KR20230123774A - Novel fusion protein derived from SARS-CoV-2 and Use thereof - Google Patents

Abstract

The present invention relates to a novel fusion protein derived from coronavirus and a use thereof, specifically, a receptor binding domain (RBD) of coronavirus (SARS-CoV-2) or a fragment thereof; and a fusion protein comprising a Kappa light chain constant region of an antibody, a gene encoding the fusion protein, a recombinant vector comprising the gene, a cell comprising the recombinant vector, and the fusion protein as an active ingredient. A vaccine composition comprising a vaccine composition, a coronavirus immunization method comprising the step of administering the vaccine composition to a subject in need of immunization, a coronavirus infection including the fusion protein, or a pharmaceutical for preventing infection-related diseases or symptoms It is about the enemy composition.
The vaccine composition containing the fusion protein according to the present invention can be usefully used as a vaccine for preventing and treating coronavirus infection by inducing an immune response capable of fighting coronavirus infection.

Description

Novel fusion protein derived from coronavirus and its use {Novel fusion protein derived from SARS-CoV-2 and Use thereof}

The present invention relates to a novel fusion protein derived from coronavirus and a use thereof, specifically, a receptor binding domain (RBD) of coronavirus (SARS-CoV-2) or a fragment thereof; and a fusion protein comprising a Kappa light chain constant region of an antibody, and a vaccine composition against coronavirus comprising the fusion protein as an active ingredient.

Coronavirus is called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), or Coronavirus Infectious Disease 19 (COVID-19) or Covid-19 (COVID-19), named Corona 19 in South Korea. do. SARS coronavirus 2 was first discovered at the Huanan Seafood Market in Wuhan on December 12, 2019. It is an RNA virus and human-to-human infection was confirmed. SARS coronavirus 2 is a virus that requires handling in a biosafety level 3 research facility (BSL-3 facility), and the reproduction index (R0) of the virus is estimated to be 1.4 to 3.9. This means that one patient can transmit the virus to at least 1.4 to a maximum of 3.9 people, that is, it is estimated that the control of infectious diseases caused by SARS coronavirus 2 is quite difficult, and as of March 31, 2020, 785,867 infected people worldwide , the number of deaths was about 37,827. Symptoms such as fever, dyspnea, kidney and liver damage, cough, and pneumonia are observed for 2 to 14 days after infection with the virus, and in the case of perjury, death may occur.

However, little is known about the etiologic mechanism of this worldwide, and a therapeutic agent has not yet been developed. In this situation, research on vaccines to prevent spread to the community is urgently needed, but the virus is a high-risk pathogen, so in the case of inactivated and live vaccines, the risk is high in the production and administration of vaccine materials to humans, especially in the case of live vaccines. It takes a very long time to attenuate and prove safety. In addition, even in the case of several vaccines under development based on recombinant proteins, there is a problem of poor expression or low productivity.

Under this background, the inventors of the present invention have made diligent efforts to develop a vaccine applicable to coronavirus in terms of versatility, commercialization, and efficacy, and have completed the present invention by developing a fusion protein for a vaccine with significantly improved productivity.

Republic of Korea Patent Publication No. 10-2021-0123234

An exemplary object of the present invention is a receptor binding domain of coronavirus (SARS-CoV-2) (Receptor Binding Domain, RBD) or a fragment thereof; And to provide a recombinant protein comprising a Kappa light chain constant region of an antibody.

Another exemplary object of the present invention is to provide a gene encoding the fusion protein, a recombinant vector containing the same, and a cell containing the same.

Another exemplary object of the present invention is to provide a vaccine composition against coronavirus, comprising the fusion protein, a fragment thereof, or a variant thereof as an active ingredient.

Another exemplary object of the present invention is to provide a method of immunization against coronavirus, comprising administering a vaccine composition to a subject in need of immunization.

Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing a coronavirus infection or a disease or symptom related to infection, comprising a fusion protein, a fragment thereof, or a variant thereof as an active ingredient.

