Fusion protein of virus-inhibiting molecule and streptavidin variant

Abstract

To provide a fusion protein of a virus-inhibiting molecule and a streptavidin variant for use in the treatment of a viral disease.SOLUTION: A fusion protein is provided in which a virus-inhibiting molecule with a molecular weight of 20,000 or less is linked to the N-terminal side and / or the C-terminal side of an amino acid sequence represented by SEQ ID NO:1 (where a part or the whole of an amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminal may be deleted) via a linker sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a fusion protein of a virus inhibitory molecule and a streptavidin mutant, and uses thereof. [Background technology]

[0002] The affinity between avidin and biotin or between streptavidin and biotin is very high (Kd=10 -15 From 10 -14 M), which is one of the strongest interactions between two biological molecules. Currently, the avidin / streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, and medicine. Drug delivery methods and pretargeting methods have been devised that combine the high binding ability of avidin / streptavidin and biotin with antibody molecules. In relation to these studies, Patent Document 1 reports a streptavidin mutant with reduced affinity for natural biotin, as well as a biotin-modified dimer that has high affinity for this streptavidin mutant with low affinity for natural biotin. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication WO2015 / 125820 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a fusion protein of a virus inhibitor molecule and a streptavidin mutant for use in the treatment of viral diseases, and further aims to provide an antiviral agent comprising the above-mentioned fusion protein. [Means for solving the problem]

[0005] As a result of intensive research to solve the above problems, the present inventors selected a molecule with a molecular weight smaller than that of an antibody as a virus-inhibiting molecule, prepared a fusion protein of the molecule with a streptavidin mutant, and found that the fusion protein can neutralize virus activity, thereby completing the present invention.

[0006] That is, according to the present invention, the following inventions are provided. <1> A fusion protein in which a virus inhibitory molecule with a molecular weight of 20,000 or less is linked via a linker sequence to the N-terminus and / or C-terminus of the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole). <2> The nucleic acid has, from the N-terminus to the C-terminus, in this order: a virus inhibitory molecule having a molecular weight of 20,000 or less, a linker sequence, and the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole). <1> A fusion protein according to claim 1. <3> The virus-inhibiting molecule is a peptide; <1> or <2> A fusion protein according to claim 1. <4> The virus-inhibiting molecule is a molecule that inhibits coronaviruses, <1> from <3> 13. A fusion protein according to any one of claims 1 to 12. <5> The virus-inhibiting molecule is a molecule that binds to the SARS-CoV-2 receptor binding domain. <1> from <4> 13. A fusion protein according to any one of claims 1 to 12. <6> The virus inhibitory molecule has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6. <1> from <5> 13. A fusion protein according to any one of claims 1 to 12. <7> the linker sequence consists of glycine residues and serine residues and has 5 to 25 amino acid residues; <1> from <6> 13. A fusion protein according to any one of claims 1 to 12. <8> The linker sequence is [(Gly) m-Ser] n (wherein m is an integer of 1 to 10, and n is an integer of 1 to 5), <1> from <7> 13. A fusion protein according to any one of claims 1 to 12. <9> having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (wherein the C-terminal amino acid sequence Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His may be deleted in part or in whole); <1> from <8> 13. A fusion protein according to any one of claims 1 to 12. <10> A nucleic acid encoding a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole). <11> <1> from <9> An antiviral agent comprising the fusion protein according to any one of the above items. Effect of the Invention

