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WO2024167312A1 - Dkk-1 protein specifically present on regulatory t cells and use thereof - Google Patents

Dkk-1 protein specifically present on regulatory t cells and use thereof Download PDF

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WO2024167312A1
WO2024167312A1 PCT/KR2024/001828 KR2024001828W WO2024167312A1 WO 2024167312 A1 WO2024167312 A1 WO 2024167312A1 KR 2024001828 W KR2024001828 W KR 2024001828W WO 2024167312 A1 WO2024167312 A1 WO 2024167312A1
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cancer
dkk
fibrosis
present
cells
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Korean (ko)
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이래경크리스티나
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Jrd Science Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a diagnostic composition for diagnosing fibrosis or cancer, a diagnostic kit, a method for providing information, and a pharmaceutical composition for preventing or treating fibrosis or cancer, comprising a formulation for measuring the expression level of DKK-1 (Dickkopf-1) protein or a gene encoding the same, which is specifically present on the surface of regulatory T cells (Treg cells) among immune cells; and a regulatory T cell comprising an extracellular surface protein of DKK-1, a fusion protein comprising an extracellular surface protein of DKK-1 and an immunoglobulin Fc region, an antibody or antigen-binding fragment specifically binding to the extracellular surface protein of DKK-1, or an epitope, and a DKK-1 inhibitor.
  • DKK-1 Dickkopf-1
  • Treg cells regulatory T cells
  • a regulatory T cell comprising an extracellular surface protein of DKK-1, a fusion protein comprising an extracellular surface protein of DKK-1 and an immunoglobulin Fc region, an antibody or antigen-binding fragment specifically
  • T cells are immune cells that are produced in the bone marrow and mature in the thymus, and they function to regulate the antibody production of B cells or to regulate the functions of innate immune cells.
  • the T cells are also called T lymphocytes and not only act as helpers for other immune cells, but also directly destroy invading substances.
  • immune cells One of the most important functions of immune cells is to suppress the immune response to antigenic substances that constitute the self surrounding the immune cell, while recognizing and inducing an immune response to non-self antigenic substances.
  • immune cells induce non-responsiveness to self-antigens through the death of cells that recognize self, mutation induction of receptors specific to self-antigens, or inactivation of immune cells that recognize self-antigens. This is called immunologic unresponsiveness or tolerance. If this self-tolerance fails, an immune response to self-antigens can be induced, resulting in disease, which is called an autoimmune disease.
  • the regulatory T cells control the function of classical T cells to induce excessive immune responses or self-tolerance.
  • the function and number of regulatory T cells are significantly reduced in autoimmune diseases and chronic inflammatory diseases, and thus the regulatory T cells do not perform their function properly. Therefore, restoring the function and number of regulatory T cells to a normal level in patients with autoimmune diseases and chronic inflammatory diseases may be one of the treatments for the diseases.
  • CD25, CTLA4, CD38, CD62L, GITR, CD45RB, etc. have been suggested through research as cell surface proteins that can target regulatory T cells, and animal and clinical trials have also been conducted, but a cell surface protein that can exclusively target regulatory T cells has not been discovered to date.
  • One object of the present invention is to provide a composition for diagnosing fibrosis or cancer, comprising an antibody or antigen-binding fragment that specifically binds to a DKK-1 extracellular surface protein or epitope.
  • Another object of the present invention is to provide a diagnostic kit for fibrosis or cancer comprising the composition.
  • Another object of the present invention is to provide a method for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein or epitope of DKK-1 present in regulatory T cells using an antibody or antigen-binding fragment that specifically binds to the surface protein or epitope.
  • Another object of the present invention is to provide a diagnostic device for fibrosis or cancer, including: (a) a measuring unit for measuring the expression level of DKK-1 or a gene encoding the protein for a biological sample obtained from a target subject; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded by the gene measured by the measuring unit.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis or cancer, comprising as an active ingredient a fusion protein comprising LRP-5, 6 and an immunoglobulin Fc region, an extracellular surface protein of DKK-1, or an antibody or antigen-binding fragment specifically binding to an epitope, or a DKK-1 inhibitor.
  • DKK-1 or “DKK1”, known as a representative negative regulator of the Wnt signaling pathway, competitively binds to LRP (Low density lipoprotein receptor-related protein) 5/6 with Wnt, or ultimately induces B-Catenin degradation and TCF-1 inactivation through endocytosis of the receptor.
  • DKK-1 is the most studied protein among the four DKK families and has a size of approximately 26 kDa composed of 266 amino acids. Looking at the structure of the protein, it interacts with domains of various receptors including LRP6 through two cysteine rich domains (CRD).
  • the currently known receptors for DKK-1 include LRP5/6, Kremen 1/2, and CKAP4, and it induces various cell signals through the above receptors.
  • DDK-1 is closely related to cell proliferation through Wnt signaling and CKAP4-PI3K-AKT signaling
  • high expression of DDK-1 in cancer indicates a negative prognosis, although the degree varies depending on the type of cancer.
  • many studies are being conducted on cancer immunotherapy targeting DDK-1.
  • recent papers have revealed the role of DDK-1 in various immune responses, and the results of studies on the influence of DDK-1 in autoimmune diseases can be confirmed.
  • DDK-1 DDK-1 secretion from platelets
  • MAPK and mTOR signaling The increase in DDK-1 under physiological conditions generates Th2 cells through the induction of C-Maf and Gata3, and causes the expression of Th2 cell-related cytokines through p38 MAPK and SGK-1. That is, DDK-1 is preferentially involved in functions associated with Th2 compared to other effector T cells.
  • DDK-1 inhibits the expression of IFN-r under Th1 polarization conditions, and simultaneously increases the expression of cytokines such as GATA3 and IL-4, IL-5, IL-10, and IL-13.
  • cytokines such as GATA3 and IL-4, IL-5, IL-10, and IL-13.
  • Foxp3 the expression of Foxp3 is down-regulated.
  • platelets are known to be a major source of TGF-B1, and in the inflammatory response environment created through the above infection, it is presumed that DDK-1, together with CXCL4, a ligand produced by platelets, lowers Foxp3 expression, thereby inhibiting TGF-B-mediated Foxp3+ Treg cell production and allowing Th2 to be properly activated.
  • nTreg cells have a high level of DDK-1 mRNA expression compared to na ⁇ ve T cells or effector CD4 T cells.
  • the generally known DDK-1 is known to bind to the receptor as a ligand in a soluble form and play its role, but DDK-1 of nTreg is expressed on the surface, and about 6% of DDK-1 is expressed in the resting state, and the expression rate increases to 14.1% when activated.
  • DDK-1 is thought to play an important role in the suppressive function of Treg.
  • Treg did not suppress the proliferation of effector CD4 T cells.
  • Treg cells from mice with reduced DDK-1 expression by more than 90% did not suppress the proliferation of effector CD4 T cells as effectively as Wild Type Treg, ultimately resulting in the development of severe colitis.
  • DDK-1 in Treg it was shown that it is expressed independently of the Wnt pathway, mTOR, and Mevalonate pathways, and that the MAPK pathway is required for activated Treg to express DDK-1.
  • DKK1 which acts as an antagonist of Wnt, indicates the possibility of erosion due to increased bone resorption when measured at high concentrations in the blood, whereas low concentrations are considered to be new bone formation.
  • RA rheumatoid arthritis
  • DKK-1 protein is highly expressed in human regulatory T cells and its expression is reduced in iTreg.
  • the DKK-1 protein is an anchored protein existing outside the human Treg cell membrane, and can then be separated into the blood and exist in a soluble state.
  • the pharmaceutical composition provided by the present invention can exert an effect on fibrosis or cancer by regulating the immune response by inhibiting the function of DKK-1.
  • the DKK-1 is one of the DKK family genes DKK1, DKK2, DKK3, and DKK4, and encodes a secreted protein.
  • the DKK protein is generally composed of 255 to 350 amino acids, and in the case of DKK-1, has a size of 24 KDa to 29 KDa, and the N-terminus exists in a form in which a sugar is bound (glycosylated).
  • the DKK-1 can be represented by the amino acid sequence of SEQ ID NO: 1, and can be encoded by the nucleotide represented by SEQ ID NO: 2 (Table 1).
  • dickkopf-1 (DKK-1) amino acid sequence MMALGAAGAT RVFVAMVAAA LGGHPLLGVS ATLNSVLNSN AIKNLPPPLG GAAGHPGSAV SAAPGILYPG GNKYQTIDNY QPYPCAEDEE CGTDEYCASP TRGGDAGVQI CLACRKRRKR CMRHAMCCPG NYCKNGICVS SDQNHFRGEI EETITESFGN DHSTLDGYSR RTTLSSKMYH TKGQEGSVCL RSSDCASGLC CARHFWS KIC KPVLKEGQVC TKHRRKGSHG LEIFQRCYCG EGLSCRIQKD HHQASNSSRL HTCQRH (SEQ ID NO: 1) dickkopf-1 (DKK-1) mRNA, complete cds (GenBank: AF177394.2) sequence atgatggctc tgggcgcagc gggagctacc cgggtctttg tcgcgatgg
  • the DKK-1 may play an important role in the development of the heart, head, and hands during embryonic development by competitively inhibiting the Wnt signal by binding more strongly to the receptor than a ligand such as Wnt3a in the Wnt signaling system involved in cell proliferation and wound healing.
  • the DKK-1 may not be secreted outside the cell as in general cases, but may be present on the surface of immune cells, particularly regulatory T cells. Through this, immune cells may help the function of regulatory T cells for self-tolerance.
  • the regulatory T cell extracellular surface protein of the DKK-1 is represented by SEQ ID NO: 3 (Table 2).
  • the regulatory T cell extracellular surface protein of DKK-1 provided in the present invention can reduce the activity of regulatory T cells by interacting with a ligand present in effector T cells.
  • the epitope of DKK1 which is a surface protein of regulatory T cells, is represented by any one of SEQ ID NOS: 4 to 36 (Table 3).
  • the "epitope” or “epitope” refers to a portion of an antigen molecule that binds to an antibody, and refers to a portion that can be recognized by an antibody.
  • antibodies do not recognize the entire antigen molecule, but only a specific portion, and even if it is the same antigen molecule, if the type of antibody is different, it can recognize different antigenic determinant portions.
  • the antigenic determinant includes an antigenic determinant of an antibody that can effectively bind to a remaining portion of a domain even when a portion of a DKK1 protein exposed to the outside of the cell is partially cleaved and released (secreted) to the outside of the cell.
  • the polypeptide that is the antigenic determinant may include all continuous or discontinuous sequences of a portion that can be bound to the DKK-1 protein according to the present invention by an antibody.
  • the polypeptide fragment that is the antigenic determinant of DKK-1 which is the surface protein of regulatory T cells provided in the present invention, may interact with a ligand present in effector T cells to reduce the activity of regulatory T cells.
  • a polynucleotide encoding an extracellular surface protein of DKK-1 represented by SEQ ID NO: 3 provided in the present invention, or an antigenic determinant of DKK-1 of any one of SEQ ID NOs: 4 to 36.
  • an expression vector into which a polynucleotide provided by the present invention is inserted is provided.
  • the "vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked.
  • a vector which refers to a circular double-stranded DNA to which additional DNA segments can be ligated.
  • a phage vector Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome.
  • Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., a bacterial vector is an episomal mammalian vector having a bacterial origin of replication).
  • vectors e.g., a non-episomal mammalian vector
  • a non-episomal mammalian vector can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome.
  • some vectors can direct the expression of genes to which they are linked in an operational manner.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors”.
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably, as the plasmid is the most commonly used form of vector.
  • specific examples of the expression vector may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, T7; and plant viruses, but are not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the target host cell.
  • the vector When introducing a vector into a host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can select and use an introduction method appropriate for the expression vector and host cell to be used.
  • the vector contains one or more selection markers, but is not limited thereto, and selection is possible based on whether or not the product is produced using a vector that does not contain a selection marker.
  • the selection marker is selected by the desired host cell, and this is done using a method already known to those skilled in the art, so the present invention is not limited thereto.
  • a tag sequence can be inserted into an expression vector and fused thereto.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art that facilitates purification can be used in the present invention.
  • the expression vector provided in the present invention provides a host cell line transfected with the expression vector.
  • the "host cell” in the present invention includes an individual cell or cell culture that can be or has been the recipient of the vector(s) for incorporation of the polypeptide insert.
  • the host cell includes progeny of a single host cell, which progeny may not necessarily be completely identical (either morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation.
  • the host cell includes a cell that has been transfected in vivo with the polypeptide(s) of the present disclosure.
  • the host cell may include a cell of mammalian, plant, insect, fungal or cellular origin, and for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); Or, it may be a plant cell, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art may be used.
  • bacterial cells such as Escherichia
  • a regulatory T cell comprising the extracellular surface protein of the DKK-1 of the present invention or an epitope of the DKK-1 is provided.
  • Regulatory T cells expressing the extracellular surface protein of the DKK-1 of the present invention or an antigenic determinant thereof on their surface can suppress the cellular activity of effector T cells through interaction with them and suppress an immune response.
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising as an effective ingredient an extracellular surface protein of the DKK-1 of the present invention or a regulatory T cell comprising an epitope of the DKK-1.
  • Regulatory T cells comprising the extracellular surface protein of the DKK1 of the present invention can suppress the cell activity of effector T cells through interaction with them and suppress immune responses.
  • regulatory T cells expressing DKK-1 protein on their cell surface can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer.
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising the DKK-1 extracellular surface protein or the antigenic determinant of the present invention as an effective ingredient.
  • the DKK-1 extracellular surface protein or the antigen determinant fragment provided in the present invention interacts with a ligand present in effector T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.
  • the "cancer” refers to or represents a physiological condition characterized by uncontrolled cell growth typically in mammals.
  • the cancer to be prevented, improved or treated in the present invention may be a solid tumor formed by a mass caused by abnormal cell growth in a solid organ, and depending on the site of the solid organ, may be gastric cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient.
  • the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.
  • the antibody can effectively prevent, improve or treat fibrosis or cancer by suppressing the activity of regulatory T cells.
  • the term "antibody” is a term known in the art and means a specific protein molecule directed against an antigenic site.
  • the antibody means an antibody that specifically binds to the protein of the present invention, and such an antibody can be produced by a conventional method by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and from the obtained protein.
  • This also includes a partial peptide that can be produced from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids.
  • the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof having antigen binding properties are also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.
  • the antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of the antibody molecule.
  • a functional fragment of an antibody molecule means a fragment that possesses at least an antigen-binding function, such as Fab, F(ab'), F(ab') 2, and Fv.
  • the antibody specifically binds to the DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.
  • the cancer may be a solid tumor formed by a mass of cells growing abnormally in a solid organ, and depending on the location of the solid organ, may be gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, and preferably, melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow, and skin, but is not limited thereto.
  • the fibrosis includes, but is not limited to, pulmonary fibrosis, liver fibrosis, cardiac fibrosis, brain fibrosis, skin fibrosis, pancreatic fibrosis, and bone fibrosis.
  • fibrosis refers to the development of excessive fibrous connective tissue within an organ or tissue. Fibrosis is characterized by the accumulation and remodeling of the extracellular matrix (ECM). Despite the clear pathognomonic and clinical differences that distinguish it from cancer, which is a mass formed by abnormal cell growth, most chronic fibrotic disorders share in common a persistent stimulus that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, particularly collagen and proteoglycans, thereby continually remodeling and destroying normal tissue architecture.
  • ECM extracellular matrix
  • the antibody can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer, by specifically binding to the DKK-1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing the activity of regulatory T cells.
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising as an active ingredient any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA specific for the DKK-1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.