Another exemplary object of the present invention is a pharmaceutical for the treatment of coronavirus infection or a disease or symptom related to infection, including an antibody that binds to the fusion protein, or blood or serum isolated from a subject to which the fusion protein is administered. It is to provide an enemy composition.

Another exemplary object of the present invention is to use an antibody that binds to the fusion protein, or blood or serum isolated from a subject to which the fusion protein is administered, to treat a coronavirus infection or a disease or symptom related to infection. It is to provide a method for treating a disease and / or symptom related to infection or infection of coronavirus, including the step of administering to a subject.

Another exemplary object of the present invention is to provide a method for preparing a vaccine composition against coronavirus comprising culturing the cells.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and another problem not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

As one aspect for achieving the above object, the present invention provides a receptor binding domain of coronavirus (SARS-CoV-2) (Receptor Binding Domain, RBD) or a fragment thereof; And it provides a recombinant protein comprising a Kappa light chain constant region of an antibody.

The term SARS coronavirus 2 (SARS-CoV-2) as used herein refers to a new type of coronavirus that first occurred in Wuhan, China in December 2019 and then spread throughout China and around the world. The SARS-CoV-2 may be a wild-type SARS-CoV-2 or a mutant virus in which a specific amino acid residue of the wild-type virus is mutated. The mutant virus may be alpha-mutant SARS-CoV-2, beta-mutant SARS-CoV-2 or delta-mutant SARS-CoV-2 virus, but is not limited thereto.

In the present invention, SARS-CoV-2 infection broadly includes not only the infection of SARS-CoV-2 itself, but also various diseases, symptoms, and symptoms (eg, respiratory disease, pneumonia, etc.) resulting from the infection of the virus. It can be understood as a concept of

In the present invention, SARS-CoV-2 has a nucleotide of 30 kb and has four important structural proteins: Nucleocapsid, Spike, Membrane, and Envelope proteins. Among them, the spike protein is an important site as it binds to the receptor of the host cell, and replication is achieved by transferring the nucleocapsid into the cell.

The term "Receptor Binding Domain (RBD)" of the present invention is a part of the spike protein and is a key region that binds to the ACE2 (Angiotensin Converting Enzyme 2) receptor of the host cell, and is a strong anti-SARS-CoV-2 infection. Because it contains multiple conformational-dependent epitopes that induce neutralizing antibodies, it could be a key target for COVID-19 treatment and vaccine development.

The RBD is a polypeptide located at positions 319 to 541 of the full-length polypeptide sequence of the spike protein of SARS-CoV-2, and in the present invention, the RBD is the RBD of the delta mutant SARS-CoV-2 (SEQ ID NO: 5), It may be RBD (SEQ ID NO: 6) of wild-type SARS-CoV-2 or RBD (SEQ ID NO: 7) of beta-mutant SARS-CoV-2.

On the other hand, an antibody is composed of two identical heavy chains and two identical light chains, and the heavy chain and the light chain each have a constant region and a variable region. The term "Kappa light chain constant region" of the present invention refers to a light chain constant region of an antibody, specifically, it may be derived from a human or a mouse, and more specifically, a human-derived immunoglobulin kappa light chain constant region (GenBank Accession No. AWK57456.1), and more specifically, it may consist of the amino acid sequence of SEQ ID NO: 8.

The term "recombinant protein" of the present invention means that one or more polypeptides are combined into a single-stranded polypeptide through a peptide bond, and one or more genes including a target gene are fused by a recombinant method to form a single polypeptide. It refers to a recombinant protein made by translation into a peptide. In the present invention, the recombinant protein is a protein or peptide sequence of interest is fused to each other, and an amino acid cleavage sequence that can be specifically recognized and cleaved by a chemical or enzyme between the desired protein or peptide sequence red cleavage sequence).

In one embodiment of the present invention, the recombinant protein may be expressed in the form of a fusion protein, and may refer to a fusion protein in which the RBD and the amino acid sequence of the kappa light chain constant region are combined. This fusion protein has the advantage of significantly improving productivity and increasing the purification yield compared to RBD alone.

