Regulatory T cell biomarkers

Abstract

The present invention relates to a biomarker for regulatory T cells.

Description

[Technical field]

[0001] Biomarkers for regulatory T cells T cell). [Background technology]

[0002] Regulatory T cells (Treg) play an important role in naturally preventing excessive inflammation and immune responses, but it has been reported that the function and number of regulatory T cells are significantly reduced in autoimmune and chronic inflammatory diseases. Therefore, for patients with immune and inflammatory diseases, it is important to produce regulatory T cells at normal levels, which could be one of the treatments for the above diseases.

[0003] In the case of cancer, regulatory T cells are widely distributed in the cancer microenvironment and inhibit the ability of natural killer cells (NK cells) and cancer-specific cytotoxic T lymphocytes to eliminate cancer cells. It has been reported that, together with immune checkpoints such as PD-1 and CTLA-4, they play an important role in the immune evasion mechanism of cancer.

[0004] Regulatory T cells are classified into naive regulatory T cells (undifferentiated Treg; nTreg), resting regulatory T cells (resting Treg; rTreg), effector regulatory T cells (eTreg) and memory regulatory T cells (mTreg) based on their developmental origin and cellular properties. Recently, Shimon Sakaguchi group classified non-Treg, nTreg / rTreg and eTreg based on the expression levels of CD4, CD45RA, Foxp3 and CD25 (CD4+CD25+CD45RA-Foxp3lo; non-Treg, CD4+CD25+CD45RA+Foxp3lo; nTreg, CD4+CD25+CD45RA-Foxp3hi; eTreg). Among the types of regulatory T cells, effector regulatory T cells and memory regulatory T cells are known to have high immunosuppressive ability. In particular, it has been reported that effector regulatory T cells are abundant in tumor infiltrating lymphocytes (TILs), which highly express the immune checkpoint CTLA-4 and have a high immunosuppressive ability. Therefore, specifically regulating effector regulatory T cells, memory regulatory T cells, and regulatory T cells in tumor infiltrating lymphocytes, which have high immunosuppressive ability among regulatory T cells, could be an effective immunotherapy.

[0005] To date, research has been conducted on genes and proteins that are specifically present in regulatory T cells, and it has been suggested that substances such as CD25, CTLA4, CD62L, CD38, CD103, GITR, and CD45RB can serve as marker substances; however, there are still no genes or proteins that can target regulatory T cells alone. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide a method for identifying regulatory T cells with an antibody or antigen-binding fragment that specifically binds to a marker specific for regulatory T cells or a fragment thereof.

[0007] Another object of the present invention is to provide a diagnostic composition for cancer, immune-related disease, or brain and nervous system disease, comprising a preparation for measuring the expression level of a marker specific to regulatory T cells or a fragment thereof, or a gene encoding the same.

[0008] Another object of the present invention is to provide a diagnostic kit comprising a diagnostic composition for cancer, immune-related disease, or cranial nervous system disease, comprising a preparation for measuring the expression level of a marker specific to regulatory T cells or a fragment thereof, or a gene encoding the same.

[0009] Another object of the present invention is to provide an information providing method for diagnosing cancer, immune-related disease or brain and nervous system disease, comprising the step of measuring the expression level of a marker specific to regulatory T cells or a fragment thereof, or a gene encoding the same, in a biological sample isolated from an individual of interest.

[0010] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those having ordinary skill in the art from the following description. [Means for solving the problem]

[0011] The "regulatory T cells" or "Tregs" of the present invention can be selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells, which have high immunosuppressive ability, but are not limited thereto.

[0012] The "nucleic acid molecule" of the present invention includes all nucleic acid molecules in which the amino acid sequence of the polypeptide provided by the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences can be produced by ORF (open reading frame), and all of these are included in the nucleic acid molecule of the present invention.

[0013] The "antibody" of the present invention refers to a substance that specifically binds to an antigen to cause an antigen-antibody reaction. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to a regulatory T cell-specific protein or its extracellular domain. The antibody of the present invention includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibody can be easily produced using techniques widely known in the art. For example, a polyclonal antibody can be produced by a method widely known in the art, including a process of injecting an antigen of the biomarker protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such a polyclonal antibody can be produced from any animal, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, etc. Monoclonal antibodies can be produced using the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519) or phage antibody library technology (see Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991), which are well known in the art. The antibodies produced by the above method are separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. The antibodies of the present invention include not only intact forms having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. The functional fragments of the antibody molecule refer to fragments that retain at least the antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv.

[0014] In the present invention, an "immunoglobulin" has a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light chain and the heavy chain contain three variable regions called complementarity determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly play a role in binding to an antigenic determinant (epitope) of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3 in order from the N-terminus, and are also named according to the chain in which a particular CDR is located. are identified.