  • the "antisense oligonucleotide” is a short length of DNA synthetic strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, and is used to achieve gene-specific inhibition in vitro as well as in vivo.
  • Antisense oligonucleotides have been proposed to block the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have also been successfully used in cell culture and animal models of diseases.
  • antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art.
  • the antisense oligonucleotides used herein include double-stranded or single-stranded DNA, double-stranded or single-stranded RNA, DNA/RNA hybrids, DNA and RNA analogs, and oligonucleotides having base, sugar or backbone modifications.
  • the oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of the oligonucleotide backbone, modifications of the sugar moieties, or modifications of the bases, which are known in the art.
  • the "siRNA (small interfering RNA)" is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and is used as an efficient gene knockdown method or gene therapy method because it can suppress the expression of a target gene.
  • siRNA molecule When an siRNA molecule is used in the present invention, it 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 a WLS mRNA sequence) are positioned on opposite sides to form a double-stranded structure, or a single-stranded structure having self-complementary sense and antisense strands.
  • siRNA is not limited to a double-stranded RNA portion in which RNAs are paired completely, and may include a portion that does not pair due to a mismatch (corresponding bases are not complementary), a bulge/protrusion (there is no base corresponding to one chain), etc.
  • the siRNA terminal structure can be either a blunt end or a sticky end as long as it can suppress the expression of the WLS gene by the RNA interference (RNAi) effect.
  • the sticky end structure can be either a 3'-terminal overhang structure or a 5'-terminal overhang structure.
  • the siRNA molecule may have a total length of 15 to 30 bases, preferably 19 to 21 bases, but is not limited thereto.
  • the "shRNA (short hairpin RNA)" is a single-stranded RNA having a length of 45 to 70 nucleotides, and when oligo DNA is synthesized to connect a 3 to 10 nucleotide linker between the sense strand of the target gene siRNA base sequence and the complementary antisense strand, and then cloned into a plasmid vector or the shRNA is expressed by inserting it into a retrovirus such as a lentivirus and an adenovirus, an shRNA having a hairpin structure with a loop is created and converted into siRNA by dicer within the cell to exhibit an RNAi effect.
  • a retrovirus such as a lentivirus and an adenovirus
  • microRNA regulates various biological processes such as development, differentiation, proliferation, preservation, and apoptosis.
  • MicroRNA generally regulates the expression of a gene encoding a target mRNA by destabilizing the target mRNA or interfering with its translation.
  • useful regulatory sequences e.g., constitutive promoter, inducible promoter, tissue-specific promoter or combinations thereof
  • useful regulatory sequences for the expression construct/vector containing the antisense oligonucleotide, siRNA, shRNA or microRNA can also be appropriately selected from those known in the art.
  • the antisense oligonucleotide, siRNA, shRNA or microRNA can specifically bind to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and inhibit their expression, thereby effectively preventing, improving or treating immune-related diseases, such as cancer.
  • the cancer may be a solid tumor formed by a mass of cells growing abnormally in a solid organ, and depending on the location of the solid organ, may be gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, and preferably, melanoma, but is not limited thereto.
  • the antisense oligonucleotide, siRNA, shRNA or microRNA can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer, by specifically binding to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing their expression.
  • prevention may include, without limitation, any act of blocking, suppressing or delaying symptoms of a disease by using the pharmaceutical composition of the present invention.
  • treatment may include without limitation any act that improves or benefits the symptoms of a disease by using the pharmaceutical composition of the present invention.
  • the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.
  • the pharmaceutical composition according to the present invention is prepared in the form of an injection and can be directly injected into a site where cancer or an immune disease has occurred, but is not limited thereto.
  • the pharmaceutical composition of the present invention is not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, and the like for injections.
  • compositions of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above.
  • pharmaceutically acceptable carriers for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses.
  • it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.
  • 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, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.
  • fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.
  • the routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
  • the pharmaceutical 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, dosage form, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and 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, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a solution, a gel, a syrup, a slurry, or a suspension.
  • the pharmaceutical composition of the present invention can additionally be administered in combination with other anticancer agents, thereby effectively inhibiting general cancer cell proliferation and cancer metastasis, and can thus be used for the treatment of cancer.
  • the above anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tycetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzum
  • the pharmaceutical composition of the present invention can additionally be administered in combination with other immunosuppressants, thereby effectively suppressing diseases caused by general hyperimmune reactions.
  • the immunosuppressant may be at least one selected from the group consisting of glucocorticoids, cyclophosphamide, cyclosporin, tacrolimus, rapamycin, type IV PDE inhibitors, p38 kinase inhibitors, azathioprine, mycophenolate mofetil, mizoribin, methotrexate, leflunomide, and brequina, but is not limited thereto.
  • a method for preventing or treating an immune-related disease comprising administering to a subject in need of treatment an antibody specific for the DKK-1 extracellular surface protein of the present invention or an antigenic determinant.
  • the subject requiring the treatment is an individual having an immune-related disease or suspected of having symptoms thereof
  • the cancer may be gastric cancer, liver cancer, glioma, 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
  • the fibrosis may be selected from the group consisting of, but not limited to, fibrosis occurring in at least one organ selected from the group consisting of the kidney, liver, lung, heart, bone or bone marrow, and skin.
  • the antibody specifically binds to the DKK-1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.
  • a method for preventing or treating an immune-related disease comprising administering to a subject in need of treatment any one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the DKK-1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.
  • the subject requiring the treatment is an individual having an immune-related disease or suspected of having symptoms thereof
  • the cancer may be gastric cancer, liver cancer, glioma, 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
  • the fibrosis may be selected from the group consisting of, but not limited to, fibrosis occurring in at least one organ selected from the group consisting of the kidney, liver, lung, heart, bone or bone marrow, and skin.
  • the antisense oligonucleotide, siRNA, shRNA or microRNA can specifically bind to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and inhibit their expression, thereby effectively preventing, improving or treating immune-related diseases, such as cancer.
  • the antisense oligonucleotide, siRNA, shRNA or microRNA can effectively prevent or treat an immune-related disease by specifically binding to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing their expression.
  • an example of the immune-related disease may be cancer such as gastric cancer, liver cancer, glioma, 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, and as another example, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • cancer such as gastric cancer, liver cancer, glioma, 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
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • administration means introducing the composition of the present invention into a patient by any appropriate method, and the route of administration of the composition of the present invention may be administered through any general route as long as it can reach the target tissue.
  • Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intracavity administration, intraperitoneal administration, and intrathecal administration may be performed, but are not limited thereto.
  • the treatment method of the present invention may include administering the antibody, antisense oligonucleotide, siRNA, shRNA, or microRNA in a pharmaceutically effective amount.
  • the effective amount may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the effective ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, administration time, administration route, and secretion rate of the composition, treatment period, and concurrently used drugs.
  • the expression inhibitor of the gene or the activity inhibitor of the protein may be administered once or several times a day at a dose of 0.01 ng/kg-10 mg/kg for siRNA, 0.01 ng/kg-10 mg/kg for antisense oligonucleotide to mRNA of the gene, 0.1 ng/kg-10 mg/kg for compound, and 0.1 ng/kg-10 mg/kg for monoclonal antibody to the protein.
  • additional administration may be performed in combination with another anticancer agent or another immunosuppressant.
  • composition for diagnosing fibrosis or cancer comprising an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient.
  • diagnosis means confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, diagnosis is confirming the presence or absence of gastric cancer and metastasis.
  • the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.
  • fibrosis or cancer can be diagnosed by measuring the expression level of DKK1 extracellular surface protein or the antigen determinant present on the surface of regulatory T cells using the antibody.
  • the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • composition for diagnosing fibrosis or cancer comprising any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for the DKK1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.
  • the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.
  • fibrosis or cancer can be diagnosed by measuring the expression level of the DKK1 extracellular surface protein present on the surface of regulatory T cells or the gene encoding the antigenic determinant using the antibody.
  • the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • a diagnostic kit for fibrosis or cancer comprising the diagnostic composition of the present invention is provided.
  • the kit refers to a set of compositions and accessories necessary for a specific purpose.
  • the kit of the present invention can diagnose the above diseases by confirming the expression level of the DKK1 extracellular surface protein, which is a diagnostic marker of immune-related diseases such as cancer, autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute and chronic inflammatory diseases, as mRNA or the expression level of its protein.
  • the kit of the present invention may include a primer, a probe, or optionally an antibody recognizing the marker for measuring the expression level of the diagnostic marker of the immune-related disease, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.
  • a method for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein or antigenic determinant of DKK1 present in regulatory T cells using an antibody specific for the extracellular surface protein or the antigenic determinant.
  • the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • a method for providing information on fibrosis or cancer by measuring the expression level of a gene encoding the DKK1 extracellular surface protein of the present invention or the antigenic determinant in a regulatory T cell using any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for the gene.
  • the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.
  • the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.
  • the present invention relates to a diagnostic device for fibrosis or cancer.
  • the diagnostic device of the present invention relates to a diagnostic device for fibrosis or cancer, comprising: (a) a measuring unit for measuring the expression level of DKK1 or a gene encoding the protein for a biological sample obtained from a target subject; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded thereby measured by the measuring unit.
  • the "target subject” refers to a subject that has developed or is likely to develop fibrosis or cancer, and may be a mammal including a human, and may be selected from the group consisting of, for example, a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep, and a goat, and is preferably a human, but is not limited thereto.
  • composition for diagnosing fibrosis or cancer comprising an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1 or a DKK-1 epitope represented by any one of SEQ ID NOS: 4 to 36.
  • a diagnostic composition wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.
  • a diagnostic composition wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.
  • a diagnostic composition wherein the cancer is a solid cancer.
  • the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem gliom
  • CNS central nervous system
  • composition for diagnosing fibrosis or cancer comprising a primer, probe or antisense nucleotide specific for a gene encoding an epitope of DKK-1 extracellular surface protein or DKK-1 represented by any one of SEQ ID NOS: 4 to 36.
  • a diagnostic composition wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.
  • a diagnostic composition wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.
  • a diagnostic composition wherein the cancer is a solid cancer.
  • the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem gliom
  • CNS central nervous system
  • a diagnostic kit for fibrosis or cancer comprising the composition is provided.
  • a method for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein of DKK-1 or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36 using an antibody or antigen-binding fragment that specifically binds to the surface protein or epitope.
  • a method for providing information wherein the DKK-1 is present on the surface of a regulatory T cell or is present in a dissolved state after being separated from the cell.
  • a method for providing information on fibrosis or cancer by measuring the expression level of a gene using any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.
  • a method for providing information wherein the DKK-1 is present on the surface of a regulatory T cell or is present in a dissolved state after being separated from the cell.
  • a method of providing information wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.
  • a method for providing information is provided, wherein the cancer is a solid cancer.
  • a method for providing information wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma,
  • a diagnostic device for fibrosis or cancer comprising: (a) a measuring unit for measuring the expression level of DKK-1 or a gene encoding the protein for a biological sample obtained from a target individual; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded thereby measured by the measuring unit.
  • a diagnostic device wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.
  • the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem gliom
  • CNS central nervous system
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising as an active ingredient an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.
  • a pharmaceutical composition wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising a DKK-1 inhibitor is provided.
  • a pharmaceutical composition wherein the DKK-1 inhibitor is selected from the group consisting of NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, and endothelin-1 (ET-1).
  • DKK-1 inhibitor is selected from the group consisting of NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, and endothelin-1 (ET-1).
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising as an active ingredient any one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA and microRNA specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.
  • a pharmaceutical composition wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.
  • a pharmaceutical composition wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.
  • a pharmaceutical composition wherein the cancer is a solid cancer.
  • the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem gliom
  • CNS central nervous system
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising as an active ingredient a fusion protein comprising an extracellular surface protein of DKK-1 present in regulatory T cells and an immunoglobulin Fc region.
  • the above fusion protein competitively binds to LRP-5, 6, which are ligands of DKK-1, and ultimately inhibits the function of DKK-1, thereby preventing or treating fibrosis or cancer.
  • Immunoglobulins comprise four polypeptide chains, two heavy chains and two light chains, which are joined by interchain disulfide bonds. Each light chain has two domains, a variable light domain (VL) and a constant light domain (CL), and each heavy chain has two domains, a variable heavy domain (VH) and a constant heavy domain (CH).
  • the constant heavy domain (CH) is made up of constant heavy domains, which are designated by numbers (e.g., CH1, CH2, CH3, etc.). Immunoglobulins are classified into different isotypes (i.e., IgG, IgM, IgA, IgD, and IgE) based on their biological properties, location in an organism, and ability to process different antigens.
  • the constant heavy domain may have three or four CH domains. Additionally, in some isotypes (IgA, IgD, and IgG), the heavy chain contains a hinge region that adds flexibility to the molecule.
  • IgG1 There are four IgG subclasses in humans (IgG1, 2, 3, 4), named according to their order of abundance in serum (IgG1 being the most abundant).
  • An IgG isotype consists of two light chains and two heavy chains, each heavy chain containing three constant heavy domains (CH1, CH2, CH3). The two heavy chains of IgG are linked to each other and to the light chain by disulfide bonds (-S-S-).
  • the antigen-binding site of IgG is located in the fragment antigen-binding region (Fab region), which includes the variable light (VL) and variable heavy (VH) domains, as well as the constant light (CL) and constant heavy (CH1) domains.
