Novel CRS fragment peptide with immunological enhancement activity and application thereof

Abstract

The invention relates to a novel CRS fragment peptide with immunological enhancement activity and application of the novel CRS fragment peptide. More specifically, the present invention relates to a novel peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto and a vaccine adjuvant, their use as a cancer therapeutic agent and an antiviral composition. The peptides disclosed herein are CRS fragment peptides disclosed herein for the first time, exhibiting anticancer activity, immunological enhancement and antiviral activity.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application Nos. 10-2022-0070283 and 10-2022-0070287, filed on Jun. 9, 2022, the disclosures of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to novel CRS fragment peptides with immune enhancing activity and uses thereof, and more specifically, to novel peptides consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto, and their uses as vaccine adjuvants, cancer therapeutic agents and antiviral agents. Background Art

[0003] Aminoacyl-tRNA synthetases (ARS or AARS) catalyze the aminoacylation of tRNA molecules and are essential for decoding genetic information during translation. Each eukaryotic tRNA synthetase consists of a core enzyme (closely related to the prokaryotic copy of the tRNA synthetase) and additional domains (attached to the amino or carboxyl terminus of the core enzyme). Therefore, there are significant differences in the composition of these enzymes between eukaryotes and prokaryotes. For example, human tyrosyl-tRNA synthetase (TyrRS) has a carboxyl terminal domain, which is absent in TyrRS molecules of prokaryotes and lower eukaryotes.

[0004] Recently, several aminoacyl-tRNA synthetases have been shown to have atypical functions that are unrelated to their involvement in the translation process. Specifically, some fragments of ARS proteins exhibit extracellular signaling activities that regulate different types of pathways outside of protein translation, showing unexpected activities that are unrelated to aminoacylation. These unexpected activities may sometimes have therapeutic effects on certain diseases, but they may also pose a risk of inducing pathological conditions in humans. For example, lysyl-tRNA synthetases (KRS) are known to have the activity of promoting cancer metastasis. In addition, the N-terminal domain of TRS is called mini-tyrosyl tRNA synthetase (mini-TRS, corresponding to amino acid residues 1-364), which is cleaved by polymorphonuclear cell elastase and plasmin, showing atypical biological activities not found in the full-length protein. In vitro, mini-TRS was shown to stimulate endothelial cell proliferation and migration, and has pro-angiogenic activity in mouse matrigel assays. The function of promoting angiogenesis is often closely related to cancer metastasis.

[0005] These unexpected activities are not observed in the natural full-length protein sequence (or show negligible effects at the natural full-length protein level), but can show significant specific activities when certain regions are isolated. In addition, these effects may have characteristics that are not suitable for therapeutic use. In order to overcome the difficulties associated with this unpredictability and utilize the therapeutic potential of these ARS family proteins, various efforts need to be made to identify the biologically relevant forms of other aminoacyl-tRNA synthetase proteins.

[0006] In addition, although vaccination is currently the best method for preventing viral diseases, the effectiveness of vaccines is an important issue because many viral serotypes (subtypes) can be produced in viral diseases. It is necessary to develop antiviral compositions that can compensate for these vaccine problems, such as compositions for preventing or treating viral infections or adjuvants that can enhance the effectiveness of viral vaccines.

[0007] In particular, in January 2020, SARS-CoV-2, identified as the cause of COVID-19, spread across the globe and was declared a global pandemic by the World Health Organization (WHO). The novel coronavirus is reported to be highly contagious and pathogenic, causing severe pneumonia symptoms in infected patients. The virus has been found to pose a serious threat, especially to the elderly and / or individuals with underlying diseases such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer. However, the development of agents that can effectively prevent and treat infection by novel viruses such as SARS-CoV-2 has not yet been completed. Summary of the invention

[0008] Issues to be resolved

[0009] The inventors of the present invention have previously discovered that fragment peptides of cysteinyl-tRNA synthetase (hereinafter referred to as "CRS") have anticancer activity and immunoenhancing activity. However, the inventors confirm that it is difficult to develop it as a drug due to the aforementioned limitations. Therefore, the inventors have conducted extensive research to develop a novel CRS fragment peptide that can overcome these limitations. As a result, the inventors found that compared with conventional CRS fragment peptides, a peptide comprising continuous amino acids from any amino acid from the 101st to the 140th amino acid in the amino acid sequence selected from SEQ ID NO: 1 to the 200th amino acid has the same anticancer activity and immunoenhancing activity, while maintaining its monomeric form, does not degrade without an affinity tag, and is very stable even at high temperatures, thereby completing the present invention.

[0010] Therefore, another object of the present invention is to provide a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, comprising the following substance as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0011] Another object of the present invention is to provide a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, which is composed of the following substances: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0012] Another object of the present invention is to provide a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, which is essentially composed of a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0013] Another object of the present invention is to provide an adjuvant composition for a viral vaccine, comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0014] Another object of the present invention is to provide a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof for use in preparing a pharmaceutical composition for antiviral use.

[0015] Another object of the present invention is to provide a method for preventing or treating viral infection, comprising administering to a subject in need thereof an effective amount of a composition comprising the following substance as an active ingredient: a peptide comprising consecutive amino acids selected from any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228, or a peptide having an amino acid sequence having 80% or higher homology with the peptide.

[0016] One object of the present invention is to provide a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto.

[0017] Another object of the present invention is to provide a polynucleotide comprising a nucleotide sequence encoding the peptide.

[0018] Another object of the present invention is to provide a vector comprising the polynucleotide.

[0019] Another object of the present invention is to provide a host cell transformed with the vector.

[0020] Another object of the present invention is to provide a vaccine adjuvant comprising one or more selected from the following:

[0021] (i) the peptide,

[0022] (ii) a polynucleotide encoding (i),

[0023] (iii) a vector comprising (ii), and

[0024] (iv) using the host cell transformed with (iii).

[0025] Another object of the present invention is to provide a vaccine composition comprising the vaccine adjuvant and an antigen.

[0026] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising one or more selected from the following (i)-(iv):

[0027] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which is composed of one or more selected from the following (i)-(iv):

[0028] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which is essentially composed of one or more selected from the following (i)-(iv):

[0029] (i) the peptide,

[0030] (ii) a polynucleotide encoding (i),

[0031] (iii) a vector comprising (ii), and

[0032] (iv) using the host cell transformed with (iii).

[0033] Means of solving the problem

[0034] In order to achieve the above-mentioned object of the present invention, the present invention provides a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, which comprises the following substance as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0035] In addition, in order to achieve the purpose of the present invention, the present invention provides a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, which is composed of a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0036] In addition, in order to achieve the purpose of the present invention, the present invention provides a pharmaceutical composition, a food composition or a quasi-drug composition for antiviral use, which is basically composed of a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0037] In order to achieve another object of the present invention, the present invention provides an adjuvant composition for a viral vaccine, which comprises the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0038] In order to achieve another object of the present invention, the present invention provides a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for antiviral use.

[0039] In order to achieve another object of the present invention, the present invention provides a method for preventing or treating viral infection, comprising administering to a subject in need thereof an effective amount of a composition comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228; or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide.

[0040] To achieve another object of the present invention, the present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto.

[0041] In order to achieve another object of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the peptide.

[0042] In order to achieve another object of the present invention, the present invention provides a vector comprising the polynucleotide.

[0043] In order to achieve another object of the present invention, the present invention provides a host cell transformed with the vector.

[0044] In order to achieve another object of the present invention, the present invention provides a vaccine adjuvant comprising one or more selected from the following:

[0045] (i) the peptide,

[0046] (ii) a polynucleotide encoding (i),

[0047] (iii) a vector comprising (ii), and

[0048] (iv) using the host cell transformed with (iii).

[0049] In order to achieve another object of the present invention, the present invention provides a vaccine composition, which comprises the vaccine adjuvant and an antigen.

[0050] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising one or more selected from the following (i)-(iv):

[0051] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which is composed of one or more selected from the following (i)-(iv):

[0052] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which is essentially composed of one or more selected from the following (i)-(iv):

[0053] (i) the peptide,

[0054] (ii) a polynucleotide encoding (i),

[0055] (iii) a vector comprising (ii), and

[0056] (iv) using the host cell transformed with (iii).

[0057] Hereinafter, the present invention will be described in detail.

[0058] The practice of the present invention, unless otherwise specified, employs conventional methods of molecular biology and recombinant DNA technology within the technical field to which the present invention belongs, and most of the descriptions are well known and are for illustrative purposes.

[0059] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

[0060] Throughout the disclosure, various aspects or conditions associated with the present invention may be presented in the form of a range. Unless otherwise indicated, the description of range values ​​includes boundary values ​​(including endpoints), which means that it includes all values ​​from the lower limit to the upper limit. The description of the range is for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the present invention. Therefore, the description of the range should be considered as not only specifically disclosing a single numerical value in the range, but also disclosing all possible sub-ranges. For example, the description of a range such as 7 to 170 should be considered as specifically disclosing a single value in the range, such as 9, 27, 35, 101 and 155, and sub-ranges such as 10 to 127, 23 to 35, 80 to 100 and 50 to 169. This applies regardless of the width of the range.

[0061] As used in this specification, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components, elements or method steps not mentioned in the composition or method. The term "consisting of..." excludes any additional elements, steps or components not explicitly mentioned. The term "consisting essentially of..." indicates that the composition or method may include additional elements or steps that do not substantially affect the basic characteristics of the composition or method.

[0062] The terms "peptide" and "protein" as used herein are used in their conventional sense to refer to the arrangement of amino acids. Although peptides are not limited to a specific length, in the context of the present invention, they generally refer to fragments of full-length proteins and may include post-translational modifications such as glycosylation, acetylation, phosphorylation, and other modifications known in the art (naturally occurring and non-naturally occurring modifications), and may be referred to as "polypeptides". The peptides and proteins of the present invention can be produced using any of a variety of known recombinant and / or synthetic techniques, and exemplary embodiments will be further described below.

[0063] The present invention arises from the discovery that CRS and CRS-derived peptides possess therapeutically relevant atypical biological activities.

[0064] In this specification, "atypical activity" generally refers to the activity that the CRS peptide of the present invention has in addition to adding cysteine ​​to the tRNA molecule. As described in detail herein, in certain embodiments, the atypical biological activity exhibited by the CRS fragment of the present invention can be selected from but not limited to anti-cancer activity, activation of innate immunity and activation of adaptive immunity.

[0065] The present invention should be understood to include not only CRS fragment peptides having at least one atypical biological activity, but also variants that substantially retain said atypical activity.

[0066] Specifically, the inventors confirmed that the novel CRS fragment peptide is active in activating both innate and adaptive immunity and is structurally very stable. The present invention discloses for the first time the region of the CRS fragment peptide.

[0067] In the present invention, the peptide is a cleavage form of the CRS protein, comprising amino acids 140 to 200 in the full-length CRS protein sequence of SEQ ID NO: 1, or amino acids having 95% or higher sequence homology thereto, and retaining at least one desired atypical biological activity.

[0068] The present invention provides a food composition for antiviral purposes, which comprises the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0069] In addition, the present invention provides a food composition for antiviral purposes, which is composed of the following substances: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0070] In addition, the present invention provides a food composition for antiviral purposes, which is basically composed of the following substances: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0071] The description of each ingredient in the food composition can be referred to the above details.

[0072] The food composition of the present invention includes all types of food in the conventional sense, including functional food, nutritional supplements, health food and food additives. These types of food compositions can be made into various forms according to conventional methods known in the art.

[0073] The food composition of the present invention may include health functional foods. The term "health functional foods" used in the present invention refers to foods manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients that have beneficial functionality to the human body. Here, "functionality" refers to obtaining effects useful for health purposes, such as regulating nutrition or physiological effects related to human body structure and function. The health functional foods of the present invention can be manufactured by methods commonly used in the art, and can include raw materials and ingredients commonly added in the art during the manufacturing process.

[0074] In addition, as long as it is identified as a health functional food, the dosage form of the health functional food can be manufactured without restriction. The food composition of the present invention can be manufactured into a variety of dosage forms, and because it uses food as a raw material, it has the advantage of not causing side effects that may be caused by long-term use of drugs, unlike general medicines. It is highly portable, and the health functional food of the present invention can be eaten as an adjuvant to enhance the improvement or treatment effect.

[0075] For example, health functional food can be consumed in the form of tea, juice, beverage, or in the form of granules, capsules, or powders. In addition, it can be mixed with known substances or active ingredients known to have preventive, ameliorative, or therapeutic effects to be manufactured in the form of a composition.