[0015] In the present invention, the "complementarity determining region (CDR)" includes three variable regions and four framework regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, sequentially from the N-terminus, and are also identified by the chain in which the particular CDR is located.

[0016] In the present invention, a "full-length antibody" is a structure having two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0017] In the present invention, an "antigen-binding fragment" refers to a fragment that retains an antigen-binding function. Examples of antigen-binding fragments include: (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and the first heavy chain constant region (CH1); (ii) an Fd fragment consisting of a VH and CH1 domain; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) an isolated CDR region; (vi) an F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that connects them to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer; and (ix) a diabody, which is a multivalent or multispecific fragment produced by gene fusion. Said antigen-binding fragments can be produced by recombinant gene techniques, such as when proteolytic enzymes, such as papain or pepsin, are used to obtain Fab or F(ab')2 fragments.

[0018] As used herein, the term "monoclonal antibody" refers to an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies, which exhibits a single binding specificity and affinity for a particular antigenic determinant (epitope).

[0019] In the present invention, "binding" or "specific binding" refers to the affinity of the present antibody or antibody composition to an antigen. In antigen-antibody binding, "specific binding" typically refers to binding with a dissociation constant (Kd) of 1×10 -5 Less than M or 1×10 -6 Less than M or 1×10 -7 If the binding is less than M, it is distinguished from non-specific background binding. Specific binding can be detected by methods known in the art, such as ELISA, surface plasmon resonance (SPR), immunoprecipitation, coprecipitation, etc., and includes appropriate controls that can distinguish specific binding from non-specific binding.

[0020] The "antibody or antigen-binding fragment" of the present invention can exist as a multimer, such as a dimer, trimer, tetramer, pentamer, etc., that contains at least a portion of the antigen-binding ability of the monomer. Such multimers also include homomultimers or heteromultimers. Antibody multimers contain multiple antigen-binding sites and therefore have superior antigen-binding ability compared to monomers. Antibody multimers also facilitate the production of multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.

[0021] In the present invention, "prevention" includes, without limitation, all actions of blocking, suppressing or delaying the symptoms of a disease using the pharmaceutical composition of the present invention.

[0022] In the present invention, "treatment" and "improvement" include, without limitation, any action that improves or benefits symptoms of a disease by examining the pharmaceutical composition of the present invention.

[0023] The "pharmaceutical composition" of the present invention can be formulated into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions by a conventional method, but is not limited thereto. The pharmaceutical composition of the present invention can contain a pharmaceutical acceptable carrier. The pharmaceutical acceptable carrier can be a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., when administered orally, and can be mixed with a buffer, a preservative, a pain-relieving agent, a solubilizer, an isotonic agent, a stabilizer, etc., when used as an injection, and can be mixed with a base, an excipient, a lubricant, a preservative, etc., when used for topical administration. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutical acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and when administered by injection, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained release preparations, etc. Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, it can contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc.

[0024] In the present invention, the "route of administration" for the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0025] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

[0026] The "dosage and dosage" of the composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and may be appropriately selected by those skilled in the art. It may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The dosage may be administered once a day or in several divided doses. The dosage does not limit the scope of the present invention in any aspect. The pharmaceutical composition according to the present invention may be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0027] The "PNA (Peptide Nucleic Acid)" of the present invention refers to an artificially synthesized polymer similar to DNA or RNA, which was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen in Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding strength and stability to DNA or RNA, and is used in molecular biology, diagnostic analysis and antisense therapy. PNA has been described in the literature [Nielsen PE, Egholm M, Berg RH, Buchardt O (December 2006) , "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide." Science 254(5037):1497-1500.

[0028] The "aptamer" of the present invention is an oligonucleic acid or peptide molecule. The general content of aptamers can be found in the literature [Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine.” J Mol Med.78(8):42630(2000);Cohen BA, Colas P, Brent R. “An artificial The details are disclosed in "Cell-cycle inhibitors isolated from a combinatorial library." Proc Natl Acad Sci USA. 95(24):142727(1998)."

[0029] The "primer" of the present invention is a fragment that recognizes a target gene sequence and includes forward and reverse primer pairs, but is preferably a primer pair that provides analytical results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, high specificity can be achieved when the primer is a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0030] The "probe" of the present invention means a substance capable of specifically binding to a target substance to be detected in a sample, and a substance capable of specifically confirming the presence of the target substance in a sample by said binding. The type of probe is not limited to a substance commonly used in the art, but may be preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably PNA. More specifically, the probe includes a biological substance derived from an organism, a substance similar thereto, or a substance produced outside the body, and may be, for example, an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and organ, a nerve cell, DNA, and RNA, and DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, etc.

