KR102225039B1 - A pharmaceutical composition for preventing or treating cd8+ memory t cell mediated diseases - Google Patents

Abstract

The present invention ^{relates to a composition for preventing, improving or treating CD8 +} memory T cell-mediated diseases, wherein the composition according to the present invention comprises ^{CD69 +} CD103 ^- CD8 ⁺ memory T ^{among CD8 +} memory T cells present in the liver. the cells are based on the group (Subpopulation) resident tissue, the CD69 ⁺ CD103 ^- CD8 ⁺ memory only T cells by inhibiting the expression level or activity of HIF-2α is present in high levels specifically CD69 ⁺ CD103 ^- CD8 ⁺ memory It can induce the death of T cells or effectively inhibit its function. Furthermore, through such an effect, it can be very usefully applied to the prevention, improvement or treatment of ^{CD8 + memory T cell mediated diseases.}

Description

A pharmaceutical composition for preventing or treating CD8+ memory T cell mediated diseases {A PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CD8+ MEMORY T CELL MEDIATED DISEASES}

The present invention relates to a pharmaceutical composition for preventing or treating ^{CD8 + memory T cell mediated diseases.}

As the liver is an immune-resistant organ, it can survive without rejection even in the case of histocompatibility complex and mismatched liver allograft, and HBV (Hepatitis B virus) and HCV (Hepatitis C virus) can easily cause persistent chronic infection. It is an organ that has a unique immunological aspect.

On the other hand, non-recirculating tissue-resident memory T (Non-recirculating tissue-resident) representing the phenotype of recent effector memory T (T _EM ) cells or CD45RA ⁺ effector memory T (CD45RA ⁺ effector memory; T _{TEMRA) cells.} memory T; T _RM ) cells have been found in a variety of peripheral organs. CD69 is expressed regardless of the presence or absence of αEintegrin (CD103), and the Sphingosine-1-phosphate receptor (S1PR1) and Kruppel-like factor 2 (KLF2) are _{These T RM} cells, which are characterized by a small presence, respond rapidly to relapsed pathogens. In addition, the T _RM cells are commonly found in non-lymphoid tissues such as skin, lungs, gastrointestinal tract and genital organs, brain, and kidney, and are found in livers of rats and humans.

Meanwhile, CD103, one of the important markers for identifying TRM cells along with CD69, is αE that forms a heterodimer with β7 integrin to recognize E-cadherin expressed in epithelial cells such as liver cells. As an integrin, it is found in a variety of immune cells, including dendritic cells, innate lymphocytes, mast cells, and T cells, and on tissue location and transforming growth factor-β (TGF-β) in ^{CD8 + T cells.} Expression increases with exposure to. Mouse liver TRM cells do not express CD103, but about 95% of ^{human liver CD69 +} CD8 ^{+ memory T cells known to have reduced cytotoxicity are CD103-} cell subset, and the remaining 5% CD103 ⁺ cell subset. Consists of. In the case of the CD103 ⁺ cell subset, it exhibits a TEM phenotype, and when stimulated by HBV stimulation, it produces cytokines such as Interleukin-2 (IL-2).

As described above, while studies on the characteristics of cells having the characteristics ^{of CD103 +} among a subset of ^{CD69 +} CD8 ⁺ memory T cells have been conducted variously, until now, the characteristics of cells having the characteristics of ^{CD103-and the prevention of related diseases or} Research on treatment is still insufficient.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating ^{CD8 + memory T cell mediated diseases.}

Another object of the present invention is to provide a food composition for preventing or improving ^{CD8 + memory T cell mediated diseases.}

Another object of the present invention is to provide a screening method for a therapeutic agent ^{for CD8 + T cell mediated diseases.}

Another object of the present invention is ^{to provide a composition for measuring CD8 +} T cell aging and a kit comprising the same.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

^{The present inventors focused on the fact that CD69 +} CD103 ^- CD8 ⁺ memory T cells with high levels of HIF-2α (Hypoxia Inducible Factors 2α) protein in the liver are tissue-resident memory T cells, and the expression or activity of the HIF-2α protein In the case of specifically inhibiting CD8 ^{+ T cells, it is possible to prevent or treat CD8 +} memory T cell-related diseases ^{by inducing the death of CD69 +} CD103 ^- CD8 ⁺ memory T cells, a group that mainly functions among liver CD8 + T cells, or inhibiting the function. It was found that the present invention was completed. Furthermore, since the CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells have a short telomere length and differentiated ends, the senescence of ^{CD8 +} memory T cells is measured by measuring the level of the presence of HIF-2α protein or a gene encoding it. The present invention has been completed by discovering that it can be done.

One embodiment of the present invention is to provide a pharmaceutical composition for preventing or treating ^{CD8 + memory T cell mediated diseases.}

The pharmaceutical composition of the present invention contains an inhibitor of the expression or activity of HIF-2α (Hypoxia Inducible Factors 2α) protein as an active ingredient.

The HIF-2α protein of the present invention is a protein encoded by the EPAS1 gene, and has a transcriptional factor activity in response to a decrease in available oxygen in a hypoxic state. The HIF-2α protein may have an amino acid sequence represented by SEQ ID NO: 1, and the EPAS1 gene may have a base sequence represented by SEQ ID NO: 2, but is not limited thereto.

The inhibitors of this invention in a population of CD8 ⁺ T cells, CD8 ⁺ CD103 ^- can induce selective apoptosis of CD69 ⁺ memory T cells, or inhibit their functions effectively.

^{The CD8 +} CD103 ^- CD69 ⁺ memory T cells of the present invention are a population that functions mainly among ^{CD8 +} T cells, and are present at a high level of HIF-2α protein in the cells compared to ^{CD8 +} CD103 ⁺ CD69 ^{+ memory T cells.} It has a characteristic. In addition, the cells maintain the activity by T-cell receptor (TCR) stimulation, the cells do not die well, the ends of the cells are differentiated, and the telomere length is short. Therefore, by inhibiting the expression or activity of HIF-2α protein present at high levels in ^{CD8 +} CD103 ^- CD69 ^{+ memory T cells, it induces selective death of CD8 +} memory T cells, or by effectively inhibiting its function, CD8 ⁺ memory It is possible to exert a preventive or therapeutic effect of a T cell mediated disease.

The inhibitor of the present invention may be included as long as it can inhibit the expression or activity of HIF-2α protein, for example, at least one selected from the group consisting of oligonucleotides, antibodies, antigen-binding fragments, aptamers, and chemical substances. It may be, but is not limited thereto.

The chemical substance of the present invention may be at least one selected from the group consisting of compounds represented by Formulas 1 to 7 below, and preferably may be a compound represented by Formula 1 below, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

The compound represented by Formula 1 is (S)-3-((2,2-difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-4-yl) )Oxy)-5-fluorobenzonitrile ((S)-3-((2,2-difluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-4-yl)oxy) -5-fluorobenzonitrile), the compound represented by Chemical Formula 2 is 7-((difluoromethyl)sulfonyl)-2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-2 ,3-dihydro-1H-inden-1-ol (7-((difluoromethyl)sulfonyl)-2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-2,3-dihydro-1H-inden -1-ol), the compound represented by Formula 3 is 7-(ethylsulfonyl)-2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-2,3-di Hydro-1H-inden-1-ol (7-(ethylsulfonyl)-2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-2,3-dihydro-1H-inden-1-ol), above The compound represented by Formula 4 is 2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-7-(methylsulfonyl)-2,3-dihydro-1H-indene-1 -Ol (2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-ol), the compound represented by Formula 5 2,2-difluoro-4-(3-fluoro-5-isocyanophenoxy)-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-amine (2,2- difluoro-4-(3-fluoro-5-isocyanophenoxy)-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-amine), the compound represented by Formula 6 is 3-((2-chloro -2-Fluorine-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-4-yl)oxy)-5-fluorobenzoni Tril (3-((2-chloro-2-fluoro-1-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-4-yl)oxy)-5-fluorobenzonitrile) and the formula 7 The compound shown is 3-((2-chloro-2-fluoro-7-(methylsulfonyl)-1-oxo-2,3-dihydro-1H-inden-4-yl)oxy)-5-fluorobenzo It may be nitrile (3-((2-chloro-2-fluoro-7-(methylsulfonyl)-1-oxo-2,3-dihydro-1H-inden-4-yl)oxy)-5-fluorobenzonitrile).

