Novel CRS fragment peptide having immunopotentiating activity and its use

Abstract

The present invention relates to a novel CRS fragment peptide having immunopotentiating activity and its use. 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 showing 95% or more sequence homology thereto, its adjuvant for vaccines, its use as a cancer therapeutic agent, and its use in antiviral compositions. The peptide disclosed in the present invention exhibits anti-cancer activity, enhancement of immune function, and antiviral activity as a CRS fragment first disclosed herein.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application Nos. 10-2022-0070283 and 10-2022-0070287, filed on June 9, 2022, and the entire specification of the applications is incorporated herein by reference.

[0002] The present invention relates to a novel CRS fragment peptide having immunostimulatory activity and its use, and more particularly, to a novel peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence showing 95% or more sequence homology therewith, and its use as a vaccine adjuvant, a cancer therapeutic agent, and an antiviral agent.

Background Art

[0003] Aminoacyl-tRNA synthetase (ARS or AARS), which catalyzes the aminoacylation of tRNA molecules, is essential for decoding genetic information during the translation process. Each of the eukaryotic tRNA synthetases consists of a core enzyme (closely related to the prokaryotic counterpart of the tRNA synthetase) and an additional domain (added to the amino or carboxyl terminus of the core enzyme). Therefore, there are significant differences in the composition of the enzymes between eukaryotes and prokaryotes. For example, human tyrosyl-tRNA synthetase (TyrRS) has a carboxyl-terminal domain that is not present in the TyrRS molecules of prokaryotes and lower eukaryotes.

[0004] In recent years, several aminoacyl-tRNA synthetases have been shown to have non-canonical functions distinct from their involvement in the translation process. That is, some fragments of ARS proteins have been found to exhibit extracellular signaling (signaling) activities that regulate different types of pathways beyond protein translation, and retain unexpected activities not related to aminoacylation. Such unexpected activities may sometimes be therapeutically exploitable against certain diseases, etc., but may also be activities that are more likely to function in inducing human disease states. As an example, lysyl-tRNA synthetase (KRS) is known to have an activity that promotes cancer metastasis. Also, mini-tyrosyl-tRNA synthetase (mini-TRS, corresponding to amino acid residues 1 to 364), which is the N-terminal domain of TRS cleaved by polymorphonuclear cell elastase and plasmin, exhibits non-canonical biological activities not seen in the full-length protein. Mini-TRS in vitro has been shown to stimulate endothelial cell proliferation and migration, and has an activity that promotes angiogenesis in the mouse matrigel assay. The function of promoting neovascularization is generally also closely related to cancer metastasis.

[0005] Thus, unexpected activities are not observed in the native full-length protein sequence (or do not show significant effects at the native full-length protein level), but when some regions are isolated, specific activities may be prominently shown, and the effects may also possess characteristics inappropriate for therapeutic use. To overcome the difficulties associated with such unpredictability and to utilize the therapeutic potential of this ARS family of proteins, various efforts are needed to investigate the biologically relevant forms of other various aminoacyl-tRNA synthetase proteins.

[0006] At present, vaccination is the best way to prevent viral diseases. However, in the case of viral diseases, problems such as the efficiency of vaccines due to the generation of many viral serotypes (subtypes) are generally raised as important issues. It is necessary to develop an antiviral composition that can compensate for such problems of vaccines, that is, a composition for preventing or treating viral infections, or an immunoadjuvant that can increase the effect of viral vaccines.

[0007] In particular, in January 2020, SARS-CoV-2, identified as the cause of COVID-19, spread around the world and was recently declared an infectious disease by the World Health Organization (WHO). The new coronavirus has been reported to be highly infectious and pathogenic, causing severe pneumonia symptoms in infected patients. It has been found that this virus can be a serious threat, especially to the elderly and / or people with underlying diseases such as cardiovascular diseases, diabetes, chronic respiratory diseases, and cancer. However, the fact remains that the development of a formulation that can effectively prevent and treat novel viral infections such as SARS-CoV-2 has not been completed.

Summary of the Invention

Problems to be Solved by the Invention

[0008] In previous studies, the present inventors found that a fragment peptide of cysteinyl-tRNA synthetase (hereinafter referred to as "CRS") has anticancer activity and immunopotentiating activity. However, it was confirmed that it is difficult to develop it as a drug due to the above limitations. Therefore, as a result of intensive research to develop a novel CRS fragment peptide that can overcome the above limitations, among the amino acid sequences of SEQ ID NO: 1, a peptide containing consecutive amino acids from any one selected from the 101st to 140th amino acids to the 200th amino acid shows anticancer activity and immunopotentiating activity equivalent to that of the conventional CRS fragment peptide described above. Moreover, it was found that it not only maintains its form as a monomer, is not degraded even without an affinity tag, and is very stable even at high temperatures, thus completing the present invention.

[0009] Therefore, another object of the present invention is a peptide comprising consecutive amino acids from any one selected from amino acids at positions 99 to 140 to any one selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutical composition, food composition or quasi-drug composition for antiviral use, which contains a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] Furthermore, another object of the present invention is to provide a pharmaceutical composition, food composition or quasi-drug composition for antiviral use, which comprises a peptide comprising consecutive amino acids from any one selected from amino acids at positions 99 to 140 to any one selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, another object of the present invention is to provide a pharmaceutical composition, food composition or quasi-drug composition for antiviral use, which consists essentially of a peptide comprising consecutive amino acids from any one selected from amino acids at positions 99 to 140 to any one selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0012] Another object of the present invention is a peptide comprising consecutive amino acids from any one selected from amino acids at positions 99 to 140 to any one selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutical composition for an immunoadjuvant for a virus vaccine, which contains a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another object of the present invention is to provide the use of a peptide comprising any one selected from the amino acids at positions 99 to 140 and any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1 for producing an antiviral pharmaceutical composition; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0014] Another object of the present invention is to provide a method for preventing or treating viral infection, which comprises administering an effective amount of a composition containing, as an active ingredient, a peptide comprising any one selected from the amino acids at positions 99 to 140 and any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; or a peptide comprising an amino acid sequence showing 80% or more homology with the peptide, to an individual in need thereof.

[0015] An 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 showing 95% or more sequence homology therewith.

[0016] Another object of the present invention is to provide a polynucleotide comprising a nucleotide sequence encoding the peptide.

[0017] Another object of the present invention is to provide a vector comprising the polynucleotide.

[0018] Another object of the present invention is to provide a host cell transformed with the vector.

[0019] Another object of the present invention is to provide a vaccine adjuvant comprising one or more selected from the group consisting of the following (i) to (iv). (i) The above peptide, (ii) A polynucleotide encoding the above (i), (iii) A vector comprising the above (ii), and (iv) A host cell transformed with the above (iii).

[0020] Another object of the present invention is to provide a vaccine composition containing the above vaccine adjuvant and antigen.

[0021] Another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment containing one or more selected from the group consisting of the following (i) to (iv).

[0022] Furthermore, another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment comprising one or more selected from the group consisting of the following (i) to (iv).

[0023] Furthermore, another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment consisting essentially of one or more selected from the group consisting of the following (i) to (iv). (i) the above peptide, (ii) a polynucleotide encoding the above (i), (iii) a vector containing the above (ii), and (iv) a host cell transformed with the above (iii).

Means for Solving the Problems

[0024] In order to achieve the above object of the present invention, the present invention provides a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the above peptide; or a pharmaceutical composition, food composition or quasi-drug composition for antiviral use containing its pharmaceutically acceptable salt as an active ingredient.

[0025] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for antiviral use, a food composition or a quasi-drug composition, which comprises a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0026] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for antiviral use, a food composition or a quasi-drug composition, which consists essentially of a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0027] In order to achieve another object of the present invention, the present invention provides an immunoadjuvant composition for a virus vaccine, which comprises a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof as an active ingredient.

[0028] In order to achieve another object of the present invention, the present invention provides the use of a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof for producing a pharmaceutical composition for antiviral use.

[0029] To achieve other objects of the present invention, the present invention provides a method for preventing or treating viral infection, which comprises administering an effective amount of a composition comprising, as an active ingredient, a peptide containing consecutive amino acids selected from any one of the amino acids at positions 99 to 140 and any one of the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; and a peptide containing an amino acid sequence showing 80% or more homology with the above peptide, to an individual in need thereof.

[0030] To achieve other objects 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 showing 95% or more sequence homology therewith.

[0031] To achieve other objects of the present invention, the present invention provides a polynucleotide containing a nucleotide sequence encoding the above peptide.

[0032] To achieve other objects of the present invention, the present invention provides a vector containing the above polynucleotide.

[0033] To achieve other objects of the present invention, the present invention provides a host cell transformed with the above vector.

[0034] To achieve other objects of the present invention, the present invention provides a vaccine adjuvant containing one or more selected from the group consisting of the following (i) to (iv). (i) The above peptide, (ii) A polynucleotide encoding the above (i), (iii) A vector containing the above (ii), and (iv) A host cell transformed with the above (iii).

[0035] To achieve other objects of the present invention, the present invention provides a vaccine composition containing the above vaccine adjuvant and an antigen.

[0036] To achieve other objects of the present invention, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising one or more selected from the group consisting of the following (i) to (iv).

[0037] Furthermore, to achieve other objects of the present invention, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising one or more selected from the group consisting of the following (i) to (iv).

[0038] Furthermore, to achieve other objects of the present invention, the present invention provides a pharmaceutical composition for cancer prevention or treatment consisting essentially of one or more selected from the group consisting of the following (i) to (iv). (i) The above peptide, (ii) A polynucleotide encoding the above (i), (iii) A vector containing the above (ii), and (iv) A host cell transformed with the above (iii).

[0039] Hereinafter, the present invention will be described in detail.

[0040] The practice of the present invention uses conventional methods of molecular biology and recombinant DNA technology within the technical field to which the present invention pertains, and most of which are known for the purpose of explanation, unless otherwise indicated to the contrary.

[0041] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0042] Throughout the content disclosed in this specification, various aspects or conditions related to the present invention can be proposed in a range format. In this specification, the description of range values includes the boundary values unless otherwise specified, that is, it means including all values from the lower limit value or more to the upper limit value or less. The description in range format is for mere convenience and simplicity, and it should not be construed as an inflexible limitation on the scope of the present invention. Therefore, the description of a range should be considered as specifically disclosing not only the individual numerical values within the range but also all possible subranges. For example, the description of a range such as 7 to 170 should be regarded as specifically disclosing the individual numerical values within the above range, such as 9, 27, 35, 101, and 155, as well as subranges such as 10 to 127, 23 to 35, 80 to 100, 50 to 169, etc. This applies regardless of the width of the range.

[0043] The term "comprising" of the present invention is used in the same way as "containing" or "characterized by", and does not exclude additional component elements or method steps not mentioned in the composition or method. Also, the term "consisting of" means excluding additional elements, steps, or components not separately described, etc. The term "essentially consisting of" means that in the scope of a composition or method, it may include materials or steps that do not substantially affect the basic characteristics in addition to the described materials or steps.

[0044] As used herein, the terms "peptide" and "protein" are used in their ordinary (conventional) sense, meaning the arrangement of amino acids. A peptide is not limited to a specific length, but in the context of the present invention generally represents a fragment of a full-length protein, and can include post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (naturally occurring and non-naturally occurring modifications), and can be represented as a "polypeptide". The peptides and proteins of the present invention can be prepared using any of a variety of known recombinant and / or synthetic techniques, and exemplary embodiments thereof are further described below.

[0045] The present invention is derived from the discovery that CRS and peptides derived from CRS retain therapeutically relevant non-canonical biological activities.

