032] Therefore, a fusion protein obtained by fusing ACE2-Fc, which is ACE2 or an ACE2 variant to which the Fc region of an antibody is conjugated, with the VHH described in the present invention can be used as a composition for neutralizing SARS-CoV-2.

[0033] This composition for neutralizing SARS-CoV-2 can be used as a pharmaceutical composition for the treatment or prevention of COVID-19 in the target population. The target population includes individuals infected with SARS-CoV-2, individuals suspected of being infected with SARS-CoV-2, individuals who have developed COVID-19, individuals suspected of developing COVID-19, and individuals at risk of developing COVID-19. Non-human animals that may be susceptible to SARS-CoV-2 infection may also be targeted.

[0034] "Treatment" refers to therapeutic measures that reduce the severity, slow the progression, or halt the progression of a target disease or disorder. In particular, in the present invention, "treatment" includes halting, slowing the progression, completely or partially alleviating symptoms, or reducing the severity of COVID-19. "Prevention" refers to preventive measures that avoid the onset or recurrence of a target disease or disorder. In particular, in the present invention, "prevention" means partially or completely avoiding the onset of COVID-19.

[0035] The pharmaceutical composition of the present invention includes an additive in addition to a fusion protein obtained by fusing ACE2-Fc, which is formed by linking ACE2 or an ACE2 variant to the Fc region of an antibody, with VHH. Examples of additives include stabilizers, preservatives, buffers, and surfactants. These additives are known in the art, and those skilled in the art can arbitrarily select appropriate additives.

[0036] The concentration of VHH in a pharmaceutical composition may vary depending on the subject's weight, symptoms, sex, and whether it is used for the treatment or prevention of COVID-19. It can be used in pharmaceutical compositions at concentrations of approximately 25 to 150 mg / mL, for example, at concentrations of approximately 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, or 150 mg / mL.

[0037] The present invention will be described below based on examples, but these examples are for illustrative purposes only and are not intended to limit the present invention.

[0038] (Example 1) Screening for anti-SARS-CoV-2 VHH 1-1. Target molecules for screening Biotinylated SARS-CoV-2 S protein, His, Avitag™, Super stable trimmer (ACROBiossystems), SARS-CoV-2 Spike RBD (L452R, T478K) Fc tag (ACROBiossystems), and Biotinylated SARS-CoV-2 Spike RBD, His, Avitag (B.1.1.529 / Omicron) (ACROBiossystems) were used as target molecules.

[0039] 1-2. Synthesis of Artificial Humanized VHH-Displaying cDNA Display 1-2-(1) Synthesis of mRNA-Linker Conjugates Using a humanized artificial VHH sublibrary (Pharmalogical® DNA library) (manufactured by Epsilon Molecular Engineering Co., Ltd.) as a template, a transcription reaction was carried out at 37°C for 30 minutes using a T7 RiboMAX Express Large Scale RNA Production System (Promega). After the transcription reaction, RQ1 RNAse-free DNase (manufactured by Promega) was added and the reaction was carried out at 37°C for 15 minutes, and the RNA was purified using RNAclean XP (Beckman Coulter). After measuring the RNA concentration using NanoPad DS-11 (Denovix), reagents were mixed to obtain a final concentration of 200 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 μM mRNA, and 1 μM cnvK linker. This mixture was heat-treated using the following program: (s1) 90°C, 1 minute, (s2) 70°C, 1 minute, (s3) 4°C. The mixture was then irradiated with 254 nm UV light using a UVP crosslinker cl-3000 until the total energy reached 406 mJ / cm2, thereby obtaining mRNA-linker conjugates.

[0040] 1-2-(2) Synthesis of cDNA display molecules After mixing the mRNA-linker conjugates of each sublibrary obtained by the above method, 1.6 mL of cell-free translation solution (Pureflex 1.0) (manufactured by GeneFrontier Co., Ltd.) was prepared from 600 μL of mRNA-linker conjugates and incubated at 37°C for 30 minutes. Then, 1 M MgCl2 and 3 M KCl were added to a final concentration of 75 mM and 900 mM, respectively, and incubated at 37°C for 1 hour. Then, 0.5 M EDTA (pH 8.0) was added to a final concentration of 70 mM, and incubated at 4°C for 5 minutes to obtain mRNA display solution (total volume 3.2 mL). The obtained mRNA display solution was purified using 1.92 mL of Dynabeads Streptavidin MyOne C1 (Thermo Scientific) according to the attached protocol. A reverse transcription reaction solution consisting of 320 μL of 5xRT Buffer (Nippon GENE), 64 μL of 25 mM dNTPs, 32 μL of Reverse Transcriptase (200 U / mL, Nippon GENE), and 1184 μL of ultrapure water was added to the mRNA display immobilized beads, and the mixture was reacted at 42°C for 1 hour. The above reaction product was eluted from the beads with 500 μL of elution buffer containing RNaseT1 (Thermo Fisher Scientific), and then the cDNA display was purified using 400 μL of His-Mag Sepharose (Cytiva), and eluted with 50 μL of His-Mag eluate to obtain an artificially humanized VHH-presenting cDNA display molecule (cDNA-linker-peptide conjugate).

[0041] 1-3. Screening for anti-SARS-CoV-2 VHH 1-3-(1) Selection for SARS-CoV-2 S protein 35 μL of 200 μg / mL Biotinylated SARS-CoV-2 S protein, His, Avitagtm, Super stable trimmer (hereinafter referred to as SAR-S-CoV-2 S trimmer) was diluted with 15 μL of PBS to prepare a 1 μM SARS-CoV2 S trimmer solution. To 50 μL of this solution, 47 μL of artificially humanized VHH-presenting cDNA display solution prepared from a 600 pmol mRNA-linker conjugate scale using the method described above, 0.5 μL of 10% Tween 20, and 2.5 μL of 5 mg / mL Heparin solution were sequentially added, and the mixture was incubated at 4°C for 30 minutes. Next, this cDNA display mixture was added to 120 μL of magnetic beads (DynabeadsMyone streptavidin C1) (manufactured by Veritas Corporation), mixed by inversion at 4°C for 30 minutes, the supernatant was removed, the magnetic beads were washed three times with PBS-T, and after elution, the cDNA display was recovered from the eluate using an Ampure XP (Beckman Coulter).