The oligonucleotide of the present invention may be, for example, an antisense oligonucleotide capable of specifically binding to the mRNA of HIF-2α, an aptamer, shRNA and siRNA, but is not limited thereto.

The oligonucleotide of the present invention is a polymer formed by polymerizing several to dozens of nucleotide sequences through covalent bonds, and is complementarily bound to mRNA to degrade mRNA or to prevent mRNA from being translated into protein. Can be. The oligonucleotide of the present invention can be easily prepared by a method known to those of ordinary skill in the art with reference to SEQ ID NO: 2, which is the base sequence of HIF-2α mRNA.

The inhibitor of the present invention may be at least one selected from the group consisting of an antibody capable of inhibiting the activity of HIF-2α protein or an antigen-binding fragment thereof and an aptamer, but is not limited thereto. Specifically, inhibiting the activity of the protein of the present invention means that the HIF-2α protein can inhibit its function as a transcription factor.

The antibody of the present invention is a protein molecule containing an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that acts as an antigen receptor that specifically recognizes and reacts when a specific antigen invades into the body. do. Further, the antibody of the present invention includes special antibodies such as humanized antibodies, human antibodies, and the like, and in addition to novel antibodies, antibodies already known in the art may be included. The antibody includes not only a complete form having a full length of two heavy and two light chains, but also a functional fragment of an antibody molecule, as long as it has the property of binding to specifically recognize the HIF-2α protein. The functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes, but is not limited to, Fab, F(ab'), F(ab')2, and Fv. The antibody of the present invention can be easily prepared by a method known to those of ordinary skill in the art with reference to SEQ ID NO: 1, which is the amino acid sequence of the HIF-2α protein.

The aptamer of the present invention refers to a single-stranded nucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures depending on its base sequence, may have a high affinity for a specific substance such as an antigen-antibody reaction, and may bind to a target molecule and play a role of inhibiting the activity of the target molecule. have. The aptamer of the present invention can be easily prepared by a method known to those of ordinary skill in the art with reference to SEQ ID NO: 1, which is the amino acid sequence of the HIF-2α protein.

^{The CD8 +} memory T cell mediated disease of the present invention is ^{a disease that can be caused by attacking normal tissues by CD8 +} memory T cells, for example, CD8 present at an abnormal level in the liver or excessively accumulated in the liver. ^{+ It} may be a liver disease caused by memory T cells, preferably the liver disease is primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, non-alcoholic Fatty liver disease (Human Nonalcoholic Fatty Liver Disease), acute hepatitis (acute hepatitis) may be at least one selected from the group consisting of liver fibrosis (Hepatic Fibrogenesis), but is not limited thereto.

^{The liver in which the CD8 +} memory T cell mediated disease of the present invention can occur is an organ having a very unique cell population related to an immune response, and is related to Kupffer cells, natural killer cells, and mucous membranes. Cells such as mucosal-associated invariant T cells can accumulate. For this reason, when ^{antigen presentation by CD8 +} memory T cells occurs locally in the liver, an autoimmune response may be induced (see Nat Rev Dis Primers. 2018 Apr 12; 4:18017). In addition, CD8 ⁺ memory T cells accumulated in the liver can induce primary biliary cirrhosis by producing inflammatory cytokines at a very high level compared to normal people (see Gastroenterology. 2014 Jul;147(1):221-232). ^{In addition, CD8 +} memory T cells are present at higher levels in diseases such as primary biliary cirrhosis and nonalcoholic fatty liver compared to normal subjects (Annu Rev Pathol. 2013 Jan 24; 8:303-30, Biomed Res Int. 2017, 2017 : 6862439 and Hepatology. 2014 Apr;59(4):1393-405). Furthermore, ^{an increase in CD8 +} T cells in rats is associated with liver fibrosis in rats (see Gastroenterology. 2004 Sep;127(3):870-82). The pharmaceutical compositions may be CD8 ⁺ CD8 ⁺ CD103 that mainly function in a population of memory T ^cells due to target the CD69 ⁺ memory T cells in a specific, leading to cell death, or be able to inhibit its function, CD8 ⁺ T cells It can be used very effectively in the prevention or treatment of such diseases mediated by.

On the other hand, the "prevention" of the present invention may include, without limitation, any action that blocks, suppresses or delays symptoms caused ^{by CD8 + memory T cell mediated diseases using the pharmaceutical composition of the present invention.} .

In addition, the "treatment" of the present invention may include, without limitation, any action that improves or benefits a symptom caused ^{by a CD8 + memory T cell mediated disease using the pharmaceutical composition of the present invention.}

The pharmaceutical composition of the present invention may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffering agents, preservatives, and painlessness in the case of injections. An agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like may be mixed and used, and for topical administration, a base agent, an excipient, a lubricant, a preservative, and the like may be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.

On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may additionally be included.

The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal. Oral or parenteral administration is preferred.

The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention depends on several factors, including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route and duration of administration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/kg per day Alternatively, it may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

Another embodiment of the present invention is to provide a food composition for preventing or improving CD8+ memory T cell mediated diseases.

The food composition of the present invention can exhibit the effect of preventing or improving ^{CD8 +} memory T cell mediated diseases by including the inhibitor of HIF-2α protein expression or activity described in the pharmaceutical composition as an active ingredient. Therefore, descriptions related to HIF-2α, expression or activity inhibitors, CD8+ memory T cell mediated diseases and prevention are omitted in order to avoid undue complexity in the specification due to repeated description.

Meanwhile, the "improvement" of the present invention may include, without limitation, any action in which symptoms caused by CD8+ memory T cell mediated diseases are improved or beneficially changed using the food composition of the present invention.

The food composition containing the inhibitor of the present invention as an active ingredient can be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, and bread. have.

When the inhibitor of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.

When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation other than including the food composition in the indicated ratio, and may contain various flavoring agents or natural carbohydrates, etc. as an additional component like a normal beverage. . Specifically, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are used. Can include. The flavoring agent may be a natural flavoring agent (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and a synthetic flavoring agent (saccharin, aspartame, etc.).

The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further include a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages.

The components of the present invention may be used independently or in combination. The ratio of the additive does not correspond to the core element of the present invention, but may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

Another embodiment of the present invention provides a method for screening a therapeutic agent ^{for CD8 + T cell mediated diseases.}

The screening method of the present invention includes, first, ^{treating CD8 +} memory T cells or a culture medium thereof with a target candidate material.

Said CD8 ⁺ memory phenotype of the T cell population of the present invention are CD69 ⁺ CD103 ^- may be a CD8 ⁺ memory T cells, the CD69 ⁺ CD103 ^{- -} CD69 ⁺ CD103 showing a phenotype of CD8 ⁺ CD8 ⁺ memory T cells, cell-terminus of Is differentiated, and the length of the telomere may be short, but is not limited thereto.

^{The CD69 +} CD103 ^- CD8 ⁺ memory T cells of the present invention may be obtained by being selected from a biological sample through a flow cytometer and cultured in vitro.

The biological sample of the present invention refers to any substance, biological body fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells ( peripheral blood mononuclear cells), leukocyte soft coat, blood (including plasma and serum), sputum, tears, mucus, nasal washes, Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid , Meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate bronchial aspirate), synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid. It does not become.

The candidate substance of the present invention ^{refers to a drug for testing whether there is an activity to treat CD8 +} memory T cell mediated diseases, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide range of compounds. And any molecule such as. These candidates also include both natural as well as synthetic materials.

The screening method of the present invention includes the step of confirming the expression level of HIF-2α protein or a gene encoding it in ^{the CD8 + memory T cells or a culture medium thereof after treatment with the candidate substance.}

The step of determining the level of the HIF-2α protein present in the present invention is a Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay method using an agent for measuring the protein level containing an antibody specific for the protein. (radioimmunoassay, RIA), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, complement fixation assay, flow cytometry It may be performed by (FACS) and protein chip, but is not limited thereto. The antibody of the present invention can be easily prepared by a method known to those of ordinary skill in the art with reference to SEQ ID NO: 1, which is the amino acid sequence of HIF-2α.

