KR20200115883A - Composition for prevention, improvement or treatment of pancreatic cancer comprising tetraspanin-2 inhibitor - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating pancreatic cancer, containing a tetraspanin-2 (TSPAN-2) inhibitor as an active component. More specifically, the present invention relates to a composition for preventing, alleviating or treating pancreatic cancer, containing an inhibitor of the expression or activity of TSPAN-2, a method for selecting a therapeutic substance for treating pancreatic cancer by measuring the expression level of TSPAN-2, and a method for providing information useful for diagnosing pancreatic cancer. The inhibitor of the expression or activity of TSPAN-2 can inhibit the growth and metastasis of pancreatic cancer cells to improve a survival rate, and TSPAN-2 expression level analysis can be usefully used in screening for pancreatic cancer therapeutic candidates and diagnosing pancreatic cancer.

Description

Composition for prevention, improvement or treatment of pancreatic cancer comprising tetraspanin-2 inhibitor as an active ingredient

The present invention relates to a composition for preventing, improving or treating pancreatic cancer comprising a tetraspanin-2 inhibitor as an active ingredient, and more specifically, an inhibitor of expression or activity of tetraspanin-2 (TSPAN-2) A composition for preventing, ameliorating or treating pancreatic cancer comprising as an active ingredient, a method of selecting a substance for treating pancreatic cancer by measuring the expression level of tetraspanin-2 (TSPAN-2), and providing useful information for pancreatic cancer diagnosis It's about how to do it.

Pancreatic cancer is a fatal cancer with a 5-year survival rate of 1-4% and a median survival period of 5 months, and has the poorest prognosis among human cancers. Since it is found in 80-90% of patients in a state in which radical resection, which expects cure at the time of diagnosis, is not possible, the prognosis is poor and treatment is mainly dependent on chemotherapy. Therefore, the development of an early diagnosis method is urgently desired than any other human cancer.

The therapeutic effect of several anticancer drugs, including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective in pancreatic cancer to date, is extremely disappointing, and the response rate to chemotherapy is only around 15%. This fact suggests that in order to improve the prognosis of pancreatic cancer patients, the development of more effective early diagnosis methods and treatments is urgently required.

On the other hand, tetraspanin-2 (TSPAN-2) is a protein encoded by the TSPAN-2 gene in humans. The protein encoded by this gene is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members are membrane proteins composed of four hydrophobic domains, transmembrane domains, and are found in almost all animal cells. The TSPAN-2 protein is involved in fundamental cytological processes including cell growth, adhesion and differentiation.

TSPAN-2 has been reported to increase in expression in breast cancer, lung cancer and uterine cancer (EP083088A2), and it has been reported that it is closely related to the invasion and movement of cancer cells, especially in the process of lung cancer (Cell Reports 7, 527-538, April 24, 2014), and the usefulness of TSPAN-2 in other carcinomas, particularly pancreatic cancer and its association with TSPAN-2, has not been reported.

Accordingly, the inventors of the present invention are continuing intensive research to detect novel genes or proteins that can effectively treat and diagnose pancreatic cancer, while TSPAN-2 is overexpressed in pancreatic cancer cell lines, and a substance capable of inhibiting its expression is The present invention was completed by discovering that it can exhibit a therapeutic effect for pancreatic cancer.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating pancreatic cancer comprising an inhibitor of expression or activity of a tetraspanin-2 (TSPAN-2) protein as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving pancreatic cancer comprising an inhibitor of expression or activity of tetraspanin-2 (TSPAN-2) protein as an active ingredient.

Another object of the present invention is (a) treating a test substance on cells expressing TSPAN-2 protein or mRNA; (b) measuring the expression level of TSPAN-2 protein or mRNA in the cells and control cells not treated with the test substance; And (c) to provide a pancreatic cancer therapeutic agent screening method comprising the step of selecting a test substance that reduces the expression level of TSPAN-2 protein or mRNA compared to the control cells as a candidate substance for the treatment of pancreatic cancer.

Another object of the present invention is to provide information necessary for diagnosis of pancreatic cancer,

(a) providing a sample of the subject; (b) measuring the expression level of TSPAN-2 protein or mRNA in the sample; And (c) comparing the expression level of the TSPAN-2 protein or mRNA with a normal person, and determining that a subject having an increased expression level of the TSPAN-2 protein or mRNA compared to the normal person has pancreatic cancer. To provide a way.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer comprising an inhibitor of expression or activity of a tetraspanin-2 (TSPAN-2) protein as an active ingredient. .

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving pancreatic cancer comprising an inhibitor of expression or activity of a tetraspanin-2 (TSPAN-2) protein as an active ingredient.

In order to achieve another object of the present invention, the present invention comprises the steps of: (a) treating a test substance on cells expressing TSPAN-2 protein or mRNA; (b) measuring the expression level of TSPAN-2 protein or mRNA in the cells and control cells not treated with the test substance; And (c) selecting a test substance that decreases the expression level of TSPAN-2 protein or mRNA compared to the control cells as a candidate substance for treating pancreatic cancer.

In order to achieve another object of the present invention, the present invention provides information necessary for diagnosing pancreatic cancer, comprising the steps of: (a) providing a sample of a subject; (b) measuring the expression level of TSPAN-2 protein or mRNA in the sample; And (c) comparing the expression level of the TSPAN-2 protein or mRNA with a normal person, and determining that a subject having an increased expression level of the TSPAN-2 protein or mRNA compared to the normal person has pancreatic cancer. Provides a way.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating pancreatic cancer comprising an inhibitor of expression or activity of a tetraspanin-2 (TSPAN-2) protein as an active ingredient.