  • the fragment crystallizable region (Fc region) of IgG is the part of the heavy chain that contains the CH2 and CH3 domains that bind to Fc receptors found on the surface of certain cells, including the neonatal Fc receptor (FcRn).
  • the heavy chain of IgG also has a hinge region (hinge) between CH1 and CH2 that separates the Fab region from the Fc region and participates in linking the two heavy chains together via a disulfide bond.
  • the structure of the hinge region contributes to the unique biological properties of each of the four IgG subclasses.
  • IgG is secreted as a small monomer that allows it to easily perfuse tissues. It is the only isotype that has a receptor (neonatal Fc receptor (FcRn)) that facilitates its passage across the human placenta to protect the fetus in the womb. IgG absorbed through the placenta provides humoral immunity to the newborn before its own immune system develops.
  • FcRn nonatal Fc receptor
  • the IgG neonatal Fc receptor (FcRn) binding site is located in the Fc region of the antibody.
  • FcRn is commonly expressed on human placental and epithelial cells and participates in the endocytic salvage pathway that protects IgG from degradation. This salvage pathway is mediated by the high pH-dependent binding affinity of IgG for FcRn at acidic pH.
  • the high affinity of IgG for FcRn at acidic pH is thought to drive binding of internalized IgG to FcRn after uptake into acidic endosomes. While most soluble proteins are directed to lysosomes after internalization, internalized FcRn-bound IgG are returned to the plasma membrane and are effectively rescued from the canonical degradation pathway.
  • IgG can dissociate from FcRn and return to the circulation. Thus, the prolonged serum half-life properties of antibodies are maintained in the Fc fragment.
  • the "immunoglobulin Fc region” means a region including a heavy chain constant region 2 (CH2) and/or a heavy chain constant region 3 (CH3) portion, excluding the heavy chain and light chain variable regions of an immunoglobulin.
  • the immunoglobulin Fc region may be a component forming a moiety of the protein complex of the present invention.
  • the immunoglobulin Fc region can affect the structural flexibility of the fusion protein to be finally manufactured, and can further increase the productivity and stability of the fusion protein, but is not limited thereto.
  • the immunoglobulin Fc region may further include a hinge portion in the heavy chain constant region, thereby affecting the structural flexibility of the final fusion protein to be manufactured, and may further increase the productivity and stability of the fusion protein, but is not limited thereto.
  • the immunoglobulin Fc region of the present invention may be an extended Fc region including part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1), excluding only the heavy and light chain variable regions of the immunoglobulin, as long as it has substantially the same or improved effect as the native type.
  • it may also be a region in which a relatively long part of the amino acid sequence corresponding to CH2 and/or CH3 is deleted.
  • the immunoglobulin Fc region of the present invention may be a dimer of 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), 6) each domain of a heavy chain constant region and a light chain constant region.
  • an immunoglobulin hinge region or a part of the hinge region
  • the immunoglobulin Fc region of the present invention includes not only a natural amino acid sequence but also a sequence derivative thereof.
  • An amino acid sequence derivative means a sequence having a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in a natural amino acid sequence.
  • amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding can be used as suitable sites for modification.
  • various types of derivatives are possible, such as those in which a site capable of forming a disulfide bond is removed, in which several amino acids at the N-terminus of a native Fc are removed, or in which a methionine residue is added to the N-terminus of a native Fc.
  • a complement binding site e.g., a C1q binding site
  • ADCC antibody dependent cell mediated cytotoxicity
  • Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).
  • the most common exchanges are between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • Modifications may also occur, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.
  • the Fc derivative described above exhibits biological activity equivalent to the Fc region of the present invention and may have enhanced structural stability of the Fc region with respect to heat, pH, etc.
  • the Fc region may be obtained from a natural type isolated from an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or may be a recombinant or a derivative thereof obtained from a transformed animal cell or microorganism.
  • the method for obtaining from a natural type may be a method of isolating the entire immunoglobulin from a human or animal body and then treating it with a protease to obtain it. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2.
  • the Fc region derived from a human or mouse is a recombinant immunoglobulin Fc region obtained from a microorganism.
  • the immunoglobulin Fc region may be a native sugar chain, a sugar chain with an increased amount compared to the native type, a sugar chain with a decreased amount compared to the native type, or a form in which the sugar chain is removed.
  • Conventional methods such as a chemical method, an enzymatic method, and a genetic engineering method using microorganisms can be used for the increase or removal of the immunoglobulin Fc sugar chain.
  • the immunoglobulin Fc region in which the sugar chain is removed from the Fc has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so that it does not induce unnecessary immune responses in vivo.
  • the form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region in which the sugar chain is removed or is non-glycosylated.
  • deglycosylation refers to an Fc region from which a sugar has been removed by an enzyme
  • aglycosylation refers to an Fc region that has not been glycosylated and is produced in a prokaryotic animal, or in a more specific embodiment, in E. coli.
  • the immunoglobulin Fc region may be of animal origin, such as human or cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, and may be of human or mouse origin as a preferred example.
  • the immunoglobulin Fc region of the present invention may be a hybrid Fc comprising an Fc region derived from IgG, IgA, IgD, IgE, IgM, heavy chain constant region 2 (CH2), heavy chain constant region 3 (CH3), a hinge, a fragment thereof, or a combination thereof, or a combination thereof.
  • a hybrid Fc comprising an Fc region derived from IgG, IgA, IgD, IgE, IgM, heavy chain constant region 2 (CH2), heavy chain constant region 3 (CH3), a hinge, a fragment thereof, or a combination thereof, or a combination thereof.
  • the term "combination” means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region, heavy chain constant region 2 (CH2) or heavy chain constant region 3 (CH3) of the same origin forms a bond with a single-chain polypeptide of different origin. That is, it is possible to produce a dimer or multimer from two or more fragments selected from the Fc region, heavy chain constant region 2 (CH2) or heavy chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD or IgE.
  • the "hybrid Fc" can be derived from a combination of human IgG subclasses or a combination of human IgD and IgG.
  • the hybrid Fc can include, for example, an IgD hinge region and a CH2 N-terminal region + an IgG4 CH2 and CH3 region, and can be used by borrowing the same hybrid Fc form disclosed in, for example, Korean Patent No. 0897938, which is incorporated herein by reference.
  • the hybrid Fc when the hybrid Fc binds to a biologically active molecule, a polypeptide, etc., it not only increases the serum half-life of the biologically active molecule, but also has the effect of increasing the expression level of the polypeptide when a nucleotide encoding an Fc-polypeptide fusion protein is expressed.
  • the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgM, IgD or IgE, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD or IgE, but is not limited thereto.
  • the immunoglobulin Fc region may be an Fc region derived from IgG or IgM which is most abundant in human blood, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG or IgM, or as another example, an Fc region derived from IgG known to enhance the half-life of a ligand binding protein, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG, or as another example, an Fc region derived from IgG1, IgG2, IgG3 or IgG4, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG1 or IgG2.
  • CH2 heavy chain constant region 2
  • CH3 heavy chain constant region 3
  • the immunoglobulin Fc region may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from human IgG1 represented by SEQ ID NO: 37, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from mouse IgG2 represented by SEQ ID NO: 38, but is not limited thereto.
  • CH2 heavy chain constant region 2
  • CH3 heavy chain constant region 3
  • mouse IgG2 represented by SEQ ID NO: 38
  • the immunoglobulin Fc region may include a hinge region derived from IgG, IgA, IgM, IgD, IgE, or Abatacept, or as another example, may include a hinge region derived from IgG, IgD or Abatacept, or may include a hinge region derived from IgG1, IgG2, IgG3, IgG4, IgD or Abatacept, but is not limited thereto.
  • the immunoglobulin Fc region may include at least one selected from the group consisting of a hinge region derived from human IgG1 represented by SEQ ID NO: 39; a hinge region derived from mouse IgG2 represented by SEQ ID NO: 40; a hinge region derived from human IgD represented by SEQ ID NO: 41; and a hinge region of Abatacept represented by SEQ ID NO: 42; thereby increasing the structural flexibility of the fusion protein to be finally manufactured and significantly improving the productivity and stability of the fusion protein, but is not limited thereto.
  • the linker when the extracellular domain and the Fc region of the DKK-1 protein are connected via a linker, the linker can be connected to the N-terminus, the C-terminus or a free radical of the Fc fragment, and can be connected to the N-terminus, the C-terminus or a free radical of the extracellular domain of the DKK-1 protein.
  • the linker is a peptide linker, the connection can occur at any site.
  • the linker can be connected to the C-terminus of the extracellular domain of the DKK-1 protein and the N-terminus of the Fc region of the immunoglobulin, or can be connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the extracellular domain of the DKK-1 protein.
  • the "linker” can reduce the interference effect between the extracellular domain of the DKK-1 protein and the immunoglobulin Fc region in the fusion protein, thereby increasing the desired activity of the extracellular domain of the DKK-1 protein in the target cell.
  • the linker in the present invention can include a sequence that can be cleaved by an enzyme that is overexpressed in the tissue or cell of the desired disease. In the case where it can be cleaved by the overexpressed enzyme as described above, the activity of the polypeptide can be effectively prevented from being reduced due to the Fc portion.
  • a peptide linker composed of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in blood more preferably a peptide linker composed of 13 amino acids located at positions 292 to 304, is a portion that is mostly exposed to the outside in the three-dimensional structure and thus has a minimal possibility of inducing an immune response in the body.
  • it is not limited thereto.
  • the linker when the linker and Fc region in the present invention are expressed separately and then linked to each other, the linker may be a cross-linking agent known in the art.
  • the crosslinking agent may be, but is not limited to, imidoesters including, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • imidoesters including, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-d
  • a pharmaceutical composition for preventing or treating fibrosis or cancer comprising a fusion protein comprising LRP-5, 6 and an immunoglobulin Fc region as an active ingredient.
  • the above fusion protein contains LRP-5, 6, which is a ligand that binds to DKK-1, LRP-5, 6 and the above fusion protein competitively bind to DKK-1, and thus fibrosis or cancer can be treated by competitively inhibiting DKK-1.
  • the above fusion protein has been described in detail in the fusion protein comprising DKK-1 and the immunoglobulin Fc region discussed above, and is omitted to avoid duplication.
  • a pharmaceutical composition wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.
  • Figure 1 shows that DKK-1 expression levels are elevated in patients with human idiopathic pulmonary fibrosis.
  • Figure 2 shows that the expression of DKK-1 was increased in the lungs stimulated with BLM.
  • Figure 3 shows that DKK-1 hypomorphic Dkk-1 d/d mice are protected from BLM-mediated lung injury.
  • FIG. 4 shows that platelets are the main source of DKK-1 histological observations.
  • FIG. 5 shows the expression of alternatively activated macrophage (AAM) markers mediated by DKK-1 in bone marrow-derived macrophages (BMDM).
  • AAM alternatively activated macrophage
  • FIG. 6 shows that deletion of the DKK1 receptor LRP6 protects mice from BLM-induced injury.
  • Figure 7 shows that delayed DKK1 antibody treatment inhibits collagen deposition in a BLM-induced lung injury model.
  • Example 1 It was confirmed that the level of DKK-1 protein expression was increased in lung tissue of human idiopathic pulmonary fibrosis (IPB) patients.
  • a human lung tissue array kit fixed in paraffin blocks was purchased (Pulmonary interstitial fibrosis tissue array, LC561; Biomax). Each lung tissue was deparaffinized with Histoclear and dehydrated. Antigens were removed with sodium citrate buffer (10 mM, pH 6.0). To inhibit endogenous peroxidase activity, the sections were blocked with 3% H2O2. Each section was blocked in 5% BSA, treated with primary antibody (Goat polyclonal anti-m/hDKK1, R&D systems; AF1096), and stained with secondary antibody at a concentration of 5 ug/mL. Antibody color development was performed using a DAB substrate kit (SK-4100; Vector Laboratories), and H&E staining was performed simultaneously to observe the images. The results are shown in Figure 1.
  • mice C57BL/6 mice were treated intranasally with 4 U/kg of BLM dissolved in 40 ⁇ L PBS. Mice aged 8 to 10 weeks were treated with BLM, and the mice were sacrificed 2 weeks later and their lungs were removed. The removed lungs were fixed in 10% formalin for at least 48 hours after lung perfusion and embedded in paraffin blocks for histological analysis. These were sectioned using a microtome. The paraffin-blocked lung tissues were subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Fig. 2.
  • BLM-induced lung fibrosis mice group was anesthetized with 1% sodium pentobarbital and administered 4 U/kg BLM (BLM, S121415; Selleckchem, Houston, TX) in 40 ⁇ l saline via the intratracheal route as reported.
  • Mice administered the same amount of saline were used as controls, and mice were sacrificed 14 days after BLM administration for lung fibrosis analysis.
  • Lungs were removed from mice, fixed in 10% formalin for at least 48 h after lung perfusion, and embedded in paraffin blocks for histological analysis. These were sectioned with a microtome. The paraffin-blocked lung tissues were subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Fig. 3.
  • Platelets were isolated from mice as previously described with some modifications. Blood was collected by cardiac puncture into 3.8% sodium citrate, pH 7.4 (2:1, by volume) and centrifuged at 250 g for 10 min to obtain platelet-rich plasma (PRP). For platelet stimulation, PRP was treated with 100 ⁇ M thrombin receptor activating peptide (SFLLRN; Sigma-Aldrich) for the indicated times and then centrifuged at 657 g for 7 min. The supernatant was collected as plasma, and the platelet pellet was also collected. For platelet release, PRP was centrifuged at 657 g for 7 min to pellet platelets.
  • SFLLRN thrombin receptor activating peptide
  • Platelets were then resuspended in Tyrode-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer and stimulated with 0.1 U/mL thrombin (Sigma-Aldrich) for 90 min. Platelet release was then collected by centrifugation at 2500 g for 10 min at 4°C.
  • Tyrode-HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • thrombin Sigma-Aldrich
  • BMDM bone marrow-derived macrophages
  • BMDM differentiation mice were sacrificed, bone marrow cells were extracted on day 0, and cultured in DMEM medium containing 20% FBS, GlutaMAX, MEM-NEAA, sodium pyruvate, 1x penicillin & streptomycin, and 20 ng/mL MCSF (macrophage colony-stimulating factor). The medium was changed every two days, and IL-4 and IL-13 were treated on day 6 to induce differentiation into M2-like macrophages.
  • Example 6 Deletion of the DKK1 receptor LRP6 protects mice from bone marrow-derived macrophage (BMDM)-induced damage.
  • BMDM bone marrow-derived macrophage
  • LRP6 fl/fl and LysM-Cre LRP6 fl/fl conditional KO mice were induced with BLM, and the degree of lung damage was compared through collagen mRNA and immunohistochemical staining.
  • deletion of the DKK1 receptor LRP6 was found to protect mice from BLM-induced damage.
  • Example 7 Delayed DKK1 antibody treatment was confirmed to inhibit collagen deposition in a BLM-induced lung injury model.
  • C57BL6 mice were treated with BLM on day 0, and 150 ⁇ g/mouse DKK-1 antibody was introduced in two administration methods to confirm the therapeutic effect of DKK-1 antibody.
  • the first administration group was treated with antibody on days -1, 1, 3, 7, 9, and 11, and the second was treated on days 7, 9, and 11.
  • both administration groups reduced the expression level of DKK-1, thereby inhibiting collagen deposition in the lungs.
  • Delayed DKK1 antibody treatment also showed sufficient therapeutic effects, inhibiting collagen deposition to a similar degree as the pre-administered group.
  • DKK-1 hypomorphic doubleridge mice reduction in DKK-1 expression protects against BLM-induced inflammation and fibrosis.
  • platelet-specific deletion of DKK-1 in DKK-1 PKO mice reduces AAM markers such as Arg1 + and CD206 + , and DKK-1 induces several proinflammatory and profibrotic markers in the absence of type 2 cytokines.
  • DKK-1 enhances IL-13-mediated AAM phenotype, and co-inhibition of STAT6 and JNK is sufficient to eliminate excessive AAM-like macrophage gene expression.
  • deletion of the DKK-1 receptor in myeloid lineage cells of Lrp6 MKO mice protects against BLM-induced inflammation and fibrosis. Delayed treatment with DKK-1 antibodies suppresses BLM-induced inflammation and fibrosis.
  • DKK-1 is an important target protein that induces the occurrence of fibrosis
  • small molecule substances or monoclonal antibodies that inhibit the function or activity of DKK-1 may be new therapeutic agents for the treatment of fibrosis
  • DKK-1 + Treg cells are important for the control of fibrosis
  • DKK-1 + Treg cells can be used as cell therapy agents.
  • DKK-1 plays an important role in solid cancer mediated by fibrosis
  • inhibition of the function of DKK-1 or DKK-1 + Treg cells or TAM induced by DKK-1 may play an important role in the prevention and treatment of these fibrosis-associated solid cancers.

