KR102554311B1 - Therapeutic protein CTP beta for treating coronavirus disease COVID-19 and use thereof - Google Patents

Abstract

The present invention relates to the therapeutic protein CTP beta for coronavirus infection COVID-19 and its use, compared to the previously known peptide (P6) that mimics the binding site of RBD and ACE2 of SARS-CoV, the therapeutic protein CTP beta of the present invention consists of a new part with the addition of a new sequence of amino acids, originally designed for interactions at the atomic level constituting amino acids to make the binding with the new epitope of SARS-CoV2 RBD stronger. In the present invention, an extended therapeutic protein CTP beta, which can additionally interact with the charged amino acids of D420 and K458 on the back side of the previously known binding interface between RBD and hACE2, was creatively designed and known in the art. A new design therapeutic protein CTP beta that can bind more strongly than peptides has been proposed, and the therapeutic protein CTP beta of the present invention has high potential for use as a future COVID-19 treatment.

Description

Therapeutic protein CTP beta for treating coronavirus disease COVID-19 and use thereof}

The present invention relates to a coronavirus infection COVID-19 therapeutic protein CTP beta (COVID-19 Therapeutic Protein Beta) and its use.

SARS-CoV2, the coronavirus that causes the COVID-19 disease, binds to the Angiotensin-converting enzyme 2 (hACE2) protein, which is mainly present in human lung epithelial cells, invades cells and transfers the genetic material of the virus into human cells. replicates and reproduces the virus. Finding strategies to prevent the coronavirus from entering human cells is a fundamental way to treat the COVID-19 pandemic.

The spike protein S1-RBD (Receptor Binding Domain) present on the surface of the corona virus binds to the hACE2 protein, and the three-dimensional structure of the binding at this time has been revealed by recent studies (Jun Lan, et al., Nature, 2020).

If you can block the combination of RBD and hACE2, you can fundamentally prevent the cell invasion of the coronavirus and treat the COVID-19 infectious disease by extremely lowering the activity of the coronavirus or eliminating its infectivity. A method of neutralizing the RBD of the coronavirus is currently being actively studied as one of the principles of therapeutic development.

As a method of neutralizing RBD, methods of pre-binding other substances to RBD instead of hACE2 to prevent RBD from binding to hACE2 on the cell surface are being studied. There is human recombinant soluble ACE2 (hrsACE2=APN01) used as a lung cancer treatment as an RBD neutralizing treatment developed or proposed based on the existing hACE2. In addition, idea-level peptide therapeutics are being proposed, and most of them are designed with a focus on mimicking only the binding site of hACE2. Accordingly, it is necessary to develop a protein therapeutic agent capable of binding to RBD more strongly than previously proposed peptide therapeutic agents.

Chinese Patent Publication CN 111349150 (published on 2020.06.30)

The present invention relates to a coronavirus infection COVID-19 treatment protein CTP beta and its use, consisting of any one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3, the receptor binding domain of coronavirus (Receptor Binding Domain (RBD) protein and a composition for diagnosing coronavirus infection containing the protein as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infection, a health functional food composition and drug for preventing or improving coronavirus infection It is intended to provide a composition for delivery.

In order to solve the above problems, the present invention consists of any one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3, and specifically binds to the receptor binding domain (RBD) of coronavirus. provides a protein that

In addition, the present invention provides a polynucleotide encoding the protein, a recombinant vector containing the polynucleotide, and a transformant (except for humans) transformed with the recombinant vector.

In addition, the present invention provides a composition for diagnosing coronavirus infection comprising the protein as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the protein as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising the protein as an active ingredient.

In addition, the present invention provides a drug delivery composition comprising the protein as an active ingredient.

The present invention relates to the therapeutic protein CTP beta for coronavirus infection COVID-19 and its use, compared to the previously known peptide (P6) that mimics the binding site of RBD and ACE2 of SARS-CoV, the therapeutic protein CTP beta of the present invention consists of a new part with the addition of a new sequence of amino acids, originally designed for interactions at the atomic level constituting amino acids to make the binding with the new epitope of SARS-CoV2 RBD stronger. In the present invention, an extended therapeutic protein CTP beta, which can additionally interact with the charged amino acids of D420 and K458 on the back side of the previously known binding interface between RBD and hACE2, was creatively designed and known in the art. A new design therapeutic protein CTP beta that can bind more strongly than peptides has been proposed, and the therapeutic protein CTP beta of the present invention has high potential for use as a future COVID-19 treatment.