The pharmaceutical composition of the present invention may be a composition comprising an expression or activity inhibitor of TSPAN-2 protein as an active ingredient, or a composition consisting of an expression or activity inhibitor of TSPAN-2 protein as an active ingredient, or effective As a component, it may be a composition essentially consisting of an inhibitor of the expression or activity of TSPAN-2 protein.

In the present specification, the term'comprising' is used in the same meaning as'including' or'characterized by', and in the composition or method according to the present invention, specifically Additional components or method steps, etc. not mentioned are not excluded. In addition, the term'consisting of' means excluding additional elements, steps, or ingredients that are not separately described. The term'essentially consisting of' means that, in the scope of the composition or method, it is possible to include substances or steps that do not substantially affect their basic properties, in addition to the substances or steps described. .

In the present specification,'treatment' refers to suppression of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction of disease progression rate, improvement of disease state, improvement, alleviation or improved prognosis do. The term'prevention' as used in the present invention means any action that suppresses the onset or delays the progression of a disease.

'Protein' is used interchangeably with'polypeptide' or'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in nature. The'fragment' of the TSPAN-2 protein refers to a peptide of a portion of the TSPAN-2 protein.

On the other hand,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the nucleotide sequence of a specific gene to the ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.

The term "TSPAN-2" as used herein is a cell surface glycoprotein receptor of about 24 to 27 kDa belonging to a member of the tetraspanin family, and plays an important role in regulating cell development, activity, growth and motility. It is known to regulate signal transduction events. In addition, it can regulate cell adhesion and cell migration, and induce platelet activation and aggregation, which are involved in platelet-induced endothelial cell proliferation. In addition, it is involved in various phenomena within cells, such as promoting muscle cell fusion and contributing to myotube maintenance.

The TSPAN-2 protein in the present invention may be derived from mammals, preferably derived from humans. Most preferably, the TSPAN-2 protein in the present invention is human TSPAN-2 isoform 1 (NP_001760.1) represented by SEQ ID NO: 1 or human TSPAN-2 isoform 2 (NP_001317241.1) represented by SEQ ID NO: 2 It is characterized by including the amino acid sequence of (NCBI Genbank accession number in parentheses). In addition, the TSPAN-2 mRNA is most preferably human TSPAN-2 mRNA transcript variant 1 (NM_001769.3) represented by SEQ ID NO: 3 or human TSPAN-2 mRNA transcript variant 2 (NM_001330312.1) represented by SEQ ID NO: 4 It may contain the nucleotide sequence of (NCBI Genbank accession number in parentheses). Features such as the coding region (exon) of the above-described human TSPAN-2 mRNA transcript variants are identified in the sequence information displayed by searching the NCBI database with the Genbank accession number listed in parentheses.

The TSPAN-2 protein expression inhibitor is in the group consisting of an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, DNAzyme, and protein nucleic acid (PNA) complementary to TSPAN-2 mRNA. It may be any one selected.

In addition, the TSPAN-2 protein activity inhibitor may be any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to TSPAN-2 protein.

According to an embodiment of the present invention, as a result of administering a TSPAN-2 protein-specific antibody to an animal model transplanted with a pancreatic cancer cell line, it was confirmed that not only the growth and metastasis of pancreatic cancer cells was inhibited, but also the survival rate of the animal was significantly increased. . Therefore, in the present invention, the TSPAN-2 activity inhibitor may preferably be an antibody that specifically binds to TSNPAN-2, but is not limited thereto.

In the present invention, the term "antibody" refers to a substance that reacts when an external substance, which is an antigen, invades while circulating in blood or lymph in the immune system of a living body, and is also called an immunoglobulin as a globulin-based protein formed in lymphoid tissue. Antibodies are proteins that B cells produce and flow into body fluids. They specifically bind to antigens. One antibody molecule has two heavy chains and two light chains, each of which is a heavy chain. And the light chain have a variable region at the end of its N-terminal. Each variable region consists of three complementarity determining regions (CDR) and four framework regions (FRs), and the complementarity determining regions determine the antigen binding specificity of the antibody, and the structure of the variable region. It exists as a relatively short peptide sequence that is maintained at the forming sites. For the purposes of the present invention, the antibody may be an antibody capable of inhibiting the activity of TSPAN-2 by binding to TSPAN-2.

The term "pancreatic cancer" as used herein refers to cancer originating from pancreatic cells. There are various types of pancreatic cancer. Pancreatic ductal adenocarcinoma, which occurs in pancreatic duct cells, accounts for about 90%. In general, pancreatic cancer means pancreatic ductal adenocarcinoma. In addition, there are cystic cancer (cystic adenocarcinoma) and endocrine tumors. About 5-10% of pancreatic cancer patients have a genetic predisposition, and about 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, which is more frequent than the 0.6% incidence of pancreatic cancer in the general population. Pancreatic cancer is a cancer with a very poor prognosis with a 5-year survival rate of less than 5%. The reason for this is that most of the cancer is found after progression, so surgical resection is possible within 20% at the time of discovery, and even if it is completely resected with the naked eye, the survival rate is not improved by micrometastasis, and the response to anticancer drugs and radiation therapy is less. Because it is low. Therefore, the most important way to improve the survival rate is to detect and operate early when there is no symptom or non-specific. The type of pancreatic cancer in the present invention is not particularly limited, and it can be interpreted that all types of pancreatic cancer described above are included.

