CN110799212A

CN110799212A - DKK1 protein specifically present in regulatory T cells and uses thereof

Info

Publication number: CN110799212A
Application number: CN201880031227.9A
Authority: CN
Inventors: 金廷镐; 金范锡
Original assignee: Goodall T Cell Co Ltd
Current assignee: Goodall T Cell Co Ltd; Good T Cells Inc
Priority date: 2017-05-10
Filing date: 2018-05-10
Publication date: 2020-02-14
Also published as: JP2020519268A; WO2018208108A1; KR20180123989A

Abstract

The present invention relates to Dickkopf-1(DKK1) protein, specific antibodies thereof, DDK1 protein being specifically present on the surface of immune cells, in particular regulatory T cells (Treg cells), and uses thereof. In the present invention, since the DKK1 protein is specifically present on the surface of immune cells, particularly regulatory T cells, the DKK1 protein can be used as a novel target for a therapeutic agent for various immune-related diseases.

Description

DKK1 protein specifically present in regulatory T cells and uses thereof

Technical Field

The present invention relates to Dickkopf-1(DKK1) protein, specific antibodies thereof, DDK1 protein being specifically present on the surface of immune cells, in particular regulatory T cells (Treg cells), and uses thereof.

Background

Among immune cells, T cells are immune cells produced in the bone marrow and matured in the thymus, and function to regulate antibody production of B cells or regulate the function of innate immune cells. T cells, also known as T lymphocytes, can not only act as a helper for other immune cells, but can also directly destroy invaders.

Among the functions of immune cells, it is most important that these immune cells exhibit suppressed immune responses to antigenic substances constituting themselves and antigenic substances around the immune cells, while recognizing non-self antigenic substances and inducing immune responses thereto. During development, immune cells do not respond to self-antigens by killing cells that recognize themselves, inducing mutations in specific receptors for self-antigens, or inactivating immune cells that recognize self-antigens, which is called immune anergy or immune tolerance. This failure of self-tolerance induces an immune response to the autoantigen, resulting in disease. This disease is known as autoimmune disease.

In the early 70's of the 19 th century, in order to induce or maintain self-tolerance, the concept of regulatory T cells was introduced, which can control the effector functions of conventional T cells (RK Gershon and k. kondo, immunology, 1970, 18: 723-37). Since then, studies to elucidate the immunological properties and functions of suppressor T cells have been ongoing.

In normal individuals, regulatory T cells control the function of conventional T cells, inducing an excessive immune response or self-tolerance. However, it has been reported that in autoimmune and chronic inflammatory diseases, the function and number of regulatory T cells are significantly reduced, and thus the regulatory T cells do not function normally. Thus, restoring normal levels of regulatory T cell function and number in patients with autoimmune and chronic inflammatory diseases may be one of the treatments for the disease.

CD25, CTLA4, CD38, CD62L, GITR, CD45RB, and the like have been suggested as cell surface proteins targeting regulatory T cells through studies, and animal and clinical trials have been performed. However, to date, no cell surface proteins have been found that target only regulatory T cells.

Technical problem

The purpose of the present invention is to provide a Dickkopf-1(DKK1) protein or an epitope thereof which is specifically present on the surface of regulatory T cells.

It is yet another object of the present invention to provide specific antibodies to DKK1 protein specifically present on the surface of regulatory T cells.

It is still another object of the present invention to provide a regulatory T cell expressing DKK1 protein on its cell surface.

It is still another object of the present invention to provide a method for screening an immunosuppressant or an immunoactivator.

It is still another object of the present invention to provide a method for preventing or treating immune-related diseases.

It is still another object of the present invention to provide a method for diagnosing immune-related diseases.

However, the technical problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned may be clearly understood by those skilled in the art from the following description.

Technical scheme for solving problems

The present inventors found Dickkopf-1(DKK1) protein that exists specifically on the surface of immune cells, particularly regulatory T cells, and screened epitopes of the protein. In addition, the present inventors found that in the case where immune cells (particularly regulatory T cells) expressing DKK1 protein were selectively screened and co-incubated with effector T cells, the activity of effector T cells was inhibited, and thus completed the present invention.

In the present invention, DKK1 is one of DKK family genes DKK1, DKK2, DKK3, DKK4, encoding a secreted protein. DKK proteins typically consist of 255 to 350 amino acids. DKK1 was 24KDa to 29KDa in size, with the N-terminus present in glycosylated form. As an example of the invention, the amino acid sequence of DKK1 may be as set forth in SEQ ID NO:1, and can be represented by seq id NO: 2.

[ Table 1]

In the present invention, DKK1 binds strongly to receptors in the Wnt signaling system involved in cell proliferation and wound recovery, thereby competitively inhibiting Wnt signaling, compared to ligands such as Wnt3 a. Thus, DKK1 may play an important role in embryonic development, such as in the development of the heart, head, hands, etc. Furthermore, DKK1 can be used to identify osteolytic bone lesions in multiple myeloma patients and can be used to grade DKK1 cancer or bone disease by increasing the level of DKK1 present in bone marrow plasma in the form of secreted proteins.

However, in the present invention, DKK1 may be present on the surface of immune cells, particularly regulatory T cells, rather than being secreted from the cells as is often the case. This may allow immune cells to play a role in the self-tolerance of regulatory T cells.

According to one embodiment of the present invention, there is provided SEQ ID NO:3, regulatory T cell outer surface protein of DKK1 (table 2).

[ Table 2]

The present invention provides regulatory T cell outer surface proteins of DKK1 that can interact with ligands present on effector T cells to reduce the activity of regulatory T cells.

According to another embodiment of the invention, there is provided an epitope of DKK1 that is a regulatory T cell surface protein, such as SEQ ID NO:4 to 36 (Table 3).