[0007] The fusion protein of the virus-inhibiting molecule of the present invention and a streptavidin mutant can be used to exert antiviral activity, thereby making it possible to treat viral diseases. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows the results of SDS-PAGE analysis of tetramer formation after adding 25 μL of denatured LCB1-Cupid in a 1.5 mL tube containing 0.4 M arginine at pH values ​​of 4.0, 5.0, 5.5, and 6.0 for 24 hours at 4°C. Comparing the bands of tetramer molecules appearing near the 75 kDa marker and monomer molecules appearing near the 25 kDa marker, the ratio of the bands of tetramer molecules is highest at pH 5.5. [Diagram 2]Figure 2 shows the results of SDS-PAGE analysis of the purity of refolded LCB1-Cupid after concentrating it and replacing the solvent with PBS. The monomer molecule band visible in Figure 1 near the 25 kDa marker has disappeared, indicating high purity. [Diagram 3] Figure 3 shows the results of analyzing the inhibition of binding of LCB1-Cupid to the SARS-CoV-2 RBD (receptor binding domain) and ACE 2 protein. Anti-RBD miniprotein diluted with PBS increased its binding to the SARS-CoV-2 RBD (receptor binding domain) in a concentration-dependent manner, inhibiting the binding of ACE 2 protein to SARS-CoV-2 RBD, while z-HER2, which does not have the ability to bind to SARS-CoV-2 RBD, did not show concentration-dependent neutralizing activity. [Figure 4] Figure 4 shows the results of three experiments examining unwinding. In lane 1, 10 μL of the concentrate (4.4 mg / mL) was mixed with 10 μL of sample buffer and applied to the lane. In lane 2, 10 μL of the concentrate diluted with PBS to 1 mg / mL was mixed with 10 μL of sample buffer and applied to the lane. [Diagram 5] Figure 5 shows the results of analyzing the inhibition of binding of LCB3-Cupid to the SARS-CoV-2 RBD (receptor binding domain) and ACE 2 protein. LCB3-Cupid (referred to as Anti-RBD miniprotein) diluted with PBS increased its binding to the SARS-CoV-2 RBD (receptor binding domain) in a concentration-dependent manner, inhibiting the binding of ACE 2 protein to SARS-CoV-2 RBD, while z-HER2, which does not have the ability to bind to SARS-CoV-2 RBD, did not show concentration-dependent neutralizing activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will now be described in further detail. The fusion protein of the present invention is a fusion protein in which a virus inhibitory molecule having a molecular weight of 20,000 or less is bound via a linker sequence to the N-terminus and / or C-terminus of the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence at the C-terminus consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His (SEQ ID NO: 5) may be deleted in part or in whole). When a virus inhibitory molecule having a molecular weight of 20,000 or less is bound via a linker sequence to the N-terminus and / or C-terminus of the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence at the C-terminus consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His-His) the virus inhibitory molecules may be the same or different.

[0010] Preferably, the fusion protein of the present invention is a fusion protein having, from the N-terminus to the C-terminus, in this order, a virus inhibitory molecule having a molecular weight of 20,000 or less, a linker sequence, and the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence at the C-terminus consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His-His may be deleted in part or in whole).

[0011] The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of a streptavidin mutant, specifically, the streptavidin mutant LISA314-V2122 described in Example 3 of International Publication WO2015 / 125820 (SEQ ID NO: 4 of International Publication WO2015 / 125820) (SEQ ID NO: 1 in the present specification).

[0012] As described above, the fusion protein of the present invention is a fusion protein of a virus inhibitory molecule having a molecular weight of 20,000 or less and a streptavidin mutant. The fusion protein of the present invention forms a tetramer due to the affinity between the amino acid sequences described in SEQ ID NO: 1. When a protein having an amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 6 is used as the virus inhibitory molecule, for example, the theoretical molecular weight of the tetramer of the fusion protein of the present invention is about 87 kDa. When a fusion protein such as that of the present invention is administered to a living body to treat a viral disease, it is important to simultaneously achieve good binding to the virus and a good clearance rate. It is assumed that the molecular weight of the tetramer described above in the present invention (about 87 kDa) is a molecular weight that can simultaneously achieve the above parameters.

[0013] The viral inhibitor molecule is preferably a peptide. The molecular weight of the virus inhibitory molecule may be 20,000 or less, but the molecular weight of the virus inhibitory molecule is generally from 4,000 to 20,000, preferably from 4,000 to 10,000, and more preferably from 4,000 to 8,000. The virus inhibitory molecule of the present invention is a molecule conceptually different from an antibody.