[0046] As used herein, "non-canonical activity" generally refers to the activities retained by the CRS peptides of the present invention, in addition to adding cysteine to tRNA molecules. As described in detail herein, in certain embodiments, the non-canonical biological activities exhibited by the CRS fragments of the present invention can be selected from the group consisting of, but not limited to, anti-cancer activity, activation of innate immunity, and activation of adaptive immunity.

[0047] It should be understood that the present invention includes not only CRS fragment peptides having at least one non-canonical biological activity, but also variants that substantially maintain the non-canonical activity, and the like.

[0048] Specifically, the inventors have investigated that the novel CRS fragment peptide exhibits the activity of activating innate and adaptive immunity and is also very stable structurally. The region of the CRS fragment peptide is first disclosed in the present invention.

[0049] In the present invention, the peptide is a cleaved form of the CRS protein, and the peptide consists of amino acids at positions 140 to 200 in the sequence of the full-length CRS protein consisting of the amino acid sequence of SEQ ID NO: 1, or amino acids showing 95% or more sequence homology therewith. It may also be to maintain (retain) at least one desired abnormal biological activity.

[0050] The present invention provides an antiviral food composition comprising a peptide containing any one continuous amino acid selected from amino acids at positions 99 to 140 and any one continuous amino acid selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof as an active ingredient.

[0051] Furthermore, the present invention provides an antiviral food composition comprising a peptide containing any one continuous amino acid selected from amino acids at positions 99 to 140 and any one continuous amino acid selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or an antiviral food composition comprising a pharmaceutically acceptable salt thereof.

[0052] Furthermore, the present invention provides an antiviral food composition consisting essentially of a peptide containing any one continuous amino acid selected from amino acids at positions 99 to 140 and any one continuous amino acid selected from amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

[0053] The description of each component in the food composition can be referred to above.

[0054] The food composition of the present invention includes all foods in the ordinary sense, including all forms such as functional foods, nutritional supplements, health foods, and food additives. The food compositions of the above types can be manufactured in various forms according to conventional methods known in the art.

[0055] The food composition of the present invention can include health functional foods. The term "health functional food" used in the present invention refers to a food manufactured and processed in forms such as tablets, capsules, powders, granules, liquid phases, and rings using raw materials and components having useful functions for the human body. Here, "functionality" means adjusting nutrients with respect to the structure and functions of the human body and obtaining useful effects for health applications such as physiological effects. The health functional foods of the present invention can be manufactured by methods commonly used in the industry, and during the above manufacturing, raw materials and components commonly added in the industry can be added for manufacturing.

[0056] In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations. Different from general pharmaceuticals, it has the advantage of having no side effects that may occur during long-term use of pharmaceuticals with food as the raw material, and is excellent in portability. The health functional food of the present invention can be ingested as an adjuvant for enhancing the improvement or therapeutic effect of liver diseases or metabolic syndrome.

[0057] For example, as a health functional food, it can be drunk in the form of tea, juice, or drink, or ingested after granulation, encapsulation, and powderization. In addition, it can be manufactured in the form of a composition by mixing with known substances or active ingredients known to have preventive, improving, or therapeutic effects on liver diseases or metabolic syndrome.

[0058] In addition, the food composition of the present invention can contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, it can include natural fruit juices, fruit juice beverages, and pulp for the production of vegetable beverages. These components can be used independently or in combination. The ratio of such additives is not very important, but it is generally selected from the range of 0.01 to 0.3 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

[0059] Moreover, the food composition of the present invention can contain various flavoring agents or natural carbohydrates, etc. as additional components like ordinary beverages. The carbohydrates are 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. As sweeteners, natural sweeteners such as thaumatin and stevia extract, synthetic sweeteners such as saccharin and aspartame, etc. can be used. The ratio of the above natural carbohydrates can generally be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention, but is not limited thereto.

[0060] The present invention also provides a pharmaceutical composition for anti-virus use, which contains, as an active ingredient, a peptide comprising any one continuous amino acid selected from the 99th to 140th amino acids and any one continuous amino acid selected from the 185th to 228th amino acids of the amino acid sequence of SEQ ID NO: 1; a peptide comprising an amino acid sequence showing 80% or more homology with the above peptide; or a pharmaceutically acceptable salt thereof.

[0061] In the "quasi-drug composition for antiviral use" according to the present invention, the quasi-drug is a preparation used for sterilization, insecticidal, and similar uses for the prevention of infectious diseases as described in Article 2, Item 7 of the Korean Pharmaceutical Affairs Act, and can mean a repellent, a remedy, a preventive agent, a control agent, or an attractant insecticide for flies, mosquitoes, etc. used for the health care of humans or animals.

[0062] Also, the quasi-drug may include external skin preparations and personal hygiene products. For example, it may be a disinfectant cleaner, nasal spray, shower foam, mouthwash (gargle), wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler, but is not limited thereto.

[0063] When using the quasi-drug composition according to the present invention as a quasi-drug additive, the composition can be added as it is or used in combination with other quasi-drugs or quasi-drug components, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

[0064] The quasi-drug composition of the present invention can be prepared, for example, in the form of a general emulsifier form and solubilizer form. For example, it can have preparations such as lotions (emulsions), creams, ointments, sprays, oil gels, gels, oils, aerosols, and soft masks, but can be used without limitation as long as it exhibits the pest control induction effect of the present invention. Also, the quasi-drug composition can be appropriately formulated and used with oils, water, surfactants, humectants, lower alcohols having 1 to 4 carbon atoms, thickeners, chelating agents, pigments, preservatives, or fragrances, etc., which are generally formulated in quasi-drug compositions for each preparation, as needed.

[0065] The present invention also provides an adjuvant composition for a viral vaccine, comprising a peptide containing any one continuous amino acid selected from the amino acids at positions 99 to 140 and any one continuous amino acid selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof as an active ingredient.

[0066] In the present invention, the adjuvant is defined as a substance capable of presenting an antigen to the immune system so that an immune response or an increase in the immune response is induced against the antigen when the viral antigen is administered together with the adjuvant. That is, the adjuvant is a substance that promotes the immune response against a viral antigen as an immune enhancer, and means a substance that is not an immunogen to the host but enhances immunity by increasing the activity of immune system cells.

[0067] According to an embodiment of the present invention, it was confirmed that the CRS fragment peptide can significantly improve the increase in the immune response of a subject when treated together with a viral antigen.

[0068] As an embodiment of the present invention, the adjuvant can induce an improved innate immune response and activation of the acquired immune response when the viral antigen and the adjuvant are administered together, as compared with that obtained with the viral antigen itself. More specifically, it can induce activation of an improved cellular immune response and a humoral immune response.

[0069] The present invention also provides the use of a peptide containing any one continuous amino acid selected from the amino acids at positions 99 to 140 and any one continuous amino acid selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof for the manufacture of an anti-viral pharmaceutical composition.

[0070] The present invention also provides a method for preventing or treating viral infection, which comprises administering an effective amount of a composition comprising, as an active ingredient, a peptide comprising consecutive amino acids selected from any one of the amino acids at positions 99 to 140 and any one of the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; and a peptide comprising an amino acid sequence having 80% or more homology with the said peptide, to an individual in need thereof.

[0071] In one embodiment of the present invention, the peptide consists of consecutive amino acids selected from any one of the amino acids at positions 99 to 140 and any one of the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1, and is characterized by containing a mutation in which cysteine, which is the 182nd amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with another amino acid. That is, it can contain a mutation in which cysteine, which is the 43rd amino acid in the amino acid sequence of SEQ ID NO: 2, is substituted with another amino acid, and by this substitution, not only can the formation of multimers of the CRS fragment peptide be suppressed, but also high stability can be maintained without degradation without affinity tags attached to the N-terminus and C-terminus.

[0072] In a preferred embodiment of the present invention, the peptide is characterized in that cysteine, which is the 182nd amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with serine. That is, it can contain a mutation in which cysteine, which is the 43rd amino acid in the amino acid sequence of SEQ ID NO: 2, is substituted with another amino acid (SEQ ID NO: 3). Most preferably, the peptide consists of SEQ ID NOs: 2 to 8, or an amino acid sequence having 80% or more homology therewith.

[0073] The peptide of the present invention can be prepared using available techniques known in the art. As an example, it can be prepared using any one of various 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 C1rC1r, complement C1s, complement Factor D, complement factor I, cucumisin, dipeptidyl peptidase IV, elastase (leukocyte), elastase (pancreatic), endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIV Protease, IGase, kallikrein tissue, leucine aminopeptidase (general), leucine aminopeptidase (cytosol), leucine aminopeptidase (microsomal), matrix metalloprotease, methionine aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E, prostate specific antigen, protease alkalophilic from Streptomyces griseus, protease from AspergillusProteases from Aspergillus, protease from Aspergillus saitoi, protease from Aspergillus sojae, protease B. licheniformis (alkaline or alcalase), protease from Bacillus polymyxa, protease from Bacillus sp, protease from Rhizopus sp., protease S, proteasomes, proteinase 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, etc. Those skilled in the art can easily determine which proteolytic enzyme is appropriate considering the chemical specificity of the fragment to be prepared.

[0074] The polypeptides described in this specification can be produced by any suitable procedure known to those skilled in the art, such as recombinant techniques. In addition to recombinant production methods, the polypeptides of the present invention can be produced by direct peptide synthesis using the solid-phase method.

[0075] The solid-phase peptide synthesis (SPPS) method can start the synthesis by binding functional units called linkers to small porous beads so as to allow the continuation of the peptide chain. Different from the liquid-phase method, the peptide is covalently bonded to the beads and prevented from dropping off by the filtration process until it is cleaved by a specific reactant such as trifluoroacetic acid (TFA). The cycle of the protection process where the N-terminal amine of the peptide attached to the solid phase binds to the N-protected amino acid unit, the deprotection process, and the coupling process where the reproduced amine group binds to a new amino acid (deprotection-wash-coupling-wash) is repeated while the synthesis is carried out. The SPPS method can be carried out in combination with microwave technology, and the microwave technology can shorten the time required for coupling and deprotection in each cycle by applying heat during the peptide synthesis process. The thermal energy can prevent the folding of the growing peptide chain or the formation of aggregates and promote chemical bonding.

[0076] Also, the peptides of the present invention can be prepared by known methods by the liquid-phase peptide synthesis method, and the peptides of the present invention can be synthesized by various methods such as a method of mixing the solid-phase synthesis method and the liquid-phase synthesis method, and the production method thereof is not limited to the means described in this specification.

[0077] Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to produce the target molecule.

[0078] The peptides provided by the present invention include variants having a sequence homology of 95% or more with the peptides. The variants mean active variants of the peptides, and such active variants mean maintaining at least one or more desired abnormal activities (for example, anti-cancer activity and immune enhancing activity) from the peptides from which they are derived. As an example of the variants, they can be splice variants, whether occurring naturally or non-naturally (generated), and the splice variants retain, for example, at least one abnormal activity described herein. As another example, the variants include one or more point mutations with respect to the peptide sequence, whether occurring naturally or non-naturally (generated), and the variant peptides retain, for example, at least one abnormal activity described herein. That is, in the present invention, the variants (or the term "active variants") are understood as functional equivalents of the peptide of SEQ ID NO: 2.

[0079] More specifically, the variants are characterized in that they are functional equivalents having a sequence homology of at least 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more along the longitudinal direction with respect to the above peptide sequence.

[0080] In one embodiment of the present invention, the peptide can be characterized by consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0081] The variant has some change in the amino acid sequence of the "peptide" and may include one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be synthetically generated using any number of techniques well known in the art, for example, by modifying or altering one or more of the peptide sequences of the present invention and evaluating their biological activities as described herein.