[0042] 1-3-(2) Preparation of an anti-SARS-CoV-2 S protein-binding DNA library The cDNA display recovered samples obtained above were subjected to PCR. Using cnvK NewYtag for polyA (SEQ ID NO: 8) and PL_T7pro (SEQ ID NO: 9), which have the following sequences, as primers, PCR was performed using PrimeSTAR Max (manufactured by Takara Bio Inc.) under conditions of annealing temperature of 60°C and extension reaction of 35 seconds. The obtained PCR products were purified using AMpure XP (manufactured by Beckman Coulter) according to the instructions for use of the product, and an anti-SARS-CoV-2 S protein-binding DNA library was prepared.

[0043] cnvK NewYtag for polyA (SEQ ID NO: 8) 5’―TTTCCACGCCGCCCCCCGTC CT―3’: PL_T7pro (SEQ ID NO: 9) 5’―GATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC―3’

[0044] 1-3-(3) Biotinylation of SARS-CoV-2 RBD delta variant (L452R, T478K) 11.8 μL of 1 mM biotinylation reagent (EZ-LinkTM Sulfo-NHS-PEG12-Biotin, manufactured by Thermo Fisher Scientific) was added to 100 μL of 5.9 μM SARS-CoV2 Spike RBD (L452R, T478K) Fc tag (manufactured by ACRO Biosystems) solution, and the reaction was carried out at room temperature for 1 hour. ZebaTM Spin Desalting Columns, 7K MWCO, 0.5 mL (manufactured by Thermo Fisher Scientific) was replaced with PBS according to the instructions, and the reaction solution was added and centrifuged at 1500 x g for 2 minutes to replace the buffer of the reaction solution with PBS to remove the unreacted biotinylation reagent, and Biotinylated SARS-CoV-2 RBD delta variant was obtained.

[0045] 1-3-(4) VHH screening of anti-SARS-CoV-2 RBD delta mutant strains Using the anti-SARS-CoV-2 S protein-binding DNA library obtained in 1-3-(2) above as a template, mRNA-linker conjugates were obtained in the same manner as in 1-2-(1) above. Then, cDNA display solutions were prepared from 20 μL of mRNA-linker conjugates in the same manner as in 1-2-(2) above. 47 μL of each cDNA display solution diluted with PBS was sequentially mixed with 50 μL of 500 nM Biotinylated SARS-CoV-2 RBD delta mutant strain solution, 0.5 μL of 10% Tween, and 2.5 μL of 5 mg / mL Heparin solution, and incubated at 4°C for 1 hour. Next, this cDNA display mixture was added to 40 μL of magnetic beads (DynabeadsMyone streptavidin C1) (Veritas), mixed by inversion at 4°C for 30 minutes, the supernatant was removed, the magnetic beads were washed three times with PBS-T, and after elution, the cDNA display was recovered from the eluate using AMpure XP (Beckman Coulter). The recovered sample was subjected to PCR in the same manner as in 1-3-(2) above, and this procedure was repeated three times.

[0046] 1-3-(5) Screening of anti-SARS-CoV-2 RBD B.1.1.529 / omicron variant VHH Using the PCR product of each variant obtained in 1-3-(4) as a template, a cDNA display solution was prepared in the same manner as in 1-3-(4). To 44.5 μL of each cDNA display solution diluted with PBS, 50 μL of a 200 nM Biotinylated SARS-CoV-2 Spike RBD, His, Avitag (B.1.1.529 / Omicron) solution, 0.5 μL of 10% Tween, and 5 μL of a 10 mg / mL BSA solution were sequentially added, and the mixture was incubated at 4°C for 1 hour. Next, 20 μL of this cDNA display mixture was added to 20 μL of magnetic beads (Dynabeads Myone streptavidin C1) (manufactured by Veritas), and the mixture was inverted and mixed at 4°C for 30 minutes. After removing the supernatant, the magnetic beads were washed 3 times with PBS-T. After elution, cDNA display was recovered from the eluate using Ampure XP (manufactured by Beckman Coulter). From the recovered cDNA display solution, after PCR amplification, this operation was carried out one more time to obtain an anti-SARS-CoV-2 RBD variant (delta & omicron) VHH antibody DNA library.

[0047] (Example 2) Identification of VM777 from an anti-SARS-CoV-2 RBD mutant (delta™ omicron) bound DNA library 2-1. Preparation of a VHH expression plasmid library The DNA library encoding VHH selected by the above screening was cloned into a plasmid vector for VHH expression. First, sequences for restriction enzyme treatment were added to the DNA library obtained by screening by PCR to obtain PCR products. The reaction solution consisted of 25 μL of PrimeSTAR MAX, 1 μL of the screening product, and 10 pmol each of PL_VHH_SfiI-NcoI_FW (SEQ ID NO: 10) and PL_VHH_BamHI-NotI_RV (SEQ ID NO: 11), which were then diluted to 50 μL with ultrapure water. The sequences of PL_VHH_SfiI-NcoI_FW and PL_VHH_BamHI-NotI_RV are as follows. The PCR program involved 25 cycles of PCR reaction with an annealing temperature of 55°C and an extension time of 15 seconds. The PCR product and the C. glutamicum expression plasmid vector were treated with the restriction enzyme BamHI at 37°C for 1 hour, and then with the restriction enzyme SfiI at 50°C for 1 hour. The reaction mixture derived from the PCR product, which would become the insert DNA, was purified using AMPureXP, and the reaction product derived from the plasmid vector was excised and purified by electrophoresis on a 1% agarose gel at 100V for 30 minutes. The purified plasmid vector DNA was dephosphorylated using the dephosphorylation enzyme Fast AP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) at 37°C for 1 hour. Subsequently, the Insert DNA and plasmid vector DNA were mixed to a molar ratio of 1:10, and a ligation reaction was performed overnight at 16°C using Ligation High (Toyobo) to obtain a plasmid library into which the selected VHH library had been introduced.