[0014] Viruses inhibited by the virus-inhibiting molecule may be RNA viruses or DNA viruses, and examples thereof include, but are not limited to, coronaviruses (SARS coronavirus, MERS coronavirus, human coronavirus, etc.), caliciviruses (feline calicivirus, Norwalk virus, etc.), influenza viruses (influenza A virus, influenza B virus, influenza C virus, etc.), human immunodeficiency viruses, herpes viruses, adenoviruses, papilloma viruses, rhinoviruses, enteroviruses, rubella viruses, Ebola viruses, measles viruses, varicella-zoster viruses, mumps viruses, arboviruses, RS viruses, hepatitis viruses (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever viruses, rabies viruses, hantaviruses, dengue viruses, Nipah viruses, lyssaviruses, noroviruses, rotaviruses, polioviruses, coxsackieviruses, parvoviruses, encephalomyocarditis viruses, polioviruses, and Zika viruses.

[0015] Among the above, coronaviruses (SARS coronavirus, MERS coronavirus, human coronavirus, etc.) are particularly preferred. Coronaviruses that infect humans include four types of human coronaviruses 229E, OC43, NL63, and HKU-1, which cause the common cold, as well as three types of coronaviruses: Severe Acute Respiratory Syndrome (SARS) coronavirus, which emerged in 2002, Middle East Respiratory Syndrome (MERS) coronavirus, which emerged in 2012, and a new coronavirus (2019-nCoV, SARS-CoV-2), which emerged in 2019. In the present invention, SARS-CoV-2 is particularly preferred as a virus. SARS-CoV-2 is classified into the same betacoronavirus genus as SARS coronavirus, and the genes of the new coronavirus are highly homologous to those of SARS coronavirus (about 80%), and further, it adsorbs to and invades human cells using a receptor (ACE1 or ACE2) similar to that of SARS coronavirus.

[0016] An example of a virus-inhibiting molecule is a peptide having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6, which can bind to the SARS-CoV-2 receptor binding domain.

[0017] The linker sequence is not particularly limited as long as it can achieve the effects of the present invention, but the number of amino acids is preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, and even more preferably 15 to 20 amino acids. A specific example of the linker sequence is a sequence consisting of glycine residues and serine residues. m -Ser] n (wherein m is an integer of 1 to 10, and n is an integer of 1 to 5) can be used. A specific example of the linker sequence can include, but is not limited to, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser.

[0018] A specific example of the fusion protein of the present invention is a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole).

[0019] The present invention further provides a nucleic acid (e.g., DNA) encoding the above-mentioned fusion protein of the present invention. Specific examples of the nucleic acid of the present invention include nucleic acids encoding a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole). An example of the nucleic acid of the present invention is a nucleic acid having the base sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9.

[0020] The nucleic acid (e.g., DNA) encoding the fusion protein of the present invention can be incorporated into a vector for use. To produce the fusion protein of the present invention, the nucleic acid encoding the fusion protein of the present invention can be incorporated into an expression vector and the expression vector can be transformed into a host to express the fusion protein of the present invention. That is, according to the present invention, there is provided a method for producing the fusion protein of the present invention, which comprises a step of expressing the nucleic acid encoding the fusion protein of the present invention in a host. The fusion protein can be preferably expressed in inclusion bodies of bacteria and then recovered.

[0021] When Escherichia coli is used as a host, the vector preferably has an origin of replication (ori) and further has a gene for selecting a transformed host (e.g., a drug resistance gene against drugs such as ampicillin, tetracycline, kanamycin, or chloramphenicol). In addition, in the case of an expression vector, it is preferable that the vector has a promoter capable of efficiently expressing the streptavidin mutant of the present invention in the host, such as the lacZ promoter or the T7 promoter. Examples of such vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP, or pET (in this case, it is preferable to use BL21, which expresses T7 RNA polymerase, as the host).

[0022] Vectors can be introduced into host cells using, for example, the calcium chloride method or electroporation. Furthermore, a tag for improving solubility, such as a sequence encoding glutathione-S-transferase, thioredoxin, or maltose-binding protein, may be added. Furthermore, a tag designed to facilitate purification, such as a polyhistidine tag, a Myc epitope, a hemagglutinin (HA) epitope, a T7 epitope, an Xpress tag, a FLAG peptide tag, or a sequence encoding other known tag sequences, may be added.

[0023] In addition to E. coli, examples of the expression vector include mammalian expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, and pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC baculovairus expression system" (Gibco BRL), and pBacPAK8), plant expression vectors (e.g., pMH1 and pMH2), animal virus expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expression vectors (e.g., pPL608 and pKTH50).