[0082] In one embodiment of the present invention, the variant includes conservative substitutions. "Conservative substitution" means a substitution in which one amino acid is replaced with another amino acid having similar properties, such that a person skilled in the art can predict that the secondary structure and hydropathic nature (hydrophobic or hydrophilic nature) of the peptide are substantially unchanged. Generally, the following groups of amino acids show conservative changes: (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.

[0083] Modifications can be made within the structure of the peptides of the present invention to obtain functional molecules encoding peptide variants or derivatives having the desired (preferred) characteristics. When it is desired to change the amino acid sequence of a peptide in order to produce a variant equivalent or improved to the peptide of the present invention, one or more codons can be changed by a person skilled in the art based on the protein codon information known in the art.

[0084] For example, without significant loss of the interactive binding ability having a structure such as a receptor, an antigen-binding site of an antibody, or a binding site on a substrate molecule, certain amino acids can be substituted with other amino acids within a protein or peptide structure. This is because, as the generally defined biological functional activity of a protein, it is due to the interactive ability and properties of the protein, and specific amino acid sequence substitutions can be made within the protein or peptide sequence, and of course within the basic DNA coding sequence, and nevertheless a protein having the same or similar properties can be obtained.

[0085] Accordingly, it is contemplated that various changes can be made to the peptide sequences disclosed above or the DNA sequences encoding such peptides without significant loss of the desired utility or activity. Such modifications can also take into account the hydropathic (hydrophobic or hydrophilic nature) index of the amino acids. The importance of the hydropathic amino acid index that confers a biological function of interacting with a protein is generally understood in the art. For example, the relative hydropathy of amino acids has been found to contribute to the secondary structure of the resulting protein, which ultimately defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobic 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).

[0086] It is known in the art that certain amino acids can be substituted by other amino acids having similar susceptibility indices or scores and a protein having similar biological activity can be obtained (i.e., still obtain a protein that is biologically functionally equivalent). In such modifications, amino acids with a susceptibility index within ±2 are preferred for substitution, amino acid substitutions with a susceptibility index within ±1 are particularly preferred, and amino acid substitutions with a susceptibility index within ±0.5 are even more particularly preferred.

[0087] It is also understood in the art that substitutions of the same amino acid can be effectively carried out based on hydrophilicity. As is 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 an amino acid can be substituted with other amino acids having similar hydrophilicity values and a biologically equivalent protein can be obtained. In such modifications, amino acid substitutions with a hydrophilicity value within ±2 are preferred, amino acid substitutions with a hydrophilicity value within ±1 are particularly preferred, and amino acid substitutions with a hydrophilicity value within ±0.5 are even more particularly preferred.

[0088] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, they can be based on the hydrophobicity, hydrophilicity, charge, size, etc. of those (relative similarity of side-chain substituents). Exemplary substitutions considering the various characteristics described above are well known to those skilled 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.

[0089] Amino acid substitutions can also be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids having uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine and tyrosine.

[0090] Furthermore, the variant may include non-conservative modifications. In a preferred embodiment, the variant peptide may differ from the native sequence by substitution, deletion or addition of five amino acids or fewer. The variant can also be modified, for example, by deletion or addition of amino acids that have a minimal effect on the secondary structure and hydropathic properties of the peptide.

[0091] The peptide may include a signal (or leader) sequence at the N-terminus of the protein, and that sequence directs the transport of the protein either during or after translation. The peptide can also be conjugated to a linker sequence or other sequence (e.g., polyHis) to facilitate the synthesis, purification or identification of the peptide, or to enhance the binding of the peptide to a solid support. For example, the peptide can be conjugated to the Fc region of an immunoglobulin.

[0092] When peptide sequences are compared, as described below, when two sequences are aligned for maximum correspondence and the amino acid sequences are identical in the two sequences, the two sequences are said to be "identical". The comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, "comparison window" means a segment of at least about 20 contiguous positions, typically 30-75, 40-50 contiguous positions, in which the sequences can be compared to a reference sequence at the same number of contiguous positions after the two sequences are optimally aligned.

[0093] Optimal alignment of the arrays for comparison can be done using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using the default parameters, for example.This program includes several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. 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, D. G. and Sharp, P. M. (1989) CABIOS 5:151 - 153; Myers, E. W. and Muller W. (1988) CABIOS 4:11 - 17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406 - 425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'l Acad., Sci. USA 80:726 - 730.

[0094] Alternatively, the optimal alignment of the arrays for comparison can be performed by the partial identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, or by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, or by the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, or by computerized execution 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.

[0095] Examples of suitable algorithms for determining percent sequence identity and percent sequence similarity can be the BLAST and BLAST 2.0 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 BLAST 2.0 can be used to determine the percent sequence homology of the polynucleotides and polypeptides of the present invention, for example, in conjunction with the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Infomation. For amino acid sequences, cumulative scores can be calculated using a scoring matrix.

[0096] The extension of word hits in all directions stops in the following cases: when the cumulative alignment score decreases from its maximum value by quantityX, when the cumulative score becomes zero or less due to the cumulative alignment of one or more negative scoring residues; or when the end of either array is reached. The parameters W, T, and X of the BLAST algorithm described above determine the sensitivity and speed of the alignment.

[0097] In one exemplary approach, the "percentage of sequence identity" is determined by comparing two optimal aligned sequences over at least 20 comparison windows, where the polypeptide sequence portion in the comparison window may include additions or deletions of 20% or less, typically 5 - 15%, or 10 - 12% compared to a reference sequence (which contains no additions or deletions). The percentage is determined by counting the number of positions where the same amino acid residue is present in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0098] The peptides provided by the present invention can be in a linear or cyclic form, which can be understood by referring to the examples of the present invention.

[0099] In the present invention, the production of cyclic peptides as the above-mentioned variants is not particularly limited as long as it is by a known peptide cyclization method known in the art. Preferably, the production of the cyclic peptides of the present invention is prepared by performing cleavage or substitution on a linear peptide such that cysteines are located at both of their ends (the N-terminus and the C-terminus), and a monosulfide bond occurs between the cysteine residues present in both ends.

[0100] The peptides provided herein contemplate the use of modified polypeptides as one embodiment, and such modified polypeptides include modifications that improve the desired properties of the polypeptides isolated as described herein. Exemplary modifications of the polypeptides of the present invention can include, but are not limited to, chemical and / or enzymatic derivatization of one or more constituent amino acids, said derivatization including side chain modifications, backbone modifications, and N-terminal and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the addition of carbohydrate or lipid moieties, cofactors, etc. An exemplary change includes the pegylation of the polypeptide.

[0101] In certain embodiments, chemoselective ligation techniques can be used to modify the peptides of the present invention, for example, by covalently attaching polymers in a site-specific and controlled manner. Such techniques typically rely on the attachment of a chemoselective anchor to the protein backbone by one of chemical or recombinant means, followed by modification to a polymer bearing a complementary linker. As a result, the assembly process and the covalent structure of the resulting protein-polymer conjugate are controlled, thereby allowing for rational optimization of drug properties such as efficacy and pharmacokinetic properties. For example, by enabling the selective attachment of PEG, their pharmacokinetic properties are improved.

[0102] The peptides of the present invention can be in the form of pharmaceutically acceptable salts. Examples of such pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, acetate, citrate, stannate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, or p-toluenesulfonate.

[0103] The present invention also provides a polynucleotide encoding the peptide.

[0104] As used herein, the terms "DNA", "polynucleotide", and "nucleic acid" refer to a DNA molecule isolated from the total genomic DNA of a particular species. Thus, a DNA fragment (portion, segment) encoding a polypeptide refers to a DNA fragment consisting of one or more coding sequences substantially isolated or purified from the total genomic DNA of the species from which the DNA fragment can be obtained. The terms "DNA fragment" and "polynucleotide" include the DNA fragment and smaller fragments thereof, and also include recombinant vectors (including, for example, plasmids, cosmids, phagemids, bacteriophages, viruses, etc.).

[0105] As will be understood by those skilled in the art, the polynucleotide sequences of the present invention have been modified to be capable of expressing or being expressed as proteins, peptides, etc., and include genomic sequences, extra-genomic sequences, sequences encoded by plasmids, and smaller engineered gene fragments, etc. Such fragments can be isolated naturally or synthetically modified by human hands.

[0106] As will be recognized by those skilled in the art, the polynucleotide can be single-stranded (coding or antisense sequence) or double-stranded, and can be a DNA molecule (genomic, cDNA or synthetic) or an RNA molecule. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention. Furthermore, the polynucleotide can be linked to other molecules and / or support materials.

[0107] The polynucleotide can include a natural sequence, or a variant, or a biological functional equivalent of its sequence. The polypeptide variant can include one or more substitutions, additions, deletions and / or insertions as further described below, and preferably, such modifications are made within a range in which the desired activity of the encoded polypeptide is not substantially reduced as compared to the unmodified polypeptide. The effect on the activity of the encoded polypeptide can generally be evaluated as described herein.

[0108] The polynucleotide provided by the present invention is not particularly limited in its specific sequence as long as it encodes the peptide of the present invention or its variant peptide, and any combination of base sequences (nucleic acid sequences) is acceptable. As an example, the peptide consisting of the amino acid sequence of SEQ ID NO: 2 can be expressed by a polynucleotide containing the base sequence represented by SEQ ID NO: 9, and the peptide consisting of the amino acid sequence of SEQ ID NO: 3 can be expressed by a polynucleotide containing the base sequence represented by SEQ ID NO: 10, but is not limited thereto.

[0109] The polynucleotide of the present invention, regardless of the length of its coding sequence itself, is combined with other DNA sequences such as, for example, a promoter, a polyadenylation signal, additional restriction enzyme sites, a multiple cloning site, other coding fragments (portions, segments), etc. As a result, its full length may vary considerably. Therefore, it is considered that polynucleotide fragments of almost all lengths can be applied, and preferably, its full length can be limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0110] Furthermore, as a result of the degeneracy of the genetic code, it will be clearly understood by those skilled in the art that there are many nucleotide sequences encoding the peptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, due to differences in codon usage, other polynucleotides (e.g., polynucleotides optimized for human and / or primate codon selection) are specifically contemplated by the present invention.

[0111] Furthermore, alleles of genes containing the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are modified as a result of one or more mutations, such as nucleotide deletions, additions and / or substitutions. The resulting mRNA and proteins can (although not necessarily) have an altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification and / or database sequence comparison).

[0112] Polynucleotides and their fusions are known in the art, available, and can be manufactured, manipulated and / or expressed using any of a well-established techniques. For example, the polynucleotide sequence encoding the peptide of the present invention, or a functional equivalent thereof, can be utilized within a recombinant DNA molecule that directs the expression of the polypeptide within a suitable host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences can be generated that encode substantially the same or functionally equivalent amino acid sequences, and these sequences can be used for cloning and expression of a given polypeptide.

[0113] As will be understood by those skilled in the art, in some cases it may be advantageous to produce nucleotide sequences (nucleotide sequences encoding polypeptides) that retain non-naturally occurring codons. For example, the preferred codons in a particular prokaryotic or eukaryotic host can be selected to produce recombinant RNA transcripts that have an increased protein expression ratio or desired properties (e.g., a longer half-life than the half-life of transcripts produced from naturally occurring sequences).

[0114] Furthermore, the polynucleotide sequences of the present invention can be manipulated using methods generally known in the art to modify peptide coding sequences for a variety of reasons, including but not limited to modifications that alter the cloning, processing, expression, and / or activity of the gene product.

[0115] Furthermore, the present invention provides a vector comprising the polynucleotide and a host cell transformed with the vector.