[0031] As used herein, "LNA (locked nucleic acids)" refers to nucleic acid analogs that contain a 2'-O, 4'-C methylene bridge. LNA nucleosides contain the common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rules. However, the "locking" of the molecule by the methylene bridge prevents LNA from forming the ideal shape for Watson-Crick binding. When LNA is included in a DNA or RNA oligonucleotide, it can pair more quickly with the complementary nucleotide strand, increasing the stability of the double helix.

[0032] "Antisense" as used herein refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize to a target sequence in an RNA by Watson-Crick base pairing, typically with the mRNA, to form an RNA:oligomer heteroduplex within the target sequence. The oligomer can have exact sequence complementarity or approximate complementarity to the target sequence.

[0033] The "kit" of the present invention may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit. The diagnostic kit may further include one or more other constituent compositions, solutions, or devices suitable for the analysis method. For example, essential elements required for performing a reverse transcription polymerase reaction may further be included. The reverse transcription polymerase reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific to the nucleic acid sequence of a control gene. Other reverse transcription polymerase reaction kits may 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-water, sterile water, etc. The diagnostic kit may include essential elements required for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, preparations, enzymes, etc. for preparing a fluorescently labeled probe. The substrate may also include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof. The diagnostic kit may include essential elements required for performing an ELISA. The ELISA kit may include an antibody specific to the protein. The antibody may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody that has high specificity and affinity for the marker protein and little cross-reactivity with other proteins. The ELISA kit may also include an antibody specific to the control protein. Other ELISA kits may include reagents capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with the antibody) and their substrates or other substances capable of binding to the antibody.

[0034] The "target individual" of the present invention refers to an individual for whom the presence or absence of onset of a disease is uncertain, and who has a high possibility of onset of a disease.

[0035] The "biological sample" of the present invention means any substance, biological fluid, tissue or cell obtained from or derived from an individual, such as whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, 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, lymphatic fluid, and the like. fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, or cerebrospinal fluid The fluid may include, but is not limited to,

[0036] In the present invention, a "test substance" includes any substance, molecule, element, compound, entity, or combination thereof, including, but not limited to, a protein, a polypeptide, a small organic molecule, It includes polysaccharides, polynucleotides, etc. It may also be a natural product, a synthetic compound, or a combination of two or more substances.

[0037] The screening method of the present invention may include searching for phage-displayed peptide clones that bind to the protein, high throughput screening (HTS) using natural product and chemical libraries, drug hit HTS, cell-based screening, or screening using DNA arrays. In this case, the composition of the present invention may contain a buffer or reaction solution that stably maintains the structure or physiological activity of the protein in addition to the protein. The composition of the present invention may also contain cells expressing the protein, or cells containing a plasmid expressing the protein under a promoter capable of regulating the transcription rate, for in vivo experiments. In the screening method of the present invention, the test subject is an individual nucleic acid, protein, other extract, natural product, compound, etc. that is presumed to have a potential as a cancer metastasis inhibitor by a conventional selection method, or that is randomly selected. Furthermore, the test substances obtained by the screening method of the present invention that exhibit the activity of enhancing gene expression or enhancing protein function, and the test substances that exhibit the activity of suppressing gene expression or suppressing protein function, can become immunosuppressant candidate substances in the former case by developing an inhibitor against the test substance, and in the latter case, can become immunosuppressant candidate substances. Such immunosuppressant candidate substances act as leading compounds in the subsequent immunosuppressant development process, and new immunosuppressants can be developed by modifying and optimizing the structure of the leading compound so that it exhibits an inhibitory effect on the function of the gene or the protein expressed therefrom.

[0038] The "siRNA (small interfering RNA)" of the present invention is a short 19-30-mer double strand RNA duplex that, when introduced into cells, exhibits the effect of suppressing the expression of only specific genes without non-specific inhibition. The mechanism of action of siRNA is known to be that it binds to RISC (RNA-induced silencing complex) in cells, excises the sense strand of the gene corresponding to the mRNA, forms a complex with an antisense stand RISC that is complementary to the sense strand, and binds to and degrades mRNA having a complementary base sequence, i.e., the mRNA of the target gene, thereby inhibiting gene expression.