The step of determining the level of the gene encoding the HIF-2α protein of the present invention is performed using a primer pair, a probe, and an anti-sense nucleotide specifically binding to the gene. , Polymerase chain reaction, fluorescence correlation spectroscopy, microarray, chip-assay, etc., but is not limited thereto. The primer pair, probe, and antisense nucleotide of the present invention can be easily prepared by a method known to those of ordinary skill in the art with reference to SEQ ID NO: 2, which is a base sequence encoding HIF-2α.

In the screening method of the present invention, when the expression level of the HIF-2α protein or the gene encoding the HIF-2α protein measured after treatment with the candidate substance is decreased compared to before treatment with the candidate substance, the candidate substance is determined as a therapeutic agent ^{for CD8 + T cell mediated diseases.} It further includes the step of.

In the screening method of the present invention, descriptions related to the CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells, CD8 ⁺ memory T cell mediated diseases, HIF-2α, etc. are omitted in order to avoid excessive complexity of the specification due to repeated description. .

Another embodiment of the present invention provides a composition for measuring ^{CD8 + T cell aging.}

The composition for measuring aging of the present invention includes an agent for measuring the level of the presence of HIF-2α protein or a gene encoding it.

The composition for measuring aging of the present invention has a short length of telomere by checking the expression level of HIF-2α protein or a gene encoding it expressed at a high level in ^{CD69 +} CD103 ^- CD8 ^{+ memory T cells described in the pharmaceutical composition. It is possible to measure aged CD8 +} memory T cells that have the characteristic of being hung and terminally differentiated.

In the composition for measuring aging of the present invention, the ^{contents related to the CD69 +} CD103 ^- CD8 ⁺ memory T cells, HIF-2α, and the like are omitted in order to avoid excessive complexity of the specification due to repeated description.

Another embodiment of the present invention provides a kit for measuring ^{CD8 + T cell senescence.}

The kit for measuring aging of the present invention confirms the expression level of the HIF-2α protein expressed at a high level in the ^{CD69 +} CD103 ^- CD8 ^{+ memory T cells described in the pharmaceutical composition or the gene encoding it, and the length of telomeres is short.} And a composition for measuring senescence for measuring aged CD8+ memory T cells having differentiated characteristics at the ends.

In the kit for measuring aging of the present invention, ^{descriptions related to the CD69 +} CD103 ^- CD8 ⁺ memory T cells, HIF-2α, and the like are omitted in order to avoid excessive complexity of the specification due to repeated description.

The kit of the present invention may be an RT-PCR kit, a DNA chip kit, a microarray or a protein chip kit, and may be prepared according to a conventional method used in the art using the composition for measuring aging. .

In addition to the composition for measuring aging, the kit of the present invention may further include one or more other constituent compositions, solutions, or devices suitable for the analysis method.

[Sequence List]

SEQ ID NO: 1: HIF-2 alpha amino acid sequence

MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLA

ISFLRTHKLLSSVCSENESEAEADQQMDNLYLKALEGFIAVVTQDGDMIFLSENISKFMG

LTQVELTGHSIFDFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRG

RTVNLKSATWKVLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLD

SKTFLSRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQNLCTKG

QVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTE

SLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKLKEEPEELAQLAPTPGDAIISLDFGNQN

FEESSAYGKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCS

TPNSPEDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDF

QLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPE

HRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTE

FLGAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQ

DLSGGDPPGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHL

PLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFESYLLPELT

RYDCEVNVPVLGSSTLLQGGDLLRALDQAT

SEQ ID NO: 2: EPAS1 gene

1 acactcgcga gcggaccgcc acacgggtcc ggtgcccgct gcgcttccgc cccagcgctc

61 ctgaggcggc cgtacaatcc tcggcagtgt cctgagactg tatggtcagc tcagcccggc

121 ctccgactcc ttccgactcc cagcattcga gccacttttt tttttctttg aaaactcaga

181 aaagtgactc cttttccagg gaaaaaggaa cttgggttcc cttctctccg tcctcttttc

241 gggtctgaca gcctccaccc actccttccc cggaccccgc ctccgcgcgc aggttcctcc

301 cagtcacctt tctccacccc cgcccccgca cctagcccgc cgcgcgccac cttccacctg

361 actgcgcggg gcgctcggga cctgcgcgca cctcggacct tcaccacccg cccgggccgc

421 ggggagcgga cgagggccac agccccccac ccgccaggga gcccaggtgc tcggcgtctg

481 aacgtctcaa agggccacag cgacaatgac agctgacaag gagaagaaaa ggagtagctc

541 ggagaggagg aaggagaagt cccgggatgc tgcgcggtgc cggcggagca aggagacgga

601 ggtgttctat gagctggccc atgagctgcc tctgccccac agtgtgagct cccatctgga

661 caaggcctcc atcatgcgac tggcaatcag cttcctgcga acacacaagc tcctctcctc

721 agtttgctct gaaaacgagt ccgaagccga agctgaccag cagatggaca acttgtacct

781 gaaagccttg gagggtttca ttgccgtggt gacccaagat ggcgacatga tctttctgtc

841 agaaaacatc agcaagttca tgggacttac acaggtggag ctaacaggac atagtatctt

901 tgacttcact catccctgcg accatgagga gattcgtgag aacctgagtc tcaaaaatgg

961 ctctggtttt gggaaaaaaa gcaaagacat gtccacagag cgggacttct tcatgaggat

1021 gaagtgcacg gtcaccaaca gaggccgtac tgtcaacctc aagtcagcca cctggaaggt

1081 cttgcactgc acgggccagg tgaaagtcta caacaactgc cctcctcaca atagtctgtg

1141 tggctacaag gagcccctgc tgtcctgcct catcatcatg tgtgaaccaa tccagcaccc

1201 atcccacatg gacatccccc tggatagcaa gaccttcctg agccgccaca gcatggacat

1261 gaagttcacc tactgtgatg acagaatcac agaactgatt ggttaccacc ctgaggagct

1321 gcttggccgc tcagcctatg aattctacca tgcgctagac tccgagaaca tgaccaagag

1381 tcaccagaac ttgtgcacca agggtcaggt agtaagtggc cagtaccgga tgctcgcaaa

1441 gcatgggggc tacgtgtggc tggagaccca ggggacggtc atctacaacc ctcgcaacct

1501 gcagccccag tgcatcatgt gtgtcaacta cgtcctgagt gagattgaga agaatgacgt

1561 ggtgttctcc atggaccaga ctgaatccct gttcaagccc cacctgatgg ccatgaacag

1621 catctttgat agcagtggca agggggctgt gtctgagaag agtaacttcc tattcaccaa

1681 gctaaaggag gagcccgagg agctggccca gctggctccc accccaggag acgccatcat

1741 ctctctggat ttcgggaatc agaacttcga ggagtcctca gcctatggca aggccatcct

1801 gcccccgagc cagccatggg ccacggagtt gaggagccac agcacccaga gcgaggctgg

1861 gagcctgcct gccttcaccg tgccccaggc agctgccccg ggcagcacca cccccagtgc

1921 caccagcagc agcagcagct gctccacgcc caatagccct gaagactatt acacatcttt

1981 ggataacgac ctgaagattg aagtgattga gaagctcttc gccatggaca cagaggccaa

2041 ggaccaatgc agtacccaga cggatttcaa tgagctggac ttggagacac tggcacccta

2101 tatccccatg gacggggaag acttccagct aagccccatc tgccccgagg agcggctctt

2161 ggcggagaac ccacagtcca ccccccagca ctgcttcagt gccatgacaa acatcttcca

2221 gccactggcc cctgtagccc cgcacagtcc cttcctcctg gacaagtttc agcagcagct

2281 ggagagcaag aagacagagc ccgagcaccg gcccatgtcc tccatcttct ttgatgccgg

2341 aagcaaagca tccctgccac cgtgctgtgg ccaggccagc acccctctct cttccatggg

2401 gggcagatcc aatacccagt ggcccccaga tccaccatta cattttgggc ccacaaagtg

2461 ggccgtcggg gatcagcgca cagagttctt gggagcagcg ccgttggggc cccctgtctc

2521 tccaccccat gtctccacct tcaagacaag gtctgcaaag ggttttgggg ctcgaggccc

2581 agacgtgctg agtccggcca tggtagccct ctccaacaag ctgaagctga agcgacagct

2641 ggagtatgaa gagcaagcct tccaggacct gagcgggggg gacccacctg gtggcagcac

2701 ctcacatttg atgtggaaac ggatgaagaa cctcaggggt gggagctgcc ctttgatgcc

2761 ggacaagcca ctgagcgcaa atgtacccaa tgataagttc acccaaaacc ccatgagggg

2821 cctgggccat cccctgagac atctgccgct gccacagcct ccatctgcca tcagtcccgg

2881 ggagaacagc aagagcaggt tccccccaca gtgctacgcc acccagtacc aggactacag

2941 cctgtcgtca gcccacaagg tgtcaggcat ggcaagccgg ctgctcgggc cctcatttga

3001 gtcctacctg ctgcccgaac tgaccagata tgactgtgag gtgaacgtgc ccgtgctggg

3061 aagctccacg ctcctgcaag gaggggacct cctcagagcc ctggaccagg ccacctgagc

3121 caggccttct acctgggcag cacctctgcc gacgccgtcc caccagcttc actctctccg

3181 tctgtttttg caactaggta tttctaacgc cagcacacta tttacaagat ggacttacct

3241 ggcagacttg cccaggtcac caagcagtgg cctttttctg agatgctcac tttattatcc

3301 ctatttttaa agtacacaat tgttttacct gttctgaaat gttcttaaat tttgtaggat

3361 ttttttcctc cccaccttca atgacttcta atttatatta tccataggtt tctctccctc

3421 cttctccttc tcacacacaa ctgtccatac taacaagttt ggtgcatgtc tgttcttctg

3481 tagggagaag ctttagcttc attttactaa aaagattcct cgttattgtt gttgccaaag

3541 agaaacaaaa atgattttgc tttccaagct tggtttgtgg cgtctccctc gcagagccct

3601 tctcgtttct tttttaaact aatcaccata ttgtaaattt cagggttttt ttttttttgt

3661 ttaagctgac tctttgctct aattttggaa aaaaagaaat gtgaagggtc aactccaacg

3721 tatgtggtta tctgtgaaag ttgcacagcg tggcttttcc taaactggtg tttttccccc

3781 gcatttggtg gattttttat tattattcaa aaacataact gagtttttta aaagaggaga

3841 aaatttatat ctgggttaag tgtttatcat atatatgggt actttgtaat atctaaaaac

3901 ttagaaacgg aaatggaatc ctgctcacaa aatcacttta agatcttttc gaagctgtta

3961 atttttctta gtgttgtgga cactgcagac ttgtccagtg ctcccacggc ctgtacggac

4021 actgtggaag gcctccctct gtcggctttt tgccatctgt gatatgccat aggtgtgaca

4081 atccgagcag tggagtcatt cagcgggagc actgcgcgct atcccctcac attctctatg

4141 tactatgtat gtatgtatta ttattattgc tgccaagagg gtctgatggc acgttgtggg

4201 gtcggggggt ggggcgggga agtgctctaa cttttcttaa ggttttgttg ctagcccttc

4261 aagtgcactg agctatgtga ctcggatggt ctttcacacg gcacatttgg acatttccag

4321 aactaccatg agatggttta gacgggaatt catgcaaatg aggggtcaaa aatggtatag

4381 tgaccccgtc cacgtcctcc aagctcacga ccttggagcc ccgtggagct ggactgagga

4441 ggaggctgca cagcgggaga gcagctggtc cagaccagcc ctgcagcccc cactcagccg

4501 gcagccagat ggccccgcaa ggcctccagg gatggcccct agccacaggc cctggctgag

4561 gtctctgggt cggtcagtga catgtaggta ggaagcactg aaaatagtgt tcccagagca

4621 ctttgcaact ccctgggtaa gagggacgac acctctggtt tttcaatacc aattacatgg

4681 aacttttctg taatgggtac aatgaagaag tttctaaaaa cacacacaaa gcacattggg

4741 ccaactattt agtaagcccg gatagactta ttgccaaaaa caaaaaatag ctttcaaaag

4801 aaatttaagt tctatgagaa attccttagt catggtgttg cgtaaatcat attttagctg

4861 cacggcatta ccccacacag ggtggcagaa cttgaagggt tactgacgtg taaatgctgg

4921 tatttgattt cctgtgtgtg ttgccctggc attaagggca ttttaccctt gcagttttac

4981 taaaacactg aaaaatattc caagcttcat attaacccta cctgtcaacg taacgatttc

5041 atgaacgtta ttatattgtc gaattcctac tgacaacatt ataactgtat gggagcttaa

5101 ctttataagg aaatgtattt tgacactggt atcttattaa agtattctga tccta

The composition according to the present invention is based on the fact that among the CD8 ⁺ memory T cells ^{present in the liver, CD69 +} CD103 ^- CD8 ⁺ memory T cells are a tissue resident population (Subpopulation), the CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells specifically By inhibiting the expression level or activity of HIF-2α present at a high level, it is possible ^{to induce the death of CD69 +} CD103 ^- CD8 ⁺ memory T cells, or to efficiently inhibit the function of the cells. Furthermore, through such an effect, it can be very usefully applied to the prevention, improvement or treatment of ^{CD8 + memory T cell mediated diseases.}

1 and 2 show the results of confirming the antigen specificity of ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells in LSMCs of transplant patients with chronic HBV infection according to an embodiment of the present invention.
3 shows the results of confirming the antigen specificity of ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells in LSMCs of patients with various viral infections according to an embodiment of the present invention.
4 and 5 show the results of confirming the specificity of the non-hepatitis virus of ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells according to an embodiment of the present invention.
6 to 10 show the results of confirming the types of genes specifically expressed in ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells according to an embodiment of the present invention.
11 shows the results of confirming the expression of genes specifically expressed in ^{CD69 +} CD8 ⁺ memory T cells according to an embodiment of the present invention through polymerase chain reaction.
12 is a result of confirming that the HIF-2α protein according to an embodiment of the present invention is present at a specifically high level in ^{CD69 +} CD103 ^- CD8 ^{+ memory T cells compared to CD69 +} CD103 ⁺ CD8 ⁺ memory T cells. Is shown.
^{13A and 13B show selective killing of CD69 +} CD103 ^- CD8 ⁺ memory T cells isolated from LSMCs of chronic HBV infected patients by treatment with HIF-2α inhibitor (PT-2385) according to an embodiment of the present invention, It shows the result of confirming the inhibitory effect of its function.
14 shows the results of confirming the phenotype of ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells in an acute viral infection according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

The following examples were reviewed and approved by the institutional review committee of Severance Hospital (Seoul, Korea), and were performed according to the principles of the Helsinki Declaration, and consent from all study subjects was obtained.

[Preparation Example 1] Isolation of mononuclear cells from liver perfusate and peripheral blood

Liver perfusate and peripheral blood were collected from 6 liver transplant patients with chronic HBV infection and then filtered. Then, the filtered liver perfusate and peripheral blood were centrifuged. Then, with Ficoll-Paque (GE Healthcare, USA) liver sinusoidal mononuclear cells (LSMCs, hereinafter referred to as'LSMCs') and peripheral blood mononuclear cells (PBMCs, hereinafter,'LSMCs'). PBMCs') were separated, and the separated LSMCs and PBMCs were prepared using 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) containing fetal bovine serum (FBS). Frozen.

[Preparation Example 2] Preparation of tumor tissue for tissue infiltrating lymphocyte analysis

Four patients with acute hepatitis A were collected by core needle biopsy, and five patients with chronic HBV were collected during liver transplantation.

For immunofluorescence staining, the tissues were fixed with 4% paraformaldehyde for 2 hours, and then dehydrated with phosphate-buffered saline (PBS) containing 20% sucrose. Then, the tissue embedded in the FSC 22 frozen section compound (Leica) was stored in a freezer at -70 °C.