[ Table 3]

As used herein, the term "epitope" refers to a portion of an antigenic molecule that binds to and can be recognized by an antibody. Typically, an antibody does not recognize the entire antigenic molecule, but only a specific portion thereof. Even the same antigen molecule can recognize different epitope regions depending on the type of antibody. The epitope for the purpose of the present invention includes an epitope in which even if a portion of DKK1 protein exposed to the outside of cells is locally cleaved and secreted to the outside of cells, the remaining partial region can be effectively bound to an antibody.

In the present invention, the polypeptide as an epitope may include continuous and discontinuous sequences of a portion of DKK1 protein according to the present invention that can bind to an antibody.

In addition, the present invention provides a polypeptide fragment as an epitope of regulatory T cell surface protein DKK1, which can interact with a ligand present in effector T cells to decrease the activity of regulatory T cells.

According to another embodiment of the invention, there is provided a polynucleotide encoding the extracellular surface protein of DKK1 shown in SEQ ID No. 3 or the DKK1 epitope shown in any one of SEQ ID NOs 4 to 36 provided by the present invention.

According to another embodiment of the present invention, there is provided an expression vector into which the polynucleotide provided by the present invention is inserted.

In the present invention, a "vector" is a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, wherein additional DNA segments can be ligated into the genome of the virus. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors useful in recombinant DNA techniques are typically in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector.

Specific examples of the expression vector of the present invention may be selected from, but are not limited to, the following groups: pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors, which are widely used commercially; sticking particles; phages, such as lambda, herringbone, M13, Mu, P1P 22, Q μ, T-even, T2, T3, T7 phages; a plant virus. As an expression vector, any expression vector known to those skilled in the art can be used in the present invention, and the expression vector is selected according to the properties of the target host cell. The vector may be introduced into the host cell by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection, or electroporation. However, the present invention is not limited thereto, and those skilled in the art can adopt and use introduction methods suitable for the expression vector and the host cell used. The vector may preferably comprise at least one selectable marker. However, the present invention is not limited thereto, and screening may be performed using a vector that does not include a screening marker, depending on whether the product is produced. The selection marker depends on the target host cell, which can be accomplished by methods known to those skilled in the art, and thus the present invention is not limited thereto.

To facilitate purification of the nucleic acid molecules of the invention, the tag sequence may be inserted into and fused to an expression vector. Labels include, but are not limited to: a hexahistidine tag, a hemagglutinin tag, a myc tag, or a flag tag, any tag known to those skilled in the art that facilitates purification can be used in the present invention.

According to another embodiment of the present invention, there is provided a host cell line transfected with the expression vector provided herein.

In the present invention, "host cell" includes a single cell or cell culture which may or may not have been the recipient of one or more vectors incorporating the polypeptide insert. Such host cells include progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Such host cells include cells transfected in vivo with one or more of the polynucleotides herein.

In the present invention, the host cell may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacterial cells such as E.coli (E.coli), Streptomyces (Streptomyces), Salmonella typhimurium (Salmonella typhimurium); fungal cells, such as yeast cells and pichia pastoris (Pichiapastoris); insect cells such as Drosophila (Drosophila) and Spodoptera (Spodoptera) Sf9 cells; animal cells, such as Chinese Hamster Ovary (CHO) cells, SP2/0 (mouse myeloma), human lymphoblastoid cells, COS, NSO (mouse myeloma), 293T, Bowes bows melanoma cells, HT-1080, Baby Hamster Kidney (BHK) cells, Human Embryonic Kidney (HEK) cells, or perc.6 (human retinal cells); or a plant cell. However, the host cell is not limited thereto, and any cell known to those skilled in the art to be usable as a host cell line may be used.

According to another embodiment of the present invention, there is provided a regulatory T cell comprising an epitope of DKK1 or an extracellular surface protein of DKK1 of the present invention.

The regulatory T cells surface-expressing the extracellular surface protein of DKK1 of the present invention or an epitope thereof can inhibit the activity of effector T cells and suppress immune response by interacting with effector T cells.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating an immune-related disease, comprising a regulatory T cell comprising an extracellular surface protein of DKK1 of the present invention or an epitope thereof as an active ingredient.

By interacting with effector T cells, regulatory T cells comprising the extracellular surface protein of DKK1 of the invention are able to inhibit the activity of effector T cells and suppress immune responses.

In the present invention, regulatory T cells expressing DKK1 protein on the cell surface can effectively prevent, improve or treat immune-related diseases caused by hyperimmune response, such as autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, and the like.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating immune-related diseases, comprising the extracellular surface protein of DKK1 of the present invention or an epitope thereof as an active ingredient.

The extracellular surface protein or epitope fragment of DKK1 provided by the present invention interacts with a ligand present on effector T cells, so that the activity of regulatory T cells can be inhibited, thereby effectively preventing, ameliorating or treating immune-related diseases such as cancer.

As used herein, the term "cancer" refers to a physiological condition in mammals characterized by cell growth that is not regulated in a typical manner. In the present invention, the cancer to be prevented, improved or treated may be a solid tumor, which is formed of agglomerates caused by abnormal growth of cells in a solid organ, and may be, but is not limited to, gastric cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, etc., depending on the location of the solid organ, preferably melanoma.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating immune-related diseases, comprising an antibody specific to the extracellular surface protein of DKK1 or an epitope thereof of the present invention as an active ingredient.

In addition, in the present invention, DKK1 extracellular surface protein may be present on the surface of regulatory T cells.

In the present invention, the antibody can inhibit the activity of regulatory T cells, thereby effectively preventing, ameliorating or treating cancer.

As used herein, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic region. An antibody for the purposes of the present invention refers to an antibody that specifically binds to a protein of the invention. Also, such antibodies can be prepared by the following method: each gene is cloned into an expression vector according to a conventional method to obtain a protein encoded by a marker gene, and the obtained protein is processed according to a conventional method. Among them, a sub-peptide that can be made from the above-mentioned protein is included, and the sub-peptide of the present invention includes at least seven amino acids, preferably nine amino acids, and more preferably twelve or more amino acids.