The composition of the present invention can be applied in vivo through various delivery methods such as liposomes and polymers, which are commonly known in the field of gene therapy.

The pharmaceutical composition for preventing or treating pancreatic cancer of the present invention may contain one or more active ingredients that exhibit a function similar to the expression or activity inhibitor of Tetraspanin-2 (TSPAN-2) according to the present invention. . If additional ingredients are included, the therapeutic effect of the composition according to the present invention may be further enhanced. When the above ingredients are added, skin safety, ease of formulation, and stability of active ingredients can be considered in combination with use.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol. In addition, it may further comprise a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods are not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above. When formulated, one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide). Side), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition of the present invention. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol (mannitol), xylitol, erythritol maltitol, celulose, methyl celulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin It can be prepared by mixing and the like. For example, tablets or sugar tablets can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, or syrups, but may include various excipients, such as wetting agents, sweetening agents, fragrances, or preservatives, in addition to water or liquid paraffin, which are simple diluents commonly used. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier and a preservative may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention may be formulated according to a method known in the art in the form of an injection, a transdermal administration, and a nasal inhalation agent together with a suitable parenteral carrier. In the case of such injections, they 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, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. An isotonic solution such as can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be additionally included. . In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, and air rolls are included. In the above, "transdermal administration" means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically applying the pharmaceutical composition to the skin.

In the case of inhalation administration, the inhibitor of the expression or activity of the TSPAN-2 protein of the present invention is used as a suitable propellant, e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Thus, it can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a formula generally known for all pharmaceutical chemistry.

The pharmaceutical composition of the present invention can provide desirable disease prevention and treatment effects when it contains an effective amount of an inhibitor of TSPAN-2 protein expression or activity. In the present specification, the term "effective amount" refers to an amount that exhibits a higher response compared to the negative control group, and preferably refers to an amount sufficient to relieve symptoms of pancreatic cancer, particularly edema. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of an inhibitor of TSPAN-2 protein expression or activity, and the remaining amount may be occupied by a pharmaceutically acceptable carrier. The effective amount of the expression or activity inhibitor of TSPAN-2 protein contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. . The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. When parenterally administered, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day based on the expression or activity inhibitor of TSPAN-2 protein, and TSPAN when administered orally. Based on the expression or activity inhibitor of the protein of -2, per 1 kg of body weight per day, preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg may be administered in an amount of 1 to several times. However, the dose of the protein expression or activity inhibitor of TSPAN-2 considers various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient, as well as the administration route and number of treatments of the pharmaceutical composition. Therefore, the effective dose for the patient is determined. Considering these points, those of ordinary skill in the art can use the TSPAN-2 protein expression or activity inhibitor as appropriate for the specific use for the prevention and treatment of pancreatic cancer. You will be able to determine the dosage. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

The pharmaceutical composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.

The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing an inhibitor of TSPAN-2 protein expression or activity as an active ingredient.

When using the pharmaceutical composition of the present invention as an external preparation for skin, additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfacial agents Skin such as activators, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators or lipid vesicles It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations. In addition, the above ingredients may be introduced in amounts generally used in the field of dermatology.

When the pharmaceutical composition of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, patch, gel, cream, or spray, but is not limited thereto.

The present invention also provides a food composition for preventing or improving pancreatic cancer comprising an inhibitor of expression or activity of a tetraspanin-2 (TSPAN-2) protein as an active ingredient.

The food composition according to the present invention can be used to prevent or improve pancreatic cancer.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives, etc., and animals including humans or livestock are eaten. To do. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage) Corn beef), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, sweets, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.), and the like, can be prepared by adding an inhibitor of the expression or activity of the TSPAN-2 protein. In addition, as a nutritional supplement, it is not limited thereto, but can be prepared by adding an inhibitor of TSPAN-2 protein expression or activity to capsules, tablets, pills, etc. In addition, the health functional food is not limited thereto, but for example, the TSPAN-2 protein expression or activity inhibitor itself is prepared in the form of tea, juice, and drink, so that it can be liquefied, granulated, It can be encapsulated and powdered for consumption. In addition, in order to use the TSPAN-2 protein expression or activity inhibitor in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. In addition, it is said that it has an effect of improving any one or more diseases selected from the group consisting of TSPAN-2 protein expression or activity inhibitors and type 1 rheumatoid arthritis, type 2 rheumatoid arthritis, gestational rheumatoid arthritis, juvenile rheumatoid arthritis, and multiple rheumatoid muscle pain. It can be prepared in the form of a composition by mixing with known known active ingredients.

When the food composition of the present invention is used as a health drink composition, the health drink composition may contain various flavoring agents or natural carbohydrates as an additional component, like a normal drink. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally 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.

When the TSPAN-2 protein expression or activity inhibitor according to the present invention is contained as an active ingredient of a food composition for preventing or improving pancreatic cancer, the amount is not particularly limited to an amount effective to achieve symptomatic improvement, but the total composition It is preferably 0.01 to 100% by weight based on the weight. The food composition of the present invention may be prepared by mixing together with an inhibitor of TSPAN-2 protein expression or activity and other active ingredients known to have an effect of improving pancreatic cancer.