[0024] When the objective is expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential to have a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and it is more preferable if it has a gene for selecting transformation into cells (for example, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0025] There is no particular limitation on the host cells into which the vector is introduced, and either prokaryotic or eukaryotic cells may be used, such as Escherichia coli and various animal cells.

[0026] When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. As animal cells, mammalian cells, for example, CHO cells, COS cells, 3T3 cells, HeLa cells, Vero cells, or insect cells, for example, Sf9, Sf21, Tn5, etc. can be used. In animal cells, CHO cells are particularly preferred when the aim is mass expression. The introduction of a vector into a host cell can be carried out, for example, by the calcium phosphate method, the DEAE dextran method, a method using cationic ribosome DOTAP (manufactured by Boehringer Mannheim), the electroporation method, lipofection, etc.

[0027] As plant cells, for example, cells derived from Nicotiana tabacum are known as a protein production system, and these can be cultured as callus. As fungal cells, yeasts such as Saccharomyces genus, e.g., Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus genus, e.g., Aspergillus niger, are known.

[0028] When prokaryotic cells are used, examples include Escherichia coli (E. coli), such as JM109, DH5α, and HB101, and also Bacillus subtilis is known.

[0029] These cells are transformed with the nucleic acid of the present invention, and the transformed cells are cultured in vitro to obtain the fusion protein of the present invention. The culture can be performed according to a known method. For example, DMEM, MEM, RPMI1640, or IMDM can be used as a culture medium for animal cells. In this case, serum supplements such as fetal calf serum (FCS) can be used in combination, or serum-free culture can be performed. The pH during culture is preferably about 6 to 8. Culture is usually performed at about 30 to 40°C for about 15 to 200 hours, and the medium is replaced, aerated, or stirred as necessary. Growth factors may also be added to promote cell proliferation.

[0030] <Antiviral agents> The fusion proteins of the present invention are useful as antiviral agents.

[0031] When a molecule that binds to a virus-associated molecule is used as the virus-inhibitory molecule, the virus activity can be neutralized by administering the fusion protein of the present invention to a patient.

[0032] Methods of administration to a patient include, but are not limited to, topical routes, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous injections), oral routes, ocular routes, sublingual routes, rectal routes, transdermal routes, intranasal routes, vaginal routes, and inhalation routes.

[0033] The fusion proteins of the invention are preferably administered in a therapeutically effective amount. The therapeutically effective amount is at least 0.5 milligrams per 60 kilograms (mg / 60kg), at least 5 mg / 60kg, at least 10 mg / 60kg, at least 20 mg / 60kg, at least 30 mg / 60kg, at least 50 mg / 60kg. For example, when administered intravenously, the therapeutically effective amount is at least 10 μg / kg, such as at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, such as at least 10 μg / kg, such as at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, such as at least 10 μg / kg, such as at least 100 μg / kg, at least 250 μg / kg, at least 500 μg / kg, at least 750 μg / kg, or at least 1000 μg / kg, such as at least 10 μg / kg, at least 100 μg / kg, at least 1000 μg / kg, at least 1000 μg / kg. In one example, the therapeutically effective amount is at least 1 μg / ml, such as at least 500 μg / ml, such as between 20 μg / ml and 100 μg / ml, such as 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, or 100 μg / ml administered in a topical solution.

[0034] The above-mentioned dosages may be administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation.

[0035] Each of the fusion proteins of the present invention can be administered alone, in the presence of a pharma- ceutically acceptable carrier, or in the presence of other therapeutic agents (such as other antiviral agents).