The form of the antibody of the present invention is not particularly limited. The antibody of the present invention includes a polyclonal antibody or a monoclonal antibody or a part thereof as long as the part has an antigen binding ability, and includes all immunoglobulin antibodies. In addition, the antibody of the present invention also includes a specific antibody, such as a humanized antibody.

Antibodies of the invention include intact forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab '), F (ab') 2, Fv, and the like.

As an example of the present invention, the antibody specifically binds to DKK1 protein expressed on the surface of regulatory T cells or an epitope thereof, and thus can inhibit the activity of regulatory T cells, thereby effectively preventing, ameliorating or treating immune-related diseases such as cancer.

In the present invention, the cancer may be a solid tumor, which is formed of agglomerates caused by abnormal growth of cells in a solid organ, and may be, but is not limited to, gastric cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, etc., depending on the location of the solid organ, and is preferably melanoma.

As another example of the present invention, the antibody specifically binds to DKK1 protein expressed on the surface of regulatory T cells or an epitope thereof, and thus can enhance the activity of regulatory T cells, thereby effectively preventing, ameliorating or treating immune-related diseases, such as autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, and the like.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for immune-related diseases, comprising any one selected from the group consisting of: antisense oligonucleotides, sirnas, shrnas, and micrornas, each specific for a gene encoding an extracellular surface protein or epitope of DKK1 of the invention.

In the present invention, antisense oligonucleotides are short synthetic DNA strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target and are used to achieve gene-specific inhibition in vivo and in vitro. It has been proposed that antisense oligonucleotides prevent the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping its expression during the transcription, translation or splicing phase. Antisense oligonucleotides have also been successfully used in cell culture and animal models of disease. Other modifications that make antisense oligonucleotides more stable and more resistant so that the oligonucleotides are not degraded are known and understood by those skilled in the art. Antisense oligonucleotides, as used herein, include duplex or single-helix DNA, duplex or single-helix RNA, DNA/RNA hybrids, DNA and RNA analogs, and oligonucleotides having base, sugar, or backbone modifications. Oligonucleotides are modified by methods known in the art to increase their stability and increase their resistance to nuclease degradation. Such modifications include, but are not limited to, modifications to the oligonucleotide backbone, sugar moieties or bases as are known in the art.

In the present invention, "siRNA (small interfering RNA)" is a nucleic acid molecule that can mediate RNA interference or gene silencing, and is used for a high-efficiency gene knockout method or a gene therapy method due to its ability to suppress expression of a target gene. In the case of using the siRNA molecule of the present invention, the siRNA molecule may have a structure in which a sense strand (a sequence corresponding to a WLS mRNA sequence) and an antisense strand (a sequence complementary to the WLS mRNA sequence) are positioned opposite to each other to form a single-stranded or double-stranded structure having complementary sense and antisense strands. siRNA is not limited to those in which double-stranded RNA portions (where RNA pairs with each other) are completely paired, but may also include unpaired portions caused by mismatches (corresponding bases are not complementary), extensions/overhangs (no base corresponds to another strand), and the like. The siRNA end structure may be a blunt end or a sticky end as long as the end allows the expression of the WLS gene to be inhibited by an RNA interference (RNAi) effect. The cohesive end structures may be 3 '-overhang structures or 5' -overhang structures. The total length of the siRNA molecule may be 15 to 30 bases, preferably 19 to 21 bases. However, the total length is not limited thereto.

In the present invention, the "shRNA (short hairpin RNA)" is a single-stranded RNA having a length of 45 to 70 nucleotides, and the shRNA is produced as follows. An oligo-DNA is synthesized in which a sense strand having a base sequence of siRNA of a target gene and an antisense strand complementary thereto are linked through a linker of 3 to 10 bases. Then, the oligomeric DNA is cloned into a plasmid vector, or shRNA is inserted into lentiviruses (retroviruses and adenoviruses); expression of the product yields a shRNA with a circular hairpin structure. The shRNA is converted into siRNA by intracellular ribonuclease (dicer), thereby exhibiting RNAi effect.

In the present invention, "microrna" (microrna) "regulates various biological processes such as development, differentiation, proliferation, conservation and apoptosis. Typically, micrornas destabilize or interrupt translation of a target mRNA, thereby regulating expression of a gene encoding the target mRNA.

In the present invention, regulatory sequences (e.g., constitutive promoters, inducible promoters, tissue-specific promoters, or combinations thereof) that can be used in expression constructs/vectors containing antisense oligonucleotides, siRNA, shRNA, or microrna are selectable by one of skill in the art.

As an example of the present invention, an antisense oligonucleotide, siRNA, shRNA or microrna specifically binds to a gene encoding DKK1 protein or an epitope thereof expressed on the surface of a regulatory T cell, thereby inhibiting the expression of the gene, thereby effectively preventing, ameliorating or treating an immune-related disease such as cancer.

As another example of the present invention, an antisense oligonucleotide, siRNA, shRNA or microrna specifically binds to a gene encoding DKK1 protein or an epitope thereof expressed on the surface of regulatory T cells, thereby enhancing the expression of the gene, thereby effectively preventing, ameliorating or treating immune-related diseases, such as autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, and the like.

In the present invention, "prevention" may include, but is not limited to, any act of blocking symptoms of a disease, or inhibiting/delaying symptoms using the pharmaceutical composition of the present invention.

Further, in the present invention, "treatment" may include, but is not limited to, any act of using the pharmaceutical composition of the present invention to alleviate or beneficially modify the symptoms of a disease.

In the present invention, the pharmaceutical composition may be in the form of a capsule, a tablet, a granule, an injection, an ointment, a powder, or granules, and the pharmaceutical composition may be targeted to a human. Preferably, the pharmaceutical composition of the present invention can be prepared in the form of an injection and directly injected into an area where cancer or immune disease occurs. However, the present invention is not limited thereto.