In addition to the above, when the food composition of the present invention is used as a health food, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH It may contain modifiers, stabilizers, preservatives, glycerin, alcohols or carbonation agents. In addition, the health food of the present invention may contain flesh for the manufacture of natural fruit juice, fruit juice beverage or vegetable beverage. These ingredients may be used independently or in combination. The proportion of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also comprises the steps of (a) treating a test substance on cells expressing TSPAN-2 protein or mRNA; (b) measuring the expression level of TSPAN-2 protein or mRNA in the cells and control cells not treated with the test substance; And (c) selecting a test substance that decreases the expression level of TSPAN-2 protein or mRNA compared to the control cells as a candidate substance for treating pancreatic cancer.

The step (a) is a step of contacting cells expressing TSPAN-2 protein or mRNA in order to determine whether the test substance to be analyzed has an activity to inhibit the expression of TSPAN-2 protein or mRNA.

In the method of the present invention,'contacting' or'treatment' is a general meaning, and two or more agents (e.g., two polypeptides) are combined, or an agent and a cell (e.g., Protein and cell). Contact can occur in vitro. For example, combining two or more agents in a test tube or other container, or combining a test agent with cells or cell lysates and a test agent. Contact can also occur in cells or in situ. For example, two polypeptides are brought into contact in a cell or cell lysate by coexpressing a recombinant polynucleotide encoding two polypeptides in a cell. In addition, a protein chip or a protein array in which the protein to be tested is arranged on the surface of a stationary bed may be used.

In addition, in the method of the present invention, the'test substance' can be used interchangeably with a test agent or agent, and any substance, molecule, element, compound ( Includes a compound, entity, or combination thereof. Examples include proteins, polypeptides, small organic molecules, polysaccharides, and polynucleotides. It may also be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances.

More specifically, test agents that can be screened by the method of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines. ), oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogues, or combinations thereof. Test formulations can be synthetic or natural. The test formulations can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Combinatorial libraries can be produced from several types of compounds that can be synthesized in a step-by-step manner. Compounds of multiple combinatorial libraries can be prepared by the encoded synthetic libraries (ESL) method (WO 95/12608, WO93/06121, WO 94/08051, WO95/395503 and WO 95/30642). Peptide libraries can be prepared by the phage display method (WO91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be directed or applied to a random chemical modification, such as acylation, alkylation, esterification, amidification, to prepare structural analogues.

In the present specification, "expression" means that a protein or nucleic acid is produced in a cell. The cells expressing TSPAN-2 may be cells that express TSPAN-2 intrinsically, or may be cells that overexpress TSPAN-2 by transforming with a recombinant expression vector containing a polynucleotide encoding TSPAN-2. . Preferably, the cells expressing the TSPAN-2 gene may be a pancreatic cancer cell line.

The step (b) is a step of measuring the gene expression level of TSPAN-2 in the cells expressing TSPAN-2 contacted with the test substance and the cells expressing TSPAN-2 without contact with the test substance.

The measurement of TSPAN-2 mRNA expression can be used without limitation, methods for confirming expression levels conventional in the art. Examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing (RNA sequencing) and the like, but are not limited thereto.

In addition, the method for measuring the expression level of TSPAN-2 protein can be used without limitation, methods known in the art, such as western blotting, dot blotting, enzyme-linked immunoassay method (enzyme-linked). immunosorbent assay), radioactive immunoassay (RIA), radioimmunoassay, octeroni immune diffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS) or protein There are chip methods and the like, but are not limited thereto.

The step (c) is a step of selecting a test substance that reduces the expression level of TSPAN-2 protein or mRNA compared to the control cell as a candidate substance for treating pancreatic cancer.

As identified by the present inventors, as the inhibition of the activity of TSPAN-2 inhibits the growth and metastasis of pancreatic cancer cells and improves the survival rate, substances that inhibit the expression of the protein or mRNA of TSPAN-2 are also TSPAN-2 It can be clearly understood that it has the potential to be used as a therapeutic agent for pancreatic cancer as it exhibits an effect of inhibiting the activity of.

Therefore, in the screening method of the present invention, the therapeutic agent for pancreatic cancer may preferably be a substance exhibiting an effect of inhibiting the activity of TSPAN-2 by lowering the expression of the protein or mRNA of TSPAN-2.

The present invention also provides a composition for diagnosing pancreatic cancer comprising an agent for measuring the expression level of TSPAN-2 protein or mRNA.

According to an embodiment of the present invention, it was confirmed that the expression of TSPAN-2 was significantly increased in the pancreatic cancer cell line when compared to the normal pancreatic cell line, and the expression of TSPAN-2 in the pancreatic sample obtained from an individual was compared with that of a normal person. Therefore, if it is increased, the individual may be suspected of having pancreatic cancer.

In the present invention,'diagnosis' means confirming the presence or characteristics of a pathological condition. The diagnosis in the present invention is to determine the presence or onset of an infectious disease by measuring the level of TSPAN-2 protein or mRNA.

When the diagnostic composition of the present invention is for measuring the expression level of the TSPAN-2 protein, the agent measuring the expression level of the TSPAN-2 protein may be an antibody that specifically binds to TSPAN-2.

Meanwhile, when the diagnostic composition of the present invention is for measuring the expression level of TSPAN-2 mRNA, the agent for measuring the expression level of TSPAN-2 mRNA may be a probe or primer pair that specifically binds to TSPAN-2 mRNA.

The'primer' is a short single-stranded oligonucleotide that acts as a starting point for DNA synthesis.