As described above, the fused recombinant protein (fusion protein) can be obtained by chemical synthesis or expression and purification by genetic recombination after two or more objects to be expressed together are determined.

In the present invention, the recombinant protein may include or consist of part or all of the amino acid sequence of any one of SEQ ID NOs: 1 to 3, and may be fragments or variants thereof. A peptide that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence above. It may be a polypeptide comprising or consisting of a sequence.

The recombinant protein may include fragments or variants of the recombinant protein. The fragment or variant may be included in the scope of the present invention if it has functional identity even if a portion of the polypeptide sequence of the recombinant protein is deleted, added, or substituted. The meaning of having the functional identity means that the recombinant protein defined by the sequence in this specification can achieve the desired effect.

In the present invention, the recombinant protein can be used for prevention or treatment of SARS-CoV-2.

In the present invention, the recombinant protein may further include a signal sequence.

As used herein, the term "signal sequence" is used to transfer the expressed recombinant protein to the endoplasmic reticulum, and may preferably consist of all or part of the amino acid sequence of SEQ ID NO: 4, but is fused with the recombinant protein. It is not limited to this, as long as it is a peptide that moves the recombinant protein to the endoplasmic reticulum when recombinant. In addition, the present invention includes a variant of SEQ ID NO: 4 as a signal sequence within the scope of the present invention. Specifically, the peptide may include an amino acid sequence having 90% or more, 95% or more, or 98% or more sequence homology with the amino acid sequence of SEQ ID NO: 4.

In the present invention, a gene encoding the recombinant protein, a recombinant vector containing the gene, and a cell containing the recombinant vector are provided.

In the present invention, the term "recombinant vector" usually refers to a double-stranded DNA fragment as a carrier into which a fragment of foreign DNA is inserted. As used herein, "foreign DNA" means DNA derived from a foreign species, or, in the case of origin from the same species, a substantially modified form from the original form, and a foreign gene is a specific target nucleic acid to be transcribed and encodes a polypeptide. do. In order to increase the expression level of the transformed gene in the host cell, the recombinant vector must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. The recombinant vector is a genetic construct containing essential regulatory elements operably linked to express the gene insert in cells of an individual, and standard recombinant DNA techniques can be used to prepare such a genetic construct. The type of recombinant vector is not particularly limited as long as it expresses the desired gene in various host cells of prokaryotic and eukaryotic cells and produces the desired protein. A vector capable of producing a large amount of an exogenous protein in a similar form is preferred. The recombinant vector preferably contains at least a promoter, a start codon, a gene encoding the desired protein, a stop codon and a terminator. In addition, DNA encoding a signal peptide, an enhancer sequence, an untranslated region (UTR) at the 5' end and 3' end of the gene of interest, a selectable marker region, or a replicable unit may be appropriately included.

In one embodiment of the present invention, a recombinant vector can be prepared by cloning a gene encoding a fusion protein using pPGXII, a highly efficient expression vector, as a host vector.

As used herein, the term “vector” refers to a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles, or simply latent genome inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host's genome or, in some cases, can integrate into the genome itself. As the plasmid is currently the most commonly used form of vector, the terms “plasmid” and “vector” are sometimes used interchangeably. However, the present invention includes other forms of vectors having functions equivalent to those known or becoming known in the art.

In the present invention, the cell containing the recombinant vector may be a recombinant cell obtained using an introduction method known in the art, such as transformation, or a transformant transformed with the recombinant vector. The type of these cells is not particularly limited, and, for example, Escherichia coli, yeast (eg, Saccharomyces cerevisiae, Pichia pastoris, etc.), animal cells (eg, CHO (Chinese Hamster Ovary) cells, HEK293 cells, Vero cells, Per. C6 cells, etc.), but may be one or more selected from the group consisting of, but is not limited thereto.