[0048] PL_VHH_SfiI-NcoI_FW (SEQ ID NO: 10) 5'-CCGGCCATGGCCACTGCGGCCGAAGTACAATTAGTTGAAT CTGGTGGTGGGCTTG-3' PL_VHH_BamHI-NotI_RV (SEQ ID NO: 11) 5'-AAAAGCGGCCGCGGATCCTGAAGAGACTGT CACCAACGTGCC-3'

[0049] 2-2. Obtaining culture supernatant containing selected VHH using Corynebacterium Each plasmid library was introduced into Corynebacterium glutamicum by electroporation to obtain transformants. The obtained transformants were inoculated into CM2G medium and cultured overnight at 30°C. Subsequently, the culture solution was subcultured into PM1S medium for VHH expression and cultured at 25°C for 72 hours to secrete and express VHH in the culture supernatant. The culture supernatant was collected by centrifugation at 3000xg, and bacterial cells were removed from the supernatant by filtering with a 0.22 μm filter.

[0050] 2-3. Single-point binding assay using biolayer interferometry. Using Octet RED384 (SARTORIUS), the VHH clones produced above were immobilized on a His1K sensor chip, and their binding activity to each target molecule was measured. SARS-CoV-2 Spike RBD (L452R, T478K) and Fc Tag (Acro Biosystems) were prepared at a concentration of 200 nM as ligands, and 70 μL of the measurement solution was added to a 384-well plate to measure the binding of each target molecule. Before measurement, the tip of the anti-penta-HIS (His1K) Biosensors (manufactured by SARTORIUS) was hydrated by immersing it in 200 μL of PBS-T (0.05% Tween 20, pH 7.4) for 10 minutes. The measurement sequence for each run was as follows: 1) Baseline step: Measurement in PBS-T for 30 seconds. 2) Loading step: Measurement in VHH diluted 50-fold with PBS-T for 60 seconds. 3) Baseline step: Measurement in PBS-T for 30 seconds. 4) Association step: Measurement in PBS-T for 100 seconds with each target molecule prepared by dilution with PBS-T. 5) Dissociation step: Measurement in PBS-T for 100 seconds. 6) Regeneration step: Measurement for 5 seconds in Glycine-HCl (pH 2.2), and measurement for 30 seconds in PBS-T. This process was repeated three times. The obtained data was processed using ForteBio Octet Date Analysis HT 11.1.2.48. Clones showing a binding response value of 0.1 nm or greater were designated as positive clones, and multiple positive clones were obtained.

[0051] 2-4. Analysis of the base sequence encoding the positive clones The base sequence of each VHH gene was identified for the positive clones selected in 2-3. Each positive clone (transformed organism) was cultured overnight at 37°C, and colony PCR was performed on the culture medium. 10 units of Exonuclease I (E. coli) (New England Biolabs) and 0.5 units of Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs) were added to the obtained PCR product, and the mixture was reacted at 37°C for 45 minutes, followed by inactivation at 80°C for 15 minutes. DNA sequencing analysis of each reaction solution was outsourced to Eurofins Genomics. As a result, we identified the nucleotide sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 6) encoding VM777, which strongly binds to RBD.

[0052] The base sequence encoding VM777 (SEQ ID NO: 7) is: gaagtacaat tagttgaatc tggtggtggg cttgtacagc caggtgggag tctgcgcctg agctgtgcag cgagtggttt cactttctct aactaccata tgtcttggtt tcgccaggca ccgggaaaag gccgtgagtt tgtggcgact atctcttggt ctggtgactc tacttactac gctgactcgg tcaaaggccg gtttaccatc agccgtgaca acgcgaagaa caccgtgtat ctccagatga attccctgcg tgctgaagat actgccgtgt actactgcgc tgcctacaaa cgtttcgcaa tccgtatcat ctacgattat tggggtcaag gcacgttggt gacagtctct tca

[0053] The amino acid sequence of VM777 (SEQ ID NO: 6) is: EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYHMSWFRQA PGKGREFVAT ISWSGDSTYY ADSVKGRFTI SRDNAKNTVY LQMNSLRAED TAVYYCAAYK RFAIRIIYDY WGQGTLVTVS S

[0054] In the amino acid sequence of VM777, 1-25 are FR1, 26-35 are CDR1, 36-49 are FR2, 50-66 are CDR2, 67-98 are FR3, 99-110 are CDR3, and 111-121 are FR4. The CDRs in the amino acid sequence of VM777 are shown below.

[0055] CDR1 amino acid sequence (SEQ ID NO: 1): GFTFSNYHMS CDR2 amino acid sequence (SEQ ID NO: 2): TISWSGDSTYYADSVKG CDR3 amino acid sequence (SEQ ID NO: 3): YKRFAIRIIYDY

[0056] The amino acid sequence enclosed in the square is the sequence of CDR1-3.

[0057] (Example 3) Production of VM777 The recombinant Corynebacterium glutamicum strain encoding VM777, prepared in 2-2 above, was inoculated into CM2G medium containing 25 μg / mL kanamycin and shaken overnight at 30°C to obtain the preculture medium. The preculture medium was inoculated at 5% into 700 μL of PM1S medium in a 96-deep-well plate and cultured with shaking at 25°C for 72 hours. After the culture was completed, the culture supernatant was centrifuged at 4,000 rpm, 4°C, and 30 minutes using a Thermo Fisher Scientific centrifuge, and the culture supernatant was collected in a tube. The collected culture supernatant was filtered using a PVDF membrane (Merck Millipore) and then purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries) according to the product's instructions for use. To elute VHH, 700 μL of His-tag elution buffer (50 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, pH 7.5) was added, and the mixture was centrifuged at 500 × g for 30 seconds using a TOMY centrifuge. The eluate was collected and used as the VHH antibody sample. SDS-PAGE was performed to confirm the purity of the purified VHH. After applying 4 μL of sample to each well, electrophoresis was performed at 150 V for 1 hour. Precision Plus Protein™ Standards (BIO-RAD) was used as the molecular weight marker. After electrophoresis, the gel was stained with Coomassie Brilliant Blue (CBB) to confirm that VHH had been synthesized in the culture supernatant.