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Abstract

The present invention relates to a Dickkopf-1 (DKK1) protein that is specifically present on the surface of immune cells, particularly regulatory T cells (Treg cells), an antibody specific thereto, 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 new target for therapeutic agents for various immune-related diseases.

Description

조절 T 세포에 특이적으로 존재하는 DKK-1 단백질 및 그 용도DKK-1 protein specifically present in regulatory T cells and its use

본 발명은 면역 세포 중에서도 특히 조절 T 세포 (regulatory T cell, Treg cell)의 표면에 특이적으로 존재하는 DKK-1 (Dickkopf-1) 단백질 또는 이를 암호화하는 유전자의 발현 수준을 측정하기 위한 제제를 포함하는 섬유증 또는 암의 진단용 조성물, 진단용 키트, 정보제공방법 및 DKK-1의 세포 외 표면 단백질을 포함하는 조절 T 세포, DKK-1의 세포 외 표면 단백질 및 면역글로불린 Fc 영역을 포함하는 융합 단백질, DKK-1의 세포 외 표면 단백질, 또는 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편 및 DKK-1 억제제를 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a diagnostic composition for diagnosing fibrosis or cancer, a diagnostic kit, a method for providing information, and a pharmaceutical composition for preventing or treating fibrosis or cancer, comprising a formulation for measuring the expression level of DKK-1 (Dickkopf-1) protein or a gene encoding the same, which is specifically present on the surface of regulatory T cells (Treg cells) among immune cells; and a regulatory T cell comprising an extracellular surface protein of DKK-1, a fusion protein comprising an extracellular surface protein of DKK-1 and an immunoglobulin Fc region, an antibody or antigen-binding fragment specifically binding to the extracellular surface protein of DKK-1, or an epitope, and a DKK-1 inhibitor.

면역 세포 중 T 세포는 골수에서 생성되어 흉선에서 성숙하는 면역 세포로, B 세포의 항체 생성을 조정하거나 선천성 면역세포들의 기능을 조정하는 기능을 한다. 상기 T 세포는 T 림프구라고도 하며 다른 면역 세포들의 조력자의 역할을 할 뿐만 아니라, 직접 침입한 물질을 파괴하기도 한다.Among immune cells, T cells are immune cells that are produced in the bone marrow and mature in the thymus, and they function to regulate the antibody production of B cells or to regulate the functions of innate immune cells. The T cells are also called T lymphocytes and not only act as helpers for other immune cells, but also directly destroy invading substances.

면역 세포의 기능 중 가장 중요한 하나는, 면역 세포를 둘러싼 자기(self)를 구성하고 있는 항원 물질에 대해선 면역 반응이 억제되는 반면, 자기가 아닌(non-self) 항원 물질에 대해서는 이를 인식하고 면역 반응을 유도하는 것이다. 면역 세포는 발달 과정에서 자기(self)를 인식하는 세포의 사멸이나, 자기 항원에 특이적인 수용체의 돌연변이 유도 또는 자기 항원을 인식하는 면역 세포의 불활성화를 통해 자기 항원에 대한 무반응을 유도하는데, 이를 면역학적 무반응성(immunologic unresponsiveness) 또는 관용(tolerance)이라 한다. 이러한 자기 관용에 실패하게 되면 자기 항원에 대한 면역 반응이 유도되어 질환이 초래될 수 있는데, 이를 자가면역질환(autoimmune disease)이라 한다.One of the most important functions of immune cells is to suppress the immune response to antigenic substances that constitute the self surrounding the immune cell, while recognizing and inducing an immune response to non-self antigenic substances. During the development process, immune cells induce non-responsiveness to self-antigens through the death of cells that recognize self, mutation induction of receptors specific to self-antigens, or inactivation of immune cells that recognize self-antigens. This is called immunologic unresponsiveness or tolerance. If this self-tolerance fails, an immune response to self-antigens can be induced, resulting in disease, which is called an autoimmune disease.

상기 자기 관용을 유도하거나 계속 유지함에 있어 1970년대 초 고식적 T 세포(conventional T cells)의 효과 기능(effector function)을 제어할 수 있는 조절 T 세포라는 개념을 도입하였으며(R. K. Gershon and K. Kondo, Immunology, 1970, 18: 723-37) 억제 T 세포의 면역학적 특성 및 기능을 규명하기 위한 연구가 꾸준히 이루어져 왔다.In order to induce or maintain the above self-tolerance, the concept of regulatory T cells that can control the effector function of conventional T cells was introduced in the early 1970s (R. K. Gershon and K. Kondo, Immunology, 1970, 18: 723-37), and research has been continuously conducted to elucidate the immunological characteristics and functions of suppressor T cells.

정상 개체에서 상기 조절 T 세포는 고식적 T 세포의 기능을 제어하여 과도한 면역 반응이나 자기 관용을 유도하지만, 자가면역질환과 만성염증질환에서 조절 T 세포의 기능과 숫자가 현저히 감소 되어있어 조절 T 세포가 제 기능을 적절히 수행하지 못하는 것이 보고되었다. 따라서 자가면역질환과 만성염증질환을 가지는 환자에서 조절 T 세포를 정상적인 수준의 기능과 숫자로 되돌리는 것이 상기 질환의 치료법 중 하나가 될 수 있다. In normal individuals, the regulatory T cells control the function of classical T cells to induce excessive immune responses or self-tolerance. However, it has been reported that the function and number of regulatory T cells are significantly reduced in autoimmune diseases and chronic inflammatory diseases, and thus the regulatory T cells do not perform their function properly. Therefore, restoring the function and number of regulatory T cells to a normal level in patients with autoimmune diseases and chronic inflammatory diseases may be one of the treatments for the diseases.

조절 T 세포를 표적화 할 수 있는 세포 표면 단백질로 CD25, CTLA4, CD38, CD62L, GITR, CD45RB 등이 연구를 통해 제시되었고 동물 및 임상 시험도 진행되어왔지만 조절 T 세포만을 단독으로 표적화할 수 있는 세포 표면 단백질은 현재까지 밝혀지지 않았다.CD25, CTLA4, CD38, CD62L, GITR, CD45RB, etc. have been suggested through research as cell surface proteins that can target regulatory T cells, and animal and clinical trials have also been conducted, but a cell surface protein that can exclusively target regulatory T cells has not been discovered to date.

본 발명의 일 목적은, DKK-1 세포 외 표면 단백질 또는 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 포함하는, 섬유증 또는 암의 진단용 조성물을 제공하는 것이다.One object of the present invention is to provide a composition for diagnosing fibrosis or cancer, comprising an antibody or antigen-binding fragment that specifically binds to a DKK-1 extracellular surface protein or epitope.

본 발명의 다른 목적은, 상기 조성물을 포함하는 섬유증 또는 암의 진단 키트를 제공하는 것이다.Another object of the present invention is to provide a diagnostic kit for fibrosis or cancer comprising the composition.

본 발명의 다른 목적은, 조절 T 세포에 존재하는 DKK-1의 세포 외 표면 단백질, 또는 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 이용하여 상기 표면 단백질 또는 에피토프의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein or epitope of DKK-1 present in regulatory T cells using an antibody or antigen-binding fragment that specifically binds to the surface protein or epitope.

본 발명의 다른 목적은, (a) 목적하는 개체로부터 얻어진 생물학적 시료에 대하여 DKK-1; 또는 상기 단백질을 암호화하는 유전자의 발현 수준을 측정하는 측정부; 및 (b) 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 암의 유무를 출력하는 검출부;를 포함하는 섬유증 또는 암의 진단기기를 제공하는 것이다.Another object of the present invention is to provide a diagnostic device for fibrosis or cancer, including: (a) a measuring unit for measuring the expression level of DKK-1 or a gene encoding the protein for a biological sample obtained from a target subject; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded by the gene measured by the measuring unit.

본 발명의 다른 목적은, LRP-5, 6 및 면역글로불린 Fc 영역을 포함하는 융합 단백질, DKK-1의 세포 외 표면 단백질, 또는 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편 또는 DKK-1 억제제를 유효 성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis or cancer, comprising as an active ingredient a fusion protein comprising LRP-5, 6 and an immunoglobulin Fc region, an extracellular surface protein of DKK-1, or an antibody or antigen-binding fragment specifically binding to an epitope, or a DKK-1 inhibitor.