As used herein, the term “transformation” refers to changes in the genetic properties of organisms by injected DNA, and “transgenic organisms (transformants)” refer to external organisms by molecular genetic methods. As an organism produced by injecting a gene, preferably an organism transformed by the recombinant vector of the present invention, the organism is not limited as long as it achieves the object of the present invention.

As another aspect for achieving the above object, the present invention provides a vaccine composition against coronavirus, comprising the recombinant protein, a fragment thereof, or a variant thereof as an active ingredient.

The terms corona virus, receptor binding domain, kappa light chain constant region and recombinant protein of the present invention are as described above.

The term "vaccine composition" of the present invention is a biological preparation containing an antigen that induces an immune response in a living body, and is an immunogen that induces immunity in a living body by injection or oral administration to a subject, such as a human or animal, for the prevention of infection. say The animal is a human or non-human animal, and the non-human animal refers to pigs, cows, horses, dogs, goats, sheep, etc., but is not limited thereto.

In the present invention, the vaccine composition may be prepared by a conventional method well known in the art, and may optionally further include various additives that can be used in preparing a vaccine in the art.

The vaccine composition according to the present invention may include the recombinant protein and a pharmaceutically acceptable carrier. For example, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, as commonly used in formulations , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

The vaccine composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injection solutions according to conventional methods. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one or more excipients, for example, starch, calcium carbonate, sucrose, etc., in the lecithin-like emulsifier. It can be prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents. can be included Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and freeze-drying agents. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous preparations and suspending agents.

The route of administration of the vaccine composition according to the present invention is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, This includes sublingual or rectal. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Vaccine compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The dose of the vaccine composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. The amount required to induce an immune response in a subject without significant side effects may vary depending on the presence of the recombinant protein used as the immunogen and any excipients.

In the present invention, the vaccine composition may further include an adjuvant.

The immune enhancer refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase or affect an immune response. Typically, a carrier or auxiliary substance for an immunogen and/or other pharmacologically active substance or composition is usually meant. Typically, the term "adjuvant" is to be interpreted in a broad sense, a broad range of substances or strategies that can enhance the immunogenicity of an antigen incorporated into or administered with the adjuvant. means (stratagem). In addition, the immune enhancer may include, but is not limited to, an immune potentiator, an antigen delivery system, or a combination thereof. The immune enhancing material may be alum, but any type of aluminum salt or polymer suitable for use as an immune enhancing material may be used in the present invention. Aluminum salts include aluminum hydroxide (Al(OH) ₃ ), aluminum phosphate (AlPO ₄ ), aluminum hydrochloride, aluminum sulfate, ammonium alum, potassium alum or aluminum silicate.

As another aspect for achieving the above object, the present invention provides a method for immunizing against coronavirus, comprising administering the vaccine composition to a subject in need of immunization.

In the present invention, the term "individual" refers to all animals, including humans, who have already been infected with or can be infected with SARS-CoV-2. By administering the vaccine composition of the present invention to a subject, the disease can be effectively prevented and treated. Specifically, the subject may include mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows, and in some cases, humans may be excluded.

In the present invention, the term "immunization" refers to antibodies, B cells, helper T cells, suppressor T cells, cytotoxic T cells, and gamma- The meaning includes, but is not limited to, one or more of the following effects: production or activation of delta T cells, displaying a therapeutic or protective immunological response in the host, thereby enhancing resistance to a new infection or reducing the clinical severity of a disease. Preferably it may be a protective immune response.

The vaccine composition of the present invention can be used for the purpose of preventing or treating coronavirus.

In the present invention, "prevention" refers to all activities that inhibit SARS-CoV-2 infection or delay the onset by administration of a vaccine composition. In addition, "treatment" refers to all activities that improve or beneficially change symptoms caused by SARS-CoV-2 infection by administration of a vaccine composition.

As another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection or a disease or symptom related to infection, comprising a fusion protein, a fragment thereof, or a variant thereof as an active ingredient. .

As another aspect for achieving the above object, the present invention provides an antibody that binds to the fusion protein, or a disease or symptom related to coronavirus infection or infection, including blood or serum isolated from an individual to whom the fusion protein is administered. It provides a pharmaceutical composition for the treatment of.