[0058] (Example 4) Evaluation of binding affinity by biolayer interferometry Using BLI, Octet® RED384 (manufactured by SARTORIUS), VM777 Wuhan type [SinoBiological, SARS-CoV-2 (2019-nCoV) Spike RBD Recombinant Protein®], Delta type [Acrobioticsem, Biotinylated SARS-CoV-2 Spike RBD (L452R, T478K), His, Avitag® (MALS verified)], B. Type 1.1.529 [Biotinylated SARS-CoV-2 SpikeRBD, His, Avitag(TM) B. 1.1.529 / Omicron) (MALS verified)], BA. Type 4 / 5 [Acrobiosystem, Biotinylated SARS-CoV-2 Spike RBD, His, Avitag (trademark) (BA.4&BA.5 / Omicron) (MALS verified)], BA. The binding affinity of type 2.75 [Acrobiotic system, Biotinylated SARS-CoV-2 Spike RBD, His, Avitag (trademark) (BA.2.75 / Omicron) (MALS verified)] to RBD was evaluated. A 96 Black Well plate was used for sensor hydration, and a 384 Well Tiltted Bottom plate (manufactured by SARTORIUS) was used for sample measurement. For the Wuhan type, a Ni-NTA biosensor was used with VM777 as the ligand and RBD as the analyte. For other mutant strains, an SA biosensor was used with each RBD mutant strain as the ligand and VM777 as the analyte for evaluation. 200 μL of PBS-T (0.05% Tween 20, pH 7.4) was placed in a 96 plate, and a Ni-NTA biosensor or SA biosensor was immersed for 10 minutes, after which it was loaded with ligand solution (10 ug / mL) for 240 seconds. After equilibration with PBS-T for 60 seconds, the samples were incubated for 120 seconds in analyte solutions prepared at various concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM).The dissociation constant was measured by immersing the sensor chip in a well containing PBS-T for 240 seconds. The obtained data was processed using ForteBio Octet Date Analysis HT 11.1.2.48, and Kon and Koff were calculated from the global fitting parameters of the 1:1 binding model to determine KD (Table 1). As a result, VM777 showed very good binding activity to all Wuhan types and each mutant strain, revealing that it has broad binding activity (Figure 1).

[0059]

[0060] (Example 5) Measurement of competitive inhibitory activity of SARS-CoV-2 Spike RBD mutant strains (L452R, E484Q, B.1.617.1 strain) against ACE2 binding. For VM777, competitive inhibitory activity of SARS-CoV-2 Spike RBD mutant strains (L452R, E484Q mutant strain) against human ACE2 binding was evaluated using Octet 384. VM777, prepared to 20 μg / mL in Kinetics Buffer (0.05% Tween20-containing PBS, pH 7.4), was immobilized on an Anti-Penta-HIS (HIS1K, Fortebio) sensor tip by incubation for 120 seconds. The tip was then immersed in a solution of SARS-CoV-2 Spike RBD recombinant protein (20 μg / mL, SPD-C525e, AcroBiossystems) as the first analyte, and binding was confirmed after 180 seconds. After that, the tip was immersed in a buffer solution for 240 seconds. Following a 30-second baseline step, the sensor tip was immersed in a well of ACE2-Fc prepared to 20 μg / mL, and the presence or absence of binding was confirmed after 180 seconds.

[0061] After confirming the coupling response between VM777, which was fixed to the sensor chip, and the first analyte RBD, we coupled it with the second analyte ACE2-Fc. VM777 did not block the coupling between ACE2 and RBD at all (Figure 2). From this, it was inferred that VM777 recognizes an epitope different from ACE2 and exhibits non-competitiveness with ACE2.

[0062] (Example 6) Analysis of the binding site of VHH to SARS-CoV-2 RBD To determine the binding site in the VM777 SARS-CoV-2 RBD mutant (Omicron strain), hydrogen / deuterium exchange mass spectrometry (HDX-MS) was performed using an automated HDX2 system (LEAP Technologies). The antigen used was SARS-CoV-2 Spike RBD (B.1.1.529 / Omicron), His Tag (MALS verified) (Acro Biosystems). The amino acid sequence of this antigen is Sequence ID No. 14, which is the amino acid sequence shown in Sequence ID No. 12 (RBD of the spike protein of the Omicron strain) with a linker-mediated His tag (Sequence ID No. 13) attached to the C-terminus. The automated HDX2 system consisted of an ultra-high-performance liquid chromatography (Water, nano Accuracy UPLC), a mass spectrometer (Waters, Synapt XS), and a LEAP PAL (Trajan Automation, NC). The final protein concentration of each evaluation sample was set to 25 μM. Reference and deuterium samples were prepared by diluting 3 μL of each evaluation sample with 57 μL of light water buffer (10 mM potassium phosphate, pH 7.0) and heavy water buffer (10 mM potassium phosphate, pD 7.0), respectively. Deuterium exchange temperatures and times were set at 20°C using five different time points: 0.5 minutes, 1 minute, 10 minutes, 60 minutes, and 240 minutes. After dilution, the protein sample was quenched with 50 μL of ice-cold quench buffer (100 mM potassium phosphate, 4 M guanidine hydrochloride, 200 mM TCEP, pH 2.3, 0°C). 90 μL of the quenched sample was injected into an HDX module, and protein digestion was performed on an online pepsin column (Waters Enzymate BEH pepsin) at a flow rate of 70 μL / min for 3 minutes. The peptide fragments were then trapped on a trap column (Waters, Acquity BEH C18 Vanguard 1.7 μm).Subsequently, the peptide was purified using an analytical column (Waters, Acquity BEH C18: 1.7 μm) under a 17-minute gradient condition (5–95% as acetonitrile) with 0.1% formic acid-containing ultrapure water / acetonitrile. After electrospraying onto Waters Synapt XS, the mass of the peptide fragment was detected by setting the mass / charge (m / z) acquisition window to 50–2000. Lock mass correction was performed by intermittent injection of 500 f / mol GluFib (785.8426 m / z, Sigma-Aldrich). The peptide was identified from MSE analysis of the SARS-CoV-2 RBD (B.1.1.529 / Omicron) strain, and the data was analyzed using ProteinLynx Global Server software (Waters). Subsequently, the identified peptides were used with DynamX 3.0 software (Waters). Deuterium uptake of each peptide was calculated by comparing the centroids of the mass envelopes of the deuterated and undeuterated samples. HDX data were analyzed by calculating and summing the difference in deuterium uptake of the same peptide between the free and complex states of RBD and VHH at all HDX time points. Since the same peptide was compared in both protein states, the inverse exchange correction factor was not applied to the HDX data analysis.