그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

Wnt signaling pathway의 대표적인 음성 조절자 (Negative Regulator)로 알려진 “DKK-1” 또는 “DKK1”은 Wnt와 경쟁적으로 LRP (Low density lipoprotein receptor-related protein) 5/6에 결합하거나, 수용체의 세포내이입 (Endocytosis)를 통해 궁극적으로 B-Catenin의 분해 (Degradation)과 TCF-1 비활성화를 유도한다. DKK-1은 총 4가지의 DKK Family 중 가장 연구가 많이 되어진 단백질로 266 아미노산으로 구성되어진 약 26kDa의 크기를 가진다. 해당 단백질의 구조를 살펴보면, 2개의 시스테인 풍부 도메인 (Cysteine Rich Domain; CRD)를 통해 LRP6을 포함한 다양한 수용체의 도메인과 상호작용을 한다. 현재까지 알려진 DKK-1의 수용체로는 LRP5/6, Kremen 1/2 그리고 CKAP4가 있으며, 위와 같은 수용체를 통해 다양한 세포 신호를 유도한다. DDK-1이 관여하는 시그널링을 살펴보면, Canonical Wnt Signaling의 Co-Receptor LRP5/6에 결합하여 시그널링을 저해하거나 반대로 시그널링을 활성화시키는 경우 CKAP4, LRP5/6와의 결합을 통해 PI3K/Akt Signaling 혹은 LRP5/6에 의한 JNK 시그널링을 유도한다.“DKK-1” or “DKK1”, known as a representative negative regulator of the Wnt signaling pathway, competitively binds to LRP (Low density lipoprotein receptor-related protein) 5/6 with Wnt, or ultimately induces B-Catenin degradation and TCF-1 inactivation through endocytosis of the receptor. DKK-1 is the most studied protein among the four DKK families and has a size of approximately 26 kDa composed of 266 amino acids. Looking at the structure of the protein, it interacts with domains of various receptors including LRP6 through two cysteine rich domains (CRD). The currently known receptors for DKK-1 include LRP5/6, Kremen 1/2, and CKAP4, and it induces various cell signals through the above receptors. When looking at the signaling involved in DDK-1, it binds to the Co-Receptor LRP5/6 of Canonical Wnt Signaling to inhibit signaling or, conversely, activate signaling, and induces PI3K/Akt Signaling or JNK signaling by LRP5/6 through binding to CKAP4 and LRP5/6.

DDK-1이 Wnt 시그널링 및 CKAP4-PI3K-AKT 시그널링을 통한 세포 증식 (Cell Proliferation)과 밀접한 관련이 있는 만큼 암의 종류에 따라 그 정도가 다르지만 암에서 DDK-1의 높은 발현은 부정적인 예후를 나타낸다고 알려져 있다. 이를 바탕으로 DDK-1을 타겟으로 하는 암 면역 치료 (Cancer Immunotherapy)에 대한 연구가 다수 진행되고 있다. 뿐만 아니라, 최근 논문에 의하면 다양한 면역 반응에서의 DDK-1의 역할이 밝혀지며, 자가면역질환에서의 DDK-1의 미치는 영향에 대한 연구 결과를 확인할 수 있다.As DDK-1 is closely related to cell proliferation through Wnt signaling and CKAP4-PI3K-AKT signaling, it is known that high expression of DDK-1 in cancer indicates a negative prognosis, although the degree varies depending on the type of cancer. Based on this, many studies are being conducted on cancer immunotherapy targeting DDK-1. In addition, recent papers have revealed the role of DDK-1 in various immune responses, and the results of studies on the influence of DDK-1 in autoimmune diseases can be confirmed.

또한, In vivo 실험 결과, 알레르기 및 Non-Healing 기생충 감염에 의한 염증 반응은 혈소판 활성을 유도하여 혈소판에서의 DDK-1 분비를 증가시켜, DDK-1이 MAPK와 mTOR 시그널링을 통해 Type 2 Cell mediated 면역반응을 유도할 수 있는 환경을 제공하는 것으로 확인되었다. 생리적 조건 (Physiological condition)에서의 DDK-1의 증가는 C-Maf와 Gata3 유도를 통하여 Th2 세포를 생성하며, p38 MAPK 와 SGK-1을 통해 Th2 세포 관련 사이토카인을 발현하게 한다. 즉, DDK-1은 다른 효과 T 세포와 비교하여 Th2와 연관된 기능에 우선적으로 관여를 한다. In vitro 실험 결과, DDK-1은 Th1 polarization condition에서 IFN-r의 발현을 억제하고, 동시에 GATA3 및 IL-4, IL-5, IL-10, IL-13등의 사이토카인 발현을 높인다. 또한, iTreg에서 IL-10의 발현을 높이는 동시에 Foxp3의 발현은 하향 조절 (down regulate) 하는 것으로 나타났다. 특히나, 혈소판은 TGF-B1을 생성해내는 주요한 원천으로 알려진 바 있는데, 위와 같은 감염을 통해 생성이 된 염증 반응 환경에서는 혈소판이 생성해내는 리간드인 CXCL4와 함께 DDK-1이 Foxp3 발현을 낮춤으로써, TGF-B mediated Foxp3+ Treg cell 생성을 억제하여, Th2가 제대로 된 활성을 갖도록 하는 것으로 추측되어 지고 있다.In addition, in vivo experimental results showed that inflammatory responses induced by allergy and non-healing parasitic infection induce platelet activation, which increases DDK-1 secretion from platelets, thereby providing an environment in which DDK-1 can induce Type 2 Cell-mediated immune responses through MAPK and mTOR signaling. The increase in DDK-1 under physiological conditions generates Th2 cells through the induction of C-Maf and Gata3, and causes the expression of Th2 cell-related cytokines through p38 MAPK and SGK-1. That is, DDK-1 is preferentially involved in functions associated with Th2 compared to other effector T cells. In vitro experimental results showed that DDK-1 inhibits the expression of IFN-r under Th1 polarization conditions, and simultaneously increases the expression of cytokines such as GATA3 and IL-4, IL-5, IL-10, and IL-13. In addition, it was shown that while increasing the expression of IL-10 in iTreg, the expression of Foxp3 is down-regulated. In particular, platelets are known to be a major source of TGF-B1, and in the inflammatory response environment created through the above infection, it is presumed that DDK-1, together with CXCL4, a ligand produced by platelets, lowers Foxp3 expression, thereby inhibiting TGF-B-mediated Foxp3+ Treg cell production and allowing Th2 to be properly activated.

nTreg 세포는 나이브 T 세포 혹은 효과 CD4 T 세포와 비교하여 높은 DDK-1 mRNA 발현 정도를 갖고 있다. 일반적으로 알려진 DDK-1은 가용성 형태로 리간드로서 수용체에 결합하여 그 역할을 하는 것으로 알려져 있으나, nTreg의 DDK-1은 표면에 발현이 되며 Resting state 에서는 약 6%의 DDK-1을 발현을 하고 있으며, 활성화시킬 시 14.1%로 발현율이 증가하는 것으로 확인이 된다. 기능적인 부분에서, DDK-1은 Treg의 억제 기능 (Suppressive)에 중요한 역할을 하는 것으로 추측이 된다. In vitro suppression assay 과정 중 DDK-1에 대한 단일 항체를 처리하였을 시 Treg이 효과 CD4 T 세포 (effector CD4 T cell)의 증식 (proliferation)을 억제하지 못하였다. 또한, Rag2 deficient mouse에 대장염 (Colitis)를 유도하여 Treg의 억제 효과를 살펴본 결과, DDK-1의 발현양을 90% 이상 줄인 Mouse의 Treg 세포는 효과 CD4 T 세포의 증식을 Wild Type의 Treg 만큼 효과적으로 억제하지 못하여, 결국 심한 대장염 (severe colitis)으로 발전을 하는 결과를 가져온다. 마지막으로, Treg 내 DDK-1의 발현 경로를 살펴본 결과 Wnt pathway, mTOR 그리고 Mevalonate pathway와 독립적으로 발현을 하며 활성화된 Treg이 DDK-1 발현을 위해서는 MAPK pathway가 요구되는 것으로 나타났다.nTreg cells have a high level of DDK-1 mRNA expression compared to naïve T cells or effector CD4 T cells. The generally known DDK-1 is known to bind to the receptor as a ligand in a soluble form and play its role, but DDK-1 of nTreg is expressed on the surface, and about 6% of DDK-1 is expressed in the resting state, and the expression rate increases to 14.1% when activated. In terms of function, DDK-1 is thought to play an important role in the suppressive function of Treg. During the in vitro suppression assay process, when a single antibody against DDK-1 was treated, Treg did not suppress the proliferation of effector CD4 T cells. In addition, when we induced colitis in Rag2 deficient mice and examined the inhibitory effect of Treg, Treg cells from mice with reduced DDK-1 expression by more than 90% did not suppress the proliferation of effector CD4 T cells as effectively as Wild Type Treg, ultimately resulting in the development of severe colitis. Finally, when we examined the expression pathway of DDK-1 in Treg, it was shown that it is expressed independently of the Wnt pathway, mTOR, and Mevalonate pathways, and that the MAPK pathway is required for activated Treg to express DDK-1.

Wnt 시그널링은 세포 증식과 더불어 조골세포 (Osteoblast)에서의 새로운 뼈 생성에 필수적인 요인으로 작용을 한다. 따라서, Wnt의 길항제 (antagonist)로 작용하는 DKK1는 혈액에서의 농도가 높게 측정될 경우 뼈의 흡수 (Resorption) 증가로 침식의 가능성을 나타내며, 반대로 낮은 농도는 새로운 뼈의 생성이라 간주된다. 이를 근거로, DKK1과 류마티스관절염 (rheumatoid arthritis, RA)의 예후 간의 관계를 살펴본 결과 병의 기간이 길어질 수록 뼈의 흡수 증가와 더불어 혈액 내 DKK1이 증가하는 결과를 확인했다. 이와 마찬가지로 또 다른 자가면역질환인 Systemic Lupus Erythematosus (SLE)에서 DDK1는 혈청 (Serum)내의 농도를 통해 Lupus Nephritis를 진단할 수 있는 양성 마커로의 가능성을 있음을 확인하였다. 이처럼 환자를 대상으로 한 임상결과에서 DDK-1은 다양한 자가면역질환의 유무를 판별할 수 있는 마커로써 작용을 할 수 있음이 확인되었지만, 어떠한 기작으로 이러한 결론을 내릴 수 있는지에 대한 구체적인 근거에 대해서는 연구가 필요하다.Wnt signaling is an essential factor for new bone formation in osteoblasts as well as cell proliferation. Therefore, DKK1, which acts as an antagonist of Wnt, indicates the possibility of erosion due to increased bone resorption when measured at high concentrations in the blood, whereas low concentrations are considered to be new bone formation. Based on this, the relationship between DKK1 and the prognosis of rheumatoid arthritis (RA) was examined and the result was that as the duration of the disease increased, bone resorption increased and DKK1 in the blood increased. Similarly, in Systemic Lupus Erythematosus (SLE), another autoimmune disease, it was confirmed that DDK1 has the potential to be a positive marker for diagnosing Lupus Nephritis through its concentration in the serum. In this way, clinical results targeting patients have confirmed that DDK-1 can act as a marker for determining the presence or absence of various autoimmune diseases, but research is needed on the specific basis for the mechanism by which this conclusion can be made.

DKK-1 단백질은 인간 조절 T세포에서 많이 발현하며, iTreg에서는 발현이 감소된다는 점이 알려져 있다.It is known that DKK-1 protein is highly expressed in human regulatory T cells and its expression is reduced in iTreg.

본 발명에서는 DKK-1의 발현이 증가하면 섬유증의 발병이 증가한다는 것을 최초로 밝혔다. 즉, 폐 섬유증 환자에게서 DKK-1의 발현이 높다는 점, DKK-1 넉아웃 마우스에서는 섬유증이 발생하지 않는다는 점을 확인하였다.In the present invention, it was first revealed that an increase in the expression of DKK-1 increases the incidence of fibrosis. That is, it was confirmed that the expression of DKK-1 was high in patients with pulmonary fibrosis, and that fibrosis did not occur in DKK-1 knockout mice.

더 나아가, 상기 DKK-1 단백질은 인간 Treg 세포막 외부에 존재하는 고정 단백질 (anchored protein)이며, 이후 혈액으로 분리되어 용해된 (Soluble) 상태로 존재할 수 있다. Furthermore, the DKK-1 protein is an anchored protein existing outside the human Treg cell membrane, and can then be separated into the blood and exist in a soluble state.

또한, 본 발명에서 제공하는 약학 조성물은 DKK-1의 기능을 억제함으로써 면역 반응을 조절하여 섬유증 또는 암에 효과를 발휘할 수 있다.In addition, the pharmaceutical composition provided by the present invention can exert an effect on fibrosis or cancer by regulating the immune response by inhibiting the function of DKK-1.

본 발명에서 상기 DKK-1은 DKK 패밀리 유전자 DKK1, DKK2, DKK3, DKK4 중 하나로, 분비 단백질을 암호화한다. 상기 DKK 단백질은 일반적으로 255에서 350개의 아미노산으로 이루어져 있고, DKK-1의 경우 24 KDa에서 29 KDa의 크기를 가지며, N 말단은 당이 결합한 형태 (glycosylated)로 존재한다. 본 발명의 일 예시로서, 상기 DKK-1은 서열번호 1의 아미노산 서열로 표시될 수 있고, 서열번호 2로 표시되는 뉴클레오티드에 의해 코딩될 수 있다 (표 1).In the present invention, the DKK-1 is one of the DKK family genes DKK1, DKK2, DKK3, and DKK4, and encodes a secreted protein. The DKK protein is generally composed of 255 to 350 amino acids, and in the case of DKK-1, has a size of 24 KDa to 29 KDa, and the N-terminus exists in a form in which a sugar is bound (glycosylated). As an example of the present invention, the DKK-1 can be represented by the amino acid sequence of SEQ ID NO: 1, and can be encoded by the nucleotide represented by SEQ ID NO: 2 (Table 1).