[0076] In addition, food composition of the present invention can contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerine, alcohol, the carbonating agent used in carbonated beverage etc. It can also contain the pulp used to make natural fruit juice, fruit juice beverage and plant beverage. These compositions can be used alone or in combination. The ratio of these additives is not particularly important, but generally selects in the range of 0.01 to 0.3 weight portion based on per 100 weight portions of food composition of the present invention, but is not limited thereto.

[0077] In addition, food composition of the present invention can contain multiple flavoring agents or natural carbohydrates as additional ingredients like conventional beverages.Carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.Natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used as sweeteners.The ratio of natural carbohydrates is generally about 0.01 to 0.04 gram per 100 milliliters of compositions of the present invention, preferably about 0.02 to 0.03 gram, but is not limited thereto.

[0078] The present invention also provides a quasi-drug composition for antiviral use, comprising the following substance as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0079] In the "quasi-drug composition for antiviral use" described in the present invention, quasi-drug refers to a preparation for preventing infectious diseases for sterilization, insecticide and similar purposes as described in Article 2, Paragraph 7 of the Korea Pharmaceutical Affairs Act, which may refer to an insect repellent, control agent, preventive agent, control agent or attractant insecticide for flies, mosquitoes, etc. used for human or animal health.

[0080] In addition, quasi-drugs may include skin external preparations and personal hygiene products. For example, it may be a disinfectant cleaner, nasal spray, shower foam, mouthwash, wet wipes, washing soap, hand sanitizer, humidifier refill, facial mask, ointment, patch or filter refill, but is not limited thereto.

[0081] When the quasi-drug composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and it may be used appropriately according to conventional methods. The blending amount of the active ingredient may be appropriately determined according to the intended use.

[0082] The quasi-drug composition of the present invention can be manufactured in the form of, for example, a general emulsified preparation and a solubilized preparation. For example, it can have dosage forms such as lotions, creams, ointments, sprays, oil gels, gels, oils, aerosols, and fumigants, but is not limited to these as long as it exhibits the pest control inducing effect of the present invention. In addition, when necessary, the quasi-drug composition can be appropriately mixed and used with oil, water, surfactants, moisturizers, lower alcohols having 1 to 4 carbon atoms, thickeners, chelating agents, pigments, preservatives, or fragrances, which are generally mixed in quasi-drug compositions.

[0083] The present invention also provides an adjuvant composition for a viral vaccine, comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof.

[0084] In the present invention, an adjuvant can be defined as a substance that can present an antigen to the immune system in a manner that induces an immune response or induces an increased immune response to the antigen when the viral antigen is administered together with the adjuvant. That is, an adjuvant is a substance that enhances the immune response to the viral antigen, which means that it is a substance that is not an immunogen to the host but enhances immunity by increasing the activity of immune system cells.

[0085] According to one embodiment of the present invention, it has been demonstrated that CRS fragment peptides can significantly enhance the increase in the immune response of a subject when treated together with a viral antigen.

[0086] In one aspect of the invention, an adjuvant when administered together with a viral antigen can induce improved activation of the innate and adaptive immune responses, more specifically, can induce improved activation of the cellular and humoral immune responses, compared to when the viral antigen itself is administered.

[0087] The present invention also provides a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for antiviral use.

[0088] The present invention also provides a method for preventing or treating viral infection, comprising administering to a subject in need thereof an effective amount of a composition comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1; or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide.

[0089] In one aspect of the present invention, the peptide is a peptide consisting of a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228, and comprises a mutation, wherein the mutation is a substitution of cysteine ​​at position 182 in the amino acid sequence of SEQ ID NO: 1 by other amino acids. That is, it may include a mutation in which cysteine ​​at position 43 in the amino acid sequence of SEQ ID NO: 2 is substituted by other amino acids, and the substitution can not only inhibit the formation of multimers of CRS fragment peptides, but also maintain high stability without degradation even when the N-terminus and the C-terminus are not connected to affinity tags.

[0090] In a preferred aspect of the present invention, the peptide is characterized in that the cysteine ​​at position 182 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine. That is, it may include a mutation in which the cysteine ​​at position 43 in the amino acid sequence of SEQ ID NO: 2 is substituted with other amino acids (SEQ ID NO: 3). Most preferably, the peptide is characterized in that it consists of the amino acid sequence of SEQ ID NO: 2 to 8 or an amino acid sequence having 80% or higher homology thereto.

[0091] The peptide of the present invention can be made using available techniques known in the art. For example, it can be made using a variety of proteolytic enzymes. Exemplary proteases include, for example, achromopeptidase, aminopeptidase, ancrod, angiotensin converting enzyme, bromelain, calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase G, carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, cathepsin B, cathepsin C, cathepsin D , cathepsin E, cathepsin G, cathepsin H, cathepsin L, chymopapain, chymase, chymotrypsin, clostripain, collagenase, complement C1r, complement C1s, complement factor D, complement factor I, cucumisin, dipeptidyl peptidase IV, leukocyte elastase (elastase, leukocyte), pancreatic elastase (elastase, pancreatic), endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A (granzyme A), granzyme B, HIV protease, IGase, kallikrein tissue, universal leucine aminopeptidase, cytosolic leucine aminopeptidase, microsomal leucine aminopeptidase, matrix metalloproteinase, methionine aminopeptidase, neutral protease, papain, pepsin, plasmin, prolidase, pronase E, prostate-specific antigen, alkaline protease from Streptomyces griseus, protease from Aspergillus, protease from Aspergillus saitoi, protease from Aspergillus sojae, alkaline protease from Bacillus licheniformis (protease B.licheniformis, alkaline or alcalase, protease from Bacillus polymyxa, protease from Bacillus sp., protease from Rhizopus sp., proteinase S, proteasome, protease from Aspergillus oryzae, proteinase 3, proteinase A, proteinase K, protein C, pyroglutamate aminopeptidase, rennin, streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase, and urokinase. One skilled in the art can readily determine which proteolytic enzyme is appropriate, depending on the chemical specificity of the fragment to be produced.

[0092] The polypeptides described herein can be prepared by any suitable method known to those skilled in the art, such as recombinant techniques. In addition to recombinant production methods, the polypeptides of the present invention can also be prepared by direct peptide synthesis using solid phase techniques.

[0093] Solid phase peptide synthesis (SPPS) can initiate the synthesis by attaching functional units called linkers to small porous beads, allowing the peptide chain to be extended. Unlike liquid phase methods, the peptide is covalently bound to the beads, preventing it from being washed away during filtration until it is cleaved by a specific reagent such as trifluoroacetic acid (TFA). The synthesis involves cycles of protection, deprotection, and coupling processes, in which the N-terminal amine of the attached peptide is bound to the N-protected amino acid unit, then deprotected and coupled to the new amino acid. This SPPS method can be performed using microwave technology, which applies heat to shorten the time required for coupling and deprotection in each cycle. The thermal energy helps prevent the unfolded peptide chain from folding or aggregating and promotes chemical bonding.

[0094] In addition, the peptides of the present invention can be synthesized by known methods using liquid phase peptide synthesis. The peptides of the present invention can be synthesized by a variety of methods, including a combination of solid phase and liquid phase synthesis methods, and the production method is not limited to the methods described herein.

[0095] Protein synthesis can be performed manually or by automation. Automated synthesis can be achieved using, for example, an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be synthesized chemically separately, and then chemical methods are used to combine the fragments to produce the target molecule.

[0096] Peptides provided by the present invention include variants with 95% or higher sequence homology with the peptides. These variants refer to active variants of peptides, which means that they retain at least one desired unconventional activity (e.g., anticancer activity and immunopotentiating activity) of the peptides derived from them. An example of such a variant can be a splice variant (whether natural or non-natural), which retains at least one unconventional activity described herein. Another example can be a variant with one or more point mutations in the peptide sequence (whether natural or non-natural), which retains at least one unconventional activity described herein. In other words, the term "active variant" in the present invention is understood to be the functional equivalent of the peptide of SEQ ID NO:2.

[0097] More specifically, the variant is characterized in that it is a functional equivalent and has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology with the aforementioned peptide sequence along its length.

[0098] In one aspect of the present invention, the peptide may consist of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0099] Variants may include any modification of the amino acid sequence of the "peptide", including one or more substitutions, deletions, additions and / or insertions. Such variants may occur naturally or may be produced synthetically by modifying or altering one or more peptide sequences of the invention and evaluating their biological activity as described herein using any of a variety of techniques known in the art.

[0100] In one embodiment of the invention, variants include conservative substitutions. "Conservative substitutions" refer to substitutions in which one amino acid is replaced with another amino acid having similar properties, such that one skilled in the art can predict that the secondary structure and hydrophilicity (hydrophobicity or hydrophilicity) of the peptide will remain essentially unchanged. Typically, the following groups of amino acids have conservative substitutions: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0101] Modifications can be made within the structure of the peptides of the present invention to obtain functional molecules encoding peptide variants or derivatives with desired properties. In order to produce equivalent or improved variants of the peptides of the present invention, one or more codons can be changed based on protein codon information known in the art.

[0102] For example, specific amino acids can be replaced within a protein or peptide structure without significant loss of interaction binding capacity, such as a receptor binding site, an antibody antigen binding site, or a substrate molecule. This is because the interaction capacity and properties of a protein determine its biological functional activity, allowing specific amino acid sequence substitutions within a protein or peptide sequence and the corresponding DNA coding sequence while still obtaining a protein with the same or similar properties.

[0103] Therefore, various changes in the disclosed peptide sequence or the DNA sequence encoding the peptide are considered without significant loss of the desired utility or activity. These modifications can also take into account the hydropathic index of amino acids. The importance of the amino acid hydropathic index is generally understood in the art, and it confers biological functions of protein interactions. For example, the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which ultimately determines its interaction with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These values ​​are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0104] It is known in the art that specific amino acids can be substituted for other amino acids having a similar hydropathic index or score to obtain a protein with similar biological activity (i.e., still obtain a biologically functionally equivalent protein). In such changes, amino acids with hydropathic indices within ±2 are preferred for substitution, amino acids within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0105] It is also understood in the art that substitutions of identical amino acids can be made effectively based on hydrophilicity. As is well known, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that one amino acid can be substituted for another amino acid having a similar hydrophilicity value and a biologically equivalent protein can be obtained. In such modifications, it is preferred to substitute amino acids having hydrophilicity values ​​within ±2, particularly preferred to substitute amino acids having hydrophilicity values ​​within ±1, and even more particularly preferred to substitute amino acids having hydrophilicity values ​​within ±0.5.

[0106] As described above, amino acid substitutions can be based on the relative similarity of the amino acid side-chain substituents, for example, based on their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various characteristics described above are well known to those of skill in the art and include the following: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0107] Amino acid substitutions may also be made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with similar hydrophilicity values ​​that do not have an electrically charged polar head group include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine, and tyrosine.

[0108] Variants can also include non-conservative changes. In preferred embodiments, variant peptides can differ from the native sequence by substitution, deletion or addition of five or fewer amino acids. Variants can also be modified by deletion or addition of amino acids that have minimal impact on the secondary structure and hydrophilicity of the peptide.

[0109] The peptide may include a signal (or leader) sequence at the N-terminus of the protein that directs co-translational transport or post-translational transport of the protein. The peptide may also be conjugated to a linker sequence or other sequence to facilitate synthesis, purification or identification of the peptide (e.g., polyhistidine), or to enhance binding to a solid support. For example, the peptide may be conjugated to the Fc region of an immunoglobulin.

[0110] As described below, when comparing peptide sequences, two sequences are considered "identical" if the sequence of amino acids in the two sequences is the same when the sequences are aligned for maximum correspondence. Comparisons between two sequences are typically performed by comparing alignments within a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to 75 or 40 to 50 consecutive positions, in which a sequence can be compared to a reference sequence after optimal alignment of the two sequences.

[0111] Sequences can be optimally aligned for comparison using, for example, the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. The program includes several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 11: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco Francisco, Calif.; Wilbur, WJ and Lipman, DJ (1983) Proc. Nat'l Acad., Sci. USA 80:726-730.

[0112] Alternatively, optimal alignment of sequences for comparison may be performed using the partial identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, or computerized versions of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.) or by inspection.

[0113] Examples of suitable algorithms for determining sequence identity and sequence similarity percentages include BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST2.0 can be used to determine the sequence homology percentages of the polynucleotides and polypeptides of the present invention, for example using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate cumulative scores.