As another aspect for achieving the above object, the present invention provides an antibody that binds to the fusion protein, or blood or serum isolated from an individual to whom the fusion protein is administered, for infection with coronavirus or a disease or symptom related to infection. Provided is a method of treating a disease and/or symptom associated with infection or infection of coronavirus, comprising administering to a subject in need of treatment.

The term "pharmaceutical composition" of the present invention means prepared for the purpose of preventing or treating SARS coronavirus 2 infection, and may be formulated and used in various forms according to conventional methods.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent according to conventional methods. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specific reference may be made to the pharmacopoeia of each country including the 'Korean Pharmacopoeia'. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, carriers, excipients and diluents that may be included in the composition of the present invention may be non-natural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. . Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the compound, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The specific formulation of the pharmaceutical composition is known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). These documents are considered as part of this specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmacologically effective amount means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. It may be determined according to the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. The dosage and frequency do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, dogs, cats, cows, horses, pigs, and humans through various routes, and may be preferably human. Any mode of administration may be expected, and may be administered by, for example, oral, intravenous, intramuscular or subcutaneous injection, but is not limited thereto.

As another aspect for achieving the above object, the present invention provides a method for preparing a vaccine composition against coronavirus comprising culturing cells containing the fusion protein.

In the present invention, the preparation method may further include culturing cells containing the fusion protein and purifying the fusion protein therefrom.

The vaccine composition containing the recombinant protein according to the present invention can be usefully used as a vaccine for preventing and treating SARS-CoV-2 virus infection by inducing an immune response capable of fighting SARS-CoV-2 infection.

Figure 1 shows the productivity of the wild-type SARS-CoV-2 receptor binding domain.
2 shows the productivity of the RBD(WT)-C _L fusion protein.
Figure 3 shows the productivity of the fusion protein of the present invention at the well level.
Figure 4 shows the productivity of the fusion proteins of the present invention at the flask level.
5 shows expression patterns of fusion proteins using Western blotting under reducing conditions and non-reducing conditions.
Figure 6 shows the productivity of the fusion protein of the present invention in the culture process.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Production of fusion protein-producing cells of the present invention and analysis of productivity

1-1. Preparation of wild type (WT) SARS-CoV-2 receptor binding domain (RBD) and transduced cells

The RBD of wild-type SARS-CoV-2 (RBD(WT), SEQ ID NO: 6) was synthesized from the SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank Accession No. NC_045512.2). The synthesized RBD gene was cloned into an expression vector (pPGXII) to prepare plasmid DNA for transduction. The plasmid DNA was transduced into CHO DG44 (dhfr-/-) cells simultaneously with the pDCH1P plasmid containing the dhfr gene.

1-2. Production of wild-type fusion protein-producing cells and analysis of productivity

The human-derived immunoglobulin kappa light chain constant region (GenBank Accession No. AWK57456.1, C _L, SEQ ID NO: 8) was ligated to prepare a synthetic gene (SEQ ID NO: 2).

CHO cells were prepared by simultaneously transducing the plasmid DNA prepared by cloning the wild-type RBD(WT)-C _L synthetic gene prepared above into an expression vector (pPGXII) and the pDCH1P plasmid containing the dhfr gene. Example 1- After culturing the initially adapted cell pool of each of the transduced cells prepared in 1 to secure a certain number of cells, the same number of cells were inoculated and cultured for 4 days in a 12 well plate to confirm productivity.

As a result, in the case of RBD (WT), expression was confirmed by dot blot, but productivity was so low that Western-blot could not be performed (Fig. 1). On the other hand, in the case of RBD(WT) _-CL , it was confirmed that productivity increased significantly compared to RBD(WT) (FIG. 2).