In addition, for flow cytometry, a mononuclear cell suspension was prepared from the tissue using a tumor dissociation kit (Miltenyi Biotec, Germany) and GentleMACS ^{™ Dissociator.}

[Preparation Example 3] T cell isolation

Using CD8 ⁺ T cell isolation kit (Miltenyi Biotec, Germany), it was isolated CD8 ⁺ T cells from PBMCs LSMCs. And, by using the CD69 ⁺ microbeads kit Ⅱ (Miltenyi Biotec, Germany), it was separated by adding CD69 ⁺ CD8 ⁺ T cells from the isolated CD8 ⁺ T cells.

[Example 1] CD69 in LSMCs of Liver Transplant Patients with Chronic HBV Infection ⁺ CD103 ^- CD8 ⁺ Confirmation of antigen specificity of memory T cells

Human leukocyte antigen (HLA) class I pentamers were used to confirm the antigen specificity of LSMCs isolated from liver transplant patients with chronic HBV infection.

Thawed LSMCs were stained using an HLA class I multimer for 15 minutes at room temperature. Then, antibodies specific for the antigen to be measured to which fluorescence is bound were reacted with the cells at room temperature for 10 minutes.

Analysis was performed using an LSR Ⅱ multicolor flow cytometer (BD Biosciences, USA), and ^{the gating of all CD8 +} T cells was TCRγ/δ ⁺ γδ T cells and TCRα7.2 ^{+ CD161 +} mucosa-related invariants. T (mucosal-associated invariant T; MAIT) cells were allowed to be excluded.

Analysis of the resultant data thus obtained was performed using FlowJo software (Treestar), and the results are shown in FIGS. 1 and 2.

As shown in Fig. 1, it was confirmed that both ^CD103- cell subset and CD103 ⁺ cell subset were present in the population ^{of CD69 +} CD8 ^{+ T cells of LSMCs specific for HBV.}

In addition, as shown in Figure 2, compared to the ^{CD103 +} cell subset among the population ^{of CD69 +} CD8 ^{+ T cells of LSMCs specific for HBV, CD103-} T, an indicator related to terminal differentiation of T cells in the cell subset. _{It was} confirmed that EMRA cells, CD57 ⁺ cells, and T-bet ^low Eomes ^hi cells were present at a higher frequency.

Meanwhile, non-hepatitis viruses such as influenza A virus (IAV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) ^{As a result of confirming a subset of CD69 +} CD8 ⁺ T cells isolated from LSMCs specific for (non-hepatotropic virus), it was confirmed that only a subset of ^{CD103 −} cells was specifically present, as shown in FIG. 3.

From the above results, the CD103 ^- cell subset is cloning-aged T cells, and ^{cell proliferation is less than that of the CD103 +} cell subset, whereas apoptosis does not occur well, resulting in the CD69 ⁺ CD8 ⁺ memory T cell subset. It can be seen that there may be many, and the CD103 ^- cell subset has hepatitis virus as well as non-hepatitis virus specificity.

[Example 2] CD69 isolated from liver tissue ⁺ CD8 ⁺ Confirmation of antigen specificity of memory T cells

In order to confirm whether the non-hepatitis virus specificity ^{of the CD103-} cell subset identified in Example 1 ^{is specific for HBV infection, Example 1 in CD69 +} CD8 ⁺ memory T cells isolated from tissue in Preparation Example 2 Flow cytometry was performed in the same manner as described in.

As shown in Fig. 4, ^{among CD69 +} CD8 ⁺ memory T cells, a ^{subset of CD103 −} cells was found to express CXCR6 as a dominant population. In addition, it was confirmed that CCR7 ^- CD45RA ⁺ T _EMRA cells were aggregated in ^{the CD103-cell subset.} These results were consistent with the results found in cells isolated from liver perfusate.

5, CD69 ⁺ CD8 ⁺ memory T cells ^{specific for CMV are all CD103-} cell subsets, whereas CD69 ⁺ CD8 ⁺ memory T cells ^{specific for HBV are CD103-} cell subset and CD103 ^{+ All of the} cell subsets were present.

From the above results, it was found that ^{among the CD69 +} CD8 ⁺ memory T cell population, only the CD103 ^- cell subset and CD103 ⁺ cell subset are specific for HBV, whereas only the ^{CD103-cell subset is specific for non-hepatitis virus.} I can.

[Example 3] CD69 ⁺ CD103 ^- CD8 ⁺ Identification of the types of genes present in memory T cells

In ^{order to identify the types of genes present in CD69 +} CD103 ^- CD8 ⁺ memory T cells, RNA sequencing and transcriptome analysis through microarray were performed.

Using the RNeasy kit (Qiagen, German), total RNA was extracted from ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells and CD69 ^- CD103 ^- CD8 ^{+ memory T cells sorted from LSMCs, respectively.} Then, after purifying and amplifying each of the total RNAs, cDNAs were synthesized through reverse transcription. Then, the cDNAs were prepared as a library for sequencing using a TruSeq RNA access library prep kit (Illumina, USA). RNA sequencing analysis was performed by Magrogen Inc. (Korea), and specific conditions are as follows. The instrument for sequencing was HiSeq2500 (Illumina, USA). As for the sequencing conditions, the RAW read was set to a condition in which the reference genome (UCSC.hg38) was aligned through the HISAT2 package, and a copy assembly was generated from the aligned read using the StringTie package, and the expression profile of the gene was 1 million mapping reads It was allowed to be presented as a fragment per kilobase of the sugar transcript.

In addition, total RNA was extracted from HLA-A*0201-CMVpp65 ₄₉₅ pentamer ⁺ CD8 ⁺ T cells sorted from LSMC, and cDNA was synthesized through reverse transcription. Then, the synthesized cDNA was labeled with a Cy3 dye and hybridized to an Agilent Human 4 X 44K microarray.

The DEGseq R package was used to identify genes with differences in expression within ^{the CD69 +} CD103 ^- CD8 ⁺ memory T cells and CD69 ⁺ CD103 ⁺ CD8 ^{+ memory T cell populations.} Here, the DAVID web tool was used to obtain functional annotation information from DEG.

In addition, by performing GSEA (Gene set enrichment analysis), a ^{set of marker genes between CD69 +} CD103 ^- CD8 ⁺ memory T cells and CD69 ⁺ CD103 ⁺ CD8 ⁺ memory T cells was confirmed. Tissue-resident gene sets and terminally differentiated T-cell gene sets were compared.

Figure 6 - As shown in Figure 9, CD69 ^- CD103 ^- CD8 ⁺ than to memory T cells, CD69 ⁺ CD103 ^- was further shown a lot of genetic traits are CD8 ⁺ memory T cells, indicating the tissue residence and final differentiation (FIG. 6 ). In addition, the result confirmed through the DEGs analysis, CD69 ^- CD103 ^- CD8 ⁺ than to memory T cells, CD69 ⁺ CD103 ^- CD8 ⁺ memory was 190 units gene significantly increased in T cells, it was confirmed that the 751 genes is decreased ( Fig. 7). In addition, as a result of gene ontology analysis and heat map analysis, ^{the activity of chemokines in CD69 +} CD103 ^- CD8 ⁺ memory T cells was increased, and it was confirmed that lymphocyte activation was decreased (FIGS. 8 and 9 ).

As shown in FIG. 10, the ^{results of RNA sequencing analysis of CD69 +} CD103 ^- CD8 ⁺ memory T cells and microarray analysis of liver sinusoidal CMV-specific T cells were significant. A list of 12 overlapping genes with increased expression was derived. Among the 12 overlapping genes, it was confirmed that only the EPAS1 gene encoding the HIF-2α protein corresponds to the only transcription factor.

From the above results, it can be seen that in CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells, the expression of the transcription factor HIF-2α protein and the EPAS1 gene encoding the protein is specifically high.

[Example 4] CD69 ⁺ CD8 ⁺ Verification of genes specifically present in memory T cells

In total CD8 ⁺ memory T cells of PBMCs, CD69 ⁺ CD8 ⁺ memory T cells of LSMCS, and CD69 ^- CD8 ⁺ memory T cells of LSMCs , the levels of HIF target genes, NDRG1 and SLC2A1 genes were quantitatively confirmed, and the results Is shown in FIG. 11. Here, the RNeasy kit (Qiagen, German) was used to extract total RNA from each of the cells, and the same method as in the real-time quantitative PCR described in Example 2 was performed to quantitatively confirm the level of the gene present. I did.