The pharmaceutical composition of the present invention can be prepared into oral preparations such as powders, granules, capsules, tablets and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and used. However, the pharmaceutical composition is not limited thereto. The pharmaceutical compositions of the present invention may also comprise a pharmaceutically acceptable carrier. As pharmaceutically acceptable carriers, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents and the like may be used for oral administration, buffers, preservatives, analgesics, solubilizers, isotonizing agents, stabilizers and the like may be used as injections, and bases, excipients, lubricants, preservatives and the like may be used for topical administration. The pharmaceutical composition of the present invention can be prepared in various ways by mixing with the above-mentioned pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical compositions may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For injection, the pharmaceutical compositions may be formulated in unit-dose ampoules or in multi-dose forms. Alternatively, the pharmaceutical composition may be formulated into a solution, suspension, tablet, capsule, sustained-release formulation, or the like.

Meanwhile, as examples of carriers, diluents or excipients suitable for preparation of formulations, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl group, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like can be used. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may also be included.

Routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to: oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal routes. Preferably, oral or parenteral administration. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intravesicular (intraburst), intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered rectally in the form of suppositories.

The pharmaceutical compositions of the present invention may vary depending on a variety of factors, including the activity of a certain compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the condition, body weight, severity of disease, pharmaceutical form, administration route and duration of the patient, and may be appropriately selected by those skilled in the art. The pharmaceutical composition may be administered in an amount of 0.0001-50mg/kg or 0.001-50mg/kg per day. Administration may be once a day or several times a day. The dosages described are not intended to limit the scope of the invention in any way. The pharmaceutical composition of the present invention may be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

In addition, the pharmaceutical composition of the present invention can be administered in combination with other anticancer agents, thereby effectively inhibiting general cancer cell proliferation and cancer metastasis, so that the pharmaceutical composition can be used for treating cancer.

Here, as the anticancer agent, one or more anticancer agents selected from the group consisting of: sinapine, imatinib, oxaliplatin, rituximab, erlotinib, nalatinib, lapatinib, gefitinib, vandetanib, nilotinib, semacrtinib, bosutinib, axitinib, cediranib, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, visfate, asparaginase, retinoic acid, hydroxycarbamide, dasatinib, asimumab, argatropizetin, oxutasin, procarbazine, prostadil, holmium nitrate chitosan (holmionite trachitosan), gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, metaalone, otazine, cytarabine, methotrexate, uracil, arabinoside, fludarabine, fluvastatin, fluacrabine, fluvastatin, cetuximab, cetrin, cetuximab, netatidine, and the like, Carbastatin, fluoropiperidine, raltitrexed, docetaxel, paclitaxel, irinotecan, belokadate, topotecan, vinorelbine, etoposide, vinblastine, idarubicin, mitomycin, bleomycin, actinomycin, pirarubicin, aclarubicin, duocarmycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, astatin, dacarbazine, thiotepa, nimustine, chlorambucil, dibromodulcitol, folinic acid, tretinoin, exemestane, aminoglutethimide, anagrelide, olaparib, nevirapine, fadrozole, tamoxifen, toremifene, testosterone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, 5FU, vorinostat, enrotitin, and carmustine. However, the anticancer agent is not limited thereto.

In addition, the pharmaceutical composition of the present invention may be administered in combination with other immunosuppressive agents, thereby effectively suppressing diseases caused by general hyperimmune response.

In the present invention, as the immunosuppressant, one or more immunosuppressants selected from the group consisting of: glucocorticoids, cyclophosphamide, cyclosporine, tacrolimus, rapamycin, type IV PDE inhibitors, p38 kinase inhibitors, azathioprine, mycophenolate mofetil, imidazoline, methotrexate, leflunomide, and brequinar. However, the immunosuppressant is not limited thereto.

According to another embodiment of the present invention, there is provided a method for preventing or treating an immune-related disease, comprising the steps of: administering to a subject in need of treatment an antibody specific for an extracellular surface protein of DKK1 or an epitope thereof of the invention.

In the present invention, the subject in need of treatment is a suspected individual having an immune-related disease or having symptoms thereof. Wherein an example of an immune-related disease can be cancer, such as gastric cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer; another example may be autoimmune diseases, graft versus host disease, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, etc.

According to another embodiment of the present invention, there is a method for preventing or treating an immune-related disease comprising the steps of: administering to a subject in need of treatment any one selected from the group consisting of: antisense oligonucleotides, sirnas, shrnas, and micrornas, each specific for a gene encoding an extracellular surface protein or epitope of DKK1 of the invention.

In one embodiment of the invention, the term "administering" or "administration" refers to introducing a composition of the invention into a patient in any suitable manner. For the route of administration, the composition of the present invention may be administered by any conventional route as long as the route allows the composition to reach the target tissue. Oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, intranasal, pulmonary, rectal, intracavity, intraperitoneal, or epidural may be performed. However, the present invention is not limited thereto. The therapeutic methods of the invention may comprise administering an antibody, antisense oligonucleotide, siRNA, shRNA or microrna in a pharmaceutically effective amount. In the present invention, the effective amount may be adjusted depending on various factors including the type of disease, the severity of disease, the type and dose of the active ingredient and other ingredients contained in the composition, the type of preparation, the age, body weight, general health, sex and diet of the patient, the administration time, the administration route, the secretion rate of the composition, the duration of treatment and the concomitant drug. In adults, the expression inhibitor of the gene or the activity inhibitor of the protein is administered in the following doses when administered once or several times a day: for siRNA, 0.01ng/kg to 10 mg/kg; 0.01ng/kg to 10mg/kg of antisense oligonucleotide against mRNA of the gene; 0.1ng/kg to 10mg/kg for the compound and 0.1ng/kg to 10mg/kg for the monoclonal antibody to the protein.

In the present invention, when administered in the above manner, another anticancer agent or immunosuppressant may be further administered in combination at the time of administration.