Figure 1 shows the bonding structure of RBD (Pink) and CTP beta.
Figure 2 shows the results of isolation and purification of the therapeutic protein CTP-β1 expressed in E. coli. (A) CTP-β1 purified from BL21(DE3) RIL cells. M: Molecular weight markers, T: Total cells after IPTG expression, S: Aqueous solution after cell disruption and P: Insoluble solution, Lanes 1 to 10: Results of CTP-β1 purified by Ni-IMAC, (B) Ion exchange chromatography Results of CTP-β1 purified by (Ion Exchange Chromatography; IEX). (C) SDS-PAGE result of pure separation and purification of CTP-β1 having a dimer structure, (D) SDS-PAGE result of final purification of CTP-β1 with a purity of 98%.
Figure 3 shows the results of isolation and purification of the therapeutic protein CTP-β2 expressed in E. coli. (A) CTP-β2 purified from BL21(DE3) RIL cells. M: Molecular weight markers, T: Total cells after IPTG expression, S: Aqueous solution after cell disruption and P: Insoluble solution, Lanes 1 to 10: Results of CTP-β2 purified by Ni-IMAC, (B) Ion exchange chromatography Results of CTP-β2 purified by (Ion Exchange Chromatography; IEX). (C) SDS-PAGE result of pure separation and purification of CTP-β2 having a dimer structure, (D) SDS-PAGE result of final purification of CTP-β2 with a purity of 99%.
Figure 4 shows the results of isolation and purification of the therapeutic protein CTP-β3 expressed in E. coli. (A) CTP-β3 purified from BL21(DE3) RIL cells. M: Molecular weight markers, T: Total cells after IPTG expression, S: Aqueous solution after cell disruption and P: Insoluble solution, Lanes 1 to 10: Results of CTP-β3 purified by Ni-IMAC, (B) Ion exchange chromatography Results of CTP-β3 purified by (Ion Exchange Chromatography; IEX). (C) SDS-PAGE result of pure separation and purification of CTP-β3 having a dimer structure, (D) SDS-PAGE result of final purification of CTP-β3 with a purity of 95%.
Figure 5 shows the mass spectrometry results of intact therapeutic protein CTP-β1.
Figure 6 shows the mass spectrometry results of intact therapeutic protein CTP-β2.
Figure 7 shows the mass spectrometry results of intact therapeutic protein CTP-β3.
Figure 8 shows the MS/MS spectrum of the sequence and N, C-terminus of the therapeutic protein CTP-β1 identified using LC-MS/MS analysis.
Figure 9 shows the MS/MS spectrum of the sequence and N, C-terminus of the therapeutic protein CTP-β2 identified using LC-MS/MS analysis.
Figure 10 shows the MS/MS spectrum of the sequence and N, C-terminus of the therapeutic protein CTP-β3 identified using LC-MS/MS analysis.
Figure 11 shows the Far UV CD spectra of hACE2, RBD, and therapeutic protein CTP beta.
12 shows changes in fluorescence intensity of MST measured by titrating RBD with hACE2 and CTP beta.
13 shows the change in cell viability by concentration when six cell lines, HEK293T, HepG2, HMC-3, MRC-5, A549, and Caki-1, were treated with the therapeutic protein CTP-β1 for 24 or 48 hours.
Figure 14 shows the change in cell viability by concentration when six cell lines, HEK293T, HepG2, HMC-3, MRC-5, A549, and Caki-1, were treated with the therapeutic protein CTP-β2 for 24 or 48 hours.
Figure 15 shows the change in cell viability by concentration when six cell lines, HEK293T, HepG2, HMC-3, MRC-5, A549, and Caki-1, were treated with the therapeutic protein CTP-β3 for 24 or 48 hours.

The present invention provides a protein that specifically binds to the Receptor Binding Domain (RBD) of coronavirus, consisting of any one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3.

In detail, the protein may inhibit the binding of RBD of coronavirus and angiotensin-converting enzyme 2 (ACE2), but is not limited thereto.

Specifically, the coronavirus may be SARS-CoV2, but is not limited thereto.

In detail, the protein may bind to D420 and K458 of SARS-CoV2 RBD, but is not limited thereto.

The protein of the present invention can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997 A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), but is not limited thereto.

In addition, the protein of the present invention can be produced by genetic engineering methods. First, a DNA sequence encoding the protein is synthesized according to a conventional method. DNA sequences can be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (eg, sold by Biosearch or AppliedBiosystems). The constructed DNA sequence is a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked to the DNA sequence to control the expression of the DNA sequence. and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow expression of the DNA sequence, and a substantially pure protein encoded by the DNA sequence is recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, 'substantially pure protein' means that the protein according to the present invention does not substantially contain any other host-derived proteins.

In the present invention, a protein consisting of any one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3 is a concept including a functional variant thereof. "Functional variant" means any similar sequence in which substitution of some amino acids occurs at amino acid positions that do not affect the properties of the protein of the present invention.

In addition, the present invention provides a polynucleotide encoding the protein.

The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides that exists in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes not only a nucleotide sequence encoding the protein, but also a sequence complementary to the sequence. The complementary sequences include sequences that are substantially complementary as well as sequences that are perfectly complementary.

In addition, the polynucleotide may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, It may be a sequence exhibiting at least 90% homology or at least 95% homology.

In addition, the present invention provides a recombinant vector comprising the polynucleotide.

In addition, the present invention provides transformants (except for humans) transformed with the recombinant vector.

In the present invention, “vector” refers to a self-replicating DNA molecule used to transfer clonal genes (or other fragments of clonal DNA).

In the present invention, "recombinant vector" refers to a plasmid, viral vector or other medium known in the art capable of expressing an inserted nucleic acid in a host cell, and is compared to conventional expression vectors known in the art. Polynucleotides encoding the proteins of the invention may be operably linked. The recombinant vector is generally operably linked to an origin of replication capable of proliferating in a host cell, one or more expression control sequences (eg, promoter, enhancer, etc.) for controlling expression, a selective marker, and an expression control sequence. It may contain polynucleotides encoding the proteins of the invention. A transformant may be one transformed by the recombinant vector.

Preferably, the transformant is a recombinant vector containing a polynucleotide encoding the protein of the present invention by methods known in the art, such as, but not limited to, transient transfection, microinjection, and transfection. Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran- mediated transfection, polybrene-mediated transfection -mediated transfection), electroporation (electropora tion), gene gun (gene gun), and can be obtained by introducing into a host cell by other known methods for introducing nucleic acids into cells (Wu et al., J. Bio.Chem., 267:963-967, 1992; Wu and Wu, J. Bio.Chem., 263:14621-14624, 1988).