[0114] Extension of the word hits in each direction ceases when: the cumulative alignment score decreases by the amount X from its maximum achieved value, the cumulative score goes to zero or lower due to the alignment of one or more negative-scoring residues, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0115] In one exemplary method, the "percentage of sequence homology" is determined by comparing two optimally aligned sequences within a comparison window of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may contain up to 20%, typically 5-15% or 10-12% additions or deletions (i.e., gaps) compared to the reference sequence (excluding additions or deletions). The percentage is calculated by determining the number of positions with identical amino acid residues in the two sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and then multiplying the result by 100 to obtain the percentage of sequence homology.

[0116] The peptides provided by the present invention may be in a linear form or a cyclic form, which can be understood with reference to the embodiments of the present invention.

[0117] In the present invention, the production of the cyclic peptide as a variant is not particularly limited to a specific cyclization method and form, as long as it is a peptide cyclization method known in the art. Preferably, the preparation of the cyclic peptide of the present invention can be carried out by cutting or replacing a linear peptide having cysteine ​​at both ends (N-terminus and C-terminus), and then forming a single sulfide bond between the cysteine ​​residues at both ends.

[0118] Peptide provided by the invention, consider using modified polypeptide on the one hand, the polypeptide of this modification comprises the modification of the desired property that improves isolated polypeptide as herein described.Exemplary modification of polypeptide of the present invention includes but is not limited to chemical derivatization and / or enzyme derivatization to one or more amino acids that form, comprises the modification of side chain modification, main chain modification and N-terminal and C-terminal, for example acetylation, hydroxylation, methylation, amidation and adding carbohydrate or lipid component and other parts.Exemplary modification comprises the PEGylation of polypeptide.

[0119] In certain aspects, chemoselective attachment techniques can be used to modify the peptides of the invention, for example, by attaching polymers in a site-specific and controlled manner. These techniques generally rely on the covalent attachment of chemoselective anchors to the protein backbone by chemical or recombinant means, followed by modification with polymers carrying complementary linkers. Thus, the assembly process and the covalent structure of the resulting protein-polymer conjugate are controlled, allowing for the rational optimization of drug properties, such as efficacy and pharmacokinetic properties. For example, allowing for the selective attachment of PEG can improve its pharmacokinetic properties.

[0120] The peptides of the present invention may include pharmaceutically acceptable salts. Examples of such pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, acetate, citrate, tartrate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate or p-toluenesulfonate.

[0121] The present invention also provides a polynucleotide encoding the peptide.

[0122] The terms "DNA", "polynucleotide" and "nucleic acid" used in this specification refer to DNA molecules isolated from the total genomic DNA of a particular species. Therefore, a DNA fragment encoding a polypeptide refers to a DNA fragment substantially isolated from the total genomic DNA of the species from which the DNA fragment can be obtained, or a DNA fragment consisting of one or more purified coding sequences. The terms "DNA fragment" and "polynucleotide" include DNA fragments and smaller fragments thereof, as well as recombinant vectors (e.g., plasmids, cosmids, phagemids, phages, viruses, etc.).

[0123] As will be appreciated by those skilled in the art, the polynucleotide sequences of the present invention are modified to express or be capable of expressing proteins, peptides, etc., including genomic sequences, extragenomic sequences, sequences encoded in plasmids, and smaller operator gene fragments. These fragments may be naturally isolated or modified by artificial synthesis.

[0124] As will be appreciated by those skilled in the art, polynucleotides can be single-stranded (coding sequence or antisense sequence) or double-stranded, can be DNA molecules (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences can be present in the polynucleotides of the present invention. In addition, polynucleotides can be connected to other molecules and / or supporting materials.

[0125] Polynucleotides may include biological functional equivalents of native sequences, variants, or sequences. Polypeptide variants may include one or more substitutions, additions, deletions, and / or insertions, as further described below, preferably in a manner that does not substantially reduce the desired activity of the encoded polypeptide compared to the unmodified polypeptide. The impact on the activity of the encoded polypeptide may generally be assessed as described herein.

[0126] The polynucleotides provided by the present invention are not particularly limited to specific sequences, as long as they can encode the peptide of the present invention or its variant peptide, and any combination of nucleotide sequences (nucleic acid sequences) is allowed. For example, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 can be expressed by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 9, and a peptide consisting of the amino acid sequence of SEQ ID NO: 3 can be expressed by a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 10, but it is not limited thereto.

[0127] The polynucleotides of the present invention, regardless of the length of their coding sequences themselves, can be combined with other DNA sequences (e.g., promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc.) to produce significantly variable overall lengths. Thus, polynucleotide segments of almost any length are considered suitable, preferably subject to the limitations of convenience of manufacture and use in the intended recombinant DNA protocol.

[0128] In addition, it is apparent to those skilled in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the peptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, due to differences in codon usage, the present invention specifically contemplates other polynucleotides (e.g., polynucleotides optimized for human and / or primate codon usage).

[0129] In addition, the allele of the gene comprising the polynucleotide sequence provided herein is within the scope of the present invention. An allele is an endogenous gene modified due to one or more variations (such as the deletion, addition and / or substitution of nucleotides). The resulting mRNA and protein may have a changed structure or function (but not necessarily). Standard techniques (such as hybridization, amplification and / or database sequence comparison) can be used to identify alleles.

[0130] Any mature technology known and available in the art can be used to make, manipulate and / or express polynucleotides and fusions thereof. For example, polynucleotide sequences encoding peptides of the present invention or functional equivalents thereof can be used in recombinant DNA molecules to direct polypeptide expression in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences can be generated and used to clone and express a given polypeptide.

[0131] As will be appreciated by those skilled in the art, in certain circumstances it may be advantageous to prepare a nucleotide sequence (encoding a polypeptide) containing non-naturally occurring codons. For example, codons that are preferred in a particular prokaryotic or eukaryotic host may be selected to produce a recombinant RNA transcript having an increased protein expression rate or a desired property (e.g., a longer half-life than transcripts produced from naturally occurring sequences).

[0132] The polynucleotide sequences of the present invention can be manipulated using methods generally known in the art to alter the peptide encoding sequence for a variety of reasons including, but not limited to, cloning, processing, expression and / or modifying the activity of the gene product.

[0133] The present invention also provides a vector comprising the polynucleotide and a host cell transformed with the vector.

[0134] In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide or its functional equivalent can be inserted into an appropriate expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted coding sequence). Using methods well known to those skilled in the art, expression vectors can be constructed, which contain the sequence encoding the polypeptide of interest and appropriate transcription and translation control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0135] Various expression vector / host systems are known and can be used to contain and express polynucleotide sequences. Expression vector / host systems include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0136] "Control elements" or "regulatory sequences" present in expression vectors are untranslated regions (enhancers, promoters, 5' and 3' untranslated regions) that interact with host cell proteins for transcription and translation. These elements may vary in strength and specificity. Depending on the vector system and host used, appropriate transcription and translation elements (including constitutive promoters and inducible promoters) may be used.

[0137] For example, in bacterial systems, inducible promoters may be used, such as the hybrid lacZ promoter of pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid (Gibco BRL, Gaithersburg, MD). In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. If it is desired to generate a cell line comprising multiple copies of the sequence encoding the polypeptide, a vector based on SV40 or EBV with an appropriate selection marker may be advantageously used.

[0138] In bacterial systems, a variety of expression vectors can be selected according to the intended use of peptide expression. For example, if a large amount is required, a vector designed to express a fusion protein that is easy to purify at a high level can be used. Such vectors include, but are not limited to, the following vectors: multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the desired polypeptide is connected in a reading frame with the sequence of the amino-terminal Met and the seven residues of the subsequent β-galactosidase within the vector to produce a hybrid protein; pIN vector (Van Heeke and Schuster, J.Biol.Chem.264:5503-5509 (1989)); etc. pGEX vectors (Promega, Madison, Wis.) can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads and then eluted in the presence of free glutathione. Proteins produced in these systems can be designed to include a cleavage site for a protease such as heparin, thrombin, or Factor Xa to release the cloned polypeptide from the GST moiety.

[0139] In yeast (Saccharomyces cerevisiae), a number of vectors containing constitutive or inducible promoters (eg, alpha factor, alcohol oxidase, and PGH) are available.

[0140] When using a plant expression vector, the expression of the sequence encoding the polypeptide can be driven by any number of promoters. For example, a viral promoter (e.g., 35S promoter and 19S promoter of CaMV) can be used alone or in combination with the ω leader sequence from TMV. Alternatively, a plant promoter (e.g., the small subunit of RUBISCO or a heat shock promoter) can be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. These techniques are well known in the art.

[0141] Insect systems can also be used to express the desired polypeptide. For example, in one system, AcNPV (Autographa californica nuclear polyhedrosis virus) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The sequence encoding the polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene, under the control of the polyhedrin promoter. Successful insertion of the sequence encoding the polypeptide will inactivate the polyhedrin gene and produce a recombinant virus lacking coat protein. The recombinant virus can then be used to infect Spodoptera frugiperda cells or Trichoplusia larvae, where the desired polypeptide can be expressed.

[0142] In mammalian host cells, there are generally many virus-based expression systems available. For example, when using an adenovirus as an expression vector, the sequence encoding the desired polypeptide can be linked to an adenoviral transcription / translation complex comprising a late promoter and a three-part leader sequence. By inserting into the non-essential E1 or E3 region of the viral genome, a live virus capable of expressing the polypeptide in infected host cells can be obtained. In addition, transcription enhancers (e.g., Rous sarcoma virus (RSV) enhancers) can be used to increase expression in mammalian host cells.

[0143] In addition, specific start signals can be used to more effectively translate the sequence of the desired polypeptide encoding. These signals include ATG start codons and adjacent sequences. When the sequence of the coded polypeptide, its start codon and upstream sequence are inserted into an appropriate expression vector, additional transcription or translation control signals may not be required. However, if only the coding sequence or a portion thereof is inserted, an exogenous translation control signal comprising an ATG start codon must be provided. The start codon must also be in the correct reading frame to ensure the translation of the entire insert. Exogenous translation elements and start codons can come from various sources (natural and synthetic). By including a suitable enhancer for the specific cell system used, expression efficiency can be enhanced (for example, as described in Scharf et al., Results Probl. Cell Differ. 20: 125-162 (1994)).

[0144] In addition, the host cell line can be selected according to the ability of the host cell line to regulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of polypeptides include but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. The post-translational processing of the "pre-original" form of the cut protein can be utilized to promote correct insertion, folding and / or function. Other host cells (for example, CHO, HeLa, MDCK, HEK293 and W138, which have specific cell components and mechanisms for such post-translational activity) can be selected to ensure accurate modification and processing of exogenous proteins.

[0145] In the long term, stable expression is usually preferred for high-yield production of recombinant proteins. For example, a cell line that stably expresses the desired polynucleotide can be transformed with an expression vector that can include viral origins of replication and / or endogenous expression elements, as well as a selectable marker gene located on the same or other vectors.

[0146] After transduction of the vector, cells can be grown in an enriched medium for 1-2 days and then transferred to a selective medium. The purpose of the selective marker is to confer resistance to the selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

[0147] Transformed cell lines can be recovered with various selection systems. Such selection systems include, but are not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., Cell 11:223-232 (1977)) and the adenine phosphoribosyl transferase gene (Lowy et al., Cell 22:817-823 (1990)), which can be used for tk-cells or aprt-cells, respectively.

[0148] In addition, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77: 3567-70 (1980)); npt confers resistance to aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150: 1-14 (1981)); als or pat confers resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively. Other selective genes are known, such as trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histidinol instead of histidine. Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin are becoming increasingly popular and are widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression caused by a specific vector system.

[0149] A variety of methods for detecting and measuring the expression of a product using polyclonal or monoclonal antibodies to a product encoded by a polynucleotide are known in the art. Examples include ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) and FACS (fluorescence activated cell sorting). These assays and other methods can be referenced to the following documents: Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158: 1211-1216 (1983). Various labels and conjugation techniques known to those skilled in the art can be used for various nucleic acid and amino acid determinations. Methods for producing labeled hybridization probes or labeled PCR probes to detect sequences associated with polynucleotides include oligonucleotide labeling, nick translation, end labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence or any portion thereof can be cloned into a vector to produce an mRNA probe. Such vectors are known and commercially available and can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases (e.g., T7, T3 or SP6) and labeled nucleotides. Various commercially available kits can be used to perform these procedures. Suitable reporter molecules or labels include radionuclides, enzymes, fluorophores, chemiluminescent agents, chromogenic agents, substrates, ligands, inhibitors, magnetic particles, and the like.