[0063] SARS-CoV-2 Spike RBD (SEQ ID NO: 12) RVQPTESIVR FPNITNLCPF DEVFNATRFA SVYAWNRKRI SNCVADYSVL YNLAPFFTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS NKLDSKVSGN YNYLYRLFRK SNLKPFERDI STEIYQAGNK PCNGVAGFNC YFPLRSYSFR PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTNLVKNK

[0064] His tag array (sequence number 13) GGGSGGGSHH HHHHHHHH

[0065] The amino acid sequence of the antigen (SEQ ID NO: 14) is: RVQPTESIVR FPNITNLCPF DEVFNATRFA SVYAWNRKRI SNCVADYSVL YNLAPFFTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS NKLDSKVSGN YNYLYRLFRK SNLKPFERDI STEIYQAGNK PCNGVAGFNC YFPLRSYSFR PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTNLVKNKG GGSGGGSHHH HHHHHHH

[0066] As a result, deuterium uptake in VM777 and the SARS-CoV-2 RBD mutant was observed for 171 peptides commonly observed in both the free and antigen-antibody bound states. Of these, the degree of deuterium exchange was calculated for 83 peptides, covering 92% of the RBD peptide sequence. A significant decrease in deuterium uptake due to VM777 binding was observed in the amino acid region from position 389 to 395 and the region from position 197 to 515 to 522 of the SARS-CoV-2 S protein, based on a modified numbering system that takes into account 69 / 70 deletions and 144 deletions (Figure 3). These sequences correspond to the amino acid region from aspartic acid at position 71 to valine at position 77 and the region from phenylalanine at position 197 to alanine at position 204 of the amino acid sequence shown in Sequence ID No. 12. Therefore, it was found that the corresponding region in RBD is the amino acid sequence containing the VM777 binding site. In other words, VM777 was found to exhibit a different binding site to RBD than known SARS-CoV-2 neutralizing antibodies (Figure 4). Specifically, it was shown that VM777 has the amino acid sequence of SEQ ID NO: 4 (positions 389-395 after numbering correction considering deletions of 69 / 70 and 144) and the amino acid sequence of SEQ ID NO: 5 (positions 515-522 after numbering correction considering deletions of 69 / 70 and 144) as its epitopes.

[0067] Epitope sequences of VM777: DLCFTNV (SEQ ID NO: 4), FELLHAPA (SEQ ID NO: 5)

[0068] (Example 7) Preparation of ACE2-Fc-VM777 Fusion Body 7-1. Construction of Expression Plasmid The ACE2-Fc-VM777 gene (SEQ ID NO: 15) was artificially synthesized by Eurofins Genomics and used to create an expression plasmid. FastDigest NheI, FastDigest ApaI, and FastDigest MunI (all from Thermo Fisher Scientific) were added to the synthesized gene, and FastDigest NheI and FastDigest ApaI were added to the expression plasmid, and the reaction was carried out at 37°C for 30 minutes. Next, the reaction mixture was placed on a 1% agarose gel containing 1x Gel Green (from Fujifilm Pure Chemical Industries, Ltd.), and electrophoresis was performed at 100V for 30 minutes. Subsequently, the FastGene Gel / PCR Extraction Kit (manufactured by Genetics Japan) was used to extract and purify the sample from the gel according to the attached instructions.

[0069] The gene of ACE2-Fc-VM777 (SEQ ID NO: 15) caatccacta tcgaggaaca ggcaaagacc ttcctggata agttcaatca cgaggcagag gatcttttct atcagagtag tcttgcgtct tggaactaca acaccaacat tactgaggag aatgtccaga atatgaacaa cgccggagac aagtggtctg ccttcctgaa agagcagtca accctggcac agatgtatcc ccttcaagag attcagaatc tgacagtaaa gctgcaactg caagccctgc aacagaatgg gtcatccgtg ctgtccgagg ataagagcaa gaggctcaac accatactga acaccatgag caccatctac tccacaggga aagtatgcaa tcccgacaat ccccaggaat gtctgttgtt ggaacctggc ttgaacgaaa ttatggccaa cagcctggac tacaatgaac gtctgtgggc ttgggagagt tggcggtcag aagttgggaa acagcttcgc cctctttacg aggaatacgt ggtgctcaag aacgagatgg cacgggcgaa tcactatgag gactacggcg actattggag gggcgattat gaggtgaacg gcgtagacgg gtacgactac agcaggggtc agcttatcga ggacgtcgaa cacaccttcg aagagatcaa acccttgtat gaacacctcc atgcctacgt gagagccaaa ctgatgaacg cttacccaag ctacattagc ccgattgggt gtctcccagc acatctgttg ggcgatatgt ggggccggtt ttggactaat ctgtatagcc tgactgttcc ttttggccag aaaccgaata tcgatgtgac ggatgctatg gttgaccagg catgggatgc tcagcgcatattcaaagagg ctgagaagtt ctttgtgtcc gtcggactgc caaatatgac gcagggattt tgggaaaact ctatgctcac cgatccagga aatgtgcaga aggccgtttg tcacccaacc gcttgggatc tggggaagg cgatttccga attctgatgt gcacgaaggt tacaatggac gacttcctga ctgcccatca tgagatgggt cacatccagt acgacatggc ctatgccgca caacccttct tgctgaggaa tggtgcgaat gagggatttc acgaagccgt gggtgagatc atgtctctta gtgcagccac acctaagcat cttaagtcca tcggtctgtt gtctccagac ttccaggaag ataacgagac tgaaatcaac tttctgctca aacaggctct gacgattgtg ggcacactcc cttttaccta tatgctggag aaatggcgct ggatggtctt taagggagaa atccccaaag accagtggat gaagaaatgg tgggaaatga agcgagagat agtgggagtg gtcgaacctg ttccccatga tgagacatat tgcgatccag cctcactctt ccacgtgtca aacgactaca gctttatacg gtactacaca agaaccctgt atcagttcca gttccaagag gccctgtgtc aagctgccaa acacgaggga ccactgcata agtgcgacat ttccaactct acagaggccg ggcagaagct gtttaacatg ctccgtctgg ggaaaagcga accctggaca ctggcactgg aaaacgtggt aggcgcgaag aatatgaacg tcagaccgct gctcaactac tttgaacctc tgtttacttg gttgaaagac cagataga atagttttgtcggctggtcc actgactgga gcccttatgc tgatgagccc aagagcagcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa ggcggtggag gttccggggg tggcggttct gaagtacaat tagttgaatc tggtggtggg cttgtacagc caggtgggag tctgcgcctg agctgtgcag cgagtggttt cactttctct aactaccata tgtcttggtt tcgccaggca ccgggaaaaggccgtgagtt tgtggcgact atctcttggt ctggtgactc tacttactac gctgactcgg tcaaaggccg gtttaccatc agccgtgaca acgcgaagaa caccgtgtat ctccagatga attccctgcg tgctgaagat actgccgtgt actactgcgc tgcctacaaa cgtttcgcaa tccgtatcat ctacgattat tggggtcaag gcacgttggt gacagtctct tca