[0039] The "shRNA (short hairpin RNA)" of the present invention has 3 to 10 base pairs between the sense and complementary nonsense siRNA base sequences of the target gene. After synthesizing oligo DNA linking a base linker, it is cloned into a plasmid vector, or the shRNA is inserted into a retrovirus, lentivirus or adenovirus, and expressed, whereby a hairpin-structured shRNA with a loop is produced, which is converted into an siRNA by Dicer in cells to show the RNAi effect, and the shRNA has the advantage of showing the RNAi effect for a relatively long period of time. The siRNA of the present invention may be in a chemically modified form to prevent rapid degradation by nucleases in vivo. Since siRNA has a double helix structure, it is a relatively stable structure compared to ribonucleic acid or antisense oligonucleotide with a single helix structure, but it is quickly degraded by nucleases in vivo, so the rate of degradation can be reduced by chemical modification. Methods for chemically modifying siRNA to make it stable and resistant to easy degradation are well known to those skilled in the art. The most commonly used method for chemically modifying siRNA is The methods used are boranophosphate or phosphorothioate modification. These substances form stable bonds between nucleosides of siRNA, which results in resistance to nucleic acid degradation. Ribonucleic acids modified with boranophosphate are characterized by poor nucleic acid degradation, but are not produced by chemical reactions, and are only synthesized by a method in which boranophosphate is incorporated into ribonucleic acids through an in vitro transcription reaction. The boranophosphate modification method is a relatively easy method, but has the disadvantage of being difficult to modify at a specific position. In contrast, the phosphorothioate modification method has the advantage of being able to introduce sulfur elements into desired positions, but severe phosphorothioation can cause problems such as reduced efficacy, toxicity, and non-specific RISC (RNA-induced silencing complex) formation. Therefore, in addition to the above two methods, a method of chemically modifying only the end position (the site beyond the 3' end) of ribonucleic acid to impart resistance to nucleases has recently become more popular. It is also known that chemical modification of the ribose ring can also enhance resistance to nucleases, and in particular, mutation of the 2'-position of ribose, which is naturally present in cells, stabilizes siRNA. However, stability can only be increased by accurately inserting a methyl group at this position, and excessive methyl groups can, conversely, cause problems such as the loss of ribonucleic acid-mediated interference. The reason for such chemical modification is also to increase the pharmacokinetic residence time in the body and to increase efficacy (Mark et.al., Molecular Therapy, 13:644-670, 2006). In addition to chemical modification methods, a safe and efficient delivery system is required to increase the intracellular delivery efficiency of siRNA. For this reason, the siRNA of the present invention is included in the pharmaceutical composition for cancer treatment in the form of a complex with a nucleic acid delivery system.

[0040] In the present invention, "cancer" refers to a physiological condition characterized by typically unregulated cell growth in mammals. In the present invention, the cancer to be prevented, improved or treated may be a solid tumor consisting of a mass generated by abnormal cell growth in a solid organ, and depending on the site of the solid organ, may be, but is not limited to, gastric cancer, liver cancer, hepatoma, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer.

[0041] In the present invention, the "immune-related disease" may be one or more selected from the group consisting of autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute or chronic inflammatory disease, but is not limited thereto. The autoimmune disease may be one or more selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa, but is not limited thereto.

[0042] In the present invention, the "cranial nervous system disease" may be a neurodegenerative disease or a neuroinflammatory disease.

[0043] In the present invention, the "neurodegenerative disease" may mean a disease caused by a decrease or loss of function of nerve cells, and the "neuroinflammatory disease" may mean a disease caused by an excessive inflammatory response of the nervous system.

[0044] In the present invention, specific examples of the neurodegenerative disease or neuroinflammatory disease include stroke, dementia, Alzheimer's disease, Parkinson's disease, and the like. Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS) The present invention may be selected from the group consisting of, but is not limited to, vascular endothelial cell carcinoma ...

[0045] In the present invention, the "method of identifying regulatory T cells" refers to a method of amplifying a specific marker present in regulatory T cells by reverse transcription polymerase chain reaction (RT-PCR) and identifying the specific marker present in regulatory T cells by fluorescence by flow cytometry (fluorescence activated cell sorting, FACS) or the like to identify or separate regulatory T cells, but is not limited thereto.

[0046] In the present invention, the "reverse transcription polymerase reaction" is a modification of the polymerase chain reaction (PCR), which is an experimental technique commonly used in molecular biology to generate multiple DNA sequences through an amplification process. However, in the reverse transcription polymerase chain reaction, RNA is first reverse transcribed by reverse transcriptase to generate cDNA, and the generated cDNA is then amplified by a conventional polymerase chain reaction or a real-time polymerase chain reaction, but is not limited thereto.

[0047] As used herein, "fluorescence activated cell sorting (FACS)" or "FACS" refers to a method for separating a population of cells into one or more subpopulations depending on the presence, absence, or level of one or more FACS-selectable polypeptides expressed by the cells. FACS relies on the optical properties, including fluorescence, of individual cells to separate cells into subpopulations.

[0048] In the present invention, the "FACS-selectable polypeptide" is a polypeptide that can be detected directly or indirectly by flow cytometry. Examples of FACS-selectable polypeptides include polypeptides that contain an extracellular domain (e.g., CD52 or CD59) that can bind to a detectable binding partner (e.g., a fluorescently labeled antibody) for indirect detection of the polypeptide by flow cytometry, and other examples of FACS-selectable polypeptides include fluorescent proteins, such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and variants thereof, including eGFP, Venus, mCherry, and mTomato, which can be directly detected by flow cytometry. FACS can be used to identify specific markers present on regulatory T cells and to separate regulatory T cells.