[0150] Host cells transformed with the desired polynucleotide sequence can be cultured under conditions suitable for expression of the protein and recovery of the protein from the cell culture. Proteins produced by recombinant cells may be secreted or contained within the cell, depending on the sequence and / or vector.

[0151] As will be appreciated by those skilled in the art, expression vectors comprising polynucleotides of the invention can be designed to include a signal sequence that directs secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to connect the sequence encoding the desired polypeptide to the sequence encoding the polypeptide domain that aids in the purification of soluble proteins.

[0152] In addition to recombinant production methods, polypeptides and fragments of the present invention can also be produced by direct peptide synthesis using solid phase techniques (Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963)). Protein synthesis can be performed manually or automatically. Automated synthesis can be achieved using, for example, an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and then combined using chemical methods to produce full-length molecules.

[0153] According to another aspect of the present invention, the polynucleotide encoding the polypeptide of the present invention can be delivered to a subject in vivo using gene therapy techniques. Gene therapy generally refers to the introduction of heterologous nucleic acids into specific cells or target cells of mammals (particularly humans) suffering from a disorder or condition requiring such treatment. The nucleic acid is introduced into selected target cells, and the heterologous DNA is expressed to produce the encoded therapeutic product.

[0154] As disclosed herein, various viral vectors suitable for gene therapy include adenovirus, herpes virus, vaccinia virus, adeno-associated virus (AAV), or preferably RNA virus, such as retrovirus. Preferably, the retroviral vector can be a mouse or avian retroviral derivative or a lentiviral vector. A preferred retroviral vector can be a lentiviral vector. Examples of retroviral vectors that can be inserted with a single exogenous gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many other retroviral vectors can integrate multiple genes. All of these vectors can include selectable marker genes to allow identification and generation of transduced cells. For example, by inserting a sequence encoding a desired zinc finger-derived DNA binding polypeptide into a viral vector together with another gene encoding a ligand for a receptor on a specific target cell, the vector can be made target specific.

[0155] Retroviral vectors can be made target specific by inserting polynucleotides encoding polypeptides. For example, targeting can be achieved by using antibodies that target retroviral vectors. Those skilled in the art are familiar with specific polynucleotide sequences that can be inserted into the retroviral genome to deliver retroviral vectors containing zinc finger nucleotide binding protein polynucleotides in a target-specific manner, and they can be easily identified without excessive experimentation.

[0156] Because recombinant retrovirus is defective, therefore need to assist to produce infectious vector particles.For example, this assistance can be provided using helper cell line, and described helper cell line comprises the plasmid of all structural genes of encoding retrovirus, and these structural genes are controlled by the regulatory sequence in LTR.These plasmids lack the nucleotide sequence that can make packaging mechanism identify RNA transcript to be packaged.The example of the helper cell line with defective packaging signal includes but is not limited to PSI.2, PA317 and PA12.These cell lines produce empty virions, because their genome is not packaged.If retroviral vector is introduced into the cell with complete packaging signal, and structural gene is replaced by other required genes, then vector can be packaged and produce vector virion.The vector virion produced by this method can then be used to infect tissue cell line (for example NIH3T3 cell), thereby produce a large amount of chimeric retroviral virions.

[0157] Non-viral gene therapy delivery techniques may also be used, such as DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun technology, electroporation, liposome methods and lipofection. Any of these methods are widely available to those skilled in the art, and are suitable for use in the present invention. Other suitable methods may also be available to those skilled in the art, and it is clearly understood that any available transfection method may be used to implement the present invention. Lipofection may be achieved by encapsulating the separated DNA molecules in liposome particles and contacting the liposome particles with the cell membrane of the target cell. Liposomes are self-assembled colloidal molecules composed of lipid bilayers formed by amphiphilic molecules such as phosphatidylserine or phosphatidylcholine, encapsulating part of the surrounding medium, and producing a hydrophilic interior surrounded by a lipid bilayer. Single or multilayer liposomes may be constructed, containing desired chemicals, drugs or separated DNA molecules as described in the present invention.

[0158] In one aspect of the invention, the virus can be characterized as an RNA virus. RNA viruses use reverse transcriptase to convert their RNA genetic information into DNA, which is then inserted into the host's DNA, allowing the host to replicate the virus' genetic information.

[0159] In the present invention, the type of RNA virus is not particularly limited, and can be selected from Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnviridae. aviridae), Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Bornaviridae, Filoviridae, Paramyxoviridae oviridae, Rhabdoviridae, Nyamiviridae, Caliciviridae, Flaviviridae, Luteoviridae, Togaviridae, Pneumoviridae, Arenaviridae, Deltavirus, and Orthomyxoviridae.

[0160] In a preferred aspect of the present invention, the RNA virus may be selected from the group consisting of influenza virus, influenza A virus H1N1 subtype, avian influenza virus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human immunodeficiency virus (HIV), retrovirus and hepatitis C virus.

[0161] In the present invention, the coronavirus can be characterized as being selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS) coronavirus.

[0162] The pharmaceutical composition for antiviral use according to the present invention can be administered as a vaccine for preventing viral infection before infection, or as a therapeutic agent for treating viral infection after infection.

[0163] The present invention also provides a vaccine adjuvant, which comprises one or more substances selected from the following:

[0164] (i) the peptide,

[0165] (ii) a polynucleotide encoding (i),

[0166] (iii) a vector comprising (ii), and

[0167] (iv) using the host cell transformed with (iii).

[0168] In the present invention, a vaccine adjuvant can be defined as a substance that presents an antigen to the immune system in a manner that induces an immune response or increases the immune response to the antigen when the antigen is administered together with the vaccine adjuvant. That is, a vaccine adjuvant is a substance that acts as an immunostimulant to enhance the immune response to an antigen, which means a substance that enhances immunity by increasing the activity of immune system cells, although it is not an immunogen of the host.

[0169] To analyze the antigen-specific immune response induced by a vaccine adjuvant, the immune response can be compared to the immune response induced in the presence of the antigen without the vaccine adjuvant. The induction can be assessed in a subject or in cells of a subject.

[0170] In the present invention, immune response can be selected from macrophages, dendritic cells, monocytes, B cells and T cell reactions. In particular, immune response can be a B cell reaction, which means that it can specifically produce antibodies against antigens. Antibodies are preferably IgG antibodies, more preferably IgG2a and / or IgG1 antibodies. Immune response can be a T cell reaction, preferably a Th1 reaction, a Th2 reaction or a balanced Th2 / Th1 reaction. Those skilled in the art recognize that it may be necessary to induce B and / or T cell reactions to control diseases, and the reactions vary from disease to disease. Alternatively, immune response can be detected by measuring the production of cytokines such as IFNγ, IL-6, TNFα or IL-10. The production of these cytokines can be assessed by ELISA, preferably as described in the embodiments.

[0171] According to one embodiment of the present invention, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto has been demonstrated to have very excellent effects in activating innate and acquired immunity, and has been demonstrated to significantly improve the increase in the subject's immune response when treated with an antigen.

[0172] Therefore, those skilled in the art can clearly understand that a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto, a polynucleotide encoding the peptide, a vector comprising the polynucleotide, and a host cell transformed with the vector will also exhibit the same physiological activity.

[0173] As one embodiment of the present invention, the vaccine adjuvant can induce better activation of innate and adaptive immune responses when administered with an antigen, more specifically, it can induce improved activation of cellular and humoral immune responses compared to when the antigen is administered alone.

[0174] As one embodiment of the present invention, detecting an antigen-specific induced immune response means at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or longer after administering the vaccine adjuvant of the present invention, or at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days or longer after administration. Detection can be assessed in a subject or a subject's cell, preferably as described in the examples.

[0175] As an embodiment of the present invention, the immune response of antigen-specific induction preferably refers to a detectable immune response to an antigen. Detectable increase means that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more, or at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days or longer, the amount of immune cells, antibodies and / or cytokines increases by at least 5%, or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200% or more after administering vaccine adjuvant and antigen to the subject. Detection can be assessed in the subject or the subject's cell, preferably as described in the examples.

[0176] The present invention also provides a vaccine composition, which comprises a vaccine adjuvant and an antigen.

[0177] In the present invention, a vaccine refers to a preparation for conferring immunity to a disease caused by an organism, which contains an antigen consisting of a whole pathogenic organism (structured or attenuated) or a component thereof, such as a protein, peptide or polysaccharide. As described above, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, or a peptide comprising an amino acid sequence having 95% or more homology with the peptide, can be used as a vaccine composition by activating the innate and acquired immune systems, significantly enhancing the immune response of a subject when administered together with an antigen.

[0178] In the present invention, the antigen contained in the vaccine composition is not particularly limited, and may be peptides, proteins, glycoproteins, glycolipids, lipids, carbohydrates, nucleic acids, polysaccharides, and viruses, bacterial cells, allergens, tissues and cells containing them. Non-limiting examples include antigens derived from pollen, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus and hepatitis F vaccine, HIV virus, influenza virus, herpes virus (HSV-1, HSV-2), anthrax, chlamydia, pneumococcus, Japanese encephalitis virus, measles virus, rubella virus, tetanus bacteria, varicella virus, SARS virus, Epstein-Barr virus, papillomavirus, Helicobacter pylori, rabies virus, West Nile virus, Hantavirus, Streptococcus, Staphylococcus, Bordetella pertussis, Mycobacterium tuberculosis, Plasmodium, poliovirus, various zoonotic infections, cancer antigens, various food allergy antigens, etc.

[0179] The antigens contained in the vaccine composition of the present invention need not be single. Considering the application of the present invention, it can induce an immune response to multiple components such as cancer cells, bacteria, viruses, etc., rather than an immune response to a single protein or peptide. In this case, it can be a mixture of multiple types of proteins or other substances that can induce an immune response, even if the type cannot be specified. In addition, actively inducing an immune response to multiple types of antigens by including multiple types of antigens is also an application form of the vaccine composition of the present invention.