As shown in FIG. 11 , EPAS1 among HIF target genes HIF1A gene and EPAS1 ^{gene specifically for CD69 +} CD8 ^{+ memory T cells compared to all CD8 +} memory T cells of PBMCs and CD69 ^- CD8 ⁺ memory T cells of LSMCs. It was confirmed that the gene and the NDRG1 and SLC2A1 genes were present at high levels.

[Example 5] CD69 ⁺ CD103 ^- CD8 ⁺ Validation of the presence level of HIF-2α in memory T cells

In order to determine whether the HIF-2α protein encoded by the EPAS1 gene is present at a higher level in which subset of CD69 ⁺ CD8 ⁺ memory T cells of ^{LSMCs CD103-} cell subset and CD103 ⁺ cell subset, the above Example 1 The same flow cytometry as the method described in was performed, and the results are shown in FIG. 12.

As shown in Figure 12, CD69 ⁺ CD103 ⁺ CD8 ⁺ memory T cells as compared to CD69 ⁺ CD103 ^- specifically in CD8 ⁺ memory T cells typically was confirmed that the HIF-2α protein present.

[Example 6] CD69 Classified from LSMCs of Chronic HBV Infection Patients ⁺ CD8 ⁺ Maintaining activity and confirming apoptosis in memory T cells

The reactivity of TCR (T cell receptor) was confirmed in ^{the CD103-} cell subset and CD103 ^{+ cell subset of the CD69 +} CD8 ⁺ memory T cells of LSMCs obtained from chronic HBV infection patients. To stimulate the cells, 1 μg/mL of HBVcore overlapping peptide was treated in RPMI culture medium containing LSMCs and 10% fetal calf serum, and cultured for 1 hour, Brefeldin A (BD Biosciences), monensin (BD Biosciences) and After the CD107a antibody (PE- or FITC-conjugated) was additionally added, it was further incubated for 5 hours. Then, after washing the cells in which the culture was completed, an antibody conjugated with fluorescence for a specific surface marker was added and cultured. Then, after performing cell permeability in the cultured cells, staining intracellular cytokines (IFN-γ, IL-2, TNF-α), flow cytometry analysis was performed, and the results are shown in FIG. It is shown in A and B.

As shown in Fig. 13A, when PT-2385, an antagonist of HIF-2α, was administered, among CD69 ⁺ CD8 ⁺ memory T cells ^{stimulated with HBVcore overlapping peptide, CD103-} cell subset ^{compared to CD103 +} cell subset. It was confirmed that the frequency of IFN-γ+ TNF+ or IL-2+ cells was remarkably low.

In addition, as shown in FIG. 13B, ^{the generation of cytokines induced by the HBVcore overlapping peptide in the CD103-} cell subset can be inhibited by administration of PT-2385, but in the CD103 ⁺ cell subset, PT-2385 The cytokine production was not inhibited despite the administration of.

Through the above results, CD8 ⁺ memory a group that is responsible for function mainly in T cells CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells and the activity maintenance of HIF-2α to the survival of the cells it can be seen that the essential.

[Example 7] CD69 in acute viral infection ⁺ CD103 ^- CD8 ⁺ Identification of the phenotype of memory T cells

^{The frequency and phenotype of CD69 +} CD103 ^- CD8 ⁺ memory T cells in LSMCs obtained from patients with type A acute virus infection were confirmed by the same method as in Example 1, and the results are shown in FIG. 14.

As shown in FIG. 14, it was confirmed that more than 95% of ^{CD69 +} CD8 ⁺ memory T cells obtained from the liver of a patient infected with the acute type A virus were a ^{subset of CD103 − cells.} In addition, compared to the ^{CD103 +} cell subset, it was confirmed that the levels of the T _EMRA cells and HIF-2α protein were increased in ^{the CD103 − cell subset.}