In addition, the present invention provides a composition for diagnosing coronavirus infection comprising the protein as an active ingredient.

Preferably, the coronavirus may be SARS-CoV2, but is not limited thereto.

In the present invention, “diagnosis” means confirming the presence or character of a pathological condition. For purposes of this invention, diagnosis is the confirmation of the presence or characteristics of a coronavirus infection.

Diagnosis of coronavirus infection using the protein of the present invention can be diagnosed by reacting the protein of the present invention with blood, urine, or directly obtained tissue or cells obtained by biopsy and detecting their binding.

In addition, in order to easily identify, detect and quantify whether the protein of the present invention binds to the RBD of the coronavirus, the protein of the present invention may be provided in a labeled state. That is, it may be provided by being linked (eg, covalent bond or cross-linking) to a detectable label. The detectable label is a color enzyme (eg, peroxidase, alkaline phosphatase), a radioactive isotope (eg, ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ⁹⁹ mTc, ³² P, ³⁵ S), Chromophores, luminescent or fluorescent substances (e.g. FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, Quantum dots may be the like.

Similarly, the detectable label may be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. Such labeling may be performed during the process of synthesizing the protein of the present invention or may be additionally performed on already synthesized proteins. If a fluorescent material is used as a detectable label, coronavirus infection can be diagnosed by fluorescence mediated tomography (FMT). For example, the protein of the present invention labeled with a fluorescent material can be circulated in the blood, and fluorescence by the protein can be observed by fluorescence tomography. If fluorescence is observed, a coronavirus infection is diagnosed.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the protein as an active ingredient.

Preferably, the coronavirus infection may be COVID-19, but is not limited thereto.

The pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, and a glidant. Alternatively, a solubilizer such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release preparations of active compounds. can The pharmaceutical composition of the present invention can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered with An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled by various factors including secretion rate, duration of treatment, and drugs used concurrently. Although not limited thereto, for example, the composition of the present invention can be administered at a dose of 0.1 ng/kg to 10 g/kg when administered once to several times a day for adults.

In addition, the present invention provides a health functional food composition for preventing or improving coronavirus infection comprising the protein as an active ingredient.

The health functional food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is used with other food or food additives in addition to the active ingredient, and according to a conventional method can be used appropriately. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it is less than the above range. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, vitamin complexes, and the like are exemplified.

In addition, the present invention provides a drug delivery composition comprising the protein as an active ingredient.

Preferably, the drug may be an antiviral agent, more preferably, the antiviral agent may be an antiviral agent for coronavirus, and the coronavirus may be SARS-CoV2, but is not limited thereto.

The protein according to the present invention can be used as an intelligent drug delivery system that selectively delivers drugs to coronaviruses. If the protein of the present invention is used for the treatment of coronavirus infection in connection with a conventionally known drug, the protein of the present invention selectively delivers the drug only to the coronavirus, thereby increasing the efficacy of the drug and significantly reducing the side effects of the drug. can

The drug is an antiviral agent, and any antiviral agent that can be linked to the protein of the present invention can be used without limitation as long as it is conventionally used for the treatment of coronavirus infection. Linkage between the antiviral agent and the protein of the present invention may be performed through methods known in the art, such as covalent linkage, crosslinking, and the like. To this end, if necessary, the protein of the present invention may be chemically modified to the extent that its activity is not lost.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

< Example 1> Coronavirus infection COVID -19 therapeutic protein CTP Beta protein 3D structure analysis

1. Preparation of three-dimensional structures of proteins

The binding structure (PDB ID: 6M0J) of hACE2-RBD registered in Protein Data Bank (PDB, https://www.rcsb.org/) was used. The structure of the P6 part was modeled using MODELLER based on the binding structure of hACE2-RBD using the sequence of 22~44-G-351~357 of hACE2.

2. Molecular Dynamics Simulation

Molecular dynamics of modeled hACE2-RBD complex, P6-RBD complex, CTP beta monomer-RBD complex, CTP beta dimer-RBD complex, P6, CTP beta monomer, and CTP beta dimer with ff14SB force field applied using AMBER18 package A simulation was performed. The simulation was performed with the pmemd.cuda program included in AMBER18, and the initial structure for the simulation was an octahedron TIP3P water box with a length of 15 Å added to the outside of the prepared protein. Periodic boundary conditions were used based on the water box, and Na+ and Cl- ions were added to neutralize the net charge. The Particle-mesh Ewald (PME) method was applied to the electromagnetic force at a distance of 9 Å. The SHAKE algorithm, which fixes the covalent bond distance of hydrogen molecules, was used, and the simulation was conducted with a time step of 2fs/step. First, a 5000-step minimization simulation was performed by applying a position restraint of 0.5 kcal/mol to the backbone structure of the protein, and a heating simulation was performed for 25 ps under NVT ensemble conditions from 10 K to 300 K by applying a position restraint of 0.1 kcal/mol. performed. Equilibrium simulations were performed under NPT ensemble conditions for 1 ns at a temperature of 300 K. The production run was performed under NPT ensemble conditions at a temperature of 300 K and a pressure of 1 bar, and simulations were performed for 500 ns in the case of RBD complex and 1 μs in the case of CTP beta alone. Simulations were performed on 5 independent trajectories for each case.

The binding energy of RBD and CTP beta was calculated with the MMGBSA algorithm using simulation snapshots between 300 and 500 ns, and entropy was calculated through normal mode analysis. The MMPBSA.py program of the AMBER18 package was used to calculate the binding free energy.