The sequence of the primer does not need to have a sequence that is completely complementary to some of the base sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to function unique to the primer. Therefore, in the present invention, the primer for measuring the expression level of TSPAN-2 mRNA does not need to have a sequence that is completely complementary to the TSPAN-2 gene sequence, and a specific section of TSPAN-2 mRNA or TSPAN-2 cDNA through DNA synthesis. It is sufficient if it has a length and complementarity suitable for the purpose of amplifying and measuring the amount of TSPAN-2 mRNA. The primers for the amplification reaction consist of a set (pair) that complementarily bind to the template (or sense) at both ends of the specific section of the TSPAN-2 mRNA to be amplified and the opposite (antisense, antisense). do. The primer can be easily designed by those skilled in the art by referring to TSPAN-2 mRNA or cDNA sequence.

The'probe' is a fragment of a polynucleotide such as RNA or DNA having a length of several to several hundred base pairs that can specifically bind to mRNA or complementary DNA (cDNA) of a specific gene. It is labeled so that the presence or absence of the target mRNA or cDNA to be bound, and the amount of expression can be confirmed. For the purpose of the present invention, a probe complementary to TSPAN-2 mRNA can be used for diagnosis of infectious inflammatory disease by performing hybridization with a sample of a subject to measure the expression level of TSPAN-2 mRNA. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

The present invention also provides a kit for diagnosis of pancreatic cancer comprising an agent for measuring the expression level of TSPAN-2 protein or mRNA.

In the diagnostic kit of the present invention, in order to measure the expression level of TSPAN-2, an antibody that selectively recognizes TSPAN-2 protein as a marker, or a primer or probe that recognizes TSPAN-2 mRNA as a marker, as well as one type suitable for analysis method Further other component compositions, solutions, or devices may be included.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. In addition, a primer specific to the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitors DEPC. -May include DEPC-water, sterilized water, etc.

The present invention also provides information necessary for diagnosing pancreatic cancer, comprising: (a) providing a sample of a subject; (b) measuring the expression level of TSPAN-2 protein or mRNA in the sample; And (c) comparing the expression level of the TSPAN-2 protein or mRNA with a normal person, and determining that a subject having an increased expression level of the TSPAN-2 protein or mRNA compared to the normal person has pancreatic cancer. Provides a way.

The sample may be selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervix, pancreatic tissue sample, or vaginal secretion and urine, preferably pancreatic tissue sample. have.

The method of measuring the expression level of TSPAN-2 protein or mRNA in step (b) of the method may be applied in the same manner as described above.

TSPAN-2 activity or expression inhibitors can inhibit the growth and metastasis of pancreatic cancer cells to improve survival, and TSPAN-2 expression level analysis can be very useful in screening for candidates for pancreatic cancer treatment and diagnosis of pancreatic cancer. have.

1 is a result of observing the expression level of TSPAN2 in normal pancreatic cell line (RNAKT-15) and pancreatic cancer cell line (Miapaca-2) using FACS (normal cell line: negative control, pancreatic cancer cell line: Tumor).
2 is a result of confirming the change in expression of the cell adhesion molecule (E-cadherin) by Western blot when the pancreatic cancer cell line was transfected with an anti-TSPAN2 antibody to suppress the expression of TSPAN2.
3 is a result of confirming the degree of necrosis of pancreatic cancer cells through H&E staining after administration of an anti-TSPAN-2 antibody to a pancreatic cancer orthotopic animal model (left: untreated control group, right: anti-TSPAN-2 antibody administration group).
4 is a result of administering an anti-TSPAN-2 antibody to a pancreatic cancer orthotopic animal model and confirming the growth of cancer cells over time.
5 is a result of administering an anti-TSPAN-2 antibody to a pancreatic cancer orthotopic animal model and confirming whether or not pancreatic cancer cells have metastasized to the liver (Control: untreated control group, Ab: anti-TSPAN-2 antibody treated group).
6 is a result of administering an anti-TSPAN-2 antibody to a pancreatic cancer orthotopic animal model and observing the survival rate of the animal over time (Control: untreated control group, Ab: anti-TSPAN-2 antibody treated group).
7 is a result of observing the survival rate of the animal over time after administering an anti-TSPAN-2 antibody to a pancreatic cancer orthotopic animal model and removing cancer tissue from the pancreas (Day 21) (Control: untreated control group, Ab: anti -TSPAN-2 antibody treatment group).

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1: Confirmation of TSPAN-2 expression in human pancreatic cancer cell line

Pancreatic cancer cells MiaPaCa-2 and normal pancreatic cells RNAKT-15 were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Thermo, Rockville, MD, USA). The separated cells were harvested in a 15L tube and centrifuged for 3 minutes at 13,000 rpm. Thereafter, the supernatant was removed and resuspended in a new culture medium. Re-suspended cells were plated in 6-well plates for two-dimensional (2D) monolayer culture, or on ultra-low adhesion surface 96-well round bottom plates. Then, centrifugation was performed at 1,800 rpm for 3 minutes to form a three-dimensional (3D) spheroid culture. This was cultured in an incubator at 37° C. for 3 days.

MiaPaCa-2 was fixed with 4% paraformaldehy at room temperature for 15 minutes, washed with PBS containing 0.1% BSA, and treated with PBS containing 0.2% Triton X-100 for 15 minutes at room temperature. MiaPaCa-2 was washed with PBS containing 0.1% BSA, transferred to a FACS tube, and reacted with the primary antibody at 4° C. for 15 minutes. As the primary antibody, mAb against TSPAN2 (TSPAN2 Ab) was used. Normal RNAKT-15 cell line was used as a negative control. After washing with FACS buffer (PBS containing 0.1% BSA and 0.05% sodium azide), FITC-labeled goat anti-mouse IgG (H+L) F(ab')2 fragment (Jackson ImmunoResearch) and 4℃ for 15 minutes Reacted for a while. After washing with FACS buffer, hFLS was analyzed with BD FACS Calibur.