[0180] The antigen contained in the vaccine composition of the present invention may preferably be a tumor antigen. The tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). Various tumor antigens and their expression patterns are well known in the art and can be selected according to the type of tumor to be treated. Non-limiting examples of tumor antigens include alpha-fetoprotein, carcinoembryonic antigen, cdk4, β-catenin, CA125, caspase-8, epithelial tumor antigens, HPV antigens, HPV16 antigens, HPV16-derived antigens, and HPV-derived antigens. CTL epitopes of E7 antigen, melanoma-associated antigen (MAGE)-1, MAGE-3, tyrosinase, surface Ig idiotype, Her-2 / neu, MUC-1, prostate-specific antigen (PSA), sialyl Tn (STn), heat shock protein, gp96, ganglioside molecules GM2, GD2, GD3, carcinoembryonic antigen (CEA), PRAME, WT1, survivin, cyclin D, cyclin E, HER2, MAGE, NY-ESO, EGF, GP100, cathepsin G, human papillomavirus (HPV )-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, Her / 2-neu antigen, chimeric Her2 antigen, prostate-specific antigen (PSA), bivalent PSA, ERG, androgen receptor (AR), PAK6, prostate stem cell antigen (PSCA), NY-ESO-1, stratum corneum chymotrypsin (SCCE) antigen, Wilms tumor antigen 1 (WT-1), HIV-1 gag, human telomerase reverse transcriptase (hTERT), proteinase 3, tyrosinase-related protein 2 (TRP2), high molecular weight Melanoma-associated antigen (HMW-MAA), synovial sarcoma X (SSX)-2, carcinoembryonic antigen (CEA), melanoma-associated antigen E (MAGE-A, MAGE1, MAGE2, MAGE3, MAGE4), interleukin-13 receptor α (IL13-Rα), carbonic anhydrase IX (CAIX), survival protein, GP100, angiogenesis antigen, ras protein, p53 protein, p97 melanoma antigen, KLH antigen, carcinoembryonic protein antigen (CEA), gp100, MART1 antigen, TRP-2, HSP-70, β-HCG ,testisin,1A01_HLA-A / m,1A02,5T4,ACRBP,AFP,AKAP4,α-actinin-_4 / m,α-methylacyl-CoA racemase,ANDR,ART-4,ARTC1 / m,AURKB,B2MG,B3GN5,B4GN1,B7H4,BAGE-1,BASI,BCL-2,bcr / abl,β-catenin / m,BING-4,BIRC7,BRCA1 / m,BY55,calreticulin,CAMEL,CASPA,caspase_8,Cathepsin_B, cathepsin_L, CD1A, CD1B, CD1C, CD1D, CD1E, CD20, CD22, CD276, CD33, CD3E, CD3Z, CD4, CD44_isotype_1, CD44_isotype_6, CD52, CD55, CD56, CD80, CD86, CD8A, CDC27 / m, CDE30, CDK4 / m, CDKN2A / m, CEA, CEAM6, CH3L2, CLCA2, CML28, CML66, COA-1 / m, coactosin-like_protein, collagen_XXIII, COX-2, CP1B1, CSAG2, CT-_9 / B RD6, CT45A1, CT55, CTAG2_isoform_LAGE-1A, CTAG2_isoform_LAGE-1B, CTCFL, Cten, cyclin_B1, cyclin_D1, cyp-B, DAM-10, DEP1A, E7, EF1A2, EFTUD2 / m, EGFR, EGLN3, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ERBB4, ERG, ETV6, EWS, EZH2, FABP7, FCGR3A_form_1, FCGR3A_form_2, FGF5, FGFR2, fibronectin, FOS, FOXP3, FUT1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8_(GAGE-2D), GASR, GnT-V, GPC3, GPNMB / m, GRM3, HAGE, hepsin, Her2 / neu , HLA-A2 / m, homeobox_NKX3.1, HOM-TES-85, HPG1, HS71A, HS71B, HST-2, hTERT, iCE, IF2B3, IL-10, IL-13Ra2, IL2-RA, IL2-RB, IL2-RG, IL-5, IMP3, ITA5, ITB 1. ITB6, kallikrein-2, kallikrein-4, KI20A, KIAA0205, KIF2C, KK-LC-1, LDLR, LGMN, LIRB2, LY6K, MAGA5, MAGA8, MAGAB, MAGE-_B1, MAGE-_E1, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1_(MAGE1), MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin_A, MART-1 / melan-A, MART-2, M C1_R, M-CSF, mesothelin, MITF, MMP1_1, MMP7, MUC-1, MUM-1 / m, MUM-2 / m, MYO1A, MYO1B, MYO1C, MYO1D, MYO1E, MYO1F, MYO1G, MYO1H, NA17, NA88-A, Neo-PAP, NFY C / m, NGEP, N-myc, NPM, NRCAM, NSE, NUF2, NY-ESO-1, OA1, OGT, OS-9, osteocalcin, osteopontin, p53, PAGE-4, PAI-1, PAI-2, PAP, PATE, PAX3, PAX5, PD1L1, PDCD1, PDEF, PECA1, PGCB, PGFRB, Pim-1_-kinase, Pin-1, PLAC1, PMEL, PML, POTE, POTEF, PRAME, PRDX5 / m, PRM2, Prostein, proteinase-3, PSA, PSB9, PSCA, PSGR, PSM, PTPRC, RAB 8A, RAGE-1, RARA, RASH, RASK, RASN, RGS5, RHAMM / CD168, RHOC, RSSA, RU1, RU2, RUNX1, S-100, SAGE, SART-1, SART-2, SART-3, SEPR, SERPINB5, SIA7F, SIA8A, SIAT9, SIRT2 / m, SOX10, SP17, SPNXA, SPXN3, SSX-1, SSX-2, SSX3, SSX-4, ST1A1, STAG2, STAMP-1, STEAP-1, survivin, survivin-2B, SYCP1, SYT -SSX-1, SYT-SSX-2, TARP, TCRg, TF2AA, TGFbeta1, TGFR2, TGM-4, TIE2, TKTL1, TPI / m, TRGV11, TRGV9, TRPC1, TRP-p8, TSG10, TSPY1, TVC_(TRGV3), TX101, tyrosinase, TYRP1, TYRP2, UPA, VEGFR1, WT1, XAGE1, α-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, β-catenin, Cdc27, CDK4, CDKN2A, COA-1,dek-can fusion protein, EFTUD2, elongation factor 2, ETV6-AML1 fusion protein, FN1, GPNMB, LDLR-fucosyltransferase AS fusion protein, HLA-A2d, HLA-A11d, hsp70-2, KIAAO205, MART2, ME1, MUM-If, MUM-2, MUM-3, neo-PAP, class I myosin, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK 、K-ras、N-ras、RBAF600、SIRT2、SNRPD1、SYT-SSX1 or SSX2 fusion protein、triosephosphate isomerase、BAGE-1、GAGE-1,2,8、GAGE-3,4,5,6,7、GnTVf、HERV-K-MEL、KK-LC-1、KM-HN-1、LAGE-1、MAGE-A1、MAGE-A2、MAGE-A3、MAGE-A4、MAGE-A6、MAGE-A9、MAGE -A10, MAGE-A12, MAGE-C2, mucin k, NA-88, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-2, SSX-4, TRAG-3, TRP2-INT2g, CEA , gp100 / Pmel17, kallikrein 4, mammaglobin-A, Melan-A / MART-1, NY-BR-1, OA1, PSA, RAB38 / NY-MEL-1, TRP-1 / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, BING-4, CPSF, cyclin D1, Ep-CAM, EphA3, FGF5, G250 / MN / CAIX, HER-2 / neu, IL13Rα2, intestinal carboxylesterase, alpha-fetoprotein, M-CSF, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, secernin 1. SOX10, STEAP1, survivin, telomerase, WT1, FLT3-ITD, BCLX(L), DKK1, ENAH(hMena), MCSP, RGS5, gastrin-17, human chorionic gonadotropin, EGFRvIII, HER2, HER2 / neu, P501, guanylate cyclase C, PAP, OVA(ovalbumin), and MART-1.

[0181] The vaccine composition of the present invention may preferably be an anticancer vaccine. In addition, the anticancer vaccine may be a vaccine for preventing cancer or a vaccine for treating cancer.

[0182] According to one aspect of the present invention, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto and a vaccine composition comprising a specific tumor antigen can inhibit the growth of cancer by pre-administering before cancer formation to activate the immune response of the subject, thereby exhibiting a preventive effect. In addition, the vaccine composition of the present invention has been demonstrated to have efficacy as a therapeutic vaccine by inhibiting the growth of cancer or killing cancer when administered to a subject after cancer formation.

[0183] In the present invention, the type of cancer is not particularly limited, and preferably, it can be selected from breast cancer, colon cancer, prostate cancer, cervical cancer, gastric cancer, skin cancer, oral cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, nasopharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, esophageal cancer, rectal cancer, liver cancer, pancreatic cancer, pancreatic neuroendocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytoma, leukemia, childhood cancer, bronchial cancer, colon cancer and ovarian cancer.

[0184] The vaccine composition of the present invention may further comprise one or more substances selected from any vaccine adjuvants and immune checkpoint inhibitors.

[0185] According to one embodiment of the present invention, it has been confirmed that the peptide of the present invention has a more significant effect in activating the immune function of the subject when combined with any additional vaccine adjuvant and / or immune checkpoint inhibitor. Specifically, this effect may be particularly desirable because it minimizes the side effects that may be produced by the administration of high doses of the substance and achieves the maximum effect with the least administration.

[0186] There is no particular limitation on the types of vaccine adjuvants that may be additionally included in the vaccine composition of the present invention. It should be understood that the types also include vaccine adjuvants currently widely used in the art or vaccine adjuvants newly developed in the future. Non-limiting examples of any vaccine adjuvant include 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG ODN, CpG7909, CyA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImmuFact IMP321, IS patch, Iscomatrix, JuviMune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, Ontak, Peptivator system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D BCG, Aquila's QS21 Stimulon, RibiDetox Quil, Superfos, Freund's, GM-CSF, cholera toxin, immune adjuvant, MF59 and cytokines, and most preferably CpG ODN.

[0187] In the present invention, immune checkpoint inhibitors are substances that block inhibitory checkpoints of the immune system. Immune checkpoints can be stimulatory or inhibitory. Blocking inhibitory immune checkpoints can activate immune system function and can be used for cancer immunotherapy [see: Pardoll, Nature Reviews. Cancer 12: 252-64 (2012)]. When tumor cells attach to specific T cell receptors, activated T cells are consumed. Immune checkpoint inhibitors prevent tumor cells from attaching to T cells, keeping T cells activated. In fact, the synergistic effect of cells and soluble components can resist damage caused by pathogens and cancer. Regulation of immune pathways involves changing the expression or functional activity of at least one component in the pathway to regulate the response of the immune system. The immune checkpoint inhibitor may be selected from: PD-1 (programmed cell death-1) antagonists, PD-L1 (programmed cell death ligand 1) antagonists, PD-L2 (programmed cell death ligand 2) antagonists, CD27 (cluster of differentiation 27) antagonists, CD28 (cluster of differentiation 28) antagonists, CD70 (cluster of differentiation 70) antagonists, CD80 (cluster of differentiation 80, also known as B7-1) antagonists, CD86 (cluster of differentiation 86, also known as B7-2) antagonists, CD137 (cluster of differentiation 137) antagonists, CD276 (cluster of differentiation 276) antagonists, KIRs (killer cell immunoglobulin-like receptor) antagonists, LAG3 (lymphocyte activation gene 3) antagonists, TNFRSF4 (tumor necrosis factor receptor superfamily, member 4, also known as CD134) antagonists, GITR (glucocorticoid-induced TNF) antagonists. FR-related protein) antagonists, GITRL (glucocorticoid-induced TNFR-related protein ligand) antagonists, 4-1BBL (4-1BB ligand) antagonists, CTLA-4 (cytolytic T lymphocyte-associated antigen-4) antagonists, A2AR (adenosine A2A receptor) antagonists, VTCN1 (V-group domain-containing T cell activation inhibitor 1) antagonists, BTLA (B and T lymphocyte attenuator) antagonists, IDO (indoleamine 2,3-dioxygenase) antagonists, TIM-3 (T cell immunoglobulin domain and mucin domain 3) antagonists, VISTA (V-domain Ig inhibitor of T cell activation) antagonists and KLRA (killer cell lectin-like receptor subfamily A) antagonists, preferably PD-1 antagonists, PD-L1 antagonists or LAG3 antagonists, most preferably PD-L1 antagonists.

[0188] In one embodiment of the present invention, the immune checkpoint inhibitor can be a PD-1 antagonist. PD-1 is a T cell co-inhibitory receptor that plays a central role in the ability of tumor cells to evade the host immune system. By blocking the interaction between PD-1 and its ligand PD-L1, PD-1 antagonists enhance immune function and mediate anti-tumor activity. Examples of PD-1 antagonists include antibodies that specifically bind to PD-1. Specific anti-PD-1 antibodies include nivolumab, pembrolizumab, STI-1014, and pidilizumab, but are not limited thereto.

[0189] In another embodiment of the present invention, the immune checkpoint inhibitor may be a PD-L1 antagonist. Examples of PD-L1 antagonists include antibodies that specifically bind to PD-L1. Specific anti-PD-L1 antibodies include avelumab, atezolizumab, durvalumab, and BMS-936559, but are not limited thereto.

[0190] In another embodiment, the immune checkpoint inhibitor may be a LAG3 antagonist. LAG3 (lymphocyte activation gene 3) is a negative co-stimulatory receptor that regulates T cell homeostasis, proliferation and activation. It has also been reported that LAG3 is involved in the inhibitory function of regulatory T cells (Treg). Most LAG3 molecules remain in the cell, near the microtubule organizing center, and are only induced after antigen-specific T cell activation [see: US2014 / 0286935]. Examples of LAG3 antagonists include antibodies that specifically bind to LAG3. Specific anti-LAG3 antibodies include GSK2831781, but are not limited thereto.

[0191] In the present invention, antibody refers to a complete monoclonal antibody, a polyclonal antibody, a multispecific antibody formed by at least two complete antibodies, and an antibody fragment, as long as they exhibit the expected biological activity. In another embodiment, antibody refers to a soluble receptor that does not retain the Fc portion of the antibody. In one embodiment, the antibody can be a humanized monoclonal antibody and a fragment thereof produced by recombinant genetic engineering.

[0192] Another class of immune checkpoint inhibitors includes polypeptides that bind to and block the PD-1 receptor on T cells without inducing inhibitory signal transduction. Such peptides include B7-DC polypeptides, B7-H1 polypeptides, B7-1 polypeptides, and B7-2 polypeptides and soluble fragments thereof, as disclosed in U.S. Pat. No. 8,114,845.