3. CTP structure of beta

Modeled CTP beta groups (CTP-β1, CTP-β2, CTP-β3) have HTH primary improved structures. When the structural ensemble in the solution was analyzed through molecular dynamics simulation of the monomer and dimer forms of the modeled structure, the structural pattern of FIG. 1 was shown.

CTP-β1 is a therapeutic protein in which K2, which will interact with D420 of RBD, and E4, which will interact with K458, are arranged at the N-terminal part. It is a therapeutic protein with an unstable secondary structure at the N-terminus.

CTP-β2 is a therapeutic protein in which E2 and E4, which interact with K458 of RBD, are arranged at the N-terminus. It is a therapeutic protein with an unstable secondary structure at the N-terminus.

CTP-β3 is a therapeutic protein in which K2, which will interact with D420 of RBD, and E4, which will interact with K458, are arranged at the N-terminus. It is a therapeutic protein with improved stability of the secondary structure of CTP-β2.

4. CTP beta- RBD's Binding free energy prediction

To confirm how well CTP beta binds to RBD, the binding energy and binding free energy were calculated (Table 1). Using molecular dynamics simulation, the structural ensemble in which hACE2, P6, and CTP beta bind to RBD, respectively, is collected, and the binding energy (ΔE) and binding free energy (ΔG) are analyzed from the structural ensemble using the MMGBSA algorithm.

It can be seen that CTPs have negative binding free energies and are stable when bound to RBD, and by interaction with the new epitope, they bind more strongly in terms of energy (ΔE) than hACE2 and P6 in simulation, and their free energy (ΔG) also binds similarly or more strongly.

Group CTP RBD + CTP monomer RBD + CTP dimer ΔE ΔG ΔE ΔG - hACE2 -74.0327 - - - - P6 -63.9558 -11.0060 - - CTP β CTP-β1 -59.4622 -12.0184 -69.4741 -12.6848 CTP-β2 -80.5331 -21.8774 -67.0206 -7.9079 CTP-β3 -70.3415 -9.0865 -96.2066 -23.6538

5. CTP Beta In- in silico Immunogenicity assay (In- in silico Immunogenecity Analysis)

In-silico immunogenicity was analyzed using the world-famous NetMHC-4.0 server (http://www.cbs.dtu.dk/services/NetMHC/). For the amino acid sequence of CTP beta, peptide-MHC class I binding was predicted for 81 different human MHC alleles in the NetMHC4.0 server and 41 alleles of animals (Monkey, Cattle, Pig and Mouse). . By searching in peptide units of 13 to 14 amino acids in length, counting the total number of peptides searched (Total) and the number of peptides expected to have strong binding (SB) and weak binding (WB) among them, and the ratio (SB + WB ) / Total was calculated to verify immunogenicity.

The lower the ratio to the total (SB+WB)/Total, the smaller the antigen-antibody reaction with other proteins when the protein of the present invention is introduced into the body (Tables 2 to 7).

Human Group CTP scan
length Total SB WB (SB+WB)/Total CTP β CTP-β1 13mer 3402 6 20 0.008 14mer 3321 9 23 0.010 CTP-β2 13mer 3402 7 22 0.009 14mer 3321 10 26 0.011 CTP-β3 13mer 3402 7 25 0.009 14mer 3321 11 30 0.012

Chimpanzee Group CTP scan
length Total SB WB (SB+WB)/Total CTP β CTP-β1 13mer 336 0 2 0.006 14mer 328 0 4 0.012 CTP-β2 13mer 336 0 2 0.006 14mer 328 0 3 0.009 CTP-β3 13mer 336 0 2 0.006 14mer 328 0 3 0.009

Rhesus macaque Group CTP scan
length Total SB WB (SB+WB)/Total CTP β CTP-β1 13mer 756 4 19 0.030 14mer 738 6 27 0.045 CTP-β2 13mer 756 4 19 0.030 14mer 738 6 27 0.045 CTP-β3 13mer 756 4 21 0.033 14mer 738 6 28 0.046

Mouse Group CTP scan
length Total SB WB (SB+WB)/Total CTP β CTP-β1 13mer 252 3 10 0.052 14mer 246 2 14 0.065 CTP-β2 13mer 252 3 10 0.052 14mer 246 2 14 0.065 CTP-β3 13mer 252 One 13 0.056 14mer 246 2 16 0.073

BoLA Group CTP scan
length Total SB WB ( SB + WB )/Total CTP β CTP - β1 13mer 252 0 0 0.000 14mer 246 0 0 0.000 CTP - β2 13mer 252 0 0 0.000 14mer 246 0 0 0.000 CTP - β3 13mer 252 0 0 0.000 14mer 246 0 0 0.000

Pig Group CTP scan
length Total SB WB (SB+WB)/Total CTP β CTP-β1 13mer 126 0 0 0.000 14mer 123 0 One 0.008 CTP-β2 13mer 126 0 0 0.000 14mer 123 0 One 0.008 CTP-β3 13mer 126 0 0 0.000 14mer 123 0 One 0.008

< Example 2> Coronavirus infection COVID -19 therapeutic protein CTP Beta's cloning

1. Strains and Media

All chemicals used for gene cloning were of analytical grade. Completed clones were transformed using DH5α Escherichia coli ( E. coli ) strains for growth, and were selected in LB medium containing ampicillin (LPS, 100 μg/ml). Cell culture was stirred at 37°C.