[0049] One embodiment of the present invention provides a method for identifying regulatory T cells using an antibody or antigen-binding fragment that specifically binds to any one protein or fragment thereof selected from the group consisting of RGS1;F5 and CD27.

[0050] In another embodiment of the invention, the protein is specifically expressed on the surface of regulatory T cells. The present invention provides a method for identifying regulatory T cells, a cell surface protein that regulates T cell function.

[0051] In another embodiment of the present invention, the regulatory T cells are cell surface proteins including one or more of the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells. The present invention provides a method for identifying regulatory T cells.

[0052] One embodiment of the present invention provides a diagnostic composition for cancer disease, comprising a preparation for measuring the expression level of any one of proteins or fragments thereof selected from the group consisting of RGS1; F5 and CD27; or a gene encoding the same.

[0053] In another embodiment of the present invention, the diagnostic composition is provided, wherein the protein is a cell surface protein specifically expressed on the surface of regulatory T cells.

[0054] In another specific example of the present invention, the diagnostic composition is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0055] In another embodiment of the present invention, a diagnostic composition is provided in which a formulation for measuring the expression level of the protein or a fragment thereof comprises one or more members selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer that specifically binds to the protein or a fragment thereof.

[0056] In another embodiment of the present invention, a diagnostic composition is provided in which a preparation for measuring the expression level of a gene encoding the protein or a fragment thereof comprises one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene.

[0057] In another embodiment of the present invention, the cancer is ovarian cancer, colon cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain cancer, anal cancer, trumpeterinary duct carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma.

[0058] Another embodiment of the present invention provides a kit for diagnosing cancer disease, comprising the above diagnostic composition.

[0059] One embodiment of the present invention provides an information providing method for diagnosing a cancer disease, comprising the step of measuring the expression level of any one of the proteins or fragments thereof selected from the group consisting of RGS1; F5 and CD27; or a gene encoding the same, in a biological sample isolated from an individual of interest.

[0060] In another embodiment of the present invention, the method for providing information is provided, wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

[0061] In another specific example of the present invention, the method for providing information is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0062] In another embodiment of the invention, the biological sample is selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, 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, and the like. In one embodiment, the method includes extracting from a tissue sample from a patient, the tissue sample being extracted from a patient's bronchial aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts or cerebrospinal fluid.

[0063] In another embodiment of the present invention, the method for measuring the expression level of the protein or a fragment thereof is performed by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-Mass spectrometry / Mass spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay).

[0064] In another embodiment of the present invention, the method for measuring the expression level of a gene encoding the protein or a fragment thereof is performed by reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting or DNA chip, thereby providing an information providing method.

[0065] In another embodiment of the present invention, the cancer is ovarian cancer, colon cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, anal cancer, trumpeterinary duct carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, or pituitary adenoma.

[0066] In one embodiment of the present invention, the present invention relates to a method for treating a patient having a disease characterized by the above-mentioned. The present invention provides a diagnostic composition for immune-related diseases, comprising a preparation for measuring

[0067] In another embodiment of the present invention, the diagnostic composition is provided, wherein the protein is a cell surface protein specifically expressed on the surface of regulatory T cells.

[0068] In another specific example of the present invention, the diagnostic composition is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0069] In another embodiment of the present invention, a diagnostic composition is provided in which a formulation for measuring the expression level of the protein or a fragment thereof comprises one or more members selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer that specifically binds to the protein or a fragment thereof.

[0070] Another embodiment of the present invention provides a diagnostic composition, wherein a preparation for measuring the expression level of a gene encoding the protein or a fragment thereof comprises one or more selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene.

[0071] In another embodiment of the present invention, the diagnostic composition is provided, wherein the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

[0072] In another embodiment of the present invention, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa.

[0073] Another embodiment of the present invention provides a kit for diagnosing immune-related diseases, comprising the above-mentioned diagnostic composition.

[0074] One embodiment of the present invention provides an information providing method for diagnosing a cancer disease, comprising the step of measuring the expression level of any one of the proteins or fragments thereof selected from the group consisting of RGS1; F5 and CD27; or a gene encoding the same, in a biological sample isolated from an individual of interest.

[0075] In another embodiment of the present invention, the method for providing information is provided, wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

[0076] In another specific example of the present invention, the method for providing information is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0077] In another embodiment of the invention, the biological sample is selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, The information providing method includes saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract or cerebrospinal fluid.

[0078] In another embodiment of the present invention, the method for measuring the expression level of the protein or a fragment thereof is performed by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-Mass spectrometry / Mass spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay).