[0036] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0037] Example 1: Preparation of LCB1-Cupid protein A conjugate was designed by linking Cupid (SEQ ID NO: 1), a streptavidin variant with low immunogenicity and no biotin binding ability described in International Publication WO2015 / 125820, and a miniprotein inhibitor (SEQ ID NO: 2) that binds to the SARS-CoV-2 receptor binding domain described in the supplement data of De novo design of picomolar SARS-CoV-2 miniprotein inhibitors, L. Cao, I. Goreshnik, B. Coventry, JB Case, L. Miller, L. Kozodoy, et al. Science 2020 Vol. 370 Issue 6515 Pages 426-431 (SEQ ID NO: 3). Based on the designed amino acid sequence described in SEQ ID NO: 3, a gene was synthesized by artificial gene synthesis. The artificially synthesized gene was optimized to Escherichia coli codons using the algorithm of an artificial gene synthesis contract company (Eurofins Genomics Co., Ltd.) (SEQ ID NO: 4). The artificially synthesized gene sequence was inserted into the E. coli expression vector pET45b(+) according to standard methods, and the ligation product was expressed as ECOS TM SONIC Competent E. coli BL21(DE3) Derived (Nippon Gene) was transformed and cloned. The cultured E. coli was miniprepped, the plasmid DNA was purified, and the correct integration of the target gene was confirmed by sequencing. The plasmid DNA was named pET45-LCB1-Cupid.

[0038] Next, in order to express the LCB1-Cupid fusion protein, E. coli transformed with the above-mentioned pET45-LCB1-Cupid, BL21(DE3)-pET45-LCB1-Cupid, was cultured. 2 mL of the cultured overnight in LB medium was inoculated into 100 mL of culture medium and cultured at 37°C. When the OD value at 600 nm reached 0.5 to 0.8, IPTG was added to a final concentration of 0.5 mM, and cultured at 37°C for 4 hours. The cells were then collected by centrifugation (7500 xg, 20 min at 4°C).

[0039] The collected cells were suspended in lysis buffer (20 mM Tris-HCl pH8.0, 2 mM MgCl2, 10 μg / mL egg white lysozyme, 2 unit / mL Benzonase) and incubated at room temperature for 30 minutes, after which DNA was further cleaved using an ultrasonicator. The treated solution was centrifuged (12,000 xg, 20 min at 4°C) to separate the insoluble fraction (inclusion body, hereafter referred to as IB) and the soluble fraction, and the IB was collected. The collected IB was then resuspended in wash buffer (20 mM Tris-HCl pH8.0), centrifuged, and the supernatant was discarded. The IB was then washed three times with ultrapure water, and the IB collected by centrifugation after three washes was resuspended in ultrapure water, dispensed into 1.5 mL tubes, centrifuged, the supernatant was discarded, and frozen at -80°C.

[0040] Denaturation buffer (6 M guanidium HCl, 200 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA; pH 8.5) was added to the IB and dissolved by pipetting. The mixture was incubated at 25°C for 1 hour with stirring, and then centrifuged (15,000 xg, 20 min at 4°C) to collect the supernatant and remove any insoluble material.

[0041] Next, to confirm the efficiency of refolding, the refolding buffer was prepared by adding 0.4 M arginine hydrochloride to McIlvaine buffer to adjust the pH to pH 4.0, pH 5.0, pH 5.5, and pH 6.0. 25 μL of the denatured solution supernatant was added to 1 mL of refolding buffer at each pH to perform refolding. The samples were incubated at 4°C, and the formation of tetramers was confirmed by SDS-PAGE electrophoresis. As shown on the left side of Figure 1, it was confirmed that tetramers were effectively formed by refolding with a refolding buffer at around pH 5.5.

[0042] To scale up the refolding, 0.5 mL of denaturing solution was added to 40 mL of pH 5.5 refolding buffer to perform refolding. After 48 hours, the protein was recovered, concentrated, and the purity was confirmed by electrophoresis (Figure 2).

[0043] Example 2: Measurement of neutralizing activity of LCB1-Cupid The neutralizing activity of purified LCB1-Cupid against SARS-CoV-2 RBD (receptor binding domain) was measured using the iFlash-2019-nCov Nab Kit (Medical and Biological Laboratories, Inc.). The iFlash-2019-nCoV Neutralizing Antibody (Nab) Assay is a paramagnetic particle chemiluminescence immunoassay (CLIA) that quantitatively measures 2019-nCoV neutralizing antibodies in human serum and plasma using the automated iFlash Immunoassay Analyzer. It is intended to be used for the evaluation of COVID-19 and for monitoring neutralizing antibodies in people who have received a COVID-19 vaccine. In this study, we prepared a dilution series of purified LCB1-Cupid and analyzed the concentration-dependent activity of 2019-nCoV neutralizing antibodies (Nab) in human serum and plasma. The iFlash-2019-nCoV neutralizing antibody (Nab) assay is a one-step competitive immunoassay using direct chemiluminescence immunoassay, and the measurement was performed according to the manual provided with the kit.