2. Plasmid Construction

After converting the amino acid sequence of the protein designed in silico into nucleotides using the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/rev_trans.html) tool, the sequences present in hACE2 and DNA sequences were determined by comparison. The synthesized DNA fragment (Bioneer, Gene Synthesis) was prepared using forward primers 5'-GGAGATATACATATGAAAAGTGAACTTGCTTCTGTGAATTAT (CTP-β1), 5'-GGAGATATACATATGGAAAGTGAACTTGCTTCTGTGAA (CTP-β2), 5'-GGAGATATACATATGGAAAGTGAACTTGCTTCTAATGTG (CTP-β3) and common reverse primer 5'-GTGGTGCTCGAGCCT. GAAGTCGCCCTTCC After each PCR amplification with EZ-Fusion™ HT Cloning Kit (Enginenomics), Nde by ligation independent cloning method I and Xho It was inserted into the pET-21a vector treated with I restriction enzyme.

3. Experimental results

The therapeutic protein CTP beta of the present invention is a therapeutic protein with a novel amino acid sequence that neutralizes or inhibits S1-RBD protein from binding to hACE2 protein, and is designed in silico. The amino acid sequence of is shown in Table 8.

Group β CTP name Sequence CTP-β1 MKSELASVNYNTNITKENVQANGEEQAKTFLDKFNHEAEDLFYQSSGLGKGDFR (SEQ ID NO: 1) CTP-β2 MESELASVNYNTNITKENVQANGEEQAKTFLDKFNHEAEDLFYQSSGLGKGDFR (SEQ ID NO: 2) CTP-β3 MESELASNVYNTNITKENVQNMNEEQAKTFLDKFNHEAEDLFYQSSGLGKGDFR (SEQ ID NO: 3)

< Example 3> Coronavirus infection COVID -19 therapeutic protein CTP Expression and purification of beta

1. Protein expression and purification materials

All chemicals used for protein expression and purification were of analytical grade. Ampicillin, 1-thio-β-d-galactopyranoside (IPTG), Sodium Phosphate-Monobasic used for protein expression and purification ), Sodium Phosphate-Dibasic, TRIS were purchased from LPS (Daejeon, Korea), Coomassie brilliant blue R-250, 2-mercaptoethanol , Chloramphenicol and Imidazole were purchased from Biosesang (Seongnam, Korea), Sodium Chloride was purchased from Deoksan (Daejeon, Korea), and all columns were purchased from GE Healthcare (Piscataway, NJ). Purchased. Phenylmethanesulfonyl Fluoride (PMSF), trizma hydrochloride, and Amicon Ultra-15 Centrifugal Filter Units were purchased from Millipore (Billerica, MA) and used.

2. Protein Expression and Purification Instruments

For protein expression, a shaker (NBS/Innova 42R) and a high-capacity high-capacity centrifuge (Beckman Coulter. Inc./AVANTI JXN-26) were used, and a protein high-speed separation system (GE Healthcare/AKTA Pure M and Start System) and protein high-speed Proteins were separated and purified using a separation system (GE Healthcare/FPLC Accessory System), an ultrasonic disruptor (Q700-Sonicator), and a refrigerated centrifuge (Epoendorf-5810R).

3. Therapeutic proteins CTP Beta expression and culture

The gene cloned into the pET-21a vector was transformed for expression using the BL21(DE3) RIL E. Coli strain, and the medium was ampicillin (100 mg/ml) and chloramphenicol (50 mg/ml). ml) was used for cell culture at 37°C using LB medium. After culturing to OD ₆₀₀ =0.6-0.8, 1M IPTG was added to a final concentration of 0.5 mM, and then expressed at 37°C for 4 hours. Cells were harvested for 20 minutes using a high-capacity centrifuge (Beckman Coulter. Inc./AVANTI JXN-26) set at 7000 RPM and 4° C. and then used immediately or stored at -80° C.

4. Therapeutic proteins CTP Beta Separation Purification

After dissolving in 10 ml Lysis buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl, 1 mM PMSF, 0.25% Tween-20 (v/v)) based on 1 g of cells, use a sonicator (Q700-Sonicator) to break the cells. shredded. In order to separate the solution and the pellet, it was conducted for 30 minutes using a high-speed centrifuge set at 18000 RPM and 4°C. A 0.45 μm syringe filter was applied to remove impurities in the aqueous solution. The solution from which impurities were removed was flowed through a HisTrap HP 5ml (GE Healthcare) column stabilized with Wash Buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl), and then Elution Buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl). , 500 mM Imidazole) was set at 0 to 100% Gradient Elution. As a result, it was confirmed that the therapeutic protein CTP beta was isolated at concentrations of 100 to 300 mM imidazole. The isolated therapeutic protein CTP beta was subjected to buffer exchange (pH 8.0, 20 mM Tris-HCl) in a cold room for 15 hours. After flowing the NaCl-free CTP beta solution through the HiTrap Q HP 5ml (GE Healthcare) column stabilized with Binding Buffer (pH 8.0, 20 mM Tris-HCl), Elution Buffer (pH 8.0, 20 mM Tris-HCl, 1.0 M NaCl ) was set from 0 to 100% Gradient Elution. As a result, it was confirmed that the therapeutic protein CTP beta was isolated at concentrations of 120 to 250 mM NaCl. Finally, using a HiLoad Superdex 75 16/600 column stabilized with pH 7.4, 10 mM Phosphate buffer, and 150 mM NaCl, the therapeutic protein CTP beta having a dimer structure was purified and purified. A was quantified at _{280 nm} using NanoDrop, and as a result of calculating the purity using the ImageJ program, >84% high purity therapeutic protein CTP beta protein was obtained (FIGS. 2 to 4).