From the above results, it can be seen that the characteristics of ^{CD69 +} CD103 ^- CD8 ^{+ memory T cells are maintained during acute viral infection.} Furthermore, it can be seen that diseases such as acute viral infection can be treated by inducing the death of ^{CD69 +} CD103 ^- CD8 ⁺ memory T cells by drugs that inhibit the gene encoding the HIF-2α protein.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea Advanced Institute of science and technology <120> A PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CD8+ MEMORY T CELL MEDIATED DISEASES <130> PDPB187266 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 870 <212> PRT <213> Homo sapiens <400> 1 Met Thr Ala Asp Lys Glu Lys Lys Arg Ser Ser Ser Glu Arg Arg Lys 1 5 10 15 Glu Lys Ser Arg Asp Ala Ala Arg Cys Arg Arg Ser Lys Glu Thr Glu 20 25 30 Val Phe Tyr Glu Leu Ala His Glu Leu Pro Leu Pro His Ser Val Ser 35 40 45 Ser His Leu Asp Lys Ala Ser Ile Met Arg Leu Ala Ile Ser Phe Leu 50 55 60 Arg Thr His Lys Leu Leu Ser Ser Val Cys Ser Glu Asn Glu Ser Glu 65 70 75 80 Ala Glu Ala Asp Gln Gln Met Asp Asn Leu Tyr Leu Lys Ala Leu Glu 85 90 95 Gly Phe Ile Ala Val Val Thr Gln Asp Gly Asp Met Ile Phe Leu Ser 100 105 110 Glu Asn Ile Ser Lys Phe Met Gly Leu Thr Gln Val Glu Leu Thr Gly 115 120 125 His Ser Ile Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Ile Arg 130 135 140 Glu Asn Leu Ser Leu Lys Asn Gly Ser Gly Phe Gly Lys Lys Ser Lys 145 150 155 160 Asp Met Ser Thr Glu Arg Asp Phe Phe Met Arg Met Lys Cys Thr Val 165 170 175 Thr Asn Arg Gly Arg Thr Val Asn Leu Lys Ser Ala Thr Trp Lys Val 180 185 190 Leu His Cys Thr Gly Gln Val Lys Val Tyr Asn Asn Cys Pro Pro His 195 200 205 Asn Ser Leu Cys Gly Tyr Lys Glu Pro Leu Leu Ser Cys Leu Ile Ile 210 215 220 Met Cys Glu Pro Ile Gln His Pro Ser His Met Asp Ile Pro Leu Asp 225 230 235 240 Ser Lys Thr Phe Leu Ser Arg His Ser Met Asp Met Lys Phe Thr Tyr 245 250 255 Cys Asp Asp Arg Ile Thr Glu Leu Ile Gly Tyr His Pro Glu Glu Leu 260 265 270 Leu Gly Arg Ser Ala Tyr Glu Phe Tyr His Ala Leu Asp Ser Glu Asn 275 280 285 Met Thr Lys Ser His Gln Asn Leu Cys Thr Lys Gly Gln Val Val Ser 290 295 300 Gly Gln Tyr Arg Met Leu Ala Lys His Gly Gly Tyr Val Trp Leu Glu 305 310 315 320 Thr Gln Gly Thr Val Ile Tyr Asn Pro Arg Asn Leu Gln Pro Gln Cys 325 330 335 Ile Met Cys Val Asn Tyr Val Leu Ser Glu Ile Glu Lys Asn Asp Val 340 345 350 Val Phe Ser Met Asp Gln Thr Glu Ser Leu Phe Lys Pro His Leu Met 355 360 365 Ala Met Asn Ser Ile Phe Asp Ser Ser Gly Lys Gly Ala Val Ser Glu 370 375 380 Lys Ser Asn Phe Leu Phe Thr Lys Leu Lys Glu Glu Pro Glu Glu Leu 385 390 395 400 Ala Gln Leu Ala Pro Thr Pro Gly Asp Ala Ile Ile Ser Leu Asp Phe 405 410 415 Gly Asn Gln Asn Phe Glu Glu Ser Ser Ala Tyr Gly Lys Ala Ile Leu 420 425 430 Pro Pro Ser Gln Pro Trp Ala Thr Glu Leu Arg Ser His Ser Thr Gln 435 440 445 Ser Glu Ala Gly Ser Leu Pro Ala Phe Thr Val Pro Gln Ala Ala Ala 450 455 460 Pro Gly Ser Thr Thr Pro Ser Ala Thr Ser Ser Ser Ser Ser Cys Ser 465 470 475 480 Thr Pro Asn Ser Pro Glu Asp Tyr Tyr Thr Ser Leu Asp Asn Asp Leu 485 490 495 Lys Ile Glu Val Ile Glu Lys Leu Phe Ala Met Asp Thr Glu Ala Lys 500 505 510 Asp Gln Cys Ser Thr Gln Thr Asp Phe Asn Glu Leu Asp Leu Glu Thr 515 520 525 Leu Ala Pro Tyr Ile Pro Met Asp Gly Glu Asp Phe Gln Leu Ser Pro 530 535 540 Ile Cys Pro Glu Glu Arg Leu Leu Ala Glu Asn Pro Gln Ser Thr Pro 545 550 555 560 Gln His Cys Phe Ser Ala Met Thr Asn Ile Phe Gln Pro Leu Ala Pro 565 570 575 Val Ala Pro His Ser Pro Phe Leu Leu Asp Lys Phe Gln Gln Gln Leu 580 585 590 Glu Ser Lys Lys Thr Glu Pro Glu His Arg Pro Met Ser Ser Ile Phe 595 600 605 Phe Asp Ala Gly Ser Lys Ala Ser Leu Pro Pro Cys Cys Gly Gln Ala 610 615 620 Ser Thr Pro Leu Ser Ser Met Gly Gly Arg Ser Asn Thr Gln Trp Pro 625 630 635 640 Pro Asp Pro Pro Leu His Phe Gly Pro Thr Lys Trp Ala Val Gly Asp 645 650 655 Gln Arg Thr Glu Phe Leu Gly Ala Ala Pro Leu Gly Pro Pro Val Ser 660 665 670 Pro Pro His Val Ser Thr Phe Lys Thr Arg Ser Ala Lys Gly Phe Gly 675 680 685 Ala Arg Gly Pro Asp Val Leu Ser Pro Ala Met Val Ala Leu Ser Asn 690 695 700 Lys Leu Lys Leu Lys Arg Gln Leu Glu Tyr Glu Glu Gln Ala Phe Gln 705 710 715 720 Asp Leu Ser Gly Gly Asp Pro Pro Gly Gly Ser Thr Ser His Leu Met 725 730 735 Trp Lys Arg Met Lys Asn Leu Arg Gly Gly Ser Cys Pro Leu Met Pro 740 745 750 Asp Lys Pro Leu Ser Ala Asn Val Pro Asn Asp Lys Phe Thr Gln Asn 755 760 765 Pro Met Arg Gly Leu Gly His Pro Leu Arg His Leu Pro Leu Pro Gln 770 775 780 Pro Pro Ser Ala Ile Ser Pro Gly Glu Asn Ser Lys Ser Arg Phe Pro 785 790 795 800 Pro Gln Cys Tyr Ala Thr Gln Tyr Gln Asp Tyr Ser Leu Ser Ser Ala 805 810 815 His Lys Val Ser Gly Met Ala Ser Arg Leu Leu Gly Pro Ser Phe Glu 820 825 830 Ser Tyr Leu Leu Pro Glu Leu Thr Arg Tyr Asp Cys Glu Val Asn Val 835 840 845 Pro Val Leu Gly Ser Ser Thr Leu Leu Gln Gly Gly Asp Leu Leu Arg 850 855 860 Ala Leu Asp Gln Ala Thr 865 870 <210> 2 <211> 5155 <212> DNA <213> Homo sapiens <400> 2 acactcgcga gcggaccgcc acacgggtcc ggtgcccgct gcgcttccgc cccagcgctc 60 ctgaggcggc cgtacaatcc tcggcagtgt cctgagactg tatggtcagc tcagcccggc 120 ctccgactcc ttccgactcc cagcattcga gccacttttt tttttctttg aaaactcaga 180 aaagtgactc cttttccagg gaaaaaggaa cttgggttcc cttctctccg tcctcttttc 240 gggtctgaca gcctccaccc actccttccc cggaccccgc ctccgcgcgc aggttcctcc 300 cagtcacctt tctccacccc cgcccccgca cctagcccgc cgcgcgccac cttccacctg 360 actgcgcggg gcgctcggga cctgcgcgca cctcggacct tcaccacccg cccgggccgc 420 ggggagcgga cgagggccac agccccccac ccgccaggga gcccaggtgc tcggcgtctg 480 aacgtctcaa agggccacag cgacaatgac agctgacaag gagaagaaaa ggagtagctc 540 ggagaggagg aaggagaagt cccgggatgc tgcgcggtgc cggcggagca aggagacgga 600 ggtgttctat gagctggccc atgagctgcc tctgccccac agtgtgagct cccatctgga 660 caaggcctcc atcatgcgac tggcaatcag cttcctgcga acacacaagc tcctctcctc 720 agtttgctct gaaaacgagt ccgaagccga agctgaccag cagatggaca acttgtacct 780 gaaagccttg gagggtttca ttgccgtggt gacccaagat ggcgacatga tctttctgtc 840 agaaaacatc agcaagttca tgggacttac acaggtggag ctaacaggac atagtatctt 900 tgacttcact catccctgcg accatgagga gattcgtgag aacctgagtc tcaaaaatgg 960 ctctggtttt gggaaaaaaa gcaaagacat gtccacagag cgggacttct tcatgaggat 1020 gaagtgcacg gtcaccaaca gaggccgtac tgtcaacctc aagtcagcca cctggaaggt 1080 cttgcactgc acgggccagg tgaaagtcta caacaactgc cctcctcaca atagtctgtg 1140 tggctacaag gagcccctgc tgtcctgcct catcatcatg tgtgaaccaa tccagcaccc 1200 atcccacatg gacatccccc tggatagcaa gaccttcctg agccgccaca gcatggacat 1260 gaagttcacc tactgtgatg acagaatcac agaactgatt ggttaccacc ctgaggagct 1320 gcttggccgc tcagcctatg aattctacca tgcgctagac tccgagaaca tgaccaagag 1380 tcaccagaac ttgtgcacca agggtcaggt agtaagtggc cagtaccgga tgctcgcaaa 1440 gcatgggggc tacgtgtggc tggagaccca ggggacggtc atctacaacc ctcgcaacct 1500 gcagccccag tgcatcatgt gtgtcaacta cgtcctgagt gagattgaga agaatgacgt 1560 ggtgttctcc atggaccaga ctgaatccct gttcaagccc cacctgatgg ccatgaacag 1620 catctttgat agcagtggca agggggctgt gtctgagaag agtaacttcc tattcaccaa 1680 gctaaaggag gagcccgagg agctggccca gctggctccc accccaggag acgccatcat 1740 ctctctggat ttcgggaatc agaacttcga ggagtcctca gcctatggca aggccatcct 1800 gcccccgagc cagccatggg ccacggagtt gaggagccac agcacccaga gcgaggctgg 1860 gagcctgcct gccttcaccg tgccccaggc agctgccccg ggcagcacca cccccagtgc 1920 caccagcagc agcagcagct gctccacgcc caatagccct gaagactatt acacatcttt 1980 ggataacgac ctgaagattg aagtgattga gaagctcttc gccatggaca cagaggccaa 2040 ggaccaatgc agtacccaga cggatttcaa tgagctggac ttggagacac tggcacccta 2100 tatccccatg gacggggaag acttccagct aagccccatc tgccccgagg agcggctctt 2160 ggcggagaac ccacagtcca ccccccagca ctgcttcagt gccatgacaa acatcttcca 2220 gccactggcc cctgtagccc cgcacagtcc cttcctcctg gacaagtttc agcagcagct 2280 ggagagcaag aagacagagc ccgagcaccg gcccatgtcc tccatcttct ttgatgccgg 2340 aagcaaagca tccctgccac cgtgctgtgg ccaggccagc acccctctct cttccatggg 2400 gggcagatcc aatacccagt ggcccccaga tccaccatta cattttgggc ccacaaagtg 2460 ggccgtcggg gatcagcgca cagagttctt gggagcagcg ccgttggggc cccctgtctc 2520 tccaccccat gtctccacct tcaagacaag gtctgcaaag ggttttgggg ctcgaggccc 2580 agacgtgctg agtccggcca tggtagccct ctccaacaag ctgaagctga agcgacagct 2640 ggagtatgaa gagcaagcct tccaggacct gagcgggggg gacccacctg gtggcagcac 2700 ctcacatttg atgtggaaac ggatgaagaa cctcaggggt gggagctgcc ctttgatgcc 2760 ggacaagcca ctgagcgcaa atgtacccaa tgataagttc acccaaaacc ccatgagggg 2820 cctgggccat cccctgagac atctgccgct gccacagcct ccatctgcca tcagtcccgg 2880 ggagaacagc aagagcaggt tccccccaca gtgctacgcc acccagtacc aggactacag 2940 cctgtcgtca gcccacaagg tgtcaggcat ggcaagccgg ctgctcgggc cctcatttga 3000 gtcctacctg ctgcccgaac tgaccagata tgactgtgag gtgaacgtgc ccgtgctggg 3060 aagctccacg ctcctgcaag gaggggacct cctcagagcc ctggaccagg ccacctgagc 3120 caggccttct acctgggcag cacctctgcc gacgccgtcc caccagcttc actctctccg 3180 tctgtttttg caactaggta tttctaacgc cagcacacta tttacaagat ggacttacct 3240 ggcagacttg cccaggtcac caagcagtgg cctttttctg agatgctcac tttattatcc 3300 ctatttttaa agtacacaat tgttttacct gttctgaaat gttcttaaat tttgtaggat 3360 ttttttcctc cccaccttca atgacttcta atttatatta tccataggtt tctctccctc 3420 cttctccttc tcacacacaa ctgtccatac taacaagttt ggtgcatgtc tgttcttctg 3480 tagggagaag ctttagcttc attttactaa aaagattcct cgttattgtt gttgccaaag 3540 agaaacaaaa atgattttgc tttccaagct tggtttgtgg cgtctccctc gcagagccct 3600 tctcgtttct tttttaaact aatcaccata ttgtaaattt cagggttttt ttttttttgt 3660 ttaagctgac tctttgctct aattttggaa aaaaagaaat gtgaagggtc aactccaacg 3720 tatgtggtta tctgtgaaag ttgcacagcg tggcttttcc taaactggtg tttttccccc 3780 gcatttggtg gattttttat tattattcaa aaacataact gagtttttta aaagaggaga 3840 aaatttatat ctgggttaag tgtttatcat atatatgggt actttgtaat atctaaaaac 3900 ttagaaacgg aaatggaatc ctgctcacaa aatcacttta agatcttttc gaagctgtta 3960 atttttctta gtgttgtgga cactgcagac ttgtccagtg ctcccacggc ctgtacggac 4020 actgtggaag gcctccctct gtcggctttt tgccatctgt gatatgccat aggtgtgaca 4080 atccgagcag tggagtcatt cagcgggagc actgcgcgct atcccctcac attctctatg 4140 tactatgtat gtatgtatta ttattattgc tgccaagagg gtctgatggc acgttgtggg 4200 gtcggggggt ggggcgggga agtgctctaa cttttcttaa ggttttgttg ctagcccttc 4260 aagtgcactg agctatgtga ctcggatggt ctttcacacg gcacatttgg acatttccag 4320 aactaccatg agatggttta gacgggaatt catgcaaatg aggggtcaaa aatggtatag 4380 tgaccccgtc cacgtcctcc aagctcacga ccttggagcc ccgtggagct ggactgagga 4440 ggaggctgca cagcgggaga gcagctggtc cagaccagcc ctgcagcccc cactcagccg 4500 gcagccagat ggccccgcaa ggcctccagg gatggcccct agccacaggc cctggctgag 4560 gtctctgggt cggtcagtga catgtaggta ggaagcactg aaaatagtgt tcccagagca 4620 ctttgcaact ccctgggtaa gagggacgac acctctggtt tttcaatacc aattacatgg 4680 aacttttctg taatgggtac aatgaagaag tttctaaaaa cacacacaaa gcacattggg 4740 ccaactattt agtaagcccg gatagactta ttgccaaaaa caaaaaatag ctttcaaaag 4800 aaatttaagt tctatgagaa attccttagt catggtgttg cgtaaatcat attttagctg 4860 cacggcatta ccccacacag ggtggcagaa cttgaagggt tactgacgtg taaatgctgg 4920 tatttgattt cctgtgtgtg ttgccctggc attaagggca ttttaccctt gcagttttac 4980 taaaacactg aaaaatattc caagcttcat attaacccta cctgtcaacg taacgatttc 5040 atgaacgtta ttatattgtc gaattcctac tgacaacatt ataactgtat gggagcttaa 5100 ctttataagg aaatgtattt tgacactggt atcttattaa agtattctga tccta 5155