[0044] The specific assay method is briefly described as follows. In the first incubation, the sample (dilution series of LCB1-Cupid: dilution series prepared using PBS) reacts with paramagnetic microparticles coated with 2019-nCoV Receptor Binding Domain (RBD) antigen to form a complex. Next, acridinium ester-labeled angiotensin-converting enzyme 2 (ACE2) conjugate is added and incubated. During this, acridinium ester-labeled ACE2 competitively binds to RBD-coated particles that have not been neutralized by LCB1-Cupid in the sample, forming a separate reaction mixture. Under a magnetic field, the magnetic particles are adsorbed to the wall of the reaction tube, and unbound substances are washed away by the washing buffer.

[0045] The triggering and measurement of the chemiluminescence signal is performed by adding a pre-trigger and trigger solution to the reaction mixture. The resulting chemiluminescence response is measured as relative light units (RLU). There is an inverse relationship between the amount of 2019-nCoV neutralizing antibodies in the sample and the RLU detected by the iFlash optics. After calculation, the measured value is output in the form of AU / mL. The cut-off value is set at 10.00 AU / mL, i.e., an output result of <10.00 AU / mL indicates negative neutralization activity, whereas ≥10.00 AU / mL indicates positive neutralization activity.

[0046] The analysis results are shown in Figure 3. A dilution series of the same concentration of the molecule that recognizes HER2 shown in Patent Document 1 was prepared and used as a negative control. It was confirmed that the neutralizing activity of LCB1-Cupid increased in a concentration-dependent manner.

[0047] Example 3: Preparation of LCB3-Cupid protein The gene was synthesized by artificial gene synthesis based on Cupid (SEQ ID NO: 1), a streptavidin variant with low immunogenicity and no biotin binding ability described in International Publication WO2015 / 125820, and LCB3 (SEQ ID NO: 6), a miniprotein inhibitor that binds to the SARS-CoV-2 receptor binding domain described in the supplement data of De novo design of picomolar SARS-CoV-2 miniprotein inhibitors, L. Cao, I. Goreshnik, B. Coventry, JB Case, L. Miller, L. Kozodoy, et al. Science 2020 Vol. 370 Issue 6515 Pages 426-431. The artificially synthesized gene was optimized to Escherichia coli codons using the algorithm of an artificial gene synthesis contract company (Eurofins Genomics Co., Ltd.) (SEQ ID NO: 7). Using the pET45-LCB1-Cupid prepared in Example 1, a plasmid vector expressing the LCB3-Cupid fusion protein (SEQ ID NO: 8) was prepared as follows. Using pET45-LCB1-Cupid as a template, PCR was performed with a primer set (21-020: CATGGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID NO: 10), 21-021: GGAGGCGGAGGGTCTGGAGGTGGCGGTTC (SEQ ID NO: 11)) to remove the LCB1 gene site and prepare a linearized pET45-Cupid cassette. Using the artificially synthesized LCB3 sequence as a template, PCR was performed with a primer set (21-024: AGGAGATATACCATGAATGACGATGAACTGCACATGCTTATG (SEQ ID NO: 12), 21-025: AGACCCTCCGCCTCCGCTCAGTAAGCGTTCTAACAGCTCTTTC (SEQ ID NO: 13)) to amplify the insert LCB3 sequence. All PCR reactions were performed using PrimeSTAR GXL (Takara Bio).The two PCR products were subjected to gel electrophoresis, gel excision and purification, and the insert LCB3 sequence was directional ligated to the linearized pET45-Cupid vector using the In-Fusion HD Cloning Kit (Takara Bio). ECOS was obtained from the ligation product. TM SONIC Competent E. coli BL21(DE3) Derived (Nippon Gene) was transformed and cloned. The cultured E. coli was miniprepped, the plasmid DNA was purified, and the correct integration of the target gene (SEQ ID NO: 9) was confirmed by sequencing. The plasmid DNA was named pET45-LCB3-Cupid.