< Example 4> Coronavirus infection COVID -19 therapeutic protein CTP Beta Mass Spectrometry

1. Mass Spectrometry Analysis

A mass spectrometer was used to determine the total mass and amino acid sequence information of the protein. Mass spectrometry of total protein was performed using a high-performance matrix laser desorption ionization mass spectrometer (Bruker, UltrafleXtreme MALDI TOF/TOF). For protein amino acid sequence analysis, a hybrid triple quadrupole linear ion trap mass spectrometer (Sciex, QTRAP) and quadrupole orbit trap mass spectrometer (Thermo scientific, Q Exactive PLUS) were used, and micro liquid chromatography (Sciex, M5 MicroLC), Q Exactive PLUS, nano-micro liquid chromatography (Waters, ACQUITY UPLC M-Class system) was used.

2. MALDI TOF Sample preparation for and MALDI TOF Intact protein analysis using

For total mass spectrometry of ACE2, RBD, and CTP beta proteins, 10 μg of protein was mixed with a protein from which small molecular compounds were removed using a Microcon centrifugal filter (Merck, MRCPRT010) and a sinapinic acid (Bruker, 8201345) matrix at a 1:1 ratio. After drying on the target plate, the experiment was conducted with the flexcontrol program of the high-performance matrix laser desorption ionization mass spectrometer. The mass value was calibrated using the starter kit for MALDI-TOF MS (Bruker, 8208241), and the spectrum was obtained with an average of 1000 laser shots based on the method provided by flexcontrol. Each spectral data was obtained using the flexanalysis program.

3. Sample preparation for LC-MS/MS analysis

For the amino acid sequence analysis of a single protein, 20 μg of RBD, hACE2, and CTP beta proteins were mixed with 100 mM triethylammonium bicarbonate (Sigma, T7408-100ML) and 2% sodium dodecyl sulfate (Duksan, 6645). After adding 10 mM of dithiothreitol (BIO-RAD, 161-0611) and processing at 56° C. for 30 minutes, 20 mM of iodoaetamide (Sigma, T1503) was added, shielded from light, and treated for 30 minutes at room temperature. Afterwards, small molecules were removed using S-trap mini columns (PROTIFI, C02-mini-80), and 2 μg of proteolytic enzymes Trypsin (Promega, V5111) and Glu-C (Promega, V1651) were added, followed by 37°C. Enzymatic digestion was performed for 16 hours. After the reaction was completed, the mixture was separated by a centrifuge, vacuum dried, and a sample was prepared by adding 0.1% trifluoroacetic acid. And, RBD and ACE2 proteins were added to the deglycosylation experiment. For the deglycosylation experiment, 100 mM triethylammonium bicarbonate and 2 μg of PNGase F (Promega, V4831) were added to the proteolytic enzyme-treated sample, followed by enzymatic digestion at 37°C for 4 hours. After the reaction was completed, proteins were separated by centrifugation using S-trap mini columns. Samples were prepared by vacuum drying and adding 0.1% trifluoroacetic acid. RBD and hACE2 proteins were tested using a quadrupole orbital mass spectrometer and nano-micro liquid chromatography. The CTP beta protein was tested using a hybrid triple quadrupole linear ion trap mass spectrometer and micro liquid chromatography.

4. LC-MS/MS Analysis: ACQUITY UPLC M-Class system - Q Exactive PLUS

All solvents used in liquid chromatography were HPLC grade solvents. Water (Duksan, 7732-18-5) and acetonitrile (Honeywell, AH015-4) were used as solvents, dimethyl sulfoxide (Sigma, 472301-1L), trifluoroacetic acid (Thermo scientific, 28904), formic acid (Thermo scientific, 28905 ) was used as a medium in the experimental solution. Nano-micro liquid chromatography was composed of solvent A (0.1% formic acid, 5% dimethyl sulfoxide in water) and solvent B (0.1% formic acid, 5% dimethyl sulfoxide in 80% acetonitrile). Acclaim™ PepMap™ 100 C18 LC Column (Thermo scientific, 164197) was used as the trapping column and EASY-Spray™ HPLC Columns (Thermo scientific, ES803A) were used as the analytical column. Liquid chromatography was conducted for 5 minutes at a flow rate of 8 μl/min and solvent A 95%, solvent B 5% conditions in the trapping step after injecting 0.5 μg of the sample. In the Analytical step, the flow rate was 250 nl/min. Solvent A 95%, solvent B 5%, after 5 minutes, the solvent B was raised sequentially from 5% to 40% for 140 minutes, and the sample was analyzed. was lowered to 5% for 5 minutes and maintained for 40 minutes to re-equilibrate. The mass value of the quadrupole orbit trap mass spectrometer was calibrated with Pierce™ LTQ Velos ESI Positive Ion Calibration Solution (Thermo scientific, 88323). The analysis method was experimented with full-MS and data dependent MS/MS methods. The calculated data was obtained using a proteome discoverer (Thermo scientific).

5. LC-MS/MS Analysis: M5 MicroLC - QTRAP

The column used for analysis was Kinetex 2.6u XB-C18 100A (Phenomenex, 00B-4496-AC). Micro liquid chromatography was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Liquid chromatography conditions were performed at a flow rate of 20 μl/min after injecting 1.5 μg of the sample. Solvent A 95%, solvent B 5%, after 5 minutes, the solvent B was raised sequentially from 5% to 40% for 45 minutes, and the sample was analyzed. was lowered to 5% for 3 minutes and then maintained for 3 minutes to re-equilibrate. The mass value of the hybrid triple quadrupole linear ion trap mass spectrometer was calibrated with a standard chemical kit (Sciex, 4406127). As for the analysis method, the experiment was conducted with the enhanced product ion method using Q3 MS and information dependent acquisition. The calculated data was converted from a wiff file to a mgf file using convert MS (proteowizard.sourceforge.net), and then the results were obtained using searchGUI and peptide-shaker (compomics.github.io).