[0193] Another class of immune checkpoint inhibitors includes compounds having a peptide portion that inhibits PD-1 signaling. Examples of such compounds are generally disclosed in the known literature.

[0194] Another class of immune checkpoint inhibitors includes inhibitors of specific metabolic enzymes, such as indoleamine 2,3-dioxygenase (IDO), which is expressed by infiltrating bone marrow cells and tumor cells. IDO enzymes inhibit immune responses by depleting amino acids required for T cell anabolism or by synthesizing specific natural ligands for cytoplasmic receptors that can alter lymphocyte function.

[0195] The compositions of the present invention (e.g., polypeptides, polynucleotides, etc.) can generally be formulated into pharmaceutically acceptable or physiologically acceptable solutions for administration to cells, tissues, or animals alone or in combination with one or more other treatment modalities. If desired (as needed), the compositions of the present invention can be administered in combination with other drugs (e.g., other proteins, polypeptides, or various pharmaceutically active drugs). There is no restriction on other components that can be included in the compositions of the present invention, as long as these additional drugs do not have a negative impact on the properties of the peptides of the present invention.

[0196] The route of administration can be oral or parenteral. Parenteral administration methods include, but are not limited to, intranasal, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration.

[0197] When the pharmaceutical composition of the present invention is administered orally, the pharmaceutical composition of the present invention can be formulated into dosage forms such as powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, and wafers with appropriate oral administration carriers according to methods known in the art. Examples of suitable carriers include sugars, such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches, such as corn starch, wheat starch, rice starch, and potato starch; celluloses, such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; fillers, such as gelatin and polyvinyl pyrrolidone. In addition, cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate can be added as a disintegrant. In addition, the pharmaceutical composition can also contain anti-caking agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives.

[0198] In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated into forms such as injections, transdermal preparations and nasal inhalants together with suitable parenteral administration carriers according to methods known in the art. For injections, they must be sterile and not contaminated by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media, including water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof and / or vegetable oils. More preferably, suitable carriers include Hank's solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% glucose. In order to prevent the injection from being contaminated by microorganisms, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and thimerosal may also be contained. In addition, in most cases, the injection may also contain isotonic agents such as sugar or sodium chloride.

[0199] For transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, pastes, liniments and aerosols are included. "Transdermal administration" refers to the topical application of the pharmaceutical composition to the skin so that an effective amount of active ingredients contained in the pharmaceutical composition is delivered to the skin. For example, the pharmaceutical composition of the present invention can be prepared in an injection form, administered by gently piercing the skin with a 30-gauge fine needle or directly applied to the skin. These preparations are described in the literature generally known in pharmaceutical chemistry.

[0200] For administration by inhalation, the compounds used in the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer, using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base (e.g., lactose or starch).

[0201] Other pharmaceutically acceptable carriers can be found in known literature.

[0202] In addition, the pharmaceutical composition of the present invention may also contain one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose or dextran), antioxidants, antibacterial agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents and / or preservatives.

[0203] Moreover, the pharmaceutical compositions of the present invention can be formulated using procedures known in the art so as to provide quick, sustained or delayed release of the active ingredient after administration to a mammal.

[0204] The pharmaceutical composition of the present invention can also be administered in combination with known substances having antiviral effects.

[0205] In the compositions of the present invention, the combination of pharmaceutically acceptable excipients and carriers is well known to those skilled in the art. In addition, well-known methods, including oral, parenteral, intravenous, intranasal, intracerebral and intramuscular administration and formulations for such administration, can be used, using appropriate doses and treatment regimens for the specific compositions described herein.

[0206] In certain applications, the pharmaceutical compositions disclosed herein can be delivered to a subject by oral administration. In this case, the composition can be formulated with an inert diluent or an absorbable edible carrier, or the composition can be encapsulated in a hard or soft shell gelatin capsule, compressed into tablets, or directly incorporated into food.

[0207] In certain cases, the pharmaceutical compositions disclosed herein may preferably be delivered parenterally, intravenously, intramuscularly or intraperitoneally, and such routes of administration may be referred to known literature. Solutions of the active compounds (as free alkali or pharmaceutically acceptable salts) may be prepared by suitable mixing with surfactants such as hydroxypropylcellulose in water. Dispersions may also be prepared in glycerol, liquid polyethylene glycol or mixtures thereof or in oil. In order to prevent microbial growth under common storage and use conditions, the formulation may include a preservative.

[0208] The pharmaceutical form suitable for injection includes sterile aqueous solution or dispersion and sterile powder, which is used to prepare sterile injection solution or dispersion temporarily. In all cases, the pharmaceutical form must be sterile, and fluidity is enough to facilitate injection. It must be stable under manufacturing and storage conditions, and prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), their suitable mixture and / or vegetable oil. Suitable fluidity can be maintained by using a coating agent (such as lecithin), maintaining the required particle size in the case of a dispersion, and using a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal). In many cases, it is preferred to include isotonic agents (such as sugar or sodium chloride). By using an absorption delay agent (such as aluminum monostearate and gelatin), extended absorption of injectable compositions can be achieved.

[0209] For example, for parenteral administration of an aqueous solution, the solution should be properly buffered as required, and the liquid diluent should first be isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous medium that can be used for this background is well known to those skilled in the art. For example, a single dose can be dissolved in 1ml isotonic NaCl solution and added to 1000ml subcutaneous perfusion solution, or injected at a proposed infusion site (for example, referring to Remington's Pharmaceutical Sciences, the 15th edition, pages 1035-1038 and 1570-1580). The dosage may vary depending on the symptoms of the subject treated with the pharmaceutical composition. Those skilled in the art can use the routine knowledge in the art to determine the appropriate dosage for individual subjects. In addition, for use in humans, the preparation must meet the sterility, pyrogenicity and general safety and purity standards required by the FDA Office of Biological Products.

[0210] Sterile injectable solutions can be prepared by adding the desired amount of active compound to an appropriate solvent together with the various other ingredients described above, followed by filtration sterilization as needed. Typically, dispersions can be prepared by adding the sterile active ingredient to a sterile solvent containing a basic dispersion medium and the other necessary ingredients described above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred manufacturing methods may include vacuum drying and freeze drying techniques, which can produce powders of the active ingredient and any other desired components from pre-sterilized solutions.

[0211] Compositions disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts can include acid addition salts (formed with the free amino groups of proteins), which can be formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid). The salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine). According to the preparation, the solution is applied in a manner suitable for the dosage form and in a therapeutically effective amount. The preparation can be easily applied in various dosage forms, such as injections or drug release capsules.

[0212] The term "carrier" as used herein includes any solvent, dispersion medium, solvent, coating, diluent, antimicrobial and antifungal agent, isotonic and absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of these media and agents for pharmaceutically active substances is well known in the art. They are used in therapeutic compositions unless they are incompatible with the active ingredient. Auxiliary active ingredients may also be included in the compositions of the present invention.

[0213] The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to humans. Methods for preparing aqueous compositions containing proteins as active ingredients are well known in the art. Typically, such compositions are prepared as injectable liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid prior to injection can also be prepared. In addition, the preparations can be emulsified.

[0214] In certain embodiments, the pharmaceutical composition can be delivered by nasal spray, inhalation and / or other aerosol delivery media. Methods for delivering genes, polynucleotides and peptide compositions directly to the lungs by nasal aerosol spray can refer to known literature. Similarly, drug delivery using nasal microparticle resins and lysophosphatidylglycerol compounds is also well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix can refer to known literature.

[0215] In certain embodiments, liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used for delivery to introduce the compositions of the present invention into appropriate cells. Specifically, the compositions of the present invention can be formulated to be encapsulated in lipid particles, liposomes, vesicles, nanospheres or nanoparticles for delivery. The preparation and use of these delivery vehicles can be carried out using known prior art.

[0216] In addition, the pharmaceutical composition of the present invention can be prepared using methods known in the art so as to provide a rapid, sustained or delayed release of the active ingredient after administration to a mammal. The pharmaceutical composition prepared in this manner can be administered in an effective amount by various routes, including oral, transdermal, subcutaneous, intravenous or intramuscular as described above.

[0217] The term "effective amount" refers to the amount of a substance that can track a diagnostic or therapeutic effect when administered to a patient, and the term "subject" may include animals, preferably mammals, especially humans, and may also include cells, tissues, and organs from animals. The subject may be a patient who needs the above-mentioned effect.

[0218] The pharmaceutical composition comprising the polypeptide of the present invention can change the content of the active ingredient according to the severity of the disease, but generally for adults, it can be administered several times a day, and the effective dose is 0.1 μg to 10,000 mg per dose, preferably 1 mg to 5,000 mg. However, the dose of the pharmaceutical composition of the present invention can be appropriately selected according to the administration route, target, target disease and its severity, age, sex, body weight, individual differences and disease state, and such techniques are known to those skilled in the art.

[0219] In another aspect, the present invention provides methods for using the compositions (e.g., polynucleotides, polypeptides, etc.) of the present invention in cells, tissues, or subjects to achieve desired cellular effects and / or therapeutic effects. The cells or tissues that can be regulated by the present invention are preferably mammalian cells or tissues, more preferably human cells or tissues. These cells or tissues can be in a healthy state or a diseased state.

[0220] The present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising one or more selected from the following (i) to (iv):

[0221] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which is composed of one or more selected from the following (i) to (iv):

[0222] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which is essentially composed of one or more selected from the following (i) to (iv):

[0223] (i) the peptide,

[0224] (ii) a polynucleotide encoding (i),

[0225] (iii) a vector comprising (ii), and

[0226] (iv) using the host cell transformed with (iii).

[0227] According to one aspect of the present invention, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto can itself exhibit the effect of inhibiting and killing tumor growth, thereby exhibiting a cancer prevention or treatment effect.

[0228] The term "treatment" in the present invention generally refers to improving cancer, viral diseases or symptoms caused by the aforementioned diseases, and may include curing the disease, substantially preventing the disease or improving the condition. It includes alleviating, curing or preventing one or most of the symptoms caused by the disease, but is not limited thereto.

[0229] Effects of the Invention

[0230] The peptide disclosed in the present invention is a CRS fragment disclosed for the first time in this specification, and exhibits anticancer activity and immune function enhancing activity.

[0231] In addition, the peptide, the polynucleotide encoding the peptide, the vector comprising the polynucleotide, the host cell transformed using the vector, or the full-length CRS protein exhibits excellent anti-cancer activity and immune function enhancing activity, making it very useful in the development of vaccine adjuvants, vaccine compositions and cancer treatment compositions.

[0232] In addition, the antiviral composition provided by the present invention not only has the effect of preventing and treating viral infections, but also has the excellent effect of assisting or enhancing immune response when administered together with viral antigens, making it very useful for preventing or treating viral infections or preparing viral vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0233] Figures 1a to 1b Results confirming the immunologically active functions of various fragment proteins from CRS (CRS = CARS1) are shown.

[0234] Figure 1a is a schematic diagram listing the protein sequences of various fragments from CRS.

[0235] Figure 1b Shown are the results of treating Thp-1 cells with PMA (50 ng / ml) for 48 hours to induce differentiation, then treating with various fragment proteins (100 nM) from CRS, and confirming the amount of TNF-α in the culture medium using ELISA 4 hours later.

[0236] Figure 2 It is a schematic diagram showing the mutation position of the fragment protein from CRS, specifically, a schematic diagram of replacing cysteine ​​at the 182nd amino acid of the helix 3-4 protein from CRS with serine.

[0237] Figure 3 The results of the confirmation of the CRS(140-200,C182S) protein expression and purification process are shown, in which the pET28aCRS(140-200,C182S) gene was transformed into BL21(DE3)codon Plus RIPL cells, and the samples obtained from each affinity chromatography purification process were subjected to SDS-PAGE electrophoresis, followed by Coomassie staining of the gel for 1 hour.

[0238] Figure 4 The results of confirming the polymorphism of CRS (140-200) and CRS (140-200, C182S) proteins are shown, specifically, the experimental results of confirming by gel filtration chromatography whether monomers appear instead of polymers formed by disulfide bonds after the amino acid cysteine ​​at position 182 in the protein purified by affinity chromatography is replaced by serine.

[0239] Figure 5 Shown are the results of confirming the charge difference between the CRS (140-200) and CRS (140-200, C182S) proteins, specifically, the results of confirming the presence of multimers and monomers by ion exchange chromatography by distinguishing the charge difference of the proteins separated by gel filtration chromatography.