구분division 서열정보Sequence information dickkopf-1 (DKK-1) 아미노산 서열dickkopf-1 (DKK-1) amino acid sequence MMALGAAGAT RVFVAMVAAA LGGHPLLGVS ATLNSVLNSN AIKNLPPPLG GAAGHPGSAV SAAPGILYPG GNKYQTIDNY QPYPCAEDEE CGTDEYCASP TRGGDAGVQI CLACRKRRKR CMRHAMCCPG NYCKNGICVS SDQNHFRGEI EETITESFGN DHSTLDGYSR RTTLSSKMYH TKGQEGSVCL RSSDCASGLC CARHFWSKIC KPVLKEGQVC TKHRRKGSHG LEIFQRCYCG EGLSCRIQKD HHQASNSSRL HTCQRH(서열번호 1)MMALGAAGAT RVFVAMVAAA LGGHPLLGVS ATLNSVLNSN AIKNLPPPLG GAAGHPGSAV SAAPGILYPG GNKYQTIDNY QPYPCAEDEE CGTDEYCASP TRGGDAGVQI CLACRKRRKR CMRHAMCCPG NYCKNGICVS SDQNHFRGEI EETITESFGN DHSTLDGYSR RTTLSSKMYH TKGQEGSVCL RSSDCASGLC CARHFWS KIC KPVLKEGQVC TKHRRKGSHG LEIFQRCYCG EGLSCRIQKD HHQASNSSRL HTCQRH (SEQ ID NO: 1) dickkopf-1 (DKK-1) mRNA, complete cds (GenBank: AF177394.2) 서열dickkopf-1 (DKK-1) mRNA, complete cds (GenBank: AF177394.2) sequence atgatggctc tgggcgcagc gggagctacc cgggtctttg tcgcgatggt agcggcggct ctcggcggcc accctctgct gggagtgagc gccaccttga actcggttct caattccaac gctatcaaga acctgccccc accgctgggc ggcgctgcgg ggcacccagg ctctgcagtc agcgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc acccgcggag gggacgcagg cgtgcaaatc tgtctcgcct gcaggaagcg ccgaaaacgc tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat gatcatagca ccttggatgg gtattccaga agaaccacct tgtcttcaaa aatgtatcac accaaaggac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt accaagcata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttactgtgga gaaggtctgt cttgccggat acagaaagat caccatcaag ccagtaattc ttctaggctt cacacttgtc agagacacta a(서열번호 2)atgatggctc tgggcgcagc gggagctacc cgggtctttg tcgcgatggt agcggcggct ctcggcggcc accctctgct gggagtgagc gccaccttga actcggttct caattccaac gctatcaaga acctgccccc accgctgggc ggcgctgcgg ggcacccagg ctctgcagtc ag cgccgcgc cgggaatcct gtacccgggc gggaataagt accagaccat tgacaactac cagccgtacc cgtgcgcaga ggacgaggag tgcggcactg atgagtactg cgctagtccc acccgcggag gggacgcagg cgtgcaaatc tgtctcgcct gcaggaagcg ccgaaaacgc tgcatgcgtc acgctatgtg ctgccccggg aattactgca aaaatggaat atgtgtgtct tctgatcaaa atcatttccg aggagaaatt gaggaaacca tcactgaaag ctttggtaat gatcatagca ccttggatgg gtattccaga agaaccacct tgtcttcaaa aatgtatcac accaaaggac aagaaggttc tgtttgtctc cggtcatcag actgtgcctc aggattgtgt tgtgctagac acttctggtc caagatctgt aaacctgtcc tgaaagaagg tcaagtgtgt accaagcata ggagaaaagg ctctcatgga ctagaaatat tccagcgttg ttactgtgga gaaggtctgt cttgccggat acaga aagat caccatcaag ccagtaattc ttctaggctt cacacttgtc agagacacta a(SEQ ID NO: 2)

본 발명에서 상기 DKK-1은 세포의 증식 및 상처 회복에 관여하는 Wnt 신호체계에서 Wnt3a와 같은 리간드보다 수용체에 강하게 결합하여 Wnt 신호를 경쟁적으로 억제하여, 배아 발달 과정에서 심장, 머리, 손 등의 발달에 중요한 역할을 할 수 있다. 단, 본 발명에서 상기 DKK-1은 일반적인 경우와 같이, 세포 밖으로 분비되지 않고, 면역 세포, 특히 조절 T 세포의 표면에 존재하는 것일 수 있다. 이를 통해 면역 세포가 자기 관용을 위한 조절 T 세포의 기능에 도움을 줄 수 있다.본 발명의 일 구현 예에 따르면, 상기 DKK-1의 조절 T 세포 외 표면 단백질은 서열번호 3으로 표시된다 (표 2).In the present invention, the DKK-1 may play an important role in the development of the heart, head, and hands during embryonic development by competitively inhibiting the Wnt signal by binding more strongly to the receptor than a ligand such as Wnt3a in the Wnt signaling system involved in cell proliferation and wound healing. However, in the present invention, the DKK-1 may not be secreted outside the cell as in general cases, but may be present on the surface of immune cells, particularly regulatory T cells. Through this, immune cells may help the function of regulatory T cells for self-tolerance. According to one embodiment of the present invention, the regulatory T cell extracellular surface protein of the DKK-1 is represented by SEQ ID NO: 3 (Table 2).

구분division 서열정보Sequence information DKK1 extracellular domainDKK1 extracellular domain TLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH(서열번호 3)TLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGL SCRIQKDHHQASNSSRLHTCQRH (SEQ ID NO: 3)

본 발명에서 제공하는 상기 DKK-1의 조절 T 세포 외 표면 단백질은, 효과 T 세포에 존재하는 리간드와 상호 작용을 하여 조절 T 세포의 활성을 저하시킬 수 있다. 본 발명의 다른 구현 예에 다르면, 조절 T 세포의 표면 단백질인 DKK1의 항원 결정기(epitope)는 서열번호 4 내지 36 중 어느 하나로 표시된다 (표 3).The regulatory T cell extracellular surface protein of DKK-1 provided in the present invention can reduce the activity of regulatory T cells by interacting with a ligand present in effector T cells. According to another embodiment of the present invention, the epitope of DKK1, which is a surface protein of regulatory T cells, is represented by any one of SEQ ID NOS: 4 to 36 (Table 3).

구분division 서열정보Sequence information 3S2K chain C Linear epitope3S2K chain C linear epitope RIQKRL(서열번호 4)RIQKRL(sequence number 4) 3S2K chain C Linear epitope3S2K chain C linear epitope HRRKGSHGLE IF(서열번호 5)HRRKGSHGLE IF(SEQ ID NO: 5) 3S2K chain C Linear epitope3S2K chain C linear epitope KGQEGSVCLR SSDCASG(서열번호 6)KGQEGSVCLR SSDCASG (SEQ ID NO: 6) 3S8V chain X Linear epitope3S8V chain X Linear epitope RIQSRL(서열번호 7)RIQSRL (SEQ ID NO: 7) 3S8V chain X Linear epitope3S8V chain X Linear epitope HRRKGSHGLE IF(서열번호 8)HRRKGSHGLE IF(SEQ ID NO: 8) 3S8V chain X Linear epitope3S8V chain X Linear epitope GEGL(서열번호 9)GEGL (SEQ ID NO: 9) 3S8V chain X Linear epitope3S8V chain X Linear epitope QEGSVCLRSS DCASG(서열번호 10)QEGSVCLRSS DCASG(SEQ ID NO: 10) 3S8V chain X Linear epitope3S8V chain X Linear epitope KEGQ(서열번호 11)KEGQ (sequence number 11) 3SOQ chain Z Linear epitope3SOQ chain Z Linear epitope NSNAIKN(서열번호 12)NSNAIKN (SEQ ID NO: 12) 5FWW chain B Linear epitope5FWW chain B Linear epitope RVPGASHIH(서열번호 13)RVPGASHIH (SEQ ID NO: 13) 5FWW chain B Linear epitope5FWW chain B Linear epitope GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGE(서열번호 14)GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGE (SEQ ID NO: 14) 5FWW chain B Linear epitope5FWW chain B Linear epitope KDHGNPPPLT GTSKTSNKL(서열번호 15)KDHGNPPPLT GTSKTSNKL(SEQ ID NO: 15) 5FWW chain B Linear epitope5FWW chain B Linear epitope FSDRINQ(서열번호 16)FSDRINQ (SEQ ID NO: 16) 5FWW chain B Linear epitope5FWW chain B Linear epitope CYVGVYWK(서열번호 17)CYVGVYWK (SEQ ID NO: 17) 5FWW chain B Linear epitope5FWW chain B Linear epitope IRDSADMVEL LDGYTHRVLA RFHGRSRPPL SFNVSLDFVI(서열번호 18)IRDSADMVEL LDGYTHRVLA RFHGRSRPPL SFNVSLDFVI (SEQ ID NO: 18) 5FWW chain B Linear epitope5FWW chain B Linear epitope LGEHNYC(서열번호 19)LGEHNYC (SEQ ID NO: 19) 5FWW chain B Linear epitope5FWW chain B Linear epitope FCGNNPDYWK YGE(서열번호 20)FCGNNPDYWK YGE (sequence number 20) 5FWW chain B Linear epitope5FWW chain B Linear epitope SQRFK(서열번호 21)SQRFK (SEQ ID NO: 21) 5FWW chain B Linear epitope5FWW chain B Linear epitope ATGRV(서열번호 22)ATGRV (SEQ ID NO: 22) 3S2K chain C Discontinuous epitope3S2K chain C Discontinuous epitope HKGSGL(서열번호 23)HKGSGL (SEQ ID NO: 23) 3S2K chain C Discontinuous epitope3S2K chain C Discontinuous epitope IQL(서열번호 24)IQL(sequence number 24) 3S2K chain C Discontinuous epitope3S2K chain C Discontinuous epitope FWIF(서열번호 25)FWIF (SEQ ID NO: 25) 3S2K chain C Discontinuous epitope3S2K chain C Discontinuous epitope EGQGEGRH(서열번호 26)EGQGEGRH (SEQ ID NO: 26) 3S2K chain C Discontinuous epitope3S2K chain C Discontinuous epitope KGQEGSVCLR SDASG(서열번호 27)KGQEGSVCLR SDASG (SEQ ID NO: 27) 3S8V chain X Discontinuous epitope3S8V chain IQSRL(서열번호 28)IQSRL (SEQ ID NO: 28) 3S8V chain X Discontinuous epitope3S8V chain HKGSGL(서열번호 29)HKGSGL (SEQ ID NO: 29) 3S8V chain X Discontinuous epitope3S8V chain QGSVCRSD(서열번호 30)QGSVCRSD (SEQ ID NO: 30) 3S8V chain X Discontinuous epitope3S8V chain FWIF(서열번호 31)FWIF (SEQ ID NO: 31) 3S8V chain X Discontinuous epitope3S8V chain EGQGGLR(서열번호 32)EGQGGLR(SEQ ID NO: 32) 3S8V chain X Discontinuous epitope3S8V chain ASG(서열번호 33)ASG (SEQ ID NO: 33) 5FWW chain B Discontinuous epitope5FWW chain B Discontinuous epitope GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGELGEHNY CPCYVGVYWK C(서열번호 34)GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGELGEHNY CPCYVGVYWK C (SEQ ID NO: 34) 5FWW chain B Discontinuous epitope5FWW chain B Discontinuous epitope AMYATGRVRV PGASHIHIRD SADMELDGYT HRVLARHGRS PPLSFNVSLD FVIFSDRINQ AQAVK(서열번호 35)AMYATGRVRV PGASHIHIRD SADMELDGYT HRVLARHGRS PPLSFNVSLD FVIFSDRINQ AQAVK (SEQ ID NO: 35) 5FWW chain B Discontinuous epitope5FWW chain B Discontinuous epitope KDHGNPPPLT GTSKTSNKLF SQRFKSGYFC GNNPDYWKYG ECFGGDG(서열번호 36)KDHGNPPPLT GTSKTSNKLF SQRFKSGYFC GNNPDYWKYG ECFGGDG (SEQ ID NO: 36)

본 발명에서 상기 "항원 결정기(Epitope)" 또는 “에피토프”란, 항원 분자에서 항체와 결합하는 부위로, 항체가 인식할 수 있는 부분을 의미한다. 일반적으로 항체는 항원 분자 전체를 인식하는 것이 아니고, 특정의 부분만을 인식하며, 동일한 항원 분자라고 하여도 항체의 종류가 달라지는 경우 다른 항원 결정기 부분을 인식할 수 있다. 본 발명의 목적상 상기 항원 결정기는 DKK1 단백질의 세포 외부로 노출되어 있는 부분이 일부가 절단되어 세포 외부로 방출(Secretion)된 경우에도, 남아있는 일부의 도메인과 효과적으로 결합할 수 있는 항체의 항원 결정기를 포함한다.본 발명에서 상기 항원 결정기인 폴리펩티드는 본 발명에 따른 상기 DKK-1 단백질에 항체가 결합할 수 있는 부분의 연속 또는 불연속 서열을 모두 포함할 수 있다.또한, 본 발명에서 제공하는 상기 조절 T 세포의 표면 단백질인 DKK-1의 항원 결정기인 폴리펩티드 단편은, 효과 T 세포에 존재하는 리간드와 상호 작용을 하여 조절 T 세포의 활성을 저하시킬 수도 있다. In the present invention, the "epitope" or "epitope" refers to a portion of an antigen molecule that binds to an antibody, and refers to a portion that can be recognized by an antibody. In general, antibodies do not recognize the entire antigen molecule, but only a specific portion, and even if it is the same antigen molecule, if the type of antibody is different, it can recognize different antigenic determinant portions. For the purpose of the present invention, the antigenic determinant includes an antigenic determinant of an antibody that can effectively bind to a remaining portion of a domain even when a portion of a DKK1 protein exposed to the outside of the cell is partially cleaved and released (secreted) to the outside of the cell. In the present invention, the polypeptide that is the antigenic determinant may include all continuous or discontinuous sequences of a portion that can be bound to the DKK-1 protein according to the present invention by an antibody. In addition, the polypeptide fragment that is the antigenic determinant of DKK-1, which is the surface protein of regulatory T cells provided in the present invention, may interact with a ligand present in effector T cells to reduce the activity of regulatory T cells.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 서열번호 3으로 표시되는 DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나의 DKK-1의 항원 결정기를 코딩하는 폴리뉴클레오티드를 제공한다. According to another embodiment of the present invention, there is provided a polynucleotide encoding an extracellular surface protein of DKK-1 represented by SEQ ID NO: 3 provided in the present invention, or an antigenic determinant of DKK-1 of any one of SEQ ID NOs: 4 to 36.