[0116] To express a desired polypeptide, a nucleotide sequence encoding the polypeptide or a functional equivalent can be inserted into a suitable expression vector (i.e., a vector containing the elements necessary for transcription and translation of the inserted coding sequence). Expression vectors can be constructed containing a sequence encoding a desired polypeptide, as well as appropriate transcriptional and translational control elements, by methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0117] A variety of expression vector / host systems are known and can be used to contain and express polynucleotide sequences. The expression vector / host systems include, but are not limited to, for example, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with virus expression vectors (e.g., baculovirus); plant cell lines transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell lines, etc.

[0118] The "control elements" or "regulatory sequences" present in the expression vector are untranslated regions (enhancers, promoters, 5' and 3' untranslated regions) that interact with host cell proteins to effect transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host used, any suitable transcriptional and translational elements (including constitutive promoters and inducible promoters) can be used.

[0119] For example, when cloning into a bacterial system, an inducible promoter such as the hybrid lacZ promoter of PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) can be used. In mammalian cell lines, promoters derived from mammalian genes or mammalian viruses are generally preferred. For encoding polypeptides, SV40- or EBV-based vectors can be usefully utilized with appropriate selectable markers when it is necessary to generate cell lines containing multiple copies of the sequence.

[0120] In the bacterial system, multiple expression vectors can be selected according to the intended use for peptide expression. For example, when large quantities are required, vectors that direct the high-level expression of fusion proteins that are easily purified can be utilized. Such vectors include, but are not limited to, the following: multifunctional E. coli cloning vectors and expression vectors, such as BLUESCRIPT ((Stratagene), in which the sequence encoding the polypeptide of interest is ligated into the vector in-frame with the amino-terminal Met of β-galactosidase and the subsequent 7-residue sequence, resulting in the production of a hybrid protein); pIN vectors (Van Heeke and Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and the like. The pGEX vector (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorbing to glutathione-agarose beads and then eluting in the presence of free glutathione. Proteins produced in such a system can be designed to contain a heparin, thrombin, or Factor Xa protease cleavage site so that the replicated polypeptide is released from the GST moiety.

[0121] In yeast (Saccharomyces cerevisiae), a number of vectors containing constitutive or inducible promoters (e.g., alpha factor, alcohol oxidase, and PGH) can be used.

[0122] When using a plant expression vector, the expression of the polypeptide-encoding sequence can be driven by any number of promoters. For example, viral promoters (e.g., the 35S promoter and 19S promoter of CaMV) can be used alone or in combination with the ω (omega) leader sequence derived from TMV. Alternatively, plant promoters (e.g., the small subunit of RUBISCO or the heat shock promoter) can be utilized. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are known in the art.

[0123] The insect system can also be used to express the desired polypeptide. For example, in one system, AcNPV (Autographa californica nuclear polyhedrosis virus) is utilized as a vector for expressing foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The polypeptide-encoding sequence can be cloned within the non-essential region of the virus and placed, for example, under the control of the polyhedrin promoter such as the polyhedrin gene. Successful insertion of the polypeptide-encoding sequence produces a recombinant virus in which the polyhedrin gene is inactivated and the coat protein is deficient. The recombinant virus can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae, where the desired polypeptide can be expressed.

[0124] In mammalian host cells, many virus-based expression systems are commonly available. For example, when an adenovirus is used as an expression vector, the sequence encoding the desired polypeptide can be ligated into the adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. By using the insertion into the non-essential E1 or E3 region of the viral genome, a viable virus capable of expressing the polypeptide in the infected host cell can be obtained. Furthermore, a transcriptional enhancer (e.g., Rous Sarcoma Virus (RSV) enhancer) can be used to increase the expression in mammalian host cells.

[0125] Furthermore, for more efficient translation of the sequence encoding the desired polypeptide, specific initiation signals can be used. Such signals include the ATG start codon and adjacent sequences. When the sequence encoding the polypeptide, its start codon, and the upstream sequence are inserted into an appropriate expression vector, additional transcriptional control signals or translational control signals may not be necessary. However, when only the unique coding sequence or only a part of it is inserted, an exogenous translational control signal containing the ATG start codon must be provided. Furthermore, the start codon must be in the correct reading frame to ensure translation of the entire insert. The exogenous translational elements and the start codon can be derived from various origins (both natural and synthetic). The efficiency of expression can be enhanced by incorporating an enhancer appropriate for the specific cell line used (e.g., the enhancer described in the literature Scharf et al., Results Probl.Cell Differ.20:125~162(1994)).

[0126] Furthermore, host cell lines can be selected according to their ability to regulate the expression of the inserted sequences or to process the expressed proteins in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can be used to facilitate correct insertion, folding, and / or function. Other host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activities) can be selected to ensure accurate modification and processing of the foreign protein.

[0127] In the long term, stable expression is generally preferred for the high-yield production of recombinant proteins. For example, cell lines that stably express the desired polynucleotide can be transformed using an expression vector that can include a viral origin of replication and / or endogenous expression elements, as well as a selectable marker gene on the same or a different vector.

[0128] After introduction of the vector, the cells are grown in enriched media for 1-2 days and then switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence enables the growth and recovery of cells in which the introduced sequence is successfully expressed. Resistant clones of stably transformed cells can be grown using tissue culture techniques appropriate for that cell type.

[0129] Cell lines transformed using multiple selection systems can be recovered. Such selection systems include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be used in tk cells or aprt cells, respectively.

[0130] Also, antimetabolite resistance, antibiotic resistance, or herbicide resistance can be used as the basis for selection. For example, dhfr that confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)); npt that confers resistance to aminoglycosides, neomycin, and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat for conferring resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes are known, such as trpB that enables cells to use indole instead of tryptophan, or hisD that enables cells to utilize histinol instead of histidine. Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin are popular and are widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression resulting from specific vector systems.

[0131] A variety of protocols are known in the art for detecting and measuring the expression of the product encoded by a polynucleotide using either a polyclonal antibody or a monoclonal antibody specific for the product encoded by the polynucleotide. Examples include ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), FACS (fluorescence activated cell sorting), and the like. The above assay methods and additional other assay methods can be referred to the following documents: Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158: 1211-1216 (1983). A variety of labeling techniques and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid assays and amino acid assays. Means for making labeled hybridization probes or labeled PCR probes for detecting sequences related to a polynucleotide include oligolabeling, nick translation, end labeling, or PCR amplification using labeled nucleotides. Alternatively, for the production of mRNA probes, the sequence or any portion thereof can be cloned into a vector. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase (e.g., T7, T3, or SP6) and labeled nucleotides. These procedures can be carried out using a variety of commercially available kits. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescence, or chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0132] Host cells transformed with the desired polynucleotide sequence can be cultured under conditions suitable for protein expression and recovery from the cell culture. The protein produced by the recombinant cells may be secreted according to its sequence and / or vector, or may be contained within the cell.

[0133] As will be understood by those skilled in the art, expression vectors containing the polynucleotides of the present invention can be designed to include signal sequences that direct the secretion of polypeptides across prokaryotic or eukaryotic cell membranes. Other recombinant constructs can be used to ligate the sequence encoding the desired polypeptide to a sequence encoding a polypeptide domain that facilitates the purification of water-soluble proteins.

[0134] In addition to recombinant production methods, the polypeptides and fragments thereof of the present invention can be manufactured by direct peptide synthesis using solid phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis can be carried out using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and then combined using chemical methods to generate the full length molecule.

[0135] According to another aspect of the present invention, the polynucleotide encoding the polypeptide of the present invention can be delivered in vivo to a subject using, for example, gene therapy techniques. Gene therapy generally refers to the transfer of a heterologous nucleic acid into specific cells, target cells, of a mammal, particularly a human, having a disorder or condition for which such treatment is needed. The nucleic acid is introduced into the selected target cells, the heterologous DNA is expressed, and the encoded therapeutic product is produced accordingly.

[0136] Among the various viral vectors available for gene therapy as disclosed in this specification, there are adenoviruses, herpesviruses, vaccinia, adeno-associated virus (AAV), or preferably RNA viruses such as retroviruses. Preferably, the retroviral vector can be a murine or avian retroviral derivative or a lentiviral vector. A preferred retroviral vector can be a lentiviral vector. Examples of retroviral vectors capable of inserting a single exogenous gene include, but are not limited to, the following: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous Sarcoma Virus (RSV). Multiple additional retroviral vectors can incorporate multiple genes. All of these vectors can contain a selectable marker gene such that transduced cells can be identified and produced. For example, by inserting a desired DNA-binding polypeptide sequence derived from a zinc finger together with another gene encoding a ligand for a receptor on a specific target cell, the vector can be made target-specific.

[0137] A retroviral vector can be target-specific, for example, by inserting a polynucleotide encoding a polypeptide. Exemplarily, targeting can be achieved by utilizing an antibody that targets the retroviral vector. Those skilled in the art are proficient in the specific polynucleotide sequences that can be inserted into the retroviral genome to enable target-specific delivery of a retroviral vector containing a zinc finger-nucleotide binding protein polynucleotide and can easily confirm it without undue experimentation.

[0138] Since recombinant retroviruses are defective, assistance is required to produce infectious vector particles. This assistance can be provided, for example, by using a helper cell line containing a plasmid encoding all the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids have lost the nucleotide sequences that enable the packaging mechanism to recognize the RNA transcripts for encapsulation. Helper cell lines lacking the packaging signal include, but are not limited to, for example, PSI.2, PA317, and PA12. Since the genomes of these cell lines are not packaged, they produce empty virions. When a retroviral vector is introduced into a cell with an intact packaging signal and the structural genes replaced by other desired genes, the vector can be packaged to produce vector virions. Next, the bacteriophages produced by this method can be used to infect tissue cell lines (e.g., NIH3T3 cells), thereby producing large amounts of chimeric retroviral virions.

[0139] In addition, "non-viral" delivery techniques for gene therapy, such as DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun technology, electroporation, liposome methods, and lipofection, can be used. Any of these methods are widely available to those skilled in the art and are suitable for use in the present invention. It is clearly understood that other suitable methods are also available to those skilled in the art and that the present invention can be achieved using any available transfection method. Lipofection can be achieved by encapsulating DNA molecules isolated within liposome particles and bringing the liposome particles into contact with the cell membrane of target cells. Liposomes are self-assembling colloidal molecules in which a lipid bilayer composed of amphiphilic molecules such as phosphatidylserine or phosphatidylcholine encapsulates a portion of the surrounding medium, resulting in the lipid bilayer surrounding a hydrophilic interior. Unilamellar or multilamellar liposomes can be constructed, resulting in the inclusion of desired chemical substances, drugs, or DNA molecules isolated as in the present invention.

[0140] In one embodiment of the present invention, the virus can be characterized as being an RNA virus. An RNA virus means a virus that uses reverse transcriptase to convert its own genetic information, which has become RNA, into DNA and inserts this between the host's DNA, causing the host to replicate the virus's genetic information instead.

[0141] In the present invention, the type of the RNA virus is not particularly limited, and it can be selected from the group consisting of viruses of the families Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Caliciviridae, Flaviviridae, Luteoviridae, Togaviridae, Pneumoviridae, Arenaviridae, Deltavirus, and Orthomyxoviridae.

[0142] In a preferred embodiment of the present invention, the RNA virus can be selected from the group consisting of 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.

[0143] In the present invention, the coronavirus can be characterized by being selected from the group consisting of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome (MERS) coronavirus.

[0144] The antiviral pharmaceutical composition according to the present invention may be administered as a preventive for viral infections, i.e., as a vaccine, before infection with the virus, or may be administered as a treatment for viral infections after viral infection, i.e., as a therapeutic agent. The present invention also provides a vaccine adjuvant comprising one or more selected from the group consisting of the following (i) to (iv): (i) the peptide, (ii) the polynucleotide encoding (i), (iii) a vector containing (ii) above, and (iv) a host cell transformed with (iii) above.