The results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that the expression of TSPAN-2 was significantly increased in the pancreatic cancer cell line when compared to the normal pancreatic cell line.

Example 2: Changes in expression of E-cadherin according to inhibition of TSPAN-2 expression in pancreatic cancer cell lines

In the case of inhibiting TSPAN2 expression in pancreatic cancer cells, in order to check whether the expression of the cell adhesion molecule (E-cadherin) changes, transfect Miapaca-2 cells with 1 or 5 μg of anti-TSPAN2 antibody, and 3 to 5 days later A cell lysate was prepared. Then, Western blot analysis was performed using anti-TSPAN2 antibody.

Specifically, after the MiaPaCa-2 cell culture was lysed, it was mixed with a loading buffer and separated by SDS-PAGE. Subsequently, the protein was transferred to a nitrocellulose membrane. Non-specific binding sites on the membrane were blocked using 5% non-fat milk powder (skim milk) for 90 minutes at room temperature. The membrane was incubated with the primary antibody E-cadherin (purchased from Cell Signaling Technology) at 4°C, and then treated with a secondary antibody (purchased by Cell Signaling Technology) at room temperature for 1 hour. The protein band on the membrane band was obtained from Santa Cruz Biotechnology Inc. Visualization was performed using the ECL Plus Blotting Detection System.

The results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that when the expression of TSPAN2 was inhibited in Miapaca-2 cells, the expression of the cell adhesion molecule E-cadherin was increased. As E-cadherin expression increases, tumor cell migration and invasion can be inhibited and metastasis can be inhibited. Therefore, these results suggest that inhibition of TSPAN2 expression can inhibit pancreatic cancer metastasis.

Example 3: Treatment effect of pancreatic cancer according to TSPAN-2 activity inhibition

(1) Preparation of pancreatic cancer orthotopic animal model and administration of anti-TSPAN-2 antibody

Miapaca-2 cells cultured with spheroid cells for 3 days were collected in a dish, and cell counts were performed to adjust the number of 1x10 ⁵ cells/200ul. A nude mouse (7 weeks old) was anesthetized and the right pancreatic area was opened 0.5cm. Miapaca-2 cells per mouse 1x10 ⁵ cells/200ul were injected into the pancreas using an insulin syringe. After the injection, the open area was closed with a surgical clip and carefully observed until the mouse awakened and recovered. The anti-TSPAN-2 antibody was administered subcutaneously according to each of the following experimental groups after transplantation of 30 ug of cancer cells.

(2) Histological analysis of pancreatic cells (H&E staining)

After breeding an orthotopic animal model transplanted with a pancreatic cancer cell line according to the above method for 2 weeks, 30ug of anti-TSPAN-2 antibody was injected once a week, a total of twice, and then the pancreatic tissue was excised, and the tissue was immersed in 4% PFA to fix the tissue. And made a paraffin block and sectioned. Thereafter, H&E staining was performed according to a conventionally reported method to perform histological analysis.

The results are shown in FIG. 3.

As shown in Fig. 3, in the pancreatic tissue of the mouse group to which the anti-TSPAN-2 antibody was administered (right figure), it was confirmed that the necrosis of cancer cells was significantly increased compared to the untreated control group.

(3) Confirmation of pancreatic cancer growth inhibitory effect

The pancreatic cancer orthotopic animal model produced according to the above method was grouped as follows.

(i) Control: PBS administered group

(ii) Group 1: Subcutaneous injection of 30 ug/mouse anti-TSPAN2 antibody, 4 times a week, once a week simultaneously with cancer cell injection

(iii) Group 2: Subcutaneous injection of 30ug/mouse anti-TSPAN-2 antibody once a week for a total of 3 times from 1 week after injection of cancer cells

(iv) Group 3: Subcutaneous injection of 30ug/mouse anti-TSPAN2 antibody twice a week once a week from 3 weeks after injection of cancer cells

The animals of each experimental group were opened at a certain time point, the size of the cancer was measured with a caliper, and the volume was calculated.

The results for this are shown in FIG. 4.

As shown in Figure 4, it was confirmed that the pancreatic cancer completely disappeared in the animal groups of groups 1 to 3 administered with the anti-TSPAN-2 antibody, and this effect is irrespective of when the antibody was administered during the growth point of pancreatic cancer. appear.

(4) Confirmation of metastasis and survival rate of pancreatic cancer

The pancreatic cancer orthotopic animal model group 1 and group 2 used in the experiment were sacrificed at the end of the experiment, and the proportion of animals in which cancer cells metastasized to the liver were found was confirmed.

The results are shown in FIG. 5.

As shown in FIG. 5, at the end of the experiment, cancer cells were confirmed to have metastasized to the liver in 80% of the animals in the untreated control group, but no liver metastasis was observed in the animal group administered with anti-TSPAN-2.

In addition, during the experimental period, the survival rates of the group 1 and group 2 animals were observed. As shown in FIG. 6, in the untreated control group 1, all animals died after about 40 days, but anti-TSPAN- 2 In the group 2 animal group administered with the antibody, no dead animals occurred until the end of the experiment.