본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 폴리뉴클레오티드가 삽입된 발현 벡터를 제공한다. According to another embodiment of the present invention, an expression vector into which a polynucleotide provided by the present invention is inserted is provided.

본 발명에서 상기 "벡터"는 어떤 핵산 분자가 연결된 또 다른 핵산을 수송할 수 있는 상기 핵산 분자이다. 벡터의 한 가지 유형은, 추가적인 DNA 세그멘트가 결찰될 수 있는 원형 이중가닥 DNA를 가리키는 "플라스미드"이다. 또 다른 유형의 벡터는 파지 벡터이다. 또 다른 유형의 벡터는 바이러스성 벡터로, 추가적인 DNA 세그멘트가 바이러스 게놈에 결찰될 수 있다. 어떤 벡터들은 그들이 유입된 숙주세포에서 자율적인 복제를 할 수 있다(예컨대, 박테리아성 벡터는 박테리아성 복제 기원을 갖는 에피솜 포유류 벡터). 기타 벡터(예컨대, 비-에피솜 포유류 벡터)는 숙주세포에 유입되면서 숙주세포의 게놈에 통합될 수 있고, 그럼으로써, 숙주 게놈과 함께 복제된다. 뿐만 아니라, 어떤 벡터는 이들이 작동차원에서 연결된 유전자의 발현을 지시할 수 있다. 이와 같은 벡터는 본원에서 "재조합 발현 벡터" 또는 단순히 "발현 벡터"라 명명된다. 일반적으로 재조합 DNA 기법에서 유용한 발현 벡터는 종종 플라스미드의 형태로 존재한다. 본 명세서에서, "플라스미드"와 "벡터"는, 플라스미드가 벡터 중 가장 통상적으로 사용되는 형태이기 때문에, 상호 교환하여 사용될 수 있다. In the present invention, the "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., a bacterial vector is an episomal mammalian vector having a bacterial origin of replication). Other vectors (e.g., a non-episomal mammalian vector) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, some vectors can direct the expression of genes to which they are linked in an operational manner. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, as the plasmid is the most commonly used form of vector.

본 발명에서 상기 발현 벡터의 구체적인 예시로는 상업적으로 널리 사용되는 pCDNA 벡터, F, R1, RP1, Col, pBR322, ToL, Ti 벡터; 코스미드; 람다, 람도이드(lambdoid), M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 등의 파아지; 식물 바이러스로 이루어진 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니며, 당업자에게 발현 벡터로 알려진 모든 발현 벡터는 본 발명에 사용 가능하고, 발현 벡터를 선택할 때에는 목적으로 하는 숙주 세포의 성질에 따른다. 숙주 세포로의 벡터 도입 시 인산칼슘 트랜스펙션, 바이러스 감염, DEAE-덱스트란 조절 트랜스펙션, 리포펙타민 트랜스펙션 또는 전기천공법에 의해 수행될 수 있으나 이에 한정되는 것은 아니며 당업자는 사용하는 발현 벡터 및 숙주 세포에 알맞은 도입 방법을 선택하여 이용할 수 있다. 바람직하게 벡터는 하나 이상의 선별 마커를 함유하나 이에 한정되지 않으며, 선별 마커를 포함하지 않은 벡터를 이용하여 생산물 생산 여부에 따라 선별이 가능하다. 선별 마커의 선택은 목적하는 숙주 세포에 의해 선별되며, 이는 이미 당업자에게 알려진 방법을 이용하므로 본 발명은 이에 제한을 두지 않는다. In the present invention, specific examples of the expression vector may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; and plant viruses, but are not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the target host cell. When introducing a vector into a host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can select and use an introduction method appropriate for the expression vector and host cell to be used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and selection is possible based on whether or not the product is produced using a vector that does not contain a selection marker. The selection marker is selected by the desired host cell, and this is done using a method already known to those skilled in the art, so the present invention is not limited thereto.

본 발명의 핵산 분자를 정제를 용이하게 하기 위하여 태그 서열을 발현 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되는 것은 아니며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. In order to facilitate purification of the nucleic acid molecule of the present invention, a tag sequence can be inserted into an expression vector and fused thereto. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art that facilitates purification can be used in the present invention.

본 발명의 또 다른 구현 예에 다르면, 본 발명에서 제공하는 상기 발현 벡터가 형질 감염된 숙주 세포주를 제공한다.According to another embodiment of the present invention, the expression vector provided in the present invention provides a host cell line transfected with the expression vector.

본 발명에서 상기 "숙주 세포"에는 폴리펩티드 삽입물의 편입을 위한 벡터(들)의 수령자(recipient)일 수 있거나 또는 수령자였던 개별적인 세포 또는 세포배양물이 포함된다. 숙주 세포에는 단일 숙주 세포의 자손이 포함되고, 상기 자손은 자연적인, 우발적인 또는 고의의 돌연변이 때문에 반드시 원래 모세포와 완전히 동일(형태학상 또는 게놈 DNA 보완체에서)하지 않을 수 있다. 숙주 세포에는 본원의 폴리펩티드(들)로 체내에서 형질주입된 세포가 포함된다.The "host cell" in the present invention includes an individual cell or cell culture that can be or has been the recipient of the vector(s) for incorporation of the polypeptide insert. The host cell includes progeny of a single host cell, which progeny may not necessarily be completely identical (either morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation. The host cell includes a cell that has been transfected in vivo with the polypeptide(s) of the present disclosure.

본 발명에 있어서, 상기 숙주 세포로는 포유동물, 식물, 곤충, 균류 또는 세포성 기원의 세포를 포함할 수 있고, 예를 들면 대장균, 스트렙토미세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 효모 세포, 피치아 파스 토리스 등의 균류세포; 드로조필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO(중국 햄스터 난소 세포, Chinese hamster ovary cells), SP2/0(생쥐 골수종), 인간 림프아구(Human lymphoblastoid), COS, NSO(생쥐 골 수종), 293T, 보우 멜라노마 세포, HT-1080, BHK(베이비 햄스터 신장세포, Baby Hamster Kidney cells), HEK(인간 배아신장 세포, Human Embryonic Kidney cells) 또는 PERC.6(인간 망막 세포)의 동물 세포; 또는 식물 세포일 수 있으나, 이에 제한되는 것은 아니며, 당업자에게 알려진 숙주 세포주로 사용 가능한 세포는 모두 이용 가능하다.In the present invention, the host cell may include a cell of mammalian, plant, insect, fungal or cellular origin, and for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); Or, it may be a plant cell, but is not limited thereto, and any cell that can be used as a host cell line known to those skilled in the art may be used.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK-1의 세포 외 표면 단백질 또는 상기 DKK-1의 에피토프를 포함하는 조절 T 세포를 제공한다. According to another embodiment of the present invention, a regulatory T cell comprising the extracellular surface protein of the DKK-1 of the present invention or an epitope of the DKK-1 is provided.

본 발명의 상기 DKK-1의 세포 외 표면 단백질 또는 이의 항원 결정기를 표면에 발현하는 조절 T 세포는 효과 T 세포와 상호 작용을 통해 그 세포 활성을 억제하며, 면역 반응을 억제할 수 있다. Regulatory T cells expressing the extracellular surface protein of the DKK-1 of the present invention or an antigenic determinant thereof on their surface can suppress the cellular activity of effector T cells through interaction with them and suppress an immune response.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK-1의 세포 외 표면 단백질 또는 상기 DKK-1의 에피토프를 포함하는 조절 T 세포를 유효 성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising as an effective ingredient an extracellular surface protein of the DKK-1 of the present invention or a regulatory T cell comprising an epitope of the DKK-1.

본 발명의 상기 DKK1의 세포 외 표면 단백질을 포함하는 조절 T 세포는 효과 T 세포와 상호 작용을 통해 그 세포 활성을 억제하며, 면역 반응을 억제할 수 있다. Regulatory T cells comprising the extracellular surface protein of the DKK1 of the present invention can suppress the cell activity of effector T cells through interaction with them and suppress immune responses.

본 발명에서 세포 표면에 DKK-1 단백질을 발현하는 조절 T 세포는 면역 관련 질환으로, 예를 들어 섬유증 또는 암 등을 효과적으로 예방, 개선 또는 치료할 수 있다. In the present invention, regulatory T cells expressing DKK-1 protein on their cell surface can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK-1 세포 외 표면 단백질 또는 상기 항원 결정기를 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising the DKK-1 extracellular surface protein or the antigenic determinant of the present invention as an effective ingredient.

본 발명에서 제공하는 상기 DKK-1 세포 외 표면 단백질 또는 상기 항원 결정기 단편은 효과 T 세포에 존재하는 리간드와 상호 작용을 하여 조절 T 세포의 활성을 억제하여, 면역 관련 질환으로, 예를 들어 섬유증 또는 암을 효과적으로 예방, 개선 또는 치료할 수 있다.The DKK-1 extracellular surface protein or the antigen determinant fragment provided in the present invention interacts with a ligand present in effector T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.

본 발명에서 상기 "암"은 포유류에서 전형적으로 조절되지 않는 세포 성장으로 특징 지어진 생리적 상태를 나타내거나 가리킨다. 본 발명에서 예방, 개선 또는 치료의 대상이 되는 암은 고형 장기(solid organ)에서 비정상적으로 세포가 성장하여 발생한 덩어리로 이루어진 고형암(solid tumor)일 수 있고, 고형 장기의 부위에 따라 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the "cancer" refers to or represents a physiological condition characterized by uncontrolled cell growth typically in mammals. The cancer to be prevented, improved or treated in the present invention may be a solid tumor formed by a mass caused by abnormal cell growth in a solid organ, and depending on the site of the solid organ, may be gastric cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기에 특이적인 항체를 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient.

또한, 본 발명에서 상기 DKK1 세포 외 표면 단백질은 조절 T 세포 표면에 존재하는 것일 수 있다. Additionally, in the present invention, the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.

본 발명에서 상기 항체는 조절 T 세포의 활성을 억제하여 섬유증 또는 암을 효과적으로 예방, 개선 또는 치료할 수 있다.In the present invention, the antibody can effectively prevent, improve or treat fibrosis or cancer by suppressing the activity of regulatory T cells.

본 발명에서 용어, "항체"란 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는, 각 유전자를 통상적인 방법에 따라 발현 벡터에 클로닝하여 상기 마커 유전자에 의해 코딩 되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함되며, 본 발명의 부분 펩티드로는, 최소한 7개의 아미노산, 바람직하게는 9개 아미노산, 더욱 바람직하게는 12개 이상의 아미노산을 포함한다. In the present invention, the term "antibody" is a term known in the art and means a specific protein molecule directed against an antigenic site. For the purpose of the present invention, the antibody means an antibody that specifically binds to the protein of the present invention, and such an antibody can be produced by a conventional method by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and from the obtained protein. This also includes a partial peptide that can be produced from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids.

본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof having antigen binding properties are also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

본 발명의 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.The antibody of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of the antibody molecule. A functional fragment of an antibody molecule means a fragment that possesses at least an antigen-binding function, such as Fab, F(ab'), F(ab') 2, and Fv.

본 발명의 일 예시로, 상기 항체는 조절 T 세포의 표면에 발현되는 DKK1 단백질 또는 이의 항원 결정기에 특이적으로 결합하여 조절 T 세포의 활성을 억제해 면역 관련 질환으로, 예를 들어 섬유증 또는 암을 효과적으로 예방, 개선 또는 치료할 수 있다. As an example of the present invention, the antibody specifically binds to the DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.

본 발명에서 상기 암은 고형 장기(solid organ)에서 비정상적으로 세포가 성장하여 발생한 덩어리로 이루어진 고형암(solid tumor)일 수 있고, 고형 장기의 부위에 따라 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the cancer may be a solid tumor formed by a mass of cells growing abnormally in a solid organ, and depending on the location of the solid organ, may be gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, and preferably, melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다. 또한, 섬유증에는 폐 섬유증, 간 섬유증, 심장 섬유증, 뇌 섬유증, 피부 섬유증, 췌장 섬유증, 골 섬유증 등이 있으나, 이에 제한되는 것은 아니다.In another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow, and skin, but is not limited thereto. In addition, the fibrosis includes, but is not limited to, pulmonary fibrosis, liver fibrosis, cardiac fibrosis, brain fibrosis, skin fibrosis, pancreatic fibrosis, and bone fibrosis.