Claims (19)

A pharmaceutical composition for the prevention or treatment of ^{CD8 +} T cell-mediated viral infections comprising an inhibitor of HIF-2α (Hypoxia Inducible Factors 2α) protein expression or activity as an active ingredient,
The inhibitor is a compound represented by the following formula (1),
The virus is Hepatitis virus, Influenza A virus (IAV), Respiratory syncytial virus (RSV), Cytomegalovirus (CMV), or Epstein-Barr virus. ; EBV) phosphorus, composition:
[Formula 1]

The method of claim 1,
Wherein the inhibitor is from CD8 ⁺ population of T cells, CD8 ⁺ CD103 ^- CD69 ⁺ induces selective apoptosis of the memory T cell, or a viral infection or a pharmaceutical composition for the treatment of diseases it is to ensure that the function is inhibited. delete delete delete delete The method of claim 1,
The hepatitis virus is hepatitis A (Hepatitis A virus; HAV), hepatitis B (Hepatitis B virus; HBV) or hepatitis C (Hepatitis C virus; HCV), a pharmaceutical composition for the prevention or treatment of viral infectious diseases. A food composition for the prevention or improvement of ^{CD8 +} T cell mediated viral infection diseases containing an inhibitor of HIF-2α (Hypoxia Inducible Factors 2α) protein expression or activity as an active ingredient,
The inhibitor is a compound represented by the following formula (1),
The virus is Hepatitis virus, Influenza A virus (IAV), Respiratory syncytial virus (RSV), Cytomegalovirus (CMV), or Epstein-Barr virus. ; EBV) phosphorus, composition:
[Formula 1]

delete The method of claim 8,
The hepatitis virus is hepatitis A (Hepatitis A virus; HAV), hepatitis B (Hepatitis B virus; HBV) or hepatitis C (Hepatitis C virus; HCV), a food composition for preventing or improving viral infectious diseases. In vitro ^{treating CD8 +} memory T cells or a target candidate material to the culture medium thereof; And
A method for screening a therapeutic agent for a ^{CD8 +} T cell mediated viral infection disease comprising the step of confirming the expression level of the HIF-2α protein or the gene encoding it after treatment with the candidate material,
The virus is Hepatitis virus, Influenza A virus (IAV), Respiratory syncytial virus (RSV), Cytomegalovirus (CMV), or Epstein-Barr virus. ; EBV), the method. The method of claim 11,
The CD8 ⁺ memory T cells are CD69 ⁺ CD103 ^- CD8 ⁺ memory T cells, the screening method of a viral infection disease therapeutic agent. The method of claim 11,
In the case where the expression level of HIF-2α protein or the gene encoding the HIF-2α protein measured after treatment with the candidate substance is decreased compared to before treatment with the candidate substance, determining the candidate substance as ^{a therapeutic agent for a CD8 +} T cell mediated viral infection disease. A method for screening a therapeutic agent for viral infectious diseases. delete The method of claim 11,
The hepatitis virus is hepatitis A (Hepatitis A virus; HAV), hepatitis B (Hepatitis B virus; HBV) or hepatitis C (Hepatitis C virus; HCV), a screening method for a therapeutic agent for viral infectious diseases. delete delete delete delete

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