[0079] In another embodiment of the present invention, the method for measuring the expression level of a gene encoding the protein or a fragment thereof is performed by reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting or DNA chip, thereby providing an information providing method.

[0080] In another embodiment of the present invention, the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

[0081] In another embodiment of the present invention, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa.

[0082] One embodiment of the present invention provides a diagnostic composition for nervous system diseases, comprising a preparation for measuring the expression level of any one of proteins or fragments thereof selected from the group consisting of RGS1; F5 and CD27; or a gene encoding the same.

[0083] In another embodiment of the present invention, the diagnostic composition is provided, wherein the protein is a cell surface protein specifically expressed on the surface of regulatory T cells.

[0084] In another specific example of the present invention, the diagnostic composition is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0085] In another embodiment of the present invention, a diagnostic composition is provided in which a formulation for measuring the expression level of the protein or a fragment thereof comprises one or more members selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer that specifically binds to the protein or a fragment thereof.

[0086] In another embodiment of the present invention, a diagnostic composition is provided in which a preparation for measuring the expression level of a gene encoding the protein or a fragment thereof comprises one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene.

[0087] In another embodiment of the present invention, the diagnostic composition is provided, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

[0088] In another embodiment of the invention, the neurodegenerative or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention provides a diagnostic composition for treating tauopathy, the disease being treated with a method for treating tauopathy, comprising administering to the patient a therapeutically effective amount of the drug selected from the group consisting of tauopathy, tauopathy, and tauopathy.

[0089] Another embodiment of the present invention provides a kit for diagnosing a central nervous system disease, comprising the above-mentioned diagnostic composition.

[0090] One embodiment of the present invention provides an information providing method for diagnosing a brain and nervous system disease, comprising the step of measuring the expression level of any one of proteins or fragments thereof selected from the group consisting of RGS1; F5 and CD27, or a gene encoding the same, in a biological sample isolated from an individual of interest.

[0091] In another embodiment of the present invention, the method for providing information is provided, wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

[0092] In another specific example of the present invention, the method for providing information is provided, wherein the regulatory T cells are cell surface proteins including one or more types selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

[0093] In another embodiment of the invention, the biological sample is selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, The information providing method includes 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, synovial fluid, joint aspirate, organ secretions, cell, cell extract or cerebrospinal fluid.

[0094] In another embodiment of the present invention, the method for measuring the expression level of the protein or a fragment thereof is performed by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-Mass spectrometry / Mass spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay).

[0095] In another embodiment of the present invention, the method for measuring the expression level of a gene encoding the protein or a fragment thereof is performed by reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting or DNA chip, thereby providing an information providing method.

[0096] In another embodiment of the present invention, there is provided a method for providing information, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

[0097] In another embodiment of the invention, the neurodegenerative or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The method of providing information is selected from the group consisting of tauopathy, tauopathy, and tauopathy. provide. Effect of the Invention

[0098] The marker protein or fragment thereof specific to the regulatory T cells of the present invention can be used to select regulatory T cells, since it is an element that can be distinguished from other immune cells. In addition, by confirming regulatory T cells expressing the marker specific to the regulatory T cells, cancer, immune-related disease, or brain and nervous system disease affected by the regulatory T cells can be diagnosed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] The present invention will now be described in detail with reference to the following examples, however, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0100] Single cell mRNA sequence analysis data from patient samples of three types of cancer (colorectal cancer, hepatocellular carcinoma, and non-small cell lung cancer) was used to apply a decision-tree-based algorithm called XGBoost (EXtreme Gradient Boosting). In this case, XGBoost used a decision-based tree based on TPM values ​​to find the optimal combination for sorting into two sets. Using this, it was possible to primarily classify which genes were related to sorting and which genes were specifically expressed in tumor Tregs.

[0101] Example 1. Screening of biomarkers for T regulatory cells in colorectal cancer (CRC) Patient samples (total of 12 patients = 8 males, 4 females) were collected from peripheral blood, fresh cancer tissue, and normal tissue at least 2 cm adjacent to the collected cancer tissue:

[0102] Next, single cell sorting and reverse transcription, amplification and sequencing were performed as described below. i) 1 mm from each tissue sample 3 A piece of tissue was excised, placed in RPMI-1640 (FBS 10%), and subjected to enzymatic digestion reaction for 30 minutes on a rotor at 37°C using the MACS tumor dissociation kit from Miltenyl Biotec. ii) The disaggregated tissue was passed through a 40 μm cell-strainer and centrifuged at 400 g for 10 minutes. iii) The supernatant was discarded and the red blood cells were lysed using RBC lysis buffer for 2 minutes. iv) After washing twice with PBS, the cell pellet was resuspended in PBS / w 1% FBS. v) PBMCs were isolated using HISTOPAQUE-1077. vi) Staining with human CD3, CD4, CD8, and CD25 antibodies was performed with single cell sorting analysis by FACS. vii) To separate the cytotoxic T cells, Th cells, and Treg cells into subsets, the cells were enriched for 7AAD-CD3+CD8+, 7AAD-CD3+CD4+CD25-, and 7AAD-CD3+CD4+CD25hi, respectively, collected in a 96-well plate pre-cooled to 4°C, and then lysed in lysis buffer. viii) cDNA was synthesized from single cell lysates by reverse transcriptase PCR using the Smart-Seq2 (Nat Protoc. 2014 Jan;9(1):171-81) method, and mRNA expression levels were measured by qPCR.