6. Intact protein mass spectrometry results

The mass of intact protein was measured using MALDI-TOF. RBD was measured to be 30 kDa. In addition to 30 kDa, the peak appeared as a dimer, such as a 60 kDa peak, a monomer with a charge (z) of 2 (m/z = 15 kDa), and a trimer (m/z = 45 kDa) with a charge (z) of 2. Considering that the mass of RBD calculated from the protein sequence is 25.9 kDa, the mass change of 4.1 kDa can be seen as caused by glycosylation. Similarly, the mass of hACE2 was measured to be about 102 kDa, and a dimer and a peak with a charge of 2 appeared together. Considering that the mass of hACE2 calculated from the protein sequence is 84.4 kDa, it can be seen that a mass change of about 17.5 kDa is caused by glycosylation.

CTP-β1 was determined to be 7.180 kD, approximately equal to the mass calculated from the sequence (FIG. 5), CTP-β2 was determined to be 7.182 kD, approximately equal to the mass calculated from the sequence (FIG. 6), and CTP-β3 was determined to be approximately equal to the mass calculated from the sequence. It was measured as 7.298 kD, which is almost the same as the calculated mass (FIG. 7).

7. Protein Sequence Verification

LC-MS/MS analysis was performed to confirm the sequences of RBD, hACE2, and CTP beta. Each protein was analyzed using proteolytic enzymes Trypsin and Glu-C, and RBD and hACE2 searched for amino acid residues where Asn was changed to Asp by PNGase F enzymatic activity, resulting in N-linked glycosylation. Found Asn.

The results of MS/MS analysis of CTP beta with confirmed N and C-terminus are shown in FIGS. 8 to 10 . Sequence analysis of the N-terminus and C-terminus is one of means for confirming the state of the protein, and if the terminus of the protein is not confirmed, it can be regarded as degraded due to a problem with the preservation state of the protein. In the case of RBD and hACE2, since the end of the protein tends not to be ID, unless it is the inaccuracy of the sequence or the effect of PTM, these are proteins that are easily degraded, so care must be taken in preservation.

< Example 5> Coronavirus infection COVID -19 therapeutic protein CTP Secondary structural stability of beta and function //validate

One. circular dichroic polarization (Circular Dichorism ; CD) experiment

Far UV circular dichroism was measured for RBD (PBS buffer), hACE2 (PBS buffer), and CTP beta. The protein concentration was 0.2 mg/ml at 20° C. using a JASCO-1500 machine and a 0.1 cm quartz cell was used. Measurement was performed at a far UV wavelength of 190-250 nm using a measurement speed of 20 nm/min, a bandwidth of 5 nm, and a digital integration time (D.I.T) of 4 seconds. The graph was shown by subtracting the data measured for protein 5 times with the data measured 5 times for the buffer.

As a result, it can be seen that hACE2 is a protein having an α-helix secondary structure, and RBD is a protein having a beta sheet secondary structure. It can be seen that CTP beta has both a helical structure and a coil structure (FIG. 11).

2. Microscale thermophoresis ( Microscale Thermophoresis ; MST) experiment

Fluorescence was attached to 100 μL of 8 μM RBD (PBS buffer with 0.05% Tween-20 added) using Monolith Protein Labeling Kit RED-NHS 2nd Generation. Ligand (hACE2 (PBS buffer), CTP beta) was prepared in 10 μL each at 16 concentrations from 16 μM to 0.488 nM using 1:1 serial dilution. 16 samples prepared by diluting fluorescent RBD to 30 nM A mixed solution of 15 nM RBD and 8 μM ~ 0.244 nM ligand was prepared by adding 10 μL of each ligand to each concentration.MST was measured 5 times for each ligand using a Monolith NT.115 machine at 60% excitation power and medium MST power. The Kd (dissociation constant) value was calculated using the Kdmodel of the MO affinity analysis program provided by Monolith, and the results of the experiment with the median Kd value among the five experiments were shown in a graph.

As a result, a change in fluorescence intensity according to a change in concentration was observed, indicating that both hACE2 and CTP beta bind to RBD. The Kd values representing the binding strength were 82 nM for CTP-β1, 42.9 nM for CTP-β2, 56.4 nM for CTP-β3, and 42.2 nM for hACE2 (FIG. 12). The Kd values of RBD and hACE2 are similar to the Kd value of 34.6 nM measured using BLI (biolayer interferometry) and SPR (Surface Plasmon Resonance) in the Science paper (Wrapp et al, Science 367, 1260-1263 (2020)). .

< Example 6> Coronavirus infection COVID -19 therapeutic protein CTP Verification of toxicity of beta

1. Therapeutic proteins CTP Cell culture for beta toxicity assay

The cell line used in the present invention was used after receiving distribution from the Korea Cell Line Bank (KCLB, Seoul, Korea). Human embryonic kidney cell line HEK293T, human liver cancer cell line HepG2, and human lung cancer cell line A549 were treated with 10% fetal bovine serum (FBS, Hyclone, USA) and 1% antibiotic (penicillin-streptomycin; P/S). . streptomycin) was added and cultured in Minimum Essential Media (MEM, Gibco, USA). Human normal lung cell line MRC-5 was supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin-streptomycin) and 25 μM Hydroxyethyl piperazine Ethane Sulfonic acid (HEPES, Hyclone, USA). ) was added to the Minimum Essential Media (MEM) was used as the culture medium. The human renal cancer cell line Caki-1 was cultured in McCoy's 5A medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin). All cell lines used were cultured in an incubator controlled at 37°C and 5% CO ₂ .