[0070] The ACE2-Fc gene (SEQ ID NO: 16) was obtained by PCR using the synthesized gene as a template. Using pNhe-ACE2-Fw (5'-GCTGGCTAGCGCCCACCATGTGGGTGG-3': SEQ ID NO: 17) and pACE2-Apa-Rv (5'-AAACGGGCCCTCCATTTTAACCCGGAGAACAGGGAGAGGC-3': SEQ ID NO: 18) as primers, and PrimeSTAR Max DNA Polymerase (Takara Bio Inc.), PCR was performed under the following conditions: denaturation temperature of 98°C for 10 seconds, annealing temperature of 55°C for 5 seconds, and extension temperature of 72°C for 30 seconds to obtain the PCR product. The obtained PCR product was purified using the Gel / PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.) according to the instructions provided. FastDigest NheI and FastDigest ApaI were added to the purified PCR product and reacted at 37°C for 30 minutes. Next, the product was purified using the Gel / PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.) according to the instructions provided.

[0071] The gene of ACE2-Fc (SEQ ID NO: 16) caatccacta tcgaggaaca ggcaaagacc ttcctggata agttcaatca cgaggcagag gatcttttct atcagagtag tcttgcgtct tggaactaca acaccaacat tactgaggag aatgtccaga atatgaacaa cgccggagac aagtggtctg ccttcctgaa agagcagtca accctggcac agatgtatcc ccttcaagag attcagaatc tgacagtaaa gctgcaactg caagccctgc aacagaatgg gtcatccgtg ctgtccgagg ataagagcaa gaggctcaac accatactga acaccatgag caccatctac tccacaggga aagtatgcaa tcccgacaat ccccaggaat gtctgttgtt ggaacctggc ttgaacgaaa ttatggccaa cagcctggac tacaatgaac gtctgtgggc ttgggagagt tggcggtcag aagttgggaa acagcttcgc cctctttacg aggaatacgt ggtgctcaag aacgagatgg cacgggcgaa tcactatgag gactacggcg actattggag gggcgattat gaggtgaacg gcgtagacgg gtacgactac agcaggggtc agcttatcga ggacgtcgaa cacaccttcg aagagatcaa acccttgtat gaacacctcc atgcctacgt gagagccaaa ctgatgaacg cttacccaag ctacattagc ccgattgggt gtctcccagc acatctgttg ggcgatatgt ggggccggtt ttggactaat ctgtatagcc tgactgttcc ttttggccag aaaccgaata tcgatgtgac ggatgctatg gttgaccagg catgggatgc tcagcgcatattcaaagagg ctgagaagtt ctttgtgtcc gtcggactgc caaatatgac gcagggattt tgggaaaact ctatgctcac cgatccagga aatgtgcaga aggccgtttg tcacccaacc gcttgggatc tggggaagg cgatttccga attctgatgt gcacgaaggt tacaatggac gacttcctga ctgcccatca tgagatgggt cacatccagt acgacatggc ctatgccgca caacccttct tgctgaggaa tggtgcgaat gagggatttc acgaagccgt gggtgagatc atgtctctta gtgcagccac acctaagcat cttaagtcca tcggtctgtt gtctccagac ttccaggaag ataacgagac tgaaatcaac tttctgctca aacaggctct gacgattgtg ggcacactcc cttttaccta tatgctggag aaatggcgct ggatggtctt taagggagaa atccccaaag accagtggat gaagaaatgg tgggaaatga agcgagagat agtgggagtg gtcgaacctg ttccccatga tgagacatat tgcgatccag cctcactctt ccacgtgtca aacgactaca gctttatacg gtactacaca agaaccctgt atcagttcca gttccaagag gccctgtgtc aagctgccaa acacgaggga ccactgcata agtgcgacat ttccaactct acagaggccg ggcagaagct gtttaacatg ctccgtctgg ggaaaagcga accctggaca ctggcactgg aaaacgtggt aggcgcgaag aatatgaacg tcagaccgct gctcaactac tttgaacctc tgtttacttg gttgaaagac cagataga atagttttgtcggctggtcc actgactgga gcccttatgc tgatgagccc aagagcagcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa

[0072] pNhe-ACE2-Fw (SEQ ID NO: 17) 5'-GCTGGCTAGCGCCACCATGTGGTGG-3' pACE2-Apa-Rv (SEQ ID NO: 18) 5'-AAACGGGCCCTCATTTACCCGGAGACAGGGAGAGGC-3'

[0073] Next, the expression plasmid and each gene were mixed in a 1:3 (molar ratio), Ligation High Ver. 2 (manufactured by TOYOBO Corporation) was added, and the mixture was reacted at 16°C for 30 minutes to construct the plasmid. Escherichia coli JM109 (manufactured by Takara Bio Corporation) was added to the obtained plasmid, seeded onto an agar plate, and incubated overnight at 37°C. Colonies that appeared on the agar plate were picked and incubated overnight at 37°C. Using FastGene Plasmid Mini Kit (manufactured by Nippon Genetics Co., Ltd.), ACE2-Fc-VM777 and ACE2-Fc expression plasmids were extracted and purified according to the attached instructions. The DNA sequencing analysis of the extracted plasmids was commissioned to Eurofins Genomics, and the DNA sequences were analyzed. Next, to obtain a transfection plasmid, Escherichia coli JM109 was transformed with ACE2-Fc-VM777 and an ACE2-Fc expression plasmid. The resulting transformants were purified using the ZymoPURE II Plasmid Midiprep kit (manufactured by Zymo Research) according to the instructions provided.