[0048] Next, in order to express the LCB1-Cupid fusion protein, E. coli transformed with the above-mentioned pET45-LCB3-Cupid, BL21(DE3)-pET45-LCB1-Cupid, was cultured. The culture solution cultured overnight in LB medium was inoculated into the culture solution and cultured at 37°C. When the OD value at 600 nm reached 0.5 to 0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 37°C for 4 hours. The cells were then collected by centrifugation (7500 xg, 20 min at 4°C).

[0049] The collected cells were suspended in lysis buffer (20 mM Tris-HCl pH8.0, 2 mM MgCl2, 10 μg / mL egg white lysozyme, 2 unit / mL Benzonase) and incubated at room temperature for 30 minutes, after which DNA was further cleaved using an ultrasonicator. The treated solution was centrifuged (12,000 xg, 20 min at 4°C) to separate the insoluble fraction (inclusion body, hereafter referred to as IB) and the soluble fraction, and the IB was collected. The collected IB was then resuspended in wash buffer (20 mM Tris-HCl pH8.0), centrifuged, and the supernatant was discarded. The IB was then washed three times with ultrapure water, and the IB collected by centrifugation after three washes was resuspended in ultrapure water, dispensed into 1.5 mL tubes, centrifuged, the supernatant was discarded, and frozen at -80°C.

[0050] To the approximately 250 mL of IB precipitated by centrifugation, 0.5 mL of denaturation buffer (6 M guanidium HCl, 200 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA; pH 8.5) was added and dissolved by pipetting. The mixture was incubated at 25°C for 1 hour with stirring, and then centrifuged (15,000 xg, 20 min at 4°C) to recover the supernatant and remove insoluble material.

[0051] The theoretical isoelectric point (pI) of LCB3-Cupid was checked on the website Expasy ProtParam (web.expasy.org / protparam / ) and found to be 5.77. Based on this, refolding was performed by adding 0.5 mL of denaturing solution to 40 mL of refolding buffer (0.1 M sodium phosphate, 0.2 M arginine-HCl, pH 5.5) for dilution and refolding. After 48 hours, the protein was recovered and concentrated, and good tetramer formation was confirmed by electrophoresis (Figure 4). After concentration, the concentration was 4.4 mg / mL, the volume was 1.32 mL, and the total protein yield was 5.8 mg.

[0052] Example 4: Measurement of neutralizing activity of LCB3-Cupid The neutralizing activity of purified LCB3-Cupid against SARS-CoV-2 RBD (receptor binding domain) was measured using the iFlash-2019-nCov Nab Kit (Medical and Biological Laboratories, Inc.). The iFlash-2019-nCoV Neutralizing Antibody (Nab) Assay is a paramagnetic particle chemiluminescence immunoassay (CLIA) that quantitatively measures 2019-nCoV neutralizing antibodies in human serum and plasma using the automated iFlash Immunoassay Analyzer. It is intended to be used for the evaluation of COVID-19 and for monitoring neutralizing antibodies in people who have received COVID-19 vaccinations. In this study, we prepared a dilution series of purified LCB1-Cupid and analyzed the concentration-dependent activity of 2019-nCoV neutralizing antibodies (Nab) in human serum and plasma. The iFlash-2019-nCoV neutralizing antibody (Nab) assay is a one-step competitive immunoassay using direct chemiluminescence immunoassay, and the measurement was performed according to the manual provided with the kit.

[0053] The specific assay method is as described in Example 2. The analysis results are shown in Figure 5. A dilution series of the same concentration of the molecule that recognizes HER2 shown in Patent Document 1 was prepared and used as a negative control. It was confirmed that the neutralizing activity of LCB3-Cupid increased in a concentration-dependent manner.