Example 4: Evaluation of survival rate after resection of pancreatic cancer tissue

After 4 weeks of transplanting the pancreatic cancer cell line, it was confirmed that liver metastasis of cancer was induced in the untreated control group. After removing cancer tissue from the pancreas of the animals in the untreated control group and the antibody-administered group, the survival rates of the group 1 and group 2 animals were observed.

As a result, as shown in FIG. 7, all died after resection of pancreatic cancer tissue in the untreated control group, but no dead animals occurred in the anti-TSPAN-2 antibody administration group. These results show that in the anti-TSPAN-2 antibody-administered group, metastasis of cancer cells did not occur, so animals could continue to survive even after the pancreatic cancer tissue was resected. However, in the untreated control group, the cancer cells were transferred to other organs even after the pancreatic cancer tissue was removed. It could be determined that metastasis occurred and died.

TSPAN-2 activity or expression inhibitors can inhibit the growth and metastasis of pancreatic cancer cells to improve survival, and TSPAN-2 expression level analysis can be very useful in screening for candidates for pancreatic cancer treatment and diagnosis of pancreatic cancer. It has excellent industrial applicability.

<110> HWANG, IN HU <120> Composition for prevention, improvement or treatment of pancreatic cancer comprising tetraspanin-2 inhibitor <130> NP19-0018 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens tetraspanin-2 isoform 1 (NP_001760.1) <400> 1 Met Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly 1 5 10 15 Phe Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly 20 25 30 Leu Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu 35 40 45 Thr Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile 50 55 60 Gly Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly 65 70 75 80 Ala Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu 85 90 95 Leu Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser 100 105 110 His Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr 115 120 125 Tyr Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys 130 135 140 Ala Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu 145 150 155 160 Gln Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe 165 170 175 Thr Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn Lys 180 185 190 Phe His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met Ile 195 200 205 Phe Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg Asn 210 215 220 Arg Glu Met 225 <210> 2 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens tetraspanin-2 isoform 2 (NP_001317241.1) <400> 2 Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly Ala Val Gln Glu Ser 1 5 10 15 Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu Leu Val Ile Phe Ala 20 25 30 Ile Glu Ile Ala Ala Ala Ile Gly Tyr Ser His Lys Asp Glu Val Ile 35 40 45 Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr Tyr Asn Lys Leu Lys Thr 50 55 60 Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys Ala Ile His Tyr Ala Leu 65 70 75 80 Asn Cys Cys Gly Leu Ala Gly Gly Val Glu Gln Phe Ile Ser Asp Ile 85 90 95 Cys Pro Lys Lys Asp Val Leu Glu Thr Phe Thr Val Lys Ser Cys Pro 100 105 110 Asp Ala Ile Lys Glu Val Phe Asp Asn Lys Phe His Ile Ile Gly Ala 115 120 125 Val Gly Ile Gly Ile Ala Val Val Met Ile Phe Gly Met Ile Phe Ser 130 135 140 Met Ile Leu Cys Cys Ala Ile Arg Arg Asn Arg Glu Met Val 145 150 155 <210> 3 <211> 1320 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens tetraspanin-2 mRNA transcript variant 1 <400> 3 cttttcccgg cacatgcgca ccgcagcggg tcgcgcgccc taaggagtgg cactttttaa 60 aagtgcagcc ggagaccagc ctacagccgc ctgcatctgt atccagcgcc aggtcccgcc 120 agtcccagct gcgcgcgccc cccagtcccg cacccgttcg gcccaggcta agttagccct 180 caccatgccg gtcaaaggag gcaccaagtg catcaaatac ctgctgttcg gatttaactt 240 catcttctgg cttgccggga ttgctgtcct tgccattgga ctatggctcc gattcgactc 300 tcagaccaag agcatcttcg agcaagaaac taataataat aattccagct tctacacagg 360 agtctatatt ctgatcggag ccggcgccct catgatgctg gtgggcttcc tgggctgctg 420 cggggctgtg caggagtccc agtgcatgct gggactgttc ttcggcttcc tcttggtgat 480 attcgccatt gaaatagctg cggccatctg gggatattcc cacaaggatg aggtgattaa 540 ggaagtccag gagttttaca aggacaccta caacaagctg aaaaccaagg atgagcccca 600 gcgggaaacg ctgaaagcca tccactatgc gttgaactgc tgtggtttgg ctgggggcgt 660 ggaacagttt atctcagaca tctgccccaa gaaggacgta ctcgaaacct tcaccgtgaa 720 gtcctgtcct gatgccatca aagaggtctt cgacaataaa ttccacatca tcggcgcagt 780 gggcatcggc attgccgtgg tcatgatatt tggcatgatc ttcagtatga tcttgtgctg 840 tgctatccgc aggaaccgcg agatggtcta gagtcagctt acatccctga gcaggaaagt 900 ttacccatga agattggtgg gattttttgt ttgtttgttt tgttttgttt gttgtttgtt 960 gtttgttttt ttgccactaa ttttagtatt cattctgcat tgctagataa aagctgaagt 1020 tactttatgt ttgtctttta atgcttcatt caatattgac atttgtagtt gagcgggggg 1080 tttggtttgc tttggtttat attttttcag ttgtttgttt ttgcttgtta tattaagcag 1140 aaatcctgca atgaaaggta ctatatttgc tagactctag acaagatatt gtacataaaa 1200 gaattttttt gtctttaaat agatacaaat gtctatcaac tttaatcaag ttgtaactta 1260 tattgaagac aatttgatac ataataaaaa attatgacaa tgtcctggac tggtaaaaaa 1320 1320 <210> 4 <211> 1437 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens tetraspanin-2 mRNA transcript variant 2 <400> 4 cttggggact gcccagccct cagctgtgtt attattcggt gataggtatt tgctaattac 60 ttccaaaagc ctcccatctg tcatcccacc cagactgcgc gcttctaatt cctcctaccc 120 cacatgctgt gcccaatgaa aagtatggtc agcgagcgaa ggtttgcaag gagacagacg 180 agggcgaaat taagccaggc ggcttccctt taaatcctcg caaagcagaa gggcccctca 240 ctctggcagc aggccttggc caaggggcct ttagccctga cgacccgggg aagagtctcc 300 caaagcagaa cgcccggtcc ggcgcccaga ccaaacgcgg gggaaccgga agggcgaggc 360 ctccaccttg ccgggattgc tgtccttgcc attggactat ggctccgatt cgactctcag 420 accaagagca tcttcgagca agaaactaat aataataatt ccagcttcta cacaggagtc 480 tatattctga tcggagccgg cgccctcatg atgctggtgg gcttcctggg ctgctgcggg 540 gctgtgcagg agtcccagtg catgctggga ctgttcttcg gcttcctctt ggtgatattc 600 gccattgaaa tagctgcggc catctgggga tattcccaca aggatgaggt gattaaggaa 660 gtccaggagt tttacaagga cacctacaac aagctgaaaa ccaaggatga gccccagcgg 720 gaaacgctga aagccatcca ctatgcgttg aactgctgtg gtttggctgg gggcgtggaa 780 cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac cgtgaagtcc 840 tgtcctgatg ccatcaaaga ggtcttcgac aataaattcc acatcatcgg cgcagtgggc 900 atcggcattg ccgtggtcat gatatttggc atgatcttca gtatgatctt gtgctgtgct 960 atccgcagga accgcgagat ggtctagagt cagcttacat ccctgagcag gaaagtttac 1020 ccatgaagat tggtgggatt ttttgtttgt ttgttttgtt ttgtttgttg tttgttgttt 1080 gtttttttgc cactaatttt agtattcatt ctgcattgct agataaaagc tgaagttact 1140 ttatgtttgt cttttaatgc ttcattcaat attgacattt gtagttgagc ggggggtttg 1200 gtttgctttg gtttatattt tttcagttgt ttgtttttgc ttgttatatt aagcagaaat 1260 ataaaagaat atttgctaga ctctagacaa gatattgtac aaggtactat cctgcaatga 1320 ttttttgtct ttaaatagat acaaatgtct atcaacttta atcaagttgt aacttatatt 1380 gaagacaatt tgatacataa taaaaaatta tgacaatgtc ctggactggt aaaaaaa 1437