[0074] 7-2. Expression of ACE2-Fc-VM777 and ACE2-Fc using Expi293F cells. Expi293F cells (Thermo Fisher Scientific) were subcultured in Expi293 Expression Medium (Thermo Fisher Scientific). Expi293F cells were seeded in 250 mL flasks (VIOLAMO) and cultured at 37°C and 8% CO2. ２ Cells were cultured under environmental conditions. The Fc-expressing plasmid, ExpiFectamine® 293 (Thermo Fisher Scientific), was suspended in Opti-MEME (Gibco) and allowed to stand at room temperature for 15 minutes. Subsequently, it was added to Expi293F cells that had been cultured overnight, and after 96 hours of culture, the culture supernatant was collected. The collected culture supernatant was filtered through a 0.22 μm filter to remove cells from the supernatant.

[0075] 7-3. Purification of ACE2-Fc-VM777 and ACE2-Fc ACE2-Fc-VM777 and ACE2-Fc were purified at 4°C using a chromatography system, AKTA pure 25 (Cytiva). The culture supernatant was diluted with an equal volume of 50 mM Tris-HCl pH 8.0, 10 mM NaCl (hereinafter sometimes abbreviated as "ion exchange buffer") and subjected to an anion exchange column, HiTrap™ Q HP (Cytiva), equilibrated with the ion exchange buffer. ACE2-Fc-VM777 and ACE2-Fc were eluted using a gradient with a buffer containing 1 M NaCl added to the ion exchange buffer at a flow rate of 1.0 mL / min. ACE2-Fc-VM777 and ACE2-Fc were collected in a 96-deep-well plate, with the increase in absorbance at 280 nm used as the fraction boundary. The obtained ACE2-Fc-VM777 and ACE2-Fc were then subjected to gel filtration using a Superdex™ 200 Increase 10 / 300 GL (Cytiva) column equilibrated with 50 mM Tris-HCl pH 8.0 and 100 mM NaCl, and eluted at a flow rate of 0.75 mL / min. ACE2-Fc-VM777 and ACE2-Fc were collected in a 96-deep-well plate, with the increase in absorbance at 280 nm used as the fraction boundary. The eluted ACE2-Fc-VM777 and ACE2-Fc were transferred to Amicon Ultra, 10 kDa (Millipore), and centrifuged at 3,500 x g for 30 minutes at 4°C to concentrate ACE2-Fc-VM777 and ACE2-Fc. The purity of the purified ACE2-Fc-VM777 and ACE2-Fc was confirmed by SDS-PAGE. SDS-PAGE was performed using a 4% concentrated, 10% separated gel, and Protein MultiColor Stable II (Biodynamics Laboratory) was used as the molecular weight marker. Using bovine serum albumin as the standard protein, the concentrations of ACE2-Fc-VM777 and ACE2-Fc were quantified by the BCA method using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0076] (Example 8) Evaluation of neutralizing activity using pseudovirus 8-1. Cell culture hACE2-hTMPRSS2-293T cells (manufactured by Vectorbuilder Inc.) were subcultured in D-MEM (High Glucose) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter referred to as "DMEM") containing 10% Fatal Bovine Serum (manufactured by Nichirei Corporation, hereinafter referred to as "FBS(+)") and 1% Penicillin-Streptomycin Mixed Solution (containing 100 units / mL penicillin G and 100 μg / mL streptomycin sulfate) (manufactured by Nacalai Tesque, hereinafter referred to as "PS(+)").

[0077] 8-2. Neutralization Activity Measurement 100 μL of hACE2-hTMPRSS2-293T cells suspended in DMEM (FBS(+), PS(+)) were placed in each 96-well white cell culture plate (Thermo Fisher Scientific) to obtain a final concentration of 2 × 10⁻⁶. ４ Sow seeds so that the cell count is 1 / 2, at 37°C and 5% CO2. ２ Cells were cultured overnight in an environmental environment. The culture medium was removed from each well, and 45 μL of each test substance (prepared in DMEM containing 2% FBS) and 45 μL of SARS-CoV2 S-pseudotyped lentivirus for Delta solution (Vectorbuilder Inc.) (5 μg / mL polyblen, prepared in DMEM containing 2% FBS) were added to each well. The cells were then cultured for a further 48 hours. After 48 hours, the cells were removed, left at room temperature for a few minutes, and then the luciferase emission intensity of the virus-introduced cells was measured using a plate reader (infinite 200Pro, TECAN Inc.) according to the product standard protocol using Bright-Glo® Luciferase Assay (Promega Inc.).

[0078] 8-3. Data Analysis: After the measurement, the data was subjected to four-parameter logistic curve analysis using GraphPad Prism 9, and the IC50 was calculated from the resulting sigmoid curve (Figure 5).

[0079] (Example 9) 9-1. Expression of VM777-kikG using E. coli BL21(DE3) Gold A gene (SEQ ID NO: 19) in which a GS linker and kikG were added to the C-terminus of VM777 was used as a plasmid to transform E. coli BL21(DE3) Gold. The resulting colonies were cultured at 37°C, and when the absorbance at 600 nm reached 0.4-0.6, 1 mM Isopropyl β-D-thiogalactopyranoside was added, and the colonies were cultured overnight at 25°C. The E. coli were recovered by centrifugation and stored at -30°C.