[0103] The T cell subsets used in the analysis were tumor Treg, peripheral Treg, Th17, effector memory T cells, and naive T cells, and the number of cells used in the analysis was 10,805 out of 11,138 single cells (data for 12,525 genes).

[0104] The analytical indicators are as follows: TPM(Transcript per million)=Reads / transcript length / kilobase / 1,000,000

[0105] The analysis was carried out in the following manner. The average TPM value for each gene expressed in each subset was calculated: (1) Tumor Treg: 1,319 cells; (2) peripheral Treg: 365 cells; (3) Th17: 229 cells; (4) T effector memory cells: 204 cells; and (5) Undifferentiated T cells: 472 cells

[0106] We distinguished tumor Treg cells from other cell subsets (peripheral Treg, Th17, T effector memory, and naive T cells): (1) Tumor Treg cluster: 1,319 single cells; and (2) non-tumor Treg cluster: 1,270 single cells;

[0107] Using XGBoost, the machine learning process proceeded as follows: (1) The TPM values ​​for all single cells were input into XGBoost to generate a conditioning algorithm that could most efficiently separate tumor Tregs from other T cell subsets, with 474 genes used as conditioning genes. (2) An algorithm using 474 genes distinguished tumor Tregs from other T cell subsets with 97.5% accuracy.

[0108] To find surface markers from the 474 selected genes, we reselected only genes with plasma membrane expression levels of 4 to 5. In the final step, we selected Treg-specific markers based on the following detailed strategy. (1) Scenario 1: Genes with higher TPM values ​​in tumor Treg cells than in undifferentiated T cells (2) Scenario 2: Genes with higher TPM values ​​in tumor Treg cells than in peripheral Treg cells (3) Scenario 3: Genes whose TPM value in peripheral Treg cells is higher than that in undifferentiated T cells (4) Scenario 4: Genes with higher TPM values ​​in peripheral Treg cells than in Th17 cells

[0109] In this scenario, the following markers were selected (see Table 1):

[0110] Example 2. Screening of regulatory T cell biomarkers in hepatocellular carcinoma (HCC) Patient samples (total of 12 patients = 4 males, 2 females) were collected from peripheral blood, fresh cancer tissue, and normal tissue at least 2 cm adjacent to the harvested cancer tissue:

[0111] Next, single cell sorting and reverse transcription, amplification and sequencing were performed as in Example 1.

[0112] However, the T cell subsets used in the analysis were tumor Tregs, peripheral Tregs, naive T cells, cytotoxic CD4 and exhausted T cells.

[0113] The number of cells used in the analysis was 5,063 single cells (data for 23,389 genes).

[0114] The analytical indexes and analytical methods were the same as in Example 1. However, the following cells were used for each subset. (1) Tumor Treg: 582 cells; (2) peripheral Treg: 261 cells; (3) undifferentiated T cells: 646 cells; and (4) Exhausted T cells: 146 cells

[0115] We differentiated tumor Treg rankings from other cell subsets (peripheral Tregs, exhausted T cells, cytotoxic CD4, and naive T cells): (1) Tumor Treg cluster: 582 single cells (2) Non-tumor Treg cluster: 1,220 single cells

[0116] Using XGBoost, the machine learning process proceeded as follows: (1) The TPM values ​​for all single cells were input into XGBoost to generate a conditioning algorithm that could most efficiently separate tumor Tregs from other T cell subsets, with 281 genes used as conditioning genes. (2) The 281-gene algorithm distinguished tumor Tregs from other T cell subsets with 97.78% accuracy.

[0117] To find surface markers from the selected genes, we reselected only genes with plasma membrane expression levels of 4 to 5. In the final step, we selected Treg-specific markers based on the following detailed strategy. (1) Scenario 1: Genes with higher TPM values ​​in tumor Treg cells than in undifferentiated T cells (2) Scenario 2: Genes whose TPM value in peripheral Treg cells is higher than that in undifferentiated T cells (3) Scenario 3: Genes with higher TPM values ​​in tumor Treg cells than in peripheral Treg cells

[0118] In this scenario, the following markers were selected (see Table 1):

[0119] Example 3. Screening of regulatory T cell biomarkers in non-small cell lung cancer (NSCLC) Patient samples (11 adenocarcinoma, 3 squamous cell carcinoma) were collected from peripheral blood and fresh cancer tissue:

[0120] Next, single cell sorting and reverse transcription, amplification and sequencing were performed as in Example 1. However, The T cell subsets used were tumor Tregs, peripheral Tregs, naive T cells, cytotoxic CD4, exhausted T cells, central memory T cells, and effector memory T cells.