According to another embodiment of the present invention, there is provided a method of screening for an immunoactivator or an immunosuppressant, comprising the steps of: (a) contacting a sample to be tested with regulatory T cells containing an extracellular surface protein or epitope of DKK 1; (b) detecting the expression level of an extracellular surface protein or epitope; and (c) determining that the sample is an immune activator or an immune inhibitor when the expression level of the detected extracellular surface protein or epitope is down-regulated or up-regulated.

In the present invention, "screening" is to select a substance having a specific target characteristic from a candidate group consisting of various substances by a specific operation or evaluation method. For the purpose of the present invention, the screening of the present invention is a screening method in which a candidate substance for treating an immune-related disease is administered to an individual suspected of having the disease, and then the expression of the protein of the present invention is decreased or increased, in which case the candidate substance is determined to be an immunosuppressive agent or an immunoactivator. Systemic methods, such as molecular biological assays, digital imaging, cytology and histology assays, can be used to determine the activity of the genes and proteins of the invention on diseased or suspected diseased tissues.

In the present invention, the expression level, amount or activity of the extracellular surface protein or epitope of DKK1 can be measured using an antibody capable of specifically binding to the extracellular surface protein or epitope of DKK 1. In the present invention, a method is used in which an antigen-antibody complex is formed between the antibody and the protein in question in a biological sample, and the antigen-antibody complex is detected.

As used herein, the term "antigen-antibody complex" refers to the combination of a protein antigen and an antibody that recognizes the antigen, the protein antigen being used to identify the presence or absence of the gene in question in a biological sample. Detection of antigen-antibody complexes can be accomplished using methods known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance-based, chemical, and other methods.

In the present invention, methods for determining or comparatively analyzing the expression level of a protein may include, but are not limited to: protein chip analysis, immunoassay, ligand binding assay, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) assay, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) assay, radioimmunoassay, radioimmunodiffusion-Ouchterlony immunodiffusion, rocket immunoelectrophoresis (rocket immunoelectrophoresis), tissue immunostaining, complement fixation assay, two-dimensional electrophoresis assay, liquid chromatography-mass spectrometry (LC-MS), liquid phase secondary mass spectrometry (LC-MS/MS), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and the like.

According to another embodiment of the present invention, there is provided a method of screening for an immunoactivator or an immunosuppressant, comprising the steps of: (a) contacting a test sample with regulatory T cells comprising an extracellular surface protein of DKK1 or an epitope-encoding gene; (b) detecting the expression level of the gene; (c) when the detected gene expression level is down-regulated or up-regulated, the sample is determined to be an immune activator or an immune inhibitor.

In the present invention, the expression level of a gene encoding an extracellular surface protein or epitope of DKK1 can be detected using a primer, probe or antisense nucleotide capable of specifically binding to the gene. Information on the extracellular surface protein or epitope of DKK1 of the present invention and its encoding gene is known. Thus, based on this information, one skilled in the art can readily design primers, probes, or antisense nucleotides that specifically bind to the gene encoding the protein.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' -hydroxyl group that can base pair with its complementary template and serve as a site for the initiation of replication of the template strand. The primers can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and at an appropriate temperature. In the present invention, cancer can be diagnosed by performing PCR amplification using sense and antisense primers against the gene encoding DKK1 extracellular surface protein and checking whether the desired product is produced. PCR conditions, the length of sense and antisense primers, can be optimized based on those known in the art.

In addition, as used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, that is capable of specifically binding to mRNA, ranging in length from as short as a few bases to as long as several hundred bases. The probe is labeled so that the presence of a particular mRNA can be identified. The probe may be in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. In the present invention, cancer can be diagnosed by hybridizing using a probe complementary to a gene encoding DKK1 extracellular surface protein and checking whether hybridization occurs. Modifications may be made based on selection of appropriate probes and hybridization conditions, as is known in the art.

The chemical synthesis of the primer or probe of the present invention can be carried out using a solid phase phosphoramidite support method or other well-known methods. Such nucleic acid sequences may also be modified using any means known in the art. Non-limiting examples of such modifications include: methylation, "capping", replacement of one or more natural nucleotides with homologues thereof, and nucleotide internal modifications, for example, modification with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay, Northern blotting, DNA chip, etc. can be used as a method for determining or comparatively analyzing the gene expression level. However, the method is not limited thereto.

According to another embodiment of the present invention, there is provided a diagnostic composition for diagnosing an immune-related disease, comprising an antibody specific to the extracellular surface protein of DKK1 or an epitope thereof of the present invention as an active ingredient.

As used herein, the term "diagnosing" refers to identifying the presence or characteristics of a pathological condition. The diagnosis used for the purpose of the present invention is to identify the occurrence and metastasis of gastric cancer.

In the present invention, by measuring the expression level of DKK1 extracellular surface protein or epitope present on the surface of regulatory T cells using an antibody, diagnosis of immune-related diseases can be performed.

As an example of the present invention, the immune-related disease may be, but is not limited to, cancer, more specifically, cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, and the like, preferably melanoma.

As another example of the present invention, the immune-related disease may be, but is not limited to, autoimmune diseases, graft-versus-host diseases, organ transplant rejection, asthma, atopic diseases, acute or chronic inflammatory diseases, and the like.

According to another embodiment of the present invention, there is provided a diagnostic composition for immune-related diseases comprising any one selected from the group consisting of: primers, probes, and antisense nucleotides, each specific for a gene encoding an extracellular surface protein or epitope of DKK1 of the invention.

In the present invention, the diagnosis of immune-related diseases can be performed by measuring the expression level of a gene encoding the extracellular surface protein or epitope of DKK1 present on the surface of regulatory T cells using an antibody.

According to another embodiment of the present invention, there is provided a diagnostic kit for immune-related diseases comprising the diagnostic composition of the present invention.