[0054] SEQ ID NO:1 (Cupid amino acid sequence) AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH

[0055] SEQ ID NO: 2 (amino acid sequence of a miniprotein inhibitor that binds to the SARS-CoV-2 receptor binding domain) DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER

[0056] SEQ ID NO:3 (LCB1-Cupid amino acid sequence) MDKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVERGGGGSGGGGSGGGGSAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH

[0057] SEQ ID NO: 4 (artificially synthesized gene sequence) ATGGACAAAGAGTGGATTCTCCAGAAAATCTACGAGATTATGCGCTTACTGGATGAACTGGGTCATGCCGAAGCAAGCATGCGTGTTTCGGATCTGATCTATGAGTTTATGAAGAAAGGCGATGAACGCCTTCTGGAAGAAGCGGAACGTTTGCTGGAAGAGGTGGAACGCGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGGTGGCGGTGGCAGTGCGGAAGCCGGTATTACCGGGACCTGGTCCGATCAACTCGGCGACACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGAATGCGGTAGGCGGTGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGCATCATCACCACCATCATTAA

[0058] Sequence number 6 (Amino acid sequence of miniprotein inhibitor: LCB3 that binds to the SARS-CoV-2 receptor binding domain) NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS

[0059] Sequence number 7 (LCB3 synthetic gene sequence) AATGACGATGAACTGCACATGCTTATGACCGATTTGGTGTATGAAGCTCTGCATTTTGCCAAAGACGAAGAGATTAAGAAACGCGTATTTCAACTGTTCGAATTGGCAGATAAAGCGTACAAGAAACAACGATCGTCAGAAACTCGAGAAAGTCGTTGAAGAACTGAAAGAGCTGTTAGAACGCTTACTGAGC

[0060] SEQ ID NO:8 (LCB3-Cupid amino acid sequence) MNDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLSGGGGSGGGGGSGGGGSAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH

[0061] SEQ ID NO: 9 (LCB3-Cupid base sequence) ATGAATGACGATGAACTGCACATGCTTATGACCGATTTGGTGTATGAAGCTCTGCATTTTGCCAAAGACGAAGAGATTAAGAAACGCGTATTTCAACTGTTCGAATTGGCAGATAAAGCGTACAAGAACAACGATCGTCAGAAACTCGAGAAAGTCGTTGAAGAACTGAAAGAGCTGTTAGAACGCTTACTGAGCGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGGTGGCGGTGGCAGTGCGGAAGCCGGTATTACCGGGACCTGGTCCGATCAACTCGGCGACACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGAATGCGGTAGGCGGTGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGCATCATCACCACCATCATTAA

Claims

1. A fusion protein in which a virus inhibitory molecule having a molecular weight of 20,000 or less is bound via a linker sequence to the N-terminus and / or C-terminus of the amino acid sequence set forth in SEQ ID NO:1 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole).

2. The fusion protein described in claim 1, having, from the N-terminus to the C-terminus, in this order, a virus inhibitory molecule having a molecular weight of 20,000 or less, a linker sequence, and the amino acid sequence set forth in SEQ ID NO: 1 (however, the amino acid sequence at the C-terminus consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His-His may be deleted in part or in whole).

3. The fusion protein of claim 1 or 2, wherein the viral inhibitory molecule is a peptide.

4. The fusion protein according to any one of claims 1 to 3, wherein the virus-inhibiting molecule is a molecule that inhibits coronavirus.

5. The fusion protein of any one of claims 1 to 4, wherein the virus inhibitory molecule is a molecule that binds to the SARS-CoV-2 receptor binding domain.

6. A fusion protein described in any one of claims 1 to 5, wherein the viral inhibitory molecule has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:

6.

7. The fusion protein according to any one of claims 1 to 6, wherein the linker sequence consists of glycine and serine residues and has 5 to 25 amino acid residues.

8. The linker sequence is [(Gly) ｍ - Ser] ｎ The fusion protein according to any one of claims 1 to 7, having an amino acid sequence represented by the following formula: (wherein m is an integer of 1 to 10, and n is an integer of 1 to 5).

9. 9. The fusion protein described in any one of claims 1 to 8, having the amino acid sequence described in SEQ ID NO: 3 or SEQ ID NO: 8 (provided that the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole).

10. A nucleic acid encoding a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (however, the amino acid sequence consisting of Pro-Ser-Ala-Ala-Ser-His-His-His-His-His-His at the C-terminus may be deleted in part or in whole).

11. An antiviral agent comprising the fusion protein according to any one of claims 1 to 9.

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