2. Therapeutic proteins CTP Cytotoxicity assay for beta

HEK293T, HepG2, HMC-3, MRC-5, A549 and Caki-1 cell lines were 3×10 ³ , 6×10 ³ , 5×10 ³ , 6×10 ³ , 5×10 ³ and The number of 5×10 ³ cells/well was dispensed into a 96-well cell culture dish and stabilized for 24 hours in an incubator controlled at 37° C. and 5% CO ₂ . In addition, a starvation culture medium in which the content of FBS was reduced from 10% to 1% in the composition of the culture medium of each cell line was prepared, replaced with a starvation culture medium, and starvation was performed for 16 hours. Thereafter, 1fg, 10fg, 100fg, 1pg, 10pg, 100pg, 1ng, 10ng, 100ng, 1μg, and 10μg of CTP beta were added to 100 μl of the culture medium, respectively, to treat the cell line, and then the cells were incubated for 24 hours and 48 hours. cultured. The viability of the cell line was measured using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). After adding 10 μl of CCK-8 reagent per 100 μl of the culture solution and incubating for 1 hour in an incubator controlled at a temperature of 37°C and 5% CO ₂ , use a SpectraMax iD3 microplate reader (Molecular Devices, San Jose, CA, USA) The optical density (OD) value was measured at the absorbance of 450 nm, and the derived value was expressed as a percentage.

3. Therapeutic proteins CTP beta in each cell line on cell viability impact

In the present invention, CCK-8 assay was performed to confirm toxicity through the effect of CTP beta on cell survival of six cell lines, HEK293T, HepG2, HMC-3, MRC-5, A549 and Caki-1. As a result, when the cell viability of each cell line was checked after treatment with CTP beta for 24 hours, no significant change could be confirmed in all 6 cell lines compared to the control group not treated with CTP beta. In addition, when the cell viability of each cell line was checked after treatment with CTP beta for 48 hours, no significant change in cell viability could be confirmed in all 6 cell lines (FIGS. 13 to 15). Overall, the cytotoxicity analysis results confirm the non-toxicity and safety of CTP beta, suggesting that CTP beta can be used for the prevention and treatment of coronavirus.

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Daegu Gyeongbuk Institute of Science and Technology KOREA BASIC SCIENCE INSTITUTE <120> Therapeutic protein CTP beta for treating coronavirus disease COVID-19 and its use <130> ADP-2020-0554 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 54 <212> PRT <213> artificial sequence <220> <223> CTP-Beta1 <400> 1 Met Lys Ser Glu Leu Ala Ser Val Asn Tyr Asn Thr Asn Ile Thr Lys 1 5 10 15 Glu Asn Val Gln Ala Asn Gly Glu Glu Gln Ala Lys Thr Phe Leu Asp 20 25 30 Lys Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Gly Leu 35 40 45 Gly Lys Gly Asp Phe Arg 50 <210> 2 <211> 54 <212> PRT <213> artificial sequence <220> <223> CTP-Beta2 <400> 2 Met Glu Ser Glu Leu Ala Ser Val Asn Tyr Asn Thr Asn Ile Thr Lys 1 5 10 15 Glu Asn Val Gln Ala Asn Gly Glu Glu Gln Ala Lys Thr Phe Leu Asp 20 25 30 Lys Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Gly Leu 35 40 45 Gly Lys Gly Asp Phe Arg 50 <210> 3 <211> 54 <212> PRT <213> artificial sequence <220> <223> CTP-Beta3 <400> 3 Met Glu Ser Glu Leu Ala Ser Asn Val Tyr Asn Thr Asn Ile Thr Lys 1 5 10 15 Glu Asn Val Gln Asn Met Asn Glu Glu Gln Ala Lys Thr Phe Leu Asp 20 25 30 Lys Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Gly Leu 35 40 45 Gly Lys Gly Asp Phe Arg 50

Claims (16)

A protein that specifically binds to the Receptor Binding Domain (RBD) of coronavirus, consisting of any one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3. The protein according to claim 1, wherein the protein inhibits the binding of RBD of coronavirus and angiotensin-converting enzyme 2 (ACE2). The protein according to claim 1, wherein the coronavirus is SARS-CoV2. The protein according to claim 1, wherein the protein binds to D420 and K458 of SARS-CoV2 RBD. A polynucleotide encoding the protein of claim 1. A recombinant vector comprising the polynucleotide of claim 5. Transformants other than humans transformed with the recombinant vector of claim 6. A composition for diagnosing coronavirus infection comprising the protein of any one of claims 1 to 4 as an active ingredient. The composition for diagnosing coronavirus infection according to claim 8, wherein the coronavirus is SARS-CoV2. delete delete delete A drug delivery composition comprising the protein of any one of claims 1 to 4 as an active ingredient. The drug delivery composition according to claim 13, wherein the drug is an antiviral agent. The drug delivery composition according to claim 14, wherein the antiviral agent is an antiviral agent against coronavirus. The drug delivery composition according to claim 15, wherein the coronavirus is SARS-CoV2.

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