As used herein, the term "kit" refers to a set of compositions and accessories required for a particular purpose. For the purposes of the present invention, the kits of the invention can be used to diagnose immune-related diseases, such as cancer, autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic diseases, or acute or chronic inflammatory diseases, by determining the expression level of DKK1 extracellular surface protein, which is a diagnostic marker for such immune-related diseases, on the mRNA or protein expression level. The following may be included in the kit of the invention: a primer or probe for detecting the expression level of a diagnostic marker for an immune related disease, or optionally an antibody recognizing the marker, and one or more other compositions, solutions or devices suitable for detection.

According to another embodiment of the present invention, there is provided a method for providing information on immune-related diseases, comprising determining the expression level of DKK1 extracellular surface protein or epitope present in regulatory T cells by using an antibody specific for the extracellular surface protein or epitope of DKK 1.

In the present invention, in the case of being diagnosed as an immune-related disease, the method may further comprise the step of administering a therapeutic agent for the immune-related disease.

According to another embodiment of the present invention, there is provided a method for providing information on immune-related diseases, comprising determining the expression level of a gene encoding a DKK1 extracellular surface protein or epitope of the present invention in regulatory T cells using any one selected from the group consisting of: a primer, a probe and an antisense nucleotide each having specificity to the gene.

Advantageous effects of the invention

In the present invention, since the DKK1 protein is specifically present on the surface of immune cells, particularly regulatory T cells, the DKK1 protein can be used as a novel target for a therapeutic agent for various immune-related diseases.

Brief description of the drawings

Fig. 1 shows the structure of DKK1 protein according to an embodiment of the present invention.

Fig. 2 shows the predicted results of an epitope of DKK1 protein according to one embodiment of the invention.

Fig. 3 shows the predicted results of the epitope of DKK1 protein according to one embodiment of the invention.

Fig. 4 shows the predicted results of the epitope of DKK1 protein according to one embodiment of the invention.

Fig. 5 shows the predicted results of epitopes in chain X of 3S8V of DKK1 protein according to one embodiment of the invention.

Fig. 6 shows the predicted results of an epitope in 3SOQ chain Z of DKK1 protein according to one embodiment of the invention.

Fig. 7 shows the predicted results of an epitope in chain 5FWW B of DKK1 protein according to one embodiment of the invention.

Fig. 8 shows the predicted results of an epitope in chain 5FWW B of DKK1 protein according to one embodiment of the invention.

FIG. 9 shows mRNA expression levels according to one embodiment of the invention.

FIG. 10 shows mRNA expression levels according to one embodiment of the invention.

Fig. 11 shows the expression of DKK1 protein on the surface of regulatory T cells according to one embodiment of the invention.

Detailed Description

According to one embodiment of the present invention, there is provided SEQ ID NO:3, DKK1, a regulatory T cell outer surface protein.

According to another embodiment of the invention, there is provided an epitope of DKK1 as regulatory T cell surface protein, as set forth in SEQ ID NO: 4-36.

According to another embodiment of the present invention, there is provided a method for providing information on immune-related diseases, comprising determining the expression level of a gene encoding a DKK1 extracellular surface protein or epitope of the present invention in regulatory T cells using any one selected from the group consisting of a gene encoding a DKK1 extracellular surface protein or epitope of the present invention specifically directed against: a primer, a probe and an antisense nucleotide, each of which is specific for the gene.

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

Examples

[ example 1]DKK1 Identification of C-terminal Structure

DKK1 is known to bind to LRP6 in the form of a secreted protein, corresponding to a ligand specific for it. Among them, the amino acid structure at position 182-266 of the C-terminal region of DKK1 is crucial for the binding of DKK1 to ligands and this structure allows DKK1 and LRP6 to have high binding ability to each other. Therefore, binding to them plays an important role in the Wnt cell signaling system. The structure of the C-terminal protein of DKK1 is shown in figure 2. 1.

[ example 2]Prediction of the amino acid sequence of DKK1 epitope

Based on the structure of DKK1 protein, epitope prediction software ABCpred (fig. 2), BCEpred (fig. 3), discotape server and Ellipro server (are used: (see below)http://tools.iedb.org/ellipro/) (FIGS. 4-8) for the above-mentioned base motifThe column is predicted. The Ellipro and discotop search engines are used because they correspond to the most reliable search engines in current predictive epitope algorithms.

The extracellular domain analyzed in example 1 was input into epitope prediction software, and then a continuous or discontinuous amino acid sequence of a predicted epitope was predicted, as shown in FIGS. 2 to 8.

As shown in fig. 2-8, 19 consecutive epitope amino acid sequences were predicted altogether, as shown in SEQ ID NO: 4-22, and predicting to obtain 14 discontinuous epitope amino acid sequences as shown in SEQ ID NO: 23-36.

[ example 3]DKK1 in regulatory T cells Identification of specific expression of mRNA

It was verified whether DKK1 protein could be used as a specific biomarker for regulatory T cells.

For validation, CD4+ T cells were isolated from mouse spleens that had been subjected to expression of C57BL/6 or Foxp3 and FRP in a conventional manner using Magnetic Activated Cell Sorting (MACS) with CD4 magnetic beads.

Subsequently, regulatory T (CD4+ CD25+ T) cells and non-regulatory T (CD4+ CD25-T) cells were separated by Fluorescence Activated Cell Sorter (FACS) using CD25 antibody. For each cell, mRNA was extracted using Trizol and gDNA was removed from genomic RNA using a gDNA extraction kit (Qiagen) according to the protocol provided by the manufacturer. The gDNA depleted mRNA was synthesized as cDNA using the BDsprint cDNA synthesis kit (Clonetech).

Real-time polymerase chain reaction (RT PCR) was performed to quantitatively identify the expression level of DKK1 mRNA in the cDNA. Real-time polymerase chain reaction was performed using SYBR Green (molecular probes) and primers shown in table 4 below according to the protocol provided by the manufacturer, for 40 cycles comprising 95 ℃ for 3 minutes, 61 ℃ for 15 seconds, 72 ℃ for 30 seconds, and relative gene expression levels were calculated using the Δ CT method and normalized using HPRT. The results are shown in FIG. 9.