[0145] In the present invention, the vaccine adjuvant can be defined as a substance that can present an antigen to the immune system so as to induce an immune response against the antigen or an increase in the immune response when the antigen is administered together with the vaccine adjuvant. That is, the vaccine adjuvant is a substance that promotes an immune response against an antigen as an immune enhancer, and means a substance that is not an immunogen to the host but enhances immunity by increasing the activity of immune system cells.

[0146] To analyze the antigen-specific induced immune response by the vaccine adjuvant, the immune response can be compared with the immune response induced in the presence of the antigen without the vaccine adjuvant. The induction can be evaluated in a subject or the cells of the subject.

[0147] In the present invention, the immune response can be one or more selected from the group consisting of macrophage, dendritic cell, monocyte, B cell, and T cell responses. In particular, the immune response can be a B cell response, that is, it means that antibodies can be specifically produced against the antigen. The antibodies are preferably IgG antibodies, more preferably IgG2a and / or IgG1 antibodies. The immune response can be a T cell response, preferably a Th1 response, a Th2 response, or a balanced Th2 / Th1 response. Those skilled in the art will recognize that the disease-dependent B and / or T cell responses may need to be induced to regulate it. In a variant, the immune response can be detected by measuring the production of cytokines such as, for example, IFNgamma, IL-6, TNFalpha, or IL-10. The production of such cytokines can be evaluated by ELISA, preferably as performed in the examples.

[0148] According to one embodiment of the present invention, it has been confirmed that the peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence showing 95% or more sequence homology thereto has a very excellent effect of activating innate immunity and acquired immunity, and it has been confirmed that when treated together with an antigen, the increase in the immune response of the subject can be significantly improved.

[0149] Therefore, it will be self - evidently understood by those skilled in the art that a peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence showing 95% or more sequence homology therewith, a polynucleotide encoding the same, a vector containing the polynucleotide, and a host cell transformed with the vector also exhibit the same physiological activity.

[0150] As one embodiment of the present invention, the vaccine adjuvant can induce an improved innate immune response and activation of the acquired immune response when administered together with the antigen, as compared with that obtained with the antigen itself. More specifically, activation of an improved cellular immune response and humoral immune response can be induced.

[0151] As one embodiment of the present invention, the detection of the antigen - specific induced immune response means that the detection occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more after administration of at least 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 more after that. The detection can preferably be evaluated in a subject or cells of the subject as performed in the examples.

[0152] In one embodiment of the present invention, the antigen-specific induced immune response preferably means a detectable immune response against the antigen. A detectable increase is achieved by administering the vaccine adjuvant and antigen to a subject, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more later, or at least 1 day later, or at least 2 days later, or at least 3 days later or at least 4 days later, or more time later, there is an increase of 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 in the amount of immune cells, antibodies and / or cytokines. The detection can be preferably evaluated in a subject or the cells of the subject as performed in the examples.

[0153] The present invention also provides a vaccine composition comprising the above vaccine adjuvant and antigen.

[0154] In the present invention, the vaccine contains an antigen consisting of a whole disease-inducing organism (the following structure or attenuation) or a component of these organisms, such as a protein, peptide or polysaccharide, and refers to a preparation used to confer immunity against the disease induced by the organism. As described above, a peptide consisting of the amino acid sequence of SEQ ID NO: 2; or a peptide containing an amino acid sequence having 95% or more homology with the peptide can activate the innate and acquired immune systems and, when administered together with an antigen, exert an effect of significantly enhancing the immune response of the subject and can be utilized as a vaccine composition.

[0155] In the present invention, the antigen contained in the vaccine composition is not particularly limited in terms of its type, and can be a peptide, protein, glycoprotein, glycolipid, lipid, carbohydrate, nucleic acid, polysaccharide, and viruses, bacterial cells, allergens, tissues, cells, etc. containing these. Non-limiting examples thereof include pollen-derived antigen, hepatitis A virus-derived antigen, hepatitis B virus-derived antigen, hepatitis C virus-derived antigen, hepatitis D virus-derived antigen, hepatitis E virus-derived antigen, hepatitis F virus-derived antigen, HIV virus-derived antigen, influenza virus-derived antigen, herpes virus (HSV-1, HSV-2)-derived antigen, Bacillus anthracis-derived antigen, Klebsiella-derived antigen, Streptococcus pneumoniae-derived antigen, Japanese encephalitis virus-derived antigen, measles virus-derived antigen, rubella virus-derived antigen, Clostridium tetani-derived antigen, varicella virus-derived antigen, SARS virus-derived antigen, Epstein-Barr virus-derived antigen, papillomavirus-derived antigen, Helicobacter pylori-derived antigen, rabies virus-derived antigen, West Nile virus-derived antigen, hantavirus-derived antigen, Streptococcus-derived antigen, Staphylococcus-derived antigen, Bordetella pertussis-derived antigen, Mycobacterium tuberculosis-derived antigen, Plasmodium-derived antigen, poliovirus-derived antigen, various zoonotic infectious disease-derived antigens, cancer antigens, various food allergy-derived antigens, and the like.

[0156] The antigen contained in the vaccine composition of the present invention does not necessarily have to be single. Considering the application of the present invention, there may be a case where an immune response is elicited against cancer cells that are not single proteins or peptides, or those composed of a plurality of components such as bacteria and viruses. In this case, it may be a plurality of types of proteins that can elicit an immune response, or a mixture whose type cannot be specified. Further, actively aiming to elicit an immune response against a plurality of types of antigens and containing a plurality of types of antigens can also be one of the usage forms of the vaccine composition of the present invention.

[0157] 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). Some tumor antigens and their expression patterns are 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 antigen, HPV antigen, HPV16 antigen, CTL epitope derived from HPV16 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 molecule 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), divalent PSA, ERG, androgen receptor (AR), PAK6, prostate stem cell antigen (PSCA), NY-ESO-1, Stratum Corneum ChymotrypticEnzyme (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, melanoma-associated antigen E (MAGE-A, MAGE1, MAGE2, MAGE3, MAGE4), interleukin-13 receptor α (IL13-Rα), carbonic anhydrase IX (CAIX), survivin, GP100, angiogenesis antigen, ras protein, p53 protein, p97 melanoma antigen, KLH antigen, carcinoembryonic antigen (CEA), gp100, MART1 antigen, TRP-2, HSP-70, beta-HCG, testisin, 1A01_HLA-A / m; 1A02; 5T4; ACRBP; AFP; AKAP4; α-actinin-_4 / m; α-methylacyl-coenzyme_A racemase; ANDR; ART-4; ARTC1 / m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr / abl; beta-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 / BRD6; CT45A1; CT55; CTAG2 isotype_LAGE-1A; CTAG2 isotype_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_Version_1; FCGR3A_Version_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; ITB1; 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-me; MART-2; MC1_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; NFYC / 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; RAB8A; 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; beta-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; myosin class I; NFYC; OGT; OS-9; pml-RAR alpha 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; IL13R alpha2; Intestinal carboxyl esterase; alpha-foetoprotein; 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, Guanylyl Cyclase C, PAP.OVA(ovalbumin) and MART-1 are included.

[0158] The vaccine composition of the present invention may preferably be an anti-cancer vaccine. Further, the anti-cancer vaccine may be a vaccine for cancer prevention or a vaccine for cancer treatment.

[0159] According to an embodiment of the present invention, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 of the present invention or an amino acid sequence showing 95% or more sequence homology therewith; and a vaccine composition containing a specific tumor antigen are administered in advance before the formation of cancer, and by activating the immune response of a subject, it can also show a preventive effect of inhibiting the growth of cancer. Further, the vaccine composition of the present invention is administered to a subject after cancer formation, shows an effect of inhibiting the growth of cancer or killing cancer, and the efficacy as a therapeutic vaccine has been confirmed.

[0160] In the present invention, the type of the cancer is not particularly limited, and it is preferably selected from the group consisting of breast cancer, colon cancer, prostate cancer, cervical cancer, gastric cancer, skin cancer, oral cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, pharyngeal head cancer, laryngeal cancer, thymoma, mesothelioma, esophageal cancer, rectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, bladder cancer, non-Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma, plasmacytic tumor, leukemia, pediatric cancer, bronchial cancer, colon cancer and ovarian cancer.

[0161] The vaccine composition of the present invention may further comprise one or more selected from the group consisting of any vaccine adjuvant and immune checkpoint inhibitor of the present invention.

[0162] According to an embodiment of the present invention, it has been confirmed that the peptide of the present invention has a more significant effect of activating the immune function of a subject when treated in combination with any additional vaccine adjuvant and / or immune checkpoint inhibitor. In particular, this effect may be particularly preferable in that it minimizes side effects that may occur by administering a high dose of a substance and can achieve the maximum effect with a minimum dose.

[0163] The type of the vaccine adjuvant that may be further included in the vaccine composition of the present invention is not particularly limited, and it should be understood that vaccine adjuvants currently widely used in the art or newly developed vaccine adjuvants in the future are also included herein. Non-limiting examples of the above-mentioned any vaccine adjuvant include 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG ODN, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM 197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D BCG, Aquila's QS21 stimulon, Ribi's Detox, Quil, Superfos, Freund's, GM-CSF, cholera toxin, immunological adjuvant, MF59 and cytokines, and most preferably may be CpG ODN.

[0164] In the present invention, the immune checkpoint inhibitor is a substance that blocks immune system inhibitor checkpoints. Immune checkpoints can be stimulatory or inhibitory. Blocking inhibitory immune checkpoints activates immune system function and can be used in cancer immunotherapy [see Pardoll, Nature Reviews.Cancer 12:252-64 (2012)]. Tumor cells eliminate activated T cells when they attach to specific T cell receptors. Immune checkpoint inhibitors prevent tumor cells from attaching to T cells, which keeps the T cells in an activated state. Indeed, the cooperative action by cells and soluble components counteracts damage by pathogens and cancer. Regulation of the immune system pathway involves changing the expression or functional activity of at least one component of the pathway to regulate the response by the immune system. The immune checkpoint inhibitor is a PD-1 (programmed cell death-1) antagonist, a PD-L1 (programmed cell death-ligand 1) antagonist, a PD-L2 (programmed cell death-ligand 2) antagonist, a CD27 (cluster of differentiation 27) antagonist, a CD28 (cluster of differentiation 28) antagonist, a CD70 (cluster of differentiation 70) antagonist, a CD80 (cluster of differentiation 80, also known as B7-1) antagonist, a CD86 (cluster of differentiation 86, also known as B7-2) antagonist,It can be selected from the group consisting of a CD137 (cluster of differentiation 86 antagonist), a CD276 (cluster of differentiation 276) antagonist, a KIRs (killer-cell immunoglobulin-like receptors) antagonist, a LAG3 (lymphocyte-activation gene 3) antagonist, a TNFRSF4 (tumor necrosis factor receptor superfamily, member 4, also known as CD1 induced TNFR-related protein) antagonist, a GITRL (glucocorticoid-induced TNFR-related protein ligand) antagonist, a 4-1BBL (4-1BB ligand) antagonist, a CTLA-4 (cytolytic T lymphocyte associated antign-4) antagonist, an A2AR (Adenosine A2A receptor) antagonist, a VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, a BTLA (B- and T-lymphocyte attenuator) antagonist, an IDO (Indoleamine 2,3-dioxygenase) antagonist, a TIM-3 (T-cell Immunoglobulin domain and Mucindomain 3) antagonist, a VISTA (V-domain Ig suppressorof T cell activation ) antagonist and a KLRA (killer cell lectin-like receptor subfamilyA) antagonist, preferably a PD-1 antagonist, a PD-L1 antagonist or a LAG3 antagonist, and most preferably a PD-L1 antagonist.,

[0165] In one embodiment of the present invention, the immune checkpoint inhibitor can be a PD-1 antagonist. The 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. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-1 antagonist enhances immune function and mediates anti-tumor activity. Examples of the PD-1 antagonist include antibodies that specifically bind to PD-1. Specific anti-PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, STI-1014, and pidilizumab.