[0121] The number of cells used in the analysis was 12,346 single cells (data for 12,394 genes).

[0122] The analytical indexes and analytical methods were the same as in Example 1. However, the following cells were used for each subset. (1) Tumor Treg: 939 cells; (2) Resting Treg: 428 cells; (3) Undifferentiated T cells: 532 cells; (4) exhausted T cells: 343 cells; (5) Central Memory T cells: 666 cells; and (6) Effector Memory T cells: 434 cells

[0123] We differentiated tumor Treg rankings from other cell subsets (peripheral Tregs, exhausted T cells, cytotoxic CD4, and naive T cells): (1) Tumor Treg cluster: 939 single cells (2) Non-tumor Treg cluster: 2,398 single cells

[0124] Using XGBoost, the machine learning process proceeded as follows: (1) The TPM values ​​for all single cells were input into XGBoost to generate a conditioning algorithm that could most efficiently separate tumor Tregs from other T cell subsets, with 548 genes used as conditioning genes. (2) The 548-gene algorithm distinguished tumor Tregs from other T cell subsets with 96.71% accuracy.

[0125] To find surface markers from the selected genes, we reselected only genes with plasma membrane expression levels of 4 to 5. In the final step, we selected Treg-specific markers based on the following detailed strategy. (1) Scenario 1: Genes with higher TPM values ​​in tumor Treg cells than in undifferentiated T cells (2) Scenario 2: Genes whose TPM value in peripheral Treg cells is higher than that in undifferentiated T cells (3) Scenario 3: Genes with higher TPM values ​​in tumor Treg cells than in peripheral Treg cells

[0126] In the above scenario, the following markers were selected (see Table 1 below).

[0127] [Table 1]

Claims

1. A method for identifying regulatory T cells using an antibody or antigen-binding fragment that specifically binds to any one protein selected from the group consisting of RGS1, F5, and CD27, or a fragment thereof.

2. The method for identifying regulatory T cells according to claim 1 , wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

3. The method for identifying regulatory T cells according to claim 1, wherein the regulatory T cells are cell surface proteins comprising one or more of the following: effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

4. A diagnostic composition for cancer disease, comprising a preparation for measuring the expression level of any one protein or fragment thereof selected from the group consisting of RGS1, F5 and CD27, or a gene encoding the same.

5. The diagnostic composition according to claim 4 , wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

6. The diagnostic composition according to claim 5, wherein the regulatory T cells are cell surface proteins comprising one or more of the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

7. 5. The diagnostic composition of claim 4, wherein the cancer is ovarian cancer, colon cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, anal cancer, trumpeterinary duct carcinoma, endometrial carcinoma, vaginal cancer, vulva carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

8. A cancer diagnosis kit comprising the diagnostic composition according to any one of claims 4 to 7.

9. A diagnostic composition for immune-related diseases, comprising a preparation for measuring the expression level of any one protein or fragment thereof selected from the group consisting of RGS1, F5, and CD27; or a gene encoding the same.

10. The diagnostic composition according to claim 9 , wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

11. The diagnostic composition according to claim 10, wherein the regulatory T cells are cell surface proteins including one or more proteins selected from the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

12. 10. The diagnostic composition of claim 9, wherein the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

13. 13. The diagnostic composition of claim 12, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa.

14. A diagnostic kit for immune-related diseases, comprising the diagnostic composition according to any one of claims 9 to 13.

15. A diagnostic composition for a nervous system disease, comprising a preparation for measuring the expression level of any one protein or fragment thereof selected from the group consisting of RGS1, F5 and CD27; or a gene encoding the same.

16. The diagnostic composition according to claim 15 , wherein the protein is a cell surface protein that is specifically expressed on the surface of regulatory T cells.

17. The diagnostic composition according to claim 16, wherein the regulatory T cells are cell surface proteins comprising one or more of the group consisting of effector regulatory T cells, memory regulatory T cells, and cancer-infiltrating lymphocyte regulatory T cells.

18. The diagnostic composition according to claim 15, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

19. The neurodegenerative or neuroinflammatory disease may be stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis (Alzheimer's disease), paraneoplastic syndrome (PFS), or other neurodegenerative disorders. syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathies.

19. The diagnostic composition of claim 18, wherein the composition is selected from the group consisting of:

20. A kit for diagnosing cranial nervous system diseases, comprising the diagnostic composition according to any one of claims 15 to 19.