[0240] Figures 6a to 6bResults confirming the thermal stability and immunological activity of fragment proteins from CRS are shown.

[0241] Figure 6a The results of analyzing the changes in the secondary structure of the protein induced by high-temperature denaturation at 100°C for 60 minutes using circular dichroism analysis are shown.

[0242] Figure 6b The results show that Thp-1 cells were treated with PMA (50 ng / ml) for 48 hours to induce differentiation, and then treated with various fragment proteins (100 nM) from CRS induced by high temperature denaturation at 100°C for 60 minutes. The amount of TNF-α in the culture medium was confirmed by ELISA 4 hours later.

[0243] Figure 7 The results of confirming the immune activity of CRS (140-200, C182S) protein are shown. Specifically, Thp-1 cells were treated with PMA (50ng / ml) for 48 hours to induce differentiation, and then treated with polymyxin B (10ug / ml), proteinase K (20ug / ml), and fragment protein from CRS, and the amount of TNF-α in the culture medium was confirmed by ELISA after 4 hours.

[0244] Figures 8a to 8c The results of confirming the antiviral effect of CARS1 (99-200, C182S) protein according to the administration route and concentration are shown ( Figure 8a A schematic diagram showing the experimental timeline in brief. Figures 8b to 8c The results are shown in which saline, CARS1 (99-200, C182S) 5 mpk, 10 mpk, and 20 mpk were administered to C57bl / 6 mice by intraperitoneal (IP) or intranasal (IN) routes on days -7 and 0, followed by intranasal administration of PR-8 influenza virus on day 0, and the survival rate (b) and body weight changes (c) over time were observed.

[0245] Figures 9a to 9b The results of confirming the antiviral effect of CARS1 (99-200, C182S) protein compared with the control group are shown ( Figure 9a is a schematic diagram briefly showing the experimental timeline. Figure 9b Shown are the results of administering saline, CARS1 (99-200, C182S) 2 mpk or 10 mpk, and oseltamivir 20 mpk to C57bl / 6 mice by the intranasal (IN) route, followed by intranasal administration of PR-8 influenza virus, and observing survival rate).

[0246] Figures 10a to 10c Results confirming the antiviral effect of CARS1 (140-200, C182S) protein are shown ( Fig.10aA schematic diagram showing the experimental timeline in brief. Figure 10b to Figure 10c The figure shows the results of administering CARS1 (106-228) and CARS1 (140-200, C182S) 10 mpk to C57bl / 6 mice by intraperitoneal route on days -7 and -3, followed by intranasal administration of PR-8 influenza virus on day 0, and observing the changes in body weight and survival rate). DETAILED DESCRIPTION

[0247] The present invention will be described in detail below in conjunction with the following examples. However, the following examples are only used to illustrate the present invention, and the present invention is not limited thereto.

[0248] Experimental methods

[0249] 1. Cloning

[0250] Using the pET28a vector containing N- and C-terminal 6x his tags, various fragment protein genes from CRS (=CARS1) were cloned. Using CRSDNA as a template, complementary primers were designed at the ends of each sequence, and PCR was performed to obtain PCR products. Next, the PCR product was inserted into the destination vector, the gene was restricted with restriction endonuclease NdeI in the N-terminal direction and XhoI in the C-terminal direction, and ligated with T4 ligase. The recombinant gene was transformed into DH5α cells to obtain the DNA of the recombinant gene. The transformed cells were cultured at 37 ° C on LB agar medium for 16 hours, and the formed colonies were inoculated into LB liquid medium and cultured at 37 ° C for 16 hours. Then, centrifuged at 3000g for 20 minutes, the supernatant was removed, and a precipitate was obtained. The precipitate was lysed using a small preparation kit to obtain DNA, and sequenced by Bioneer to confirm that it matched the designed sequence.

[0251] 2. Affinity Chromatography Purification

[0252] BL21-codon plus cells were transformed with DNA of various proteins from CRS, and colonies were inoculated into culture medium and grown. Cells were grown in LB until OD 600 reached 0.5, and protein expression was induced for 16 hours at 4 ° C using 0.5mM IPTG. Cell pellets were obtained by centrifugation, and cells were lysed by ultrasonic treatment in 50mM Tris buffer pH 7.5 containing 300mM NaCl. Then, the supernatant was obtained by centrifugation at 20,000g for 30 minutes. It was poured onto a column containing Ni-NTA resin. The washing step was performed with 50mM Tris pH 7.5 containing 300mM NaCl, 5% glycerol and 15mM imidazole. The protein was eluted from the column with 10 ml of elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 300 mM imidazole) and endotoxin was removed using TX-114 (reference: Endotoxin removal from protein solutions by phase separation using Triton X-114). Appropriate protein from the LAL assay of less than 0.04 EU / mg was used throughout the experiment.

[0253] 3. Gel Filtration Chromatography Purification

[0254] After connecting the Superdex 75 10 / 300GL column to the AKTA pure device, the column was washed with 3 column volumes of 70% ethanol and 1N NaOH. To stabilize the column, 2 column volumes of 50mMTris buffer pH7.5 containing 150mM NaCl and 0.1mM EDTA were passed through the column. 500μl of protein obtained by affinity chromatography was loaded into a 1ml syringe and injected into the AKTA injection valve. The protein was eluted at different retention times according to the molecular weight. Then, the protein eluted at the same retention time was subjected to SDS-PAGE, and the presence of the target protein was confirmed by Coomassie Brilliant Blue staining.

[0255] 4. Ion Exchange Chromatography Purification

[0256] After connecting the Hitrap Q column to the AKTA pure device, wash the column with 3 column volumes of 70% ethanol and 1N NaOH. In order to stabilize the column, pass the column with 5 column volumes of 20mM Tris buffer pH 7.5. Load 500μl of protein obtained by gel filtration chromatography into a 1ml syringe and inject it into the AKTA injection valve. Then, set the flow rate to 5ml / min, pass it with 20mM Tris buffer pH 7.5 containing 1M NaCl, and obtain the eluted sample in the tube over time.

[0257] 5.ELISA

[0258] THP1-PMA cells were assayed to confirm CRS-induced cytokine secretion. Each cell type was plated at 5x10 cells / mL in a 24-well plate. 5 Cells / ml were treated overnight, and each well was replaced with serum-free medium for 2 hours before drug treatment. For RAW264.7 and THP1-PMA, 100 nM protein was treated for 4 hours. For BMM and BMDC cells, 100 nM protein was treated for 24 hours. The supernatant was centrifuged at 500 g for 10 minutes and ELISA was performed using IL-6, TNF-α, IL-12p70 and IL-10 ELISA kits (BD).

[0259] 6. Circular dichroism analysis

[0260] CD measurements were performed on proteins dissolved in 50 mM Tris buffer containing 300 mM NaCl using a chirascan V100 in the wavelength range of 190-260 nm and a bandwidth of 1 nm. A quartz sample cell with a 1 mm optical path length was used and the proteins to be measured differed in terms of heat-induced denaturation. Spectral analysis was performed to analyze changes in the secondary structure of the proteins.

[0261] 7. Antiviral analysis

[0262] (1) Affinity chromatography purification (bacterial cells)

[0263] BL21-codon plus cells were transformed with DNA of various proteins from CARS1, and colonies were inoculated into culture medium and grown. Cells were grown in LB until OD 600 reached 0.5, and protein expression was induced using 0.5mM IPTG at 4 ° C for 16 hours. Cell pellets were obtained by centrifugation, and cells were lysed by ultrasonic treatment in 50mM Tris buffer pH 7.5 containing 300mM NaCl. Then, the supernatant was obtained by centrifugation at 20,000g for 30 minutes. It was poured onto a column containing Ni-NTA resin. The washing step was performed using 50mM Tris (pH 7.5) containing 300mM NaCl, 5% glycerol and 15mM imidazole. The protein was eluted from the column using 10 ml of elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 300 mM imidazole) and endotoxins were removed using TX-114 (reference: Endotoxin removal from protein solutions by phase separation using Triton X-114). Appropriate protein from the LAL assay of less than 0.04 EU / mg was used throughout the experiment.

[0264] (2) Trained immune mouse model

[0265] (2-1) Influenza virus

[0266] C57BL / 6 mice were purchased from DooYeol Biotech. Female C57BL / 6 mice aged 6-8 weeks were subcutaneously injected with 5, 10, and 20 mg / kg CARS1 (99-200, C182S) protein on the dorsal side on days -7 and -3. 50 (50% lethal dose) PR8 influenza virus was administered intranasally. Body weight was measured daily from the day of virus administration, and survival graphs were recorded. In a comparative experiment using oseltamivir, oseltamivir was orally administered once a day for 5 days after virus infection.

[0267] (2-2) Coronavirus

[0268] hACE-2-introduced C57BL / 6 mice were purchased from DooYeol Biotech. Female hACE-2-introduced C57BL / 6 mice aged 6-8 weeks were injected intranasally with 10 mg / kg CARS1 (99-200, C182S) protein on days -7 and -3. On day 0, 5 lethal doses of highly lethal SARS-CoV-2 (S clade) virus were administered intranasally. Body weight was measured daily from the date of virus administration, survival graphs were recorded, and virus titers were analyzed.

[0269] (3) Protein blotting

[0270] Hek 293T cells were cultured at 3-5 x 10 5 Cells / well were distributed into 6-well plates and cultured for 24 hours. Then, 1μg S-PP-GSAS-Foldon DNA and 2μl Terbofect were mixed evenly and reacted at room temperature for 15-20 minutes. Then the mixture was slowly added dropwise to the cells. After 4 hours, the culture medium was removed, fresh culture medium was added, and the cells were cultured for 24 hours. Take 1ml of culture medium and centrifuge at 500g for 10 minutes at 4℃. Take the supernatant and centrifuge at 10000g for 30 minutes at 4℃. Separate 880μl of supernatant, carefully mix with 120μl TCA (trichloroacetic acid solution), and react at 4℃ for one day. Then centrifuge at 4℃ and 18000g for 15 minutes. When a precipitate is formed, completely remove the supernatant and dry at room temperature for 1 hour. Then add 50μl 0.1M Hepes buffer (pH 8.0) to dissolve the precipitate, add 12.5μl 5X sample buffer, and boil at 100℃ for 10 minutes. The samples were then electrophoresed on an 8% polyacrylamide gel and transferred to an Immobilon P PVDF membrane. Anti-Myc antibody and tubulin antibody were then applied and the target protein was detected using Abclon ECL solution.

[0271] (4) Affinity chromatography purification (animal cells)

[0272] Resuspend ExpiCHO-S cells in ExpiCHO Expression Medium and culture in a shaking incubator until they reach 4 × 10 6 Up to 6×10 6 One day before transfection, subculture the cells to 3×10 6 -4×10 6 The next day, dilute the cells to 25 ml with fresh medium to reach 6 × 10 6 cells / ml. For transfection, shake ExpiFectamine CHO reagent 4-5 times, take 80μl and mix with 920μl OptiPRO SFM solution, and then slowly aspirate 2-3 times. Mix 20μg of DNA for transfection with 1ml OptiPRO SFM solution and shake slowly. Before reaching 5 minutes, slowly add the solution mixed with ExpiFectamine CHO reagent to the DNA solution and shake slowly. After about 3 minutes of reaction at room temperature, slowly add the solution mixed with ExpiFectamine CHO reagent and DNA to the culture flask containing cells. Then place the culture flask containing cells in an environment of 37℃ and 8% CO2. 18-22 hours after culture, slowly add 150μl ExpiFectamine CHO Enhancer and 6ml ExpiCHO Feed, and culture cells under the same conditions. On the 10th day after transfection, remove all the cell culture medium and place it in a 50ml centrifuge tube, and centrifuge it at 5000g for 30 minutes at 4℃. Take the supernatant and pass it through a 0.22 μm filter (Acrodisc syringe filter). During affinity chromatography, 3.5 ml of Ni-NTA resin was added to a glass column, and a 50 mM Tris binding buffer (pH 8.0) containing 300 mM NaCl and 5% glycerol was passed through the resin at 10 times the resin volume. The previously separated supernatant was loaded onto a glass column. 100 ml of A wash buffer containing 15 mM imidazole in the binding buffer was passed through the glass column, and 5 ml of B wash buffer containing 30 mM imidazole in the binding buffer was added. Subsequently, 10 ml of elution buffer containing 300 mM imidazole in the binding buffer was loaded into the column, and the eluate was collected in a 10 ml conical tube. The eluate was placed in a dialysis cassette and dialyzed for 4 hours at 4° C. in 1.5 L of a storage buffer (50 mM Tris, pH 8.0) containing 300 mM NaCl and 15% glycerol. The storage buffer was then replaced with fresh storage buffer (1.5 L) and dialysis was continued for 16 hours.