[0166] In another embodiment of the present invention, the immune checkpoint inhibitor can be a PD-L1 antagonist. Examples of the PD-L1 antagonist include antibodies that specifically bind to PD-L1. Specific anti-PD-L1 antibodies include, but are not limited to, avelumab, atezolizumab, durvalumab, and BMS-936559.

[0167] In another embodiment, the immune checkpoint inhibitor can be a LAG3 antagonist. LAG3, lymphocyte activation gene 3, is a negative co-stimulatory receptor that regulates T cell homeostasis, proliferation, and activation. Furthermore, LAG3 has been reported to be involved in the inhibitory function of regulatory T cells (Tregs). Most of the LAG3 molecules are retained in cells close to the microtubule organizing center and are induced only after antigen-specific T cell activation [see: US 2014 / 0286935]. Examples of the LAG3 antagonist include antibodies that specifically bind to LAG3. Specific anti-LAG3 antibodies include, but are not limited to, GSK2831781.

[0168] In the present invention, the antibodies mean intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, as long as they exhibit the desired biological activity. In another embodiment, the antibody means 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 fragments thereof prepared by recombinant genetic engineering.

[0169] Another class of immune checkpoint inhibitors includes polypeptides that bind to and block the PD-1 receptor on T cells without causing inhibitor 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. Patent No. 8,114,845.

[0170] Another class of immune checkpoint inhibitors includes compounds having a peptide moiety that inhibits PD-1 signaling. Examples of such compounds are disclosed in generally known literature.

[0171] Another class of immune checkpoint inhibitors includes inhibitors of specific metabolic enzymes such as indoleamine 2,3-dioxygenase (IDO), which are expressed by infiltrating myeloid cells and tumor cells. The IDO enzyme inhibits the immune response by depleting amino acids required for assimilation in T cells or through the synthesis of specific natural ligands for cytoplasmic receptors that can alter lymphocyte function.

[0172] The compositions of the present invention (e.g., polypeptides, polynucleotides, etc.) can generally be formulated (formulated) in a pharmaceutically acceptable or physiologically acceptable solution, alone or in combination with one or more other therapeutic methods, for administration to cells, tissues or animals. Optionally, the compositions of the present invention can be administered in combination with other agents (e.g., other proteins, polypeptides, or various pharmaceutically active agents, etc.). There are no restrictions on other components that can be included in the compositions of the present invention in fact, as long as the additional agents do not adversely affect the properties of the above-mentioned peptides of the present invention.

[0173] The administration route 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.

[0174] When the pharmaceutical composition of the present invention is administered orally, the pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, suspensions, wafers, etc. according to methods known in the art together with suitable oral carriers. Examples of suitable carriers can include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and 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, polyvinylpyrrolidone, etc. Also, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, etc. can be added as disintegrants. Furthermore, the pharmaceutical composition may further contain anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives, etc.

[0175] Furthermore, when administered parenterally, the pharmaceutical composition of the present invention can be formulated according to methods known in the art in the form of injections, transdermal administration agents, and nasal inhalants together with suitable parenteral carriers. In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, as suitable carriers, Hank's solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose can be used. To protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. can be further included. Furthermore, the injection may further contain an isotonic agent such as sugar or sodium chloride in most cases.

[0176] In the case of transdermal administration agents, forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, aerosols, etc. are included. The above "transdermal administration" means that the pharmaceutical composition is locally administered to the skin and an effective amount of the active ingredient contained in the pharmaceutical composition is delivered to the skin. For example, the pharmaceutical composition of the present invention can be prepared as an injection preparation and administered by lightly terminating the skin with a thin 30-gauge injection needle or directly applying it to the skin. These preparations are described in the literature generally known in pharmaceutical chemistry.

[0177] In the case of inhalants, the compounds used according to the present invention can be conveniently supplied in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0178] As other pharmaceutically acceptable carriers, those described in known literature can be referred to.

[0179] Furthermore, the pharmaceutical composition according to the present invention may further contain at least one buffering agent (e.g., saline or PBS), carbohydrate (e.g., glucose, mannose, sucrose or dextran), antioxidant, bacteriostatic agent, chelating agent (e.g., EDTA or glutathione), adjuvant (e.g., aluminum hydroxide), suspending agent, thickening agent and / or preservative.

[0180] Furthermore, the pharmaceutical composition of the present invention can be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

[0181] Also, the pharmaceutical composition of the present invention can be administered in combination with a known substance having an antiviral effect.

[0182] In the compositions of the present invention, the formulation of pharmaceutically acceptable excipients and carriers is well known to those skilled in the art. Furthermore, appropriate dosing and treatment regimens for the use of the specific compositions described herein can be determined using methods well known to those skilled in the art and can include, for example, oral, parenteral, intravenous, intranasal, intracerebral and intramuscular administrations and formulations (formulations) therefor.

[0183] In certain applications, the pharmaceutical compositions disclosed herein can be delivered by oral administration to a subject. As such, the compositions can be formulated with an inert diluent or with an absorbable edible carrier, or the compositions can be encapsulated in hard-shell or soft-shell gelatin capsules, or the compositions can be compressed into tablets, or the compositions can be directly included in foods.

[0184] In certain circumstances, the pharmaceutical compositions disclosed herein may desirably be delivered parenterally, intravenously, intramuscularly, or intraperitoneally, and such routes of administration can refer to those described in known literature. Solutions of the active compound (as the free base or as a pharmaceutically acceptable salt) can be prepared by appropriately mixing in water with a surfactant such as hydroxypropylcellulose. Further, dispersions can be prepared in glycerol, liquid polyethylene glycol, or mixtures thereof, or in oils. To prevent the growth of microorganisms under ordinary conditions of storage and use, the preparations can contain a preservative.

[0185] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions, and sterile powders that enable the extemporaneous preparation of sterile injection solutions or dispersions. In any case, the pharmaceutical form should be sterile and should be fluid enough to be easily injectable. It should be stable under production and storage conditions and should be preservative against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), their suitable mixtures and / or vegetable oils. For example, by using coating agents such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants, appropriate fluidity can be maintained. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents (such as sugars or sodium chloride, etc.). Prolonged absorption of injectable compositions can be achieved by utilizing agents that delay absorption (such as aluminum monostearate and gelatin, etc.) in the composition.

[0186] For parenteral administration of an aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media that can be used in this regard are known to those skilled in the art. For example, a single dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid, or can be injected into the proposed injection site (for example, reference can be made to Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035 - 1038 and 1570 - 1580). Depending on the condition of the subject being treated with the pharmaceutical composition, some variation in dosage will inherently occur. A person skilled in the art can determine the dosage suitable for an individual subject based on the conventional knowledge in the art. Further, for administration to humans, the preparation needs to meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA Office of Biologics standards.

[0187] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, optionally with various other ingredients enumerated above, and then filtering to sterilize. Generally, dispersions can be prepared by incorporating the sterilized various active ingredients in a sterile vehicle comprising a basic dispersion medium and other required ingredients described above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of manufacture can be vacuum - drying and freeze - drying techniques, which produce a powder of the active ingredient, along with any additional desired ingredients, from the previously sterile - filtered solution.

[0188] The compositions disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), and the acid addition salts are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid, etc.) or organic acids (acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Free carboxyl groups and the salts formed can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, etc.) and organic bases (isopropylamine, trimethylamine, histidine, procaine, etc.). Depending on the formulation, the solution is administered in a manner appropriate for the dosage formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms such as, for example, injection solutions, drug release capsules, etc.

[0189] As used herein, the term "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffer solutions, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. They are used in therapeutic compositions, except in cases where any conventional media or agents are incompatible with the active ingredient. Auxiliary active ingredients can also be included in the compositions of the present invention.

[0190] The expression "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic or similar untoward reactions when administered to humans. Methods for manufacturing aqueous compositions containing proteins as active ingredients are well known in the art. Typically, such compositions are prepared as injectable liquid solutions or suspensions; or solid forms suitable for dissolving or suspending in a liquid prior to injection can also be manufactured. Further, the product can be an emulsion.

[0191] In certain embodiments, the pharmaceutical composition can be delivered by intranasal spray, inhalation and / or other aerosol delivery vehicles. Methods for directly delivering gene, polynucleotide and peptide compositions to the lungs via nasal aerosol sprays can refer to those described in the known literature. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidylglycerol compounds is well known in the field of pharmacy. Similarly, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix can refer to those described in the known literature.

[0192] In certain embodiments, the delivery can be effected by the use of liposomes, nanocapsules, microparticles (microspheres), microspheres, lipid particles, vesicles, etc. for introducing the composition of the present invention into appropriate cells. In particular, the composition of the present invention can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc. and formulated for delivery. The formulation and the use of such delivery vehicles can be carried out using known prior art.

[0193] Furthermore, the pharmaceutical composition of the present invention can be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The pharmaceutical composition formulated as described above can be administered in an effective amount through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular as described above.

[0194] As used above, "effective amount" refers to the amount of substance that enables the tracking of diagnostic or therapeutic effects when administered to a patient. The "individual" can be an animal, preferably a mammal, particularly an animal including a human, and can be cells, tissues, organs, etc. derived from an animal. The individual can be a patient in need of an effect.

[0195] The pharmaceutical composition containing the polypeptide of the present invention can vary the content of the active ingredient depending on the degree of the disease. Usually, when based on an adult, it can be repeatedly administered several times a day at an effective dose of 0.1 μg to 10,000 mg, preferably 1 mg to 5,000 mg per administration. However, the dosage of the pharmaceutical composition according to the present invention can be appropriately selected according to the administration route, administration subject, target disease and its severity, age, gender, body weight, individual differences and disease state, and such techniques are known to those skilled in the art.

[0196] In another aspect, the present invention provides a method of using the composition of the present invention (e.g., polynucleotide, polypeptide, etc.) for a cell, tissue or subject to achieve a desired cell effect and / or therapeutic effect. The cells or tissues that can be controlled by the present invention can preferably be mammalian cells or tissues, more preferably human cells or tissues. Such cells or tissues can be in a healthy state or in a diseased state. The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising one or more selected from the group consisting of the following (i) to (iv): Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising one or more selected from the group consisting of the following (i) to (iv): Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer consisting essentially of one or more selected from the group consisting of the following (i) to (iv): (i) the above peptide, (ii) a polynucleotide encoding (i) above, (iii) a vector containing (ii) above, and (iv) The host cell transformed in the above (iii).

[0197] According to one embodiment of the present invention, peptides consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence showing 95% or more sequence homology therewith all exhibit the effect of suppressing tumor growth and causing cell death by themselves, and can show a cancer prevention or treatment effect.

[0198] The "treatment" of the present invention comprehensively refers to improving cancer, viral diseases or symptoms caused by the diseases, which can include curing the diseases, or practically preventing, or improving symptoms, and includes, but is not limited to, alleviating, curing, or preventing one or most symptoms resulting from the diseases.

Effects of the Invention

[0199] The peptides disclosed in the present invention exhibit anti-cancer activity and immune function enhancing activity as the CRS fragments first disclosed herein.

[0200] In addition, the peptides, polynucleotides encoding them, vectors containing the polynucleotides, host cells transformed with the vectors, or full-length CRS proteins are very excellent in anti-cancer activity and immune function enhancing activity, and can be very usefully used for the development of vaccine adjuvants, vaccine compositions and cancer treatment compositions.