[ Table 4]

As shown in fig. 9, the expression of DKK family genes and Wnt genes was hardly found in regulatory T cells. However, it has been determined that the expression of DKK1 gene is very high. The results indicate that DKK1 is likely to be used as a biomarker for regulatory T cells.

[ example 4]]DKK1 in immune cells Identification of mRNA expression

Among the CD4+ T cells isolated in example 3, Treg (CD4+ RFP +) cells, natural T cells and effector T cells were isolated using CD62L antibody (eBioscience) and a fluorescence-activated cell sorter CD8+ T cells and B cells were isolated using an antibody against CD8 β chain and an antibody against CD19, followed by incubation with or without CD3 and CD28 antibodies (eBioscience), or under conditions that can differentiate into CD4+ T cell subsets.

As shown in fig. 10, DKK1 gene of the present invention was expressed only at high level in regulatory T cells. Among the same regulatory T cells, a group of cells treated with CD3 and CD28 antibodies showed higher DKK1 expression; DKK1 expression was observed to be higher in natural regulatory T cells (natural Tregs) generated in the thymus compared to induced regulatory T cells (induced Tregs) obtained by differentiation of differentiation signals into peripherally located (periphery) regulatory T cells in the body, or by experimental differentiation into regulatory T cells.

[ example 5]Identification of specific expression of DKK1 protein in regulatory T cells

From the following viewpoints: in order to be a target for cell therapy, DKK1 protein must be expressed on the surface of regulatory T cells so that targeted therapy can be performed more efficiently. It was identified whether DKK1 protein was expressed on the surface of regulatory T cells.

Each of the differentiated T cell subsets of example 4 was stained with anti-CD 4-APC and anti-DKK 1-PE antibodies, and the expression level of DKK1 was detected on the surface of each cell using a Fluorescence Activated Cell Sorter (FACS). The results are shown in FIG. 11.

As shown in fig. 11, regulatory T cells (CD4+ Foxp +) expressed DKK1 at higher levels when T cell receptor signaling was activated compared to other effector T cells that did not express Foxp 3.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and changes may be made without departing from the technical spirit of the present invention described in the claims.

Industrial applicability

Sequence Listing free text

1, SEQ ID NO: dickkopf-1(DKK-1) amino acid sequence

2, SEQ ID NO: dickkopf-1(DKK-1) mRNA, complete cds sequence (GenBank: AF177394.2)

3 DKK1 extracellular domain of SEQ ID NO

TLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH

4:3S2K chain C linear epitope of SEQ ID NO

RIQKRL

5:3S2K chain C linear epitope of SEQ ID NO

HRRKGSHGLE IF

6:3S2K chain C linear epitope of SEQ ID NO

KGQEGSVCLR SSDCASG

7:3S8V chain X linear epitope of SEQ ID NO

RIQSRL

Linear epitope of chain X of SEQ ID NO 8:3S8V

HRRKGSHGLE IF

9:3S8V chain X linear epitope of SEQ ID NO

GEGL

10:3S8V chain X linear epitope of SEQ ID NO

QEGSVCLRSS DCASG

11:3S8V chain X linear epitope of SEQ ID NO

KEGQ

12:3S8V chain X linear epitope of SEQ ID NO

NSNAIKN

Chain B linear epitope of SEQ ID NO 13:5FWW

RVPGASHIH

Chain B linear epitope of SEQ ID NO. 14:5FWW

GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGE

Chain B linear epitope of SEQ ID NO 15:5FWW

KDHGNPPPLT GTSKTSNKL

16:5FWW chain B Linear epitope

FSDRINQ

Chain B linear epitope of SEQ ID NO 17:5FWW

CYVGVYWK

Chain B linear epitope of SEQ ID NO 18:5FWW

IRDSADMVEL LDGYTHRVLA RFHGRSRPPL SFNVSLDFVI

Chain B linear epitope of SEQ ID NO 19:5FWW

LGEHNYC

Chain B linear epitope of SEQ ID NO 20:5FWW

FCGNNPDYWK YGE

Chain B linear epitope of SEQ ID NO 21:5FWW

SQRFK

Chain B linear epitope of SEQ ID NO. 22:5FWW

ATGRV

Discontinuous epitope of chain C of SEQ ID NO. 23:3S2K

HKGSGL

24:3S2K chain C discontinuous epitope

IQL

Discontinuous epitope of chain C of SEQ ID NO 25:3S2K

FWIF

26:3S2K chain C discontinuous epitope of SEQ ID NO

EGQGEGRH

Discontinuous epitope of chain C of SEQ ID NO 27:3S2K

KGQEGSVCLR SDASG

Discontinuous epitope of chain X of SEQ ID NO 28:3S8V

IQSRL

SEQ ID NO 29:3S8V chain X discontinuous epitope

HKGSGL

30:3S8V chain X discontinuous epitope of SEQ ID NO

QGSVCRSD

31:3S8V chain X discontinuous epitope

FWIF

32:3S8V chain X discontinuous epitope of SEQ ID NO

EGQGGLR

33:3S8V chain X discontinuous epitope

ASG

Chain B discontinuous epitope of SEQ ID NO. 34:5FWW

GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGELGEHNY CPCYVGVYWK C

35:5FWW chain B discontinuous epitope

AMYATGRVRV PGASHIHIRD SADMELDGYT HRVLARHGRS PPLSFNVSLD FVIFSDRINQAQAVK

Chain B discontinuous epitope of SEQ ID NO 36:5FWW

KDHGNPPPLT GTSKTSNKLF SQRFKSGYFC GNNPDYWKYG ECFGGDG

Sequence listing

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gctatcaaga acctgccccc accgctgggc ggcgctgcgg ggcacccagg ctctgcagtc 180

agcgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac 240

cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc 300

acccgcggag gggacgcagg cgtgcaaatc tgtctcgcct gcaggaagcg ccgaaaacgc 360

tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct 420

tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat 480

gatcatagca ccttggatgg gtattccaga agaaccacct tgtcttcaaa aatgtatcac 540

accaaaggac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt 600

tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt 660

accaagcata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttactgtgga 720

gaaggtctgt cttgccggat acagaaagat caccatcaag ccagtaattc ttctaggctt 780

cacacttgtc agagacacta a 801

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Claims

1. A method of screening for an immune activator or immune inhibitor comprising the steps of:

(a) contacting a test sample with regulatory T cells comprising a Dickkopf-1(DKK1) extracellular surface protein or epitope as set forth in SEQ ID NO: 4-36;