Example 2: Analysis of wild-type and variant fusion protein productivity

2-1. Manufacture of mutant fusion protein-producing cells

The receptor binding domain (RBD, SEQ ID NO: 5) of the delta-mutant SARS-CoV-2 to which the signal sequence (MGWSCIILFLVATATGVHS, SEQ ID NO: 4) is attached to 5' and the RBD of the beta-mutant SARS-CoV-2 (SEQ ID NO: 4) 7) Human-derived immunoglobulin kappa light chain constant region in each gene (GenBank Accession No. AWK57456.1, _CL , SEQ ID NO: 8) A synthetic gene was prepared by ligating the gene. The amino acid sequence of each synthetic gene is shown in Table 1.

synthetic gene RBD amino acid sequence variation compared to WT sequence number RBD(Delta)-C _L L452R, T478K One RBD(Beta)-C _L K417N, E484K, N501Y 3

The two prepared synthetic genes were cloned into an expression vector (pPGXII) to prepare plasmid DNA for transduction. CHO DG44 (dhfr-/-) adapted to suspension culture was transfected with each RBD-C _L expression plasmid DNA and the pDCH1P plasmid containing the dhfr gene.

2-2. Analysis of wild-type and variant fusion protein productivity at the well level

Wild-type (RBD(WT) _-CL ), delta mutant (RBD(Delta) _-CL ) and beta-mutant (RBD(beta) _-CL ) fusion protein producing cells. During the manufacturing process, the initially adapted cell population (Pool) grows When a certain number of cells were secured, the same number of cells were inoculated into a 12-well plate and cultured for 4 days to compare and analyze productivity. As a result of the analysis, it was confirmed that the productivity of the delta mutant (RBD(Delta) _-CL ) was significantly higher than that of the wild type (RBD(WT) _-CL ). Beta mutation (RBD (beta) -C _L ) also confirmed that productivity was significantly lower than that of delta mutation (Fig. 3, Table 2).

recombinant protein Productivity (mg/L) RBD(WT)-C _L 0.3 RBD(Beta)-C _L 5.2 RBD(Delta)-C _L 31.6

2-3. Analysis of wild-type and variant fusion protein productivity at the flask level

The productivity of the fusion protein at the flask level was confirmed. Specifically, when a sufficient number of cells were secured using cells obtained from the initially adapted cell group (Pool), the same number of cells were inoculated into a 125 mL Erlenmeyer flask, batch culture was performed for 10 days, and productivity was measured through ELISA analysis. compared. As a result of the analysis, the highest productivity was confirmed in the case of RBD(Delta)-C _L , equal to the expression level at the well level, followed by RBD(beta)-C _L , RBD(WT)-C _L , in order. Productivity was confirmed (FIG. 4, Table 3).

recombinant protein Productivity (mg/L) RBD(WT)-C _L 0.9 RBD(Beta)-C _L 71.0 RBD(Delta)-C _L 417.7

Western blotting was performed to compare the pattern of expressed proteins using the culture medium obtained from the batch culture. As a result of the analysis, the expression patterns of the three RBD(WT)-C _L , RBD(beta)-C _L and RBD(Delta)-C _L proteins were found to be similar in both non-reducing and reducing conditions (FIG. 5).

2-4. Analysis of wild-type and mutant fusion protein productivity after development of culture process

In addition, the productivity of the fusion protein was confirmed after the development of the culture process. Specifically, in order to secure a cell line derived from a single cell from the initially adapted cell group (Pool), single clonal selection was performed according to a limiting dilution method. Among the single cell lines, a cell line with excellent productivity was selected to develop a culture process for mass production. Fed-batch culture was performed by applying a combination of commercial media and media additives and various culture temperatures. As a result of comparative analysis of the productivity of the most productive conditions based on the fed-batch culture results, the highest productivity (>2.5 g / L) was confirmed in the case of RBD (Delta)-C _L , followed by RBD (beta ) _-CL , RBD (WT) _-CL productivity was confirmed in the order (Fig. 6).