[0201] In addition, the anti-viral composition provided by the present invention not only has the effect of preventing and treating viral infections, but also has an extremely excellent effect of assisting or enhancing the immune response when administered together with viral antigens, and can be very usefully used for the production of viral infection prevention or treatment agents, or viral vaccines.

Brief Description of the Drawings

[0202]

Figure 1a

Figure 1b

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6a

Figure 6b

Figure 7

Figure 8a

Figure 8b

Figure 8c

Figure 9a

Figure 9b

Figure 10a

Figure 10b

Figure 10c

Mode for Carrying Out the Invention

[0203] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

[0204] Experimental Method 1. Cloning Several fragment protein genes derived from CRS (= CARS1) were cloned using the pET28a vector containing 6x his tags at the N- and C-termini. Using CRS DNA as a template, primers complementary to the ends of each sequence were prepared, and a PCR reaction was carried out to obtain PCR products. Next, in order to insert the PCR products into the target vector, the gene was restricted using NdeI as the restriction enzyme in the N-terminal direction and XhoI as the restriction enzyme in the C-terminal direction, and ligated using T4 ligase. To obtain the DNA of the recombinant gene, the recombinant gene prepared using the transformed cell DH5α was transformed. After culturing the transformed cells in LB agar medium at 37 °C for 16 hours, the formed colonies were inoculated into LB liquid medium and cultured at 37 °C for 16 hours. Then, after centrifuging at 3000g for 20 minutes to remove the supernatant, a precipitate was obtained. After decomposing the precipitate using a Mini prep kit to obtain DNA, sequencing was carried out via Biionia to confirm that it was identical to the designed sequence.

[0205] 2. Affinity Chromatography Purification BL21-CodonPlus cells were transformed with the DNA of several CRS-derived proteins, and colonies were inoculated into the medium and grown. Large-scale cells were grown in LB until the OD 600 reached 0.5, and protein expression was induced using 0.5 mM IPTG at 4 °C for 16 hours. Cell pellets were obtained from centrifugation and sonicated and disrupted in 50 mM Tris buffer pH 7.5 containing 300 mM NaCl. Then, centrifugation was performed at 20,000 g for 30 minutes to obtain the supernatant. This was poured onto a column containing Ni-NTA resin. A washing step was performed with 50 mM Tris, pH 7.5, containing 300 mM NaCl, 5% glycerol, and 15 mM imidazole. The protein was separated 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 (REF: Removal of endotoxinfrom protein solutions separation using Triton X-114). Titrated proteins of 0.04 EU / mg or less from LAL analysis were used in all experiments.

[0206] 3. Gel filtration chromatography purification After installing a Superdex 75 10 / 300 GL column on an AKTA pure instrument, the column was washed three times the column volume with 70% ethanol and 1N NaOH. For column stabilization, 50 mM Tris buffer pH 7.5 containing 150 mM NaCl and 0.1 mM EDTA was flowed through twice the column volume. 500 μl of the protein obtained by affinity chromatography was filled into a 1 ml syringe and then inserted into the AKTA injection valve. The proteins eluted with different retention times according to their molecular weights. Then, the proteins eluted at the same retention time were subjected to SDS-PAGE, and the presence of the target protein was confirmed by coomassie blue staining. 4. Ion exchange chromatography purification After installing the Hitrap Q column on the AKTA pure instrument, the column was washed three times with the column volume using 70% ethanol and 1N NaOH. To stabilize the column, 20 mM Tris buffer at pH 7.5 was passed through the column at five times the column volume. After filling 500 μl of the protein obtained by gel filtration chromatography into a 1 ml syringe, it was inserted into the AKTA injection valve. Then, after setting the flow rate to 5 ml / min, 20 mM Tris buffer at pH 7.5 containing 1 M NaCl was passed through, and the elution sample was obtained in a tube over time.

[0207] 5. ELISA THP1-PMA cells were tested to confirm cytokine secretion by CRS. Each cell type was treated at 5x10 5 cells / ml in a 24-well plate overnight, and each well was changed to serum-free medium 2 hours before drug treatment. In the case of RAW264.7 and THP1-PMA, 100 nM of the protein was treated for 4 hours. In the case of BMM and BMDC cells, 100 nM concentration of the protein was treated for 24 hours. The supernatant was centrifuged at 500 g for 10 minutes, and ELISA was performed using an IL-6, TNF-α, IL-12 p70, and IL-10 ELISA Set (BD).

[0208] 6. Circular dichroism analysis CD measurements of the protein dissolved in 50 mM Tris buffer containing 300 mM NaCl were performed on a chirascan V100 under wavelength band conditions of 190 - 260 nm and a bandwidth of 1 nm. A 1 mm pathlength quartz sample cell was used, and there were differences in the proteins to be measured in terms of induction of high-temperature denaturation. Spectral analysis was used to analyze changes in the secondary structure of the protein.

[0209] 7. Antiviral analysis (1) Affinity chromatography purification (bacterial cells) BL21-CodonPlus cells were transformed with the DNA of several proteins derived from CARS1, and colonies were inoculated into the medium and grown. Large-scale cells were grown in LB until the OD 600 reached 0.5, and protein expression was induced using 0.5 mM IPTG at 4 °C for 16 hours. Cell pellets were obtained by centrifugation and disrupted by sonication in 50 mM Tris buffer pH 7.5 containing 300 mM NaCl. Then, centrifugation was performed at 20,000 g for 30 minutes to obtain the supernatant. This was poured onto a column containing Ni-NTA resin. A washing step was performed with 50 mM Tris, pH 7.5, containing 300 mM NaCl, 5% glycerol, and 15 mM imidazole. The protein was separated 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 (REF: Removal of endotoxinfrom protein solutions separation using Triton X-114). Titrated proteins of 0.04 EU / mg or less from LAL analysis were used in all experiments.

[0210] (2) Trained immune mouse model (2-1) Influenza virus C57BL / 6 mice were purchased from Doyal Biotech. Female C57BL / 6 mice, 6 - 8 weeks old, were injected subcutaneously on the back with 5, 10, and 20 mg / kg of CARS1 (99-200, C182S) protein on day - 7 and day - 3. On day - 0, the PR8 influenza virus was administered intranasally at the LD 50 (50% lethal dose). Body weight was measured daily at 1-day intervals from the day of virus administration, and a survival graph was recorded. Also, in the comparative experiment using Oseltamivir, Oseltamivir was administered orally once a day for 5 days after virus infection.

[0211] (2-2) Coronavirus C57BL / 6 mice transfected with hACE-2 were purchased from Duoyal Biotech. Female C57BL / 6 mice transfected with hACE-2, aged 6 - 8 weeks, were intranasally injected with 10 mg / kg of CARS1 (99 - 200, C182S) protein on day - 7 and day - 3 respectively. On Day - 0, a highly lethal SARS - Cov2 (S clade) virus was intranasally administered at 5 lethal doses. Body weight was measured daily at one - day intervals from the day of virus administration, a survival graph was recorded, and the virus titer was analyzed.

[0212] (3) Western blot Hek 293T cells were dispensed into a 6 - well plate at 3 - 5×10 5 cells / well and cultured for 24 hours. Then, 1 μg of S - PP - GSAS - Foldon DNA and 2 μl of Terbofect were well mixed and reacted at room temperature for 15 - 20 minutes. Then, the mixture was slowly added drop by drop to the cells. After 4 hours, the medium was removed, the plate was filled with fresh medium, and cultured for 24 hours. After collecting 1 ml of the culture solution, it was centrifuged at 500 g for 10 minutes at 4°C. The supernatant was then centrifuged at 10000 g for 30 minutes at 4°C. 880 μl of the supernatant was separated, carefully mixed with 120 μl of TCA (Trichloroacetic acid solution), and reacted at 4°C for one day. Then, after centrifuging at 18000 g for 15 minutes at 4°C to form a precipitate, all the supernatant was removed and dried at room temperature for 1 hour. Next, 50 μl of 0.1M Hepes buffer at pH 8.0 was added to loosen the precipitate, then 12.5 μl of 5× sample buffer was added and boiled at 100°C for 10 minutes. Then, it was electrophoresed on an 8% polyacrylamide gel and transferred to an Immobilon P PVDF membrane. Then, anti - Myc antibody and tubulin antibody were treated, and the target protein was confirmed using an Apclone ECL solution.

[0213] (4) Affinity chromatography purification (animal cells) After releasing Expi CHO-S cells using ExpiCHO Expression medium, the cells were cultured in a shaking incubator until they reached 4×10 6 -6 ×10 6 cells / ml. One day before transformation, the cells were passaged at 3×10 6 -4×10 6 cells / ml. The next day, the cells were adjusted to 6×10 6It was diluted to 25 ml using fresh medium at [X] cells / ml. After gently shaking the ExpiFectamine CHO Reagent about 4 - 5 times for transfection, 80 μl was taken and mixed with 920 μl of OptiPRO SFM solution, then pipetted gently about 2 - 3 times. 20 μg of DNA used for transfection was mixed with 1 ml of OptiPRO SFM solution and shaken gently. Before 5 minutes elapsed, the solution mixed with ExpiFectamine CHO reagent was slowly added to the solution mixed with DNA and shaken gently. After reacting at room temperature for about 3 minutes, the solution mixed with ExpiFectamine CHO reagent and DNA was slowly added to the flask containing cells. Next, the flask containing cells was cultured in an incubator at 37°C and 8% CO₂. After placing it in the incubator, within 18 - 22 hours, 150 μl of ExpiFectamine CHO Enhancer and 6 ml of ExpiCHO Feed were slowly added and cultured under the same conditions. On the 10th day after transfection, all the cell culture medium was taken, then put into a 50 ml centrifuge tube, and then centrifuged at 4°C and 5000 g for 30 minutes. Then, the supernatant was taken and passed through a 0.22 μm filter (Acrodisc syringe filter). For affinity chromatography, 3.5 ml of Ni - NTA resin was put into a glass column, and 50 mM Tris binding buffer at pH 8.0 containing 300 mM Nacl and 5% glycerol was flowed through at 10 times the volume of the resin. Next, all the previously separated supernatant was inoculated into the glass column. Then, 100 ml of Wash Buffer A with 15 mM imidazole added to the binding buffer was flowed into the glass column, and further 5 ml of Wash Buffer B with 30 mM imidazole added to the binding buffer was flowed in. Then, 10 ml of elution buffer with 300 mM imidazole added to the binding buffer was inoculated into the column, and the eluate was received in a 10 ml conical tube.Next, the eluate was placed in a semipermeable membrane cassette and put into a container containing 1.5 L of a pH 8.0 50 mM Tris storage buffer containing 300 mM NaCl and 15% glycerol. After reacting for 4 hours under the condition of 4°C, the previously contained storage buffer was discarded, replaced with 1.5 L of a new storage buffer, and then reacted for 16 hours.

[0214] 8. Production of Experimental Substances The CRS fragment peptides used in the present invention and their sequence information are as follows, and these peptides were produced according to conventionally known methods. SEQ ID NO: 2: CRS(140 - 200) SEQ ID NO: 3: CRS(140 - 200, C182S) SEQ ID NO: 4: CRS(106 - 228) SEQ ID NO: 5: CRS(101 - 200) SEQ ID NO: 6: CRS(119 - 200) SEQ ID NO: 7: CRS(99 - 200) SEQ ID NO: 8: CRS(99 - 200, C182S)

[0215] The numbers in parentheses of the above sequences mean the amino acid numbers in the full-length CRS protein of SEQ ID NO: 1. Hereinafter, in the experimental results, CARS1 is interpreted as being the same as the above CRS.