Claims (12)

A pharmaceutical composition for the prevention or treatment of pancreatic cancer comprising an inhibitor of expression or activity of tetraspanin-2 (TSPAN-2) protein as an active ingredient.
The method of claim 1, wherein the TSPAN-2 protein expression inhibitor is composed of an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, DNAzyme, and protein nucleic acid (PNA) complementary to TSPAN-2 mRNA. A pharmaceutical composition, characterized in that any one selected from the group.
The method of claim 1, wherein the TSPAN-2 protein activity inhibitor is any selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to TSPAN-2 protein. Pharmaceutical composition, characterized in that one
A food composition for preventing or improving pancreatic cancer, comprising as an active ingredient an inhibitor of expression or activity of tetraspanin-2 (TSPAN-2) protein.
(a) treating cells expressing TSPAN-2 protein or mRNA with a test substance;
(b) measuring the expression level of TSPAN-2 protein or mRNA in the cells and control cells not treated with the test substance; And
(C) a pancreatic cancer therapeutic agent screening method comprising the step of selecting a test substance that reduces the expression level of TSPAN-2 protein or mRNA compared to the control cells as a candidate substance for treating pancreatic cancer.
The method of claim 5, wherein the mRNA expression level of TSPAN-2 is RT-PCR, quantitative or semi-quantitative RT-PCR (Quentitative or semi-Quentitative RT-PCR), quantitative or semi-quantitative real-time RT-PCR (Quentitative or semi -Quentitative real-time RT-PCR), Northern blot (northern blot), a screening method characterized in that it is measured using any one method selected from the group consisting of DNA chip (chip) and RNA chip.
The method of claim 5, wherein the protein expression level of TSPAN-2 is Western blot, ELISA, radioimmunoassay method, radioimmuno diffusion method, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, A screening method, characterized in that it is measured using any one method selected from the group consisting of complement fixation analysis, FACS, and protein chip.
A composition for diagnosing pancreatic cancer comprising an agent for measuring the expression level of TSPAN-2 protein or mRNA.
The composition of claim 8, wherein the agent for measuring the expression level of the TSPAN-2 protein is an antibody that specifically binds to TSPAN-2.
The composition of claim 8, wherein the agent for measuring the expression level of TSPAN-2 mRNA is a probe or primer pair that specifically binds to TSPAN-2 mRNA.
To provide information necessary for the diagnosis of pancreatic cancer,
(a) providing a sample of the subject;
(b) measuring the expression level of TSPAN-2 protein or mRNA in the sample; And
(c) comparing the expression level of the TSPAN-2 protein or mRNA with a normal person, and determining that a subject whose expression level of the TSPAN-2 protein or mRNA is increased compared to the normal person has pancreatic cancer. .
The method of claim 11, wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervix, pancreatic tissue samples, or vaginal secretions and urine. .

Images