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

<110> Pangen Biotech Inc. <120> Novel fusion protein derived from SARS-CoV-2 and Use It <130> KPA2021-321 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 349 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 1 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn 20 25 30 Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe 35 40 45 Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala 50 55 60 Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys 65 70 75 80 Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val 85 90 95 Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala 100 105 110 Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp 115 120 125 Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser 130 135 140 Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser 145 150 155 160 Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala 165 170 175 Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro 180 185 190 Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro 195 200 205 Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr 210 215 220 Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val 225 230 235 240 Asn Phe Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 245 250 255 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 260 265 270 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 275 280 285 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 290 295 300 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 305 310 315 320 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 325 330 335 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 340 345 <210> 2 <211> 349 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn 20 25 30 Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe 35 40 45 Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala 50 55 60 Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys 65 70 75 80 Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val 85 90 95 Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala 100 105 110 Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp 115 120 125 Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser 130 135 140 Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser 145 150 155 160 Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala 165 170 175 Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro 180 185 190 Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro 195 200 205 Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr 210 215 220 Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val 225 230 235 240 Asn Phe Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 245 250 255 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 260 265 270 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 275 280 285 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 290 295 300 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 305 310 315 320 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 325 330 335 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 340 345 <210> 3 <211> 349 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn 20 25 30 Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe 35 40 45 Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala 50 55 60 Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys 65 70 75 80 Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val 85 90 95 Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala 100 105 110 Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp 115 120 125 Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser 130 135 140 Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser 145 150 155 160 Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala 165 170 175 Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro 180 185 190 Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro 195 200 205 Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr 210 215 220 Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val 225 230 235 240 Asn Phe Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 245 250 255 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 260 265 270 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 275 280 285 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 290 295 300 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 305 310 315 320 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 325 330 335 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 340 345 <210> 4 <211> 19 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 5 <211> 223 <212> PRT <213> SARS-CoV-2 <400> 5 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 6 <211> 223 <212> PRT <213> SARS-CoV-2 <400> 6 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 7 <211> 223 <212> PRT <213> SARS-CoV-2 <400> 7 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 8 <211> 107 <212> PRT <213> Homo sapiens <400> 8 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims (16)

Receptor Binding Domain (RBD) of coronavirus (SARS-CoV-2) or a fragment thereof; and a Kappa light chain constant region of an antibody.
According to claim 1,
A fusion protein comprising all or part of the amino acid sequence of any one of SEQ ID NOs: 1 to 3.
According to claim 1,
A fusion protein comprising at least one mutation in the amino acid sequence of any one of SEQ ID NOs: 1 to 3.
According to claim 1,
Wherein the receptor binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 5 to 7, the fusion protein.
According to claim 1,
The kappa light chain constant region of the antibody comprises the amino acid sequence of SEQ ID NO: 8, the fusion protein.
According to claim 1,
Wherein the fusion protein further comprises a signal sequence, the fusion protein.
A gene encoding the fusion protein according to claim 1.
A recombinant vector comprising the gene according to claim 7.
A cell comprising the gene according to claim 7 or the recombinant vector according to claim 8.
A vaccine composition against coronavirus, comprising the fusion protein of claim 1, a fragment thereof, or a variant thereof as an active ingredient.
According to claim 10,
The vaccine composition is characterized in that it further comprises an immune enhancer (Adjuvant), the vaccine composition.
A method of immunization against coronavirus, comprising administering the vaccine composition of claim 10 to a subject in need of immunization.
A pharmaceutical composition for preventing a coronavirus infection or infection-related disease or symptom, comprising the fusion protein of claim 1, a fragment thereof, or a variant thereof as an active ingredient.
A pharmaceutical composition for treating coronavirus infection or a disease or symptom related to infection, comprising an antibody binding to the fusion protein of claim 1 or blood or serum isolated from an individual to whom the fusion protein is administered.
Administering an antibody that binds to the fusion protein of claim 1, or blood or serum separated from an individual to whom the fusion protein is administered, to a subject in need of treatment for infection or a disease or symptom related to infection with coronavirus A method of treating a disease and/or symptom associated with infection or infection with a coronavirus.
A method for preparing a vaccine composition against coronavirus comprising the step of culturing the cells of claim 9.