[0216] Experimental Results 1. Confirmation of Immunoreactive Sites of Fragment Proteins Derived from CRS To identify the immunoreactive sites of the fragment protein containing amino acids 106 to 228 of CRS discovered in previous studies, the protein secondary structure was analyzed to prepare several fragment protein genes containing each helix. After culturing and purifying the protein, it was treated with Thp-1 cells differentiated using PMA.

[0217] As a result, it was confirmed that proteins without helix 4 could not induce immunoreactivity when compared with LPS. It was confirmed that proteins having SEQ ID NOs: 1, 2, 3, and 4 containing helices 3 and 4 induced immunoreactivity (Figure 1a). 2. Confirmation of the production stability of the CRS-derived fragment protein CRS(140-200, C182S)

[0218] Based on the above experimental results, in order to suppress the multimer formation by cysteine at position 182 of the fragment protein containing amino acids from position 140 to 200 derived from CRS, a gene with cysteine at position 182 replaced by serine was designed and cloned, and the protein was produced using the gene through cells for transformation of BL21 DE3 codon plus RIPL (Figure 2).

[0219] As a result, the degradation problem reported in previous studies was not observed (Figure 3). Also, when comparing the protein with cysteine at position 182 unsubstituted, it was confirmed by gel filtration chromatography and ion exchange chromatography that it did not form multimers relatively (Figures 4 - 5). 3. Confirmation of the thermal stability of the CRS-derived fragment protein CRS(140-200, C182S)

[0220] Based on the experimental results, the thermal stability of the protein with cysteine at position 182 replaced by serine, which contains amino acids from position 140 to 200 derived from CRS, was confirmed by circular dichroism analysis and ELISA.

[0221] As a result, when compared with the CRS-derived fragment protein induced to undergo high-temperature denaturation, it was confirmed that the secondary structure maintained a level equivalent to that of the protein containing CRS from position 106 to 228, which was reported to have thermal stability in previous studies (Figure 6a). Then, when the CRS-derived fragment protein induced to undergo high-temperature denaturation was treated with Thp-1 cells differentiated with PMA and the amount of TNF-α in the medium was measured to observe the immune activity, the result showed that the function of inducing immune activity could be maintained despite inducing high-temperature denaturation, which means that its function was not lost at high temperatures due to thermal stability (Figure 6b).

[0222] 4. Confirmation of the ability of the CRS-derived fragment protein CRS(140-200, C182) to induce immune cell activity Based on the above experimental results, to confirm that the ability of the CRS-derived fragment protein CRS(140-200, C182S) to induce immune cell activity is an inherent function of the protein, after treatment with polymyxin b (10 ug / ml), known as an LPS inhibitor, and proteinase K (20 ug / ml), a proteolytic enzyme, respectively, it was treated with Thp-1 differentiated with PMA.

[0223] As a result, in the polymyxin b treatment group, in the case of LPS, it was observed that the activity decreased, but the activity of the CRS-derived fragment protein did not decrease. This means that the induced immune activity is not due to LPS. And in the case of the proteinase K treatment group, it was observed that the immune activity of the CRS-derived fragment protein decreased compared with LPS, which means that the induced immune activity is due to the protein. Therefore, the ability of the CRS-derived fragment protein CRS(140-200, C182S) to induce immune activity means that it is not due to LPS contamination but is a function of the protein.

[0224] 5. Confirmation of antiviral effect (1) Confirmation of the antiviral effect by the administration route and concentration of the CARS1(99-200, C182S) protein Since the greatest changes shown by virus infection are known as weight loss and a decrease in survival rate, to confirm the antiviral efficacy, it is necessary to confirm how much the weight can recover to the pre-infection weight compared with the control group and whether survival is maintained over time. Also, since the entry routes of each virus are different, the administration route through which the candidate substance should be injected varies depending on the target virus.

[0225] Intracellular reprogramming by the ability of CARS1 to induce immune cell activity causes secondary stimuli and begins to affect innate immune memory. Such a process is called trained immunity, and it is reported that it can regulate immune homeostasis, tolerance, etc., and thus equip a defense mechanism against viruses. Therefore, we decided to confirm whether the defense ability against virus infection occurs through trained immunity by the CARS1-derived fragment protein.

[0226] Therefore, in order to confirm the difference in efficacy depending on the administration route of the CARS1(99-200, C182S) protein, the inventors injected proteins at concentrations of 5, 10, and 20 mpk (mg / kg) using two administration routes, intraperitoneal and intranasal administration, to confirm the antiviral efficacy. C57bl / 6 mice were preferentially administered the CARS1(99-200, C182S) protein on day-7 and day-3, and then the PR-8 influenza virus was injected by intranasal administration on day-0 (Figure 8a).

[0227] As a result, in the case of intraperitoneal administration, it was confirmed that the survival rate increased at 10 and 20 mpk compared to saline, and the body weight change was also maintained high at the two concentrations (Figures 8b - 8c). In the case of intranasal administration, the survival rate increased at 5, 10, and 20 mpk compared to saline, and in the case of body weight change, it was maintained higher than saline at all three concentrations, and it was confirmed that 10 mpk was the highest.

[0228] (2)Confirmation of the efficacy of the CARS1(99-200, C182S) protein against influenza virus compared to the control group According to the above results, Oseltamivir (Tamiflu), which is known as an influenza virus therapeutic agent, was used as a comparison group for the antiviral efficacy of CARS1 (99-200, C182S) protein. After preferentially administering CARS1 (99-200, C182S) protein to C57bl / 6 mice on day -7 and day -3, PR-8 influenza virus was injected by intranasal administration on day -0. In the case of Oseltamivir, viral infection was allowed to progress after oral administration 4 hours before PR-8 influenza virus administration (Figure 9a).

[0229] As a result, it was confirmed that the survival rate increased at 2 and 10 mpk of Saline vs CARS1 (99-200, C182S) protein, and at 10 mpk, it maintained a survival rate comparable to that of oseltamivir used as a comparison group (Figure 9b).

[0230] (3) Confirmation of the anti-coronavirus efficacy of CARS1 (99-200, C182S) protein against the comparison group After preferentially administering CARS1 (99-200, C182S) protein to C57bl / 6 mice transfected with hACE-2 on day -7 and day -3, SARS-Cov2 (S clade) virus was injected by intranasal administration on day -0 (Figure 10a).

[0231] As a result, there was no weight loss at 10 mpk of Saline vs CARS1 (99-200, C182S) protein (Figure 10b), and it was confirmed that all individuals survived (Figure 10c). Then, as a result of sacrificing animals on day 4 and day 6 for lung tissue analysis, in the Saline vs CARS1 (99-200, C182S) protein treatment group, the Cov-2 virus titer was significantly decreased (Figure 10d).

[0232] (4) Confirmation of the antiviral ability of CARS1 (140-200, C182S) protein According to the above results, to compare the antiviral ability using CARS1(140-200, C182S) protein, which is the minimum unit form in which the active part of CARS1(99-200, C182S) protein is maintained, with CARS1(106-228), each protein was injected into C57bl / 6 mice at 10 mpk by intraperitoneal administration, and after PR-8 influenza virus was injected by intranasal administration on day-0, body weight change and survival rate were confirmed (Figure 11a).

[0233] As a result, weight gain was observed in both Saline control CARS1(106-228) and CARS1(140-200, C182S) proteins (Figure 11b), and it was also confirmed that the survival rate increased in both proteins compared to Saline control (Figure 11c).

Industrial Applicability

[0234] The peptide disclosed in the present invention exhibits anti-cancer activity and immune function enhancing activity as the CRS fragment first disclosed herein. In addition, the peptide, the polynucleotide encoding it, the vector containing the polynucleotide, the host cell transformed with the vector, or the full-length CRS protein is extremely excellent in anti-cancer activity and immune function enhancing activity, and can be very usefully utilized for the development of vaccine adjuvants, vaccine compositions, and cancer treatment compositions. The anti-viral composition provided by the present invention not only has the effect of preventing and treating viral infections, but also has an extremely excellent effect of assisting or enhancing the immune response when administered together with a viral antigen, and can be very usefully utilized for the production of a preventive or therapeutic agent for viral infections or a viral vaccine, and has very high industrial applicability.

Claims

**Claim 1** A pharmaceutical composition for antiviral use, comprising as an active ingredient a peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof. **Claim 2** The pharmaceutical composition according to claim 1, wherein the peptide contains a mutation in which cysteine, which is the 182nd amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with another amino acid. **Claim 3** The pharmaceutical composition according to claim 2, wherein the other amino acid is serine. **Claim 4** The pharmaceutical composition according to claim 1, wherein the peptide is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 8. **Claim 5** The pharmaceutical composition according to claim 1, wherein the virus is selected from the group consisting of viruses of the families Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Bornaviridae, Filoviridae, Paramyxoviridae, 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 the group consisting of 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. The pharmaceutical composition according to claim 6, wherein the coronavirus is selected from the group consisting of 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 as an active ingredient a peptide containing consecutive amino acids from any one selected from the amino acids at positions 99 to 140 to any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more 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. An external pharmaceutical composition for antiviral use, comprising as an active ingredient a peptide containing consecutive amino acids from any one selected from the amino acids at positions 99 to 140 to any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical preparation according to claim 10, wherein the quasi-drug is a disinfectant cleaner, nasal spray, shower foam, mouthwash (gargle), wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

12. A peptide containing consecutive amino acids from any one selected from the 99th to 140th amino acids to any one selected from the 185th to 228th amino acids in the amino acid sequence of SEQ ID NO: 1; a peptide containing an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof as an active ingredient, an immunoadjuvant composition for a virus vaccine.

13. The immunoadjuvant composition for a virus vaccine according to claim 12, wherein the virus is selected from the group consisting of influenza virus, novel influenza A 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.

14. The immunoadjuvant composition for a virus vaccine according to claim 12, wherein the coronavirus is selected from the group consisting of 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 consecutive amino acids from any one selected from the amino acids at positions 99 to 140 to any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1 for producing an antiviral pharmaceutical composition; a peptide comprising an amino acid sequence showing 80% or more homology with the peptide; or a pharmaceutically acceptable salt thereof.

16. A method for preventing or treating viral infection, characterized by administering an effective amount of a composition comprising, as an active ingredient, a peptide comprising consecutive amino acids from any one selected from the amino acids at positions 99 to 140 to any one selected from the amino acids at positions 185 to 228 in the amino acid sequence of SEQ ID NO: 1; or a peptide comprising an amino acid sequence showing 80% or more homology with the peptide, to an individual in need thereof.

17. A peptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence showing 95% or more sequence homology therewith.

18. The peptide according to claim 17, characterized in that the peptide contains a mutation in which cysteine, which is the 43rd amino acid in the amino acid sequence of SEQ ID NO: 2, is replaced with another amino acid.

19. The peptide according to claim 18, characterized in that the other amino acid is serine.

20. A polynucleotide containing a base sequence encoding the peptide according to claim 17.

21. The polynucleotide according to claim 20, characterized in that the polynucleotide consists of the base sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

22. A vector containing the polynucleotide according to claim 20.

23. A host cell transformed with the vector according to claim 22.

24. A vaccine adjuvant comprising one or more selected from the group consisting of the following (i) to (iv). (i) The peptide according to claim 1, (ii) A polynucleotide encoding (i) above, (iii) A vector containing (ii) above, and (iv) A host cell transformed with (iii) above.

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

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

27. The vaccine composition according to claim 25, wherein the anti-cancer vaccine is a cancer preventive vaccine or a cancer therapeutic vaccine.

28. A pharmaceutical composition for cancer prevention or cancer treatment comprising one or more selected from the group consisting of the following (i) to (iv): (i) The peptide according to claim 1, (ii) A polynucleotide encoding the above (i), (iii) A vector containing the above (ii), and (iv) A host cell transformed with the above (iii).

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