[0273] 8. Preparation of Experimental Materials

[0274] The CRS fragment peptides used in the present invention and their sequence information are as follows, and these peptides are prepared according to conventional methods:

[0275] SEQ ID NO:2:CRS(140-200)

[0276] SEQ ID NO:3: CRS(140-200, C182S)

[0277] SEQ ID NO:4:CRS(106-228)

[0278] SEQ ID NO:5:CRS(101-200)

[0279] SEQ ID NO:6:CRS(119-200)

[0280] SEQ ID NO:7:CRS(99-200)

[0281] SEQ ID NO:8: CRS(99-200, C182S)

[0282] The numbers in brackets of each sequence represent the amino acid numbers in the full-length CRS protein of SEQ ID NO: 1. In the following experimental results, CARS1 is interpreted as being the same as the above-mentioned CRS.

[0283] Experimental Results

[0284] 1. Identification of immunoreactive sites of CRS-derived fragment proteins

[0285] In order to identify the immunologically active site of the fragment protein containing amino acids 106 to 228 of CRS found in previous studies, several fragment protein genes including each helix were constructed by analyzing the secondary structure of the protein. After culturing and purifying the proteins, they were treated with Thp-1 cells differentiated with PMA.

[0286] The results confirmed that the protein excluding helix 4 did not induce immune activity compared to LPS. It was confirmed that the protein having sequences 1, 2, 3 and 4 (including helices 3 and 4) induced immune activity ( Figure 1a ).

[0287] 2. Confirmation of production stability of CRS-derived fragment protein CRS (140-200, C182S)

[0288] Based on the above experimental results, a gene was designed and cloned in which cysteine ​​at position 182 was substituted with serine to inhibit the formation of polymers caused by cysteine ​​at position 182 in the fragment protein containing amino acids 140 to 200 of CRS. The protein was produced by transformation using BL21DE3 codon plus RIPL cells ( Figure 2 ).

[0289] As a result, no degradation issues reported in previous studies were observed ( Figure 3 ). It was also confirmed by gel filtration chromatography and ion exchange chromatography that the protein was relatively non-multimerizing compared to the protein without the cysteine ​​substitution at position 182 ( Figures 4 to 5 ).

[0290] 3. Confirmation of thermal stability of CRS-derived fragment protein CRS (140-200, C182S)

[0291] Based on the above experimental results, the thermal stability of the protein in which cysteine ​​at position 182 in the fragment protein containing amino acids 140 to 200 of CRS was replaced by serine was confirmed by circular dichroism analysis and ELISA.

[0292] The results confirmed that, compared with the CRS-derived fragment protein induced by high temperature denaturation, its secondary structure was maintained at the same level as that of the protein containing amino acids 106 to 228 of CRS reported to be thermostable in previous studies ( Figure 6a In addition, when the CRS-derived fragment protein induced by high temperature denaturation was treated with Thp-1 cells differentiated with PMA, and the amount of TNF-α in the culture medium was measured to observe the immune activity, it was found that despite the high temperature denaturation, the function of inducing immune activity was still maintained, indicating that due to thermal stability, the function is not lost at high temperatures ( Figure 6b ).

[0293] 4. Confirmation of the ability of CRS-derived fragment protein CRS (140-200, C182) to induce immune cell activation

[0294] Based on the above experimental results, in order to confirm whether the ability of CRS-derived fragment protein CRS (140-200, C182S) to induce immune cell activation is a protein-specific function, it was treated with LPS inhibitor polymyxin B (10ug / ml) and protein degrading enzyme proteinase K (20ug / ml), and then treated with Thp-1 differentiated with PMA.

[0295] The results showed that in the polymyxin b treated group, the activity decreased in the case of LPS, but the activity of the fragment protein derived from CRS did not decrease, which means that the induced immune activity was not caused by LPS. In addition, in the case of the proteinase K treated group, the immune activity of the fragment protein from CRS was observed to be reduced compared with LPS, which means that the induced immune activity was caused by protein. Therefore, the immune activity inducing ability of the fragment protein CRS (140-200, C182S) from CRS is not caused by LPS contamination, but the function of the protein.

[0296] 5. Verification of antiviral efficacy

[0297] (1) Confirmation of antiviral efficacy by administration route and concentration of CARS1 (99-200, C182S) protein

[0298] The most significant changes caused by known viral infections are weight loss and decreased survival. To confirm antiviral efficacy, it is necessary to examine how much weight can be restored to pre-infection weight compared to the control group and whether survival is maintained over time. In addition, since each virus has a different entry route, the route of administration of the candidate substance should also vary depending on the target virus.

[0299] The immune cell activation capacity of CARS1 induces intracellular reprogramming, which causes secondary stimulation and affects innate immune memory. This process is called trained immunity, which can regulate immune homeostasis and tolerance, and it has been reported that defense mechanisms against viruses can be established in this way. Therefore, it was decided to verify whether trained immunity induced by CARS1-derived fragment proteins acquires defense capabilities against viral infections.

[0300] Therefore, the inventors confirmed the antiviral efficacy by injecting CARS1 (99-200, C182S) protein at concentrations of 5, 10 and 20 mpk (mg / kg) using both intraperitoneal and intranasal routes to examine the efficacy differences according to the route of administration. CARS1 (99-200, C182S) protein was administered to C57bl / 6 mice on days -7 and -3, and PR-8 influenza virus ( Figure 8a ).

[0301] Results showed that in the case of intraperitoneal administration, survival was increased at 10 and 20 mpk compared with saline, and body weight changes were also maintained at higher levels at both concentrations ( Figures 8b to 8c ). In the case of intranasal administration, the survival rate at concentrations of 5, 10, and 20 mpk was higher than that of saline, and the body weight changes at the three concentrations were maintained at a level higher than that of saline, with 10 mpk being the highest.

[0302] (2) Confirmation of the antiviral efficacy of CARS1 (99-200, C182S) protein compared with the control group

[0303] According to the above results, the influenza virus treatment drug oseltamivir (Tamiflu) was used as a control group to compare the antiviral efficacy of CARS1 (99-200, C182S) protein. CARS1 (99-200, C182S) protein was administered to C57bl / 6 mice on days -7 and -3, and PR-8 influenza virus was administered intranasally on day 0. For oseltamivir, it was orally administered 4 hours before the administration of PR-8 influenza virus, and then the virus infection was performed ( Figure 9a ).

[0304] The results showed that the survival rate increased at 2 and 10 mpk CARS1 (99-200, C182S) protein compared with saline, and at 10 mpk, the survival rate was maintained at a level similar to that of oseltamivir used as a control group ( Figure 9b ).

[0305] (3) Confirmation of the antiviral efficacy of CARS1 (99-200, C182S) protein against coronavirus compared with the control group

[0306] CARS1 (99-200, C182S) protein was administered to hACE-2-introduced C57bl / 6 mice on days -7 and -3, and SARS-Cov-2 (S clade) virus was intranasally administered on day 0 ( Fig.10a ).

[0307] Results showed no weight loss at 10 mpk CARS1(99-200,C182S) protein compared to saline ( Fig.10b ), all individuals survive ( Fig.10c In addition, when the animals were sacrificed on days 4 and 6 and lung tissues were analyzed, Cov-2 virus titers were significantly reduced in the CARS1 (99-200, C182S) protein-treated group compared with saline ( Fig.10d ).

[0308] (4) Confirmation of the antiviral ability of CARS1 (140-200, C182S) protein

[0309] Based on the above results, in order to compare the antiviral ability of the minimal unit form of CARS1 (99-200, C182S) protein that retains the active part with that of CARS1 (106-228), each protein was intraperitoneally administered to C57bl / 6 mice at 10 mpk, and PR-8 influenza virus was intranasally administered on day 0, and then the body weight change and survival rate were examined ( Fig.10a ).

[0310] The results showed that compared with saline, both CARS1(106-228) and CARS1(140-200,C182S) proteins were observed to increase body weight ( Fig.10b ), and the survival rate of both proteins was also increased compared with saline ( Fig.10c ).

Claims

1. A pharmaceutical composition for antiviral use, comprising as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the peptide comprises a mutation, wherein the mutation is a substitution of the cysteine ​​at position 182 in the amino acid sequence of SEQ ID NO: 1 by other amino acids.

3. The pharmaceutical composition according to claim 2, wherein the other amino acid is serine. 4 . The pharmaceutical composition according to claim 1 , wherein the peptide is selected from the amino acid sequences of SEQ ID NOs: 2 to 8.

5. The pharmaceutical composition according to claim 1, wherein the virus is selected from the group consisting of Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Maraviridae. naviridae), Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Bornaviridae, Filoviridae, Paramyxoviridae oviridae, Rhabdoviridae, Nyamiviridae, Caliciviridae, Flaviviridae, Luteoviridae, Togaviridae, Pneumoviridae, Arenaviridae, Deltavirus, and Orthomyxoviridae.

6. The pharmaceutical composition according to claim 1, wherein the virus is selected from influenza virus, influenza A virus subtype H1N1, avian influenza virus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human immunodeficiency virus (HIV), retrovirus and hepatitis C virus.

7. A pharmaceutical composition according to claim 6, wherein the coronavirus is selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS) coronavirus.

8. A food composition for antiviral use, comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof.

9. The food composition according to claim 8, wherein the food composition is a health functional food.

10. A quasi-drug composition for antiviral use, comprising as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

11. The quasi-drug composition according to claim 10, wherein the quasi-drug is a disinfectant cleaner, a nasal spray, a shower foam, a mouthwash, a wet wipe, a washing soap, a hand sanitizer, a humidifier refill, a facial mask, an ointment, a patch or a filter refill.

12. An adjuvant composition for a viral vaccine, comprising the following substances as active ingredients: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or more homology with the peptide, or a pharmaceutically acceptable salt thereof.

13. The adjuvant composition for a viral vaccine according to claim 12, wherein the virus is selected from influenza virus, influenza A virus H1N1 subtype, avian influenza virus, rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, human immunodeficiency virus (HIV), retrovirus and hepatitis C virus.

14. An adjuvant composition for a viral vaccine according to claim 12, wherein the coronavirus is selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS) coronavirus.

15. Use of a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 to any amino acid selected from amino acids 185 to 228 in the amino acid sequence of SEQ ID NO: 1, or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide, or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for antiviral use.

16. A method for preventing or treating viral infection, comprising administering to a subject in need thereof an effective amount of a composition comprising as an active ingredient: a peptide comprising a continuous amino acid sequence of any amino acid selected from amino acids 99 to 140 in the amino acid sequence of SEQ ID NO: 1 to any amino acid selected from amino acids 185 to 228; or a peptide comprising an amino acid sequence having 80% or higher homology with the peptide.

17. A peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 95% or higher sequence homology thereto. 18 . The peptide according to claim 17 , wherein the peptide comprises a mutation, wherein the mutation is a substitution of cysteine ​​at position 43 in the amino acid sequence of SEQ ID NO: 2 with other amino acids.

19. The peptide according to claim 18, wherein the other amino acid is serine.

20. A polynucleotide comprising a nucleotide sequence encoding the peptide of claim 17.

21. The polynucleotide according to claim 20, wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:

10.

22. A vector comprising the polynucleotide of claim 20.

23. A host cell transformed with the vector of claim 22.

24. A vaccine adjuvant comprising one or more selected from the following: (i) the peptide according to claim 1, (ii) a polynucleotide encoding (i), (iii) a vector comprising (ii), and (iv) using the host cell transformed with (iii).

25. A vaccine composition comprising the vaccine adjuvant of claim 24 and an antigen.

26. The vaccine composition according to claim 25, wherein the vaccine is an anti-cancer vaccine.

27. The vaccine composition according to claim 25, wherein the anti-cancer vaccine is a vaccine for cancer prevention or a vaccine for cancer treatment.

28. A pharmaceutical composition for preventing or treating cancer, comprising one or more selected from the following: (i) the peptide according to claim 1, (ii) a polynucleotide encoding (i), (iii) a vector comprising (ii), and (iv) using the host cell transformed with (iii).

Images