(b) detecting the expression level of said extracellular surface protein or epitope; and

(c) determining that the sample is an immune activator or an immune inhibitor if the expression level of the extracellular surface protein or epitope is detected as down-regulated or up-regulated.

2. The method of claim 1, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

wherein the expression level of the extracellular surface protein or epitope is detected using an antibody capable of specifically binding to the extracellular surface protein or epitope.

3. The method of claim 1, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

wherein the DKK1 extracellular surface protein is shown as SEQ ID NO:3, respectively.

4. The method of claim 1, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

wherein the expression level of the extracellular surface protein or epitope is detected using a method selected from the group consisting of: protein chip analysis, immunoassay, ligand binding assay, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) assay, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) assay, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis (rocket immunoelectrophoresis), tissue immunostaining, complement fixation assay, two-dimensional electrophoresis assay, liquid chromatography-mass spectrometry (LC-MS), liquid phase secondary mass spectrometry (LC-MS/Ms), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

5. A method of screening for an immune activator or immune inhibitor comprising the steps of:

(a) contacting a test sample with regulatory T cells comprising a gene encoding a Dickkopf-1(DKK1) extracellular surface protein or an epitope as set forth in SEQ ID NO: 4-36;

(b) detecting the expression level of said gene; and

(c) determining that the sample is an immune activator or an immune inhibitor in the event that the expression level of the gene is detected as down-regulated or up-regulated.

6. The method of claim 5, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

wherein the expression level of the gene is detected using a primer, a probe or an antisense nucleotide capable of specifically binding to the gene.

7. The method of claim 5, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

8. The method of claim 5, wherein the first and second light sources are selected from the group consisting of a red light source, a green light source, and a blue light source,

wherein the expression level of the gene is detected using a method selected from the group consisting of: reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay, Northern blot and DNA chip method.

9. A polypeptide as set forth in SEQ ID NO:4-36, or a Dickkopf-1(DKK1) epitope as shown in any one of claims 4-36.

10. A pharmaceutical composition for preventing or treating immune-related diseases comprising

As an active ingredient, regulatory T cells (Treg cells) containing the amino acid sequence of SEQ ID NO:3 (DKK1) extracellular surface protein of Dickkopf-1.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable salt thereof,

wherein the immune-related disease is selected from the group consisting of: autoimmune diseases, graft versus host disease, organ transplant rejection, asthma, atopic diseases and acute or chronic inflammatory diseases.

12. A pharmaceutical composition for preventing or treating immune-related diseases comprising

As an active ingredient, regulatory T cells (Treg cells) containing the amino acid sequence of SEQ ID NO:4-36, or a Dickkopf-1(DKK1) epitope as shown in any one of claims 4-36.

13. The pharmaceutical composition according to claim 12, wherein the compound is selected from the group consisting of,

14. A pharmaceutical composition for preventing or treating immune-related diseases comprising

An antibody specific for a DKK1 extracellular surface protein or epitope as set forth in SEQ ID NO: 4-36.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable salt thereof,

wherein the immune-related disease is cancer.

16. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable salt thereof,

17. A pharmaceutical composition for immune-related diseases comprising

Any one selected from the group consisting of: antisense oligonucleotides, siRNA, shRNA and microRNA, which respectively have specificity to DKK1 extracellular surface protein or epitope coding genes, wherein the epitope is shown as SEQ ID NO: 4-36.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutically acceptable salt thereof,

wherein the immune-related disease is cancer.

19. The pharmaceutical composition according to claim 17, wherein the pharmaceutically acceptable salt thereof,

20. A diagnostic composition for immune related diseases comprising:

DKK1 extracellular surface protein or SEQ ID NO: 4-36.

21. A diagnostic composition for immune related diseases comprising:

a primer, probe or antisense nucleotide specific for DKK1 extracellular surface protein or an epitope-encoding gene, said epitope being as set forth in SEQ ID NO: 4-36.

22. A diagnostic kit for immune related diseases comprising:

the pharmaceutical composition of claim 20 or 21.

23. A method of providing information about an immune-related disease, comprising:

determining the expression level of DKK1 extracellular surface protein or epitope present in regulatory T cells by using an antibody specific for the DKK1 extracellular surface protein or epitope as set forth in SEQ ID NO: 4-36.

24. A method of providing information about an immune-related disease, comprising:

the expression level of DKK1 extracellular surface protein or epitope-encoding gene present in regulatory T cells is determined by using any one selected from the group consisting of primers, probes and antisense nucleotides each having specificity for the gene, the epitope being as set forth in SEQ ID NO: 4-36.

25. A method for preventing or treating an immune-related disease, comprising the steps of:

administering to a subject in need of treatment an antibody specific for a DKK1 extracellular surface protein or epitope of the invention.

26. A method for preventing or treating an immune-related disease, comprising the steps of:

administering to a subject in need of treatment any one selected from the group consisting of: antisense oligonucleotides, sirnas, shrnas, and micrornas, each specific for a gene encoding an extracellular surface protein or epitope of DKK1 of the invention.