[0080] The gene of VM777-kikG (SEQ ID NO: 19) gaagtacaat tagttgaatc tggtggtggg cttgtacagc caggtgggag tctgcgcctg agctgtgcag cgagtggttt cactttctct aactaccata tgtcttggtt tcgccaggca ccgggaaaag gccgtgagtt tgtggcgact atctcttggt ctggtgactc tacttactac gctgactcgg tcaaaggccg gtttaccatc agccgtgaca acgcgaagaa caccgtgtat ctccagatga attccctgcg tgctgaagat actgccgtgt actactgcgc tgcctacaaa cgtttcgcaa tccgtatcat ctacgattat tggggtcaag gcacgttggt gacagtctct tcaggtggag gtggatccgg aggaggcggt agtggcggcg gtggctcttc cgttatcacc agcgaaatga agatggagct gcgtatggag ggcgccgtga acggtcacaa attcgtgatt acgggtaaag gctccggcca accgtttgaa ggtatccaga atatggatct gaccgtcatc gaaggtggcc cactgccgtt cgccttcgac attctgacca cggtttttga ctacggcaac cgtgtttttg tgaaatatcc ggaggaaatc gtggactact tcaaacagag cttcccggag ggctatagct gggaacgttc catgtcctat gaggatggtg ggatctgcct cgcgaccaac aacatcacca tgaagaagga cggctctaat tgtttcgtct acgagatccg cttcgacggc gtcaacttcc cggcaaatgg tccggttatg cagcgtaaaa cggtgaagtg ggagccgagc accgaaaaaa tgtatgtgcg cgacggtgttctgaagggcg atgttaacat ggcgcttctg ttgcaaggtg gcggccacta tcgttgcgat tttcgcacta cgtacaaagc gaagaaggtg gttcagctgc ctgattacca ttttgtggac catcgtattg agattaccag ccatgacaaa gactacaata aagttaagct gtatgagcac gctaaggcgc acagcggtct gccgcgtttg gcgaag

[0081] 9-2. Purification of VM777-kikG E. coli was suspended in 50 mM Tris-HCl pH 7.5 and 300 mM NaCl containing the complete™ EDTA-free protease inhibitor cocktail (Roche), and the cells were disrupted by sonication. The cell extract was transferred to a centrifuge tube and centrifuged at 27,000 x g for 30 minutes at 4°C to collect the soluble fraction. Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the collected soluble fraction and mixed at 4°C for 1 hour. The mixed solution was packed into the column, and the support was washed with 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 50 mM Imidazole. Then, VM777-kikG was eluted with 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 500 mM Imidazole. The eluted VM777-kikG was transferred to Amicon Ultra, 10 kDa (Millipore), and concentrated by centrifugation at 14,000 x g for 30 minutes at 4°C. Next, Zeba Spin Desalting Columns, 7K MWCO (Thermo Fisher Scientific) were replaced with PBS according to the instructions, and concentrated EN777-kikG was added and replaced with PBS. The purity of the purified VM777-kikG was confirmed by SDS-PAGE. The concentration of VM777-kikG was quantified using the BCA method with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using bovine serum albumin as the standard protein.

[0082] 9-3. Virus detection test using VM777-kikG ACE2-Fc protein was prepared in PBS to a concentration of 2 μg / mL and added to each well of a 96-well immunoplate (Thermo Fisher Scientific). The plate was left standing overnight at 4°C. The solution was removed from each well and washed three times with a PBS solution containing 0.05% Tween-20 (PBS-T). A blocking solution (2% FBS-containing DMEM) was then added. After standing at room temperature for 1 hour, the solution was removed from each well and washed twice with PBS-T solution. A solution of SARS-CoV2 S-pseudotyped lentivirus for Delta (Vectorbuilder, Inc.) (prepared in 5 μg / mL polyblen, 2% FBS-containing DMEM), which had been pre-suspended with VM777-kikG, was added to each well and allowed to stand at room temperature for 1 hour. This lentivirus is a pseudotyped lentivirus that has the coronavirus S protein as its outer sheath. After removing the solution from each well, the wells were washed three times with PBS, and each well was filled with PBS solution and observed using a fluorescence microscope (Olympus, Inc.). As a result, fluorescence originating from the immobilized ACE2-Fc / virus / VM777-kikG complex was observed in the wells to which the VM777-kikG and virus suspension solution had been added (Figure 6).

Claims

1. A VHH that specifically binds to the RBD (receptor-binding domain) of the SARS-CoV-2 spike protein and has at least one of the following characteristics: (a) it binds to the Wuhan-Hu-1, delta, and omicron strains of SARS-CoV-2; (b) CDR1, CDR2, and CDR3 each contain an amino acid sequence having at least 90% homology to the amino acid sequences shown in SEQ ID NOs. 1, 2, and 3, and / or; (c) it recognizes the amino acid sequence shown in SEQ ID NO: 4 and / or the amino acid sequence shown in SEQ ID NO:

5.

2. A VHH that binds to the RBD (receptor-binding domain) of SARS-CoV2, wherein CDR1, CDR2, and CDR3 each contain amino acid sequences having at least 90% homology to the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

3. An antibody or its antigen-binding fragment that recognizes the amino acid sequence shown in SEQ ID NO: 4 and / or the amino acid sequence shown in SEQ ID NO: 5, or VHH.

4. A composition for detecting SARS-CoV2, comprising the VHH described in claim 1.

5. A method for detecting SARS-CoV-2, comprising the steps of: contacting SARS-CoV-2 contained in a sample solution with the detection composition described in claim 4 to bind the SARS-CoV-2 to the detection composition; and detecting the presence of SARS-CoV-2 by the presence of the detection composition.

6. The detection method according to claim 5, wherein the step of binding SARS-CoV2 to the detection composition is performed by contacting the sample solution with a binding molecule that binds to SARS-CoV2, which is fixed to a solid phase, to bind the SARS-CoV2 in the sample solution to the binding molecule, and then further contacting the detection composition with the SARS-CoV2 bound to the binding molecule.

7. The detection method according to claim 6, wherein the binding molecule is (a) an antibody that binds to SARS-CoV2, (b) ACE2 or an ACE2 variant that binds to SARS-CoV2, or (c) ACE2-Fc obtained by linking ACE2 or an ACE2 variant that binds to SARS-CoV2 to the Fc region of the antibody.

8. A fusion protein comprising (a) (i) an ACE2 variant having binding affinity to ACE2 or SARS-CoV2, or (ii) ACE2-Fc obtained by linking ACE2 to the Fc region of an antibody, or an ACE2 variant-Fc obtained by linking an ACE2 variant having binding affinity to SARS-CoV2 to the Fc region of an antibody, and (b) the VHH described in claim 1.

9. A composition for neutralizing SARS-CoV-2, comprising the fusion protein described in claim 8.

10. The composition according to claim 9, which is a pharmaceutical composition for the treatment or prevention of COVID-19.

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