본 발명에서 용어, "섬유증 (fibrosis)"은 기관 또는 조직내에 과도한 섬유성 결합조직이 발생되는 것을 말한다. 섬유증은 세포외 기질 (ECM)의 축적 및 개조를 특징으로 한다. 이와 같이 비정상적으로 세포가 성장하여 발생한 덩어리인 암과 규별되는 명백한 병인적 및 임상적 차이를 가짐에도 불구하고, 대부분의 만성 섬유증 장애는 성장 인자, 단백질분해 효소, 혈관신생 인자 및 섬유생성 사이토카인의 생산을 지속시키는 지속적 자극을 공통으로 지니며, 이들 인자는 함께 결합 조직 요소들, 특히 콜라겐 및 프로테오글라이칸의 침착을 자극시켜 정상 조직 구조를 지속적으로 리모델링시키고 파괴시킨다.As used herein, the term "fibrosis" refers to the development of excessive fibrous connective tissue within an organ or tissue. Fibrosis is characterized by the accumulation and remodeling of the extracellular matrix (ECM). Despite the clear pathognomonic and clinical differences that distinguish it from cancer, which is a mass formed by abnormal cell growth, most chronic fibrotic disorders share in common a persistent stimulus that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, particularly collagen and proteoglycans, thereby continually remodeling and destroying normal tissue architecture.

본 발명의 다른 예시로, 상기 항체는 조절 T 세포의 표면에 발현되는 DKK-1 단백질 또는 이의 항원 결정기에 특이적으로 결합하여 조절 T 세포의 활성을 증가시킴에 따라 면역 관련 질환으로, 예를 들어 섬유증 또는 암 등을 효과적으로 예방, 개선 또는 치료할 수 있다. As another example of the present invention, the antibody can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer, by specifically binding to the DKK-1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing the activity of regulatory T cells.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK-1 세포 외 표면 단백질 또는 상기 항원 결정기를 코딩하는 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 마이크로RNA으로 이루어진 군에서 선택되는 어느 하나를 유효 성분으로 포함하는, 섬유증 또는 암 예방 또는 치료용 약학 조성물을 제공한다. According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising as an active ingredient any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA specific for the DKK-1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.

본 발명에서 상기 "안티센스 올리고뉴클레오타이드"는 특정 DNA 또는 RNA 타겟에 대해 안티센스(또는 상보)인 짧은 길이의 DNA 합성 가닥(또는 DNA 아날로그)으로서, 생체 내뿐만 아니라 생체 외에서도 유전자-특이적 억제를 달성하기 위해 사용된다. 안티센스 올리고뉴클레오타이드는 타겟에 결합하고 전사, 번역 또는 스플라이싱의 단계에서 발현을 멈추게 함으로써 DNA 또는 RNA 타겟에 의해 인코드된 단백질의 발현을 막기 위해 제안되었다. 안티센스 올리고뉴클레오타이드는 세포 배양 및 질환의 동물 모델에서도 성공적으로 이용되어 왔다. 올리고뉴클레오타이드가 분해되지 않도록 더욱 안정하고 저항적이 되게 하기 위한 안티센스 올리고뉴클레오타이드의 또 다른 변형이 당업자에게 알려져 있고 이해된다. 여기서 사용된 안티센스 올리고뉴클레오타이드는 이중나선 또는 단일나선 DNA, 이중나선 또는 단일나선 RNA, DNA/RNA 하이브리드, DNA 및 RNA 아날로그 및 염기, 당 또는 백본 변형을 지닌 올리고뉴클레오타이드를 포함한다. 올리고뉴클레오타이드는 안정성을 증가시키고, 뉴클레아제 분해에 대한 저항성을 증가시키기 위해 당분야에 알려진 방법에 의해 변형된다. 이들 변형은 당분야에 알려져 있는 올리고뉴클레오타이드 백본의 변형, 당 모이어티의 변형 또는 염기의 변형을 포함하나 이에 제한되지는 않는다.In the present invention, the "antisense oligonucleotide" is a short length of DNA synthetic strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, and is used to achieve gene-specific inhibition in vitro as well as in vivo. Antisense oligonucleotides have been proposed to block the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides have also been successfully used in cell culture and animal models of diseases. Other modifications of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. The antisense oligonucleotides used herein include double-stranded or single-stranded DNA, double-stranded or single-stranded RNA, DNA/RNA hybrids, DNA and RNA analogs, and oligonucleotides having base, sugar or backbone modifications. The oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of the oligonucleotide backbone, modifications of the sugar moieties, or modifications of the bases, which are known in the art.

본 발명에서 상기 "siRNA(small interfering RNA)"는 RNA 방해 또는 유전자 사일런싱(silencing)을 매개할 수 있는 핵산 분자로서, 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 녹다운 방법 또는 유전자 치료 방법으로 사용된다. 본 발명에서 siRNA 분자가 이용되는 경우, 센스 가닥(WLS mRNA 서열에 상응하는 서열)과 안티센스 가닥(WLS mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조 또는 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다. siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 불일치(대응하는 염기가 상보적이지 않음), 팽창/돌출(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. siRNA 말단 구조는 WLS 유전자의 발현을 RNA 간섭(RNA interference, RNAi) 효과에 의하여 억제할 수 있는 것이면 평활 말단 혹은 점착 말단 모두 가능하다. 점착 말단 구조는 3'-말단 돌출 구조와 5'-말단 돌출 구조 모두 가능하다. 상기 siRNA분자는 이에 제한되지는 않으나, 전체 길이가 15 내지 30 염기, 바람직하게는 19 내지 21 염기일 수 있다.In the present invention, the "siRNA (small interfering RNA)" is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and is used as an efficient gene knockdown method or gene therapy method because it can suppress the expression of a target gene. When an siRNA molecule is used in the present invention, it 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 a WLS mRNA sequence) are positioned on opposite sides to form a double-stranded structure, or a single-stranded structure having self-complementary sense and antisense strands. siRNA is not limited to a double-stranded RNA portion in which RNAs are paired completely, and may include a portion that does not pair due to a mismatch (corresponding bases are not complementary), a bulge/protrusion (there is no base corresponding to one chain), etc. The siRNA terminal structure can be either a blunt end or a sticky end as long as it can suppress the expression of the WLS gene by the RNA interference (RNAi) effect. The sticky end structure can be either a 3'-terminal overhang structure or a 5'-terminal overhang structure. The siRNA molecule may have a total length of 15 to 30 bases, preferably 19 to 21 bases, but is not limited thereto.

본 발명에서 상기 "shRNA(short hairpin RNA)"는 45 내지 70 뉴클레오타이드의 길이를 가지는 단일가닥의 RNA로서, 타겟유전자 siRNA 염기서열의 센스가닥과 상보적인 안티센스가닥 사이에 3-10개의 염기 링커를 연결하는 올리고 DNA를 합성한 후, 플라스미드 벡터에 클로닝하거나 또는 shRNA를 레트로바이러스인 렌티바이러스 및 아데노 바이러스에 삽입하여 발현시키면 루프가 있는 헤어핀 구조의 shRNA가 만들어지고 세포내의 다이서(dicer)에 의해 siRNA로 전환되어 RNAi 효과를 나타낸다.In the present invention, the "shRNA (short hairpin RNA)" is a single-stranded RNA having a length of 45 to 70 nucleotides, and when oligo DNA is synthesized to connect a 3 to 10 nucleotide linker between the sense strand of the target gene siRNA base sequence and the complementary antisense strand, and then cloned into a plasmid vector or the shRNA is expressed by inserting it into a retrovirus such as a lentivirus and an adenovirus, an shRNA having a hairpin structure with a loop is created and converted into siRNA by dicer within the cell to exhibit an RNAi effect.

본 발명에서 상기 "마이크로 RNA(microRNA)"는 발생, 분화, 증식, 보존 및 아폽토시스 등 다양한 생물학적 과정을 조절한다. 마이크로 RNA는 일반적으로 타겟 mRNA를 불안정하게 하거나, 번역을 방해함으로써 타겟 mRNA를 코딩하는 유전자의 발현을 조절한다.In the present invention, the "microRNA" regulates various biological processes such as development, differentiation, proliferation, preservation, and apoptosis. MicroRNA generally regulates the expression of a gene encoding a target mRNA by destabilizing the target mRNA or interfering with its translation.

본 발명에서 상기 안티센스 올리고 뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA를 지닌 발현 컨스트럭트/벡터에 유용한 조절 서열(예를 들어, 구성적 프로모터, 유도성 프로모터, 조직 특이적 프로모터 또는 그의 결합) 역시 당분야에서 공지된 내용으로부터 적절히 선택할 수 있다.In the present invention, useful regulatory sequences (e.g., constitutive promoter, inducible promoter, tissue-specific promoter or combinations thereof) for the expression construct/vector containing the antisense oligonucleotide, siRNA, shRNA or microRNA can also be appropriately selected from those known in the art.

본 발명의 일 예시로, 상기 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA는 조절 T 세포의 표면에 발현되는 DKK1 단백질 또는 이의 항원 결정기를 코딩하는 유전자에 특이적으로 결합하여 이들의 발현을 억제해 면역 관련 질환으로, 예를 들어 암을 효과적으로 예방, 개선 또는 치료할 수 있다. As an example of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA can specifically bind to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and inhibit their expression, thereby effectively preventing, improving or treating immune-related diseases, such as cancer.

본 발명에서 상기 암은 고형 장기(solid organ)에서 비정상적으로 세포가 성장하여 발생한 덩어리로 이루어진 고형암(solid tumor)일 수 있고, 고형 장기의 부위에 따라 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the cancer may be a solid tumor formed by a mass of cells growing abnormally in a solid organ, and depending on the location of the solid organ, may be gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, and preferably, melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA는 조절 T 세포의 표면에 발현되는 DKK1 단백질 또는 이의 항원 결정기를 코딩하는 유전자에 특이적으로 결합하여 이들의 발현을 증가시킴에 따라 면역 관련 질환으로, 예를 들어 섬유증 또는 암 등을 효과적으로 예방, 개선 또는 치료할 수 있다. As another example of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA can effectively prevent, improve or treat immune-related diseases, such as fibrosis or cancer, by specifically binding to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing their expression.

본 발명에서, "예방"은 본 발명의 약학 조성물을 이용하여 질환의 증상을 차단하거나, 그 증상을 억제 또는 지연시키는 모든 행위라면 제한없이 포함할 수 있다. In the present invention, “prevention” may include, without limitation, any act of blocking, suppressing or delaying symptoms of a disease by using the pharmaceutical composition of the present invention.

또한, 본 발명에서, "치료"는 본 발명의 약학 조성물을 이용하여 질환의 증상이 호전되거나 이롭게 되는 모든 행위라면 제한없이 포함할 수 있다.Additionally, in the present invention, “treatment” may include without limitation any act that improves or benefits the symptoms of a disease by using the pharmaceutical composition of the present invention.

본 발명에 있어서, 상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 바람직하게는 본 발명에 따른 약학 조성물은 주사제 형태로 제조되어 암 또는 면역 질환이 발생된 부위에 직접적으로 주사 될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans. Preferably, the pharmaceutical composition according to the present invention is prepared in the form of an injection and can be directly injected into a site where cancer or an immune disease has occurred, but is not limited thereto.

본 발명의 약학 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사 용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여 시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형화할 수 있다.The pharmaceutical composition of the present invention is not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, and the like for injections. In the case of topical administration, may include bases, excipients, lubricants, and preservatives. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.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, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

본 발명에 따른 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. 본원에 사용된 용어 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.The routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

본 발명의 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약무 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50mg/kg 또는 0.001 내지 50mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical 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, dosage form, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and 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, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a solution, a gel, a syrup, a slurry, or a suspension.

본 발명의 약학 조성물은 추가로 다른 항암제와 병용 투여할 수 있으며, 이를 통해서 일반적인 암세포 증식 및 암 전이를 효과적으로 억제하여 암의 치료에 사용될 수 있다.The pharmaceutical composition of the present invention can additionally be administered in combination with other anticancer agents, thereby effectively inhibiting general cancer cell proliferation and cancer metastasis, and can thus be used for the treatment of cancer.

여기서 상기 항암제로는 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 소라페닙, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투모맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 카페시타빈, 데시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르모퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈블라스틴, 이다루비신, 미토마이신, 블레로마이신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테모졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 올라파립, 나벨빈, 파드라졸, 타목시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴, 5FU, 보리노스텟, 엔티노스텟 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상을 사용할 수 있으나, 이에 제한되는 것은 아니다. Here, the above anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tycetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, Doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagarbine, enocitabine, flutamide, capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vinblastine, idarubicin, mitomycin, bleromycin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, One or more selected from the group consisting of nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, olaparib, navelbine, padrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, 5FU, vorinostat, entinostat, and carmustine may be used, but is not limited thereto.

본 발명의 약학 조성물은 추가로 다른 면역 억제제와 병용 투여할 수 있으며, 이를 통해서 일반적인 과면역 반응으로 인한 질환 효과적으로 억제할 수 있다.The pharmaceutical composition of the present invention can additionally be administered in combination with other immunosuppressants, thereby effectively suppressing diseases caused by general hyperimmune reactions.

본 발명에서 상기 면역 억제제로는 글루코코르티코이드(Glucocorticoids), 사이클로포스파미드(Cyclophosphamid), 사이클로스포린(Cyclosporin), 타크롤리무(Tacrolimu), 라파마이신(Rapamycin), Ⅳ형 PDE 억제제(Type Ⅳ PDE inhibitors), p38 키나제 억제제(p38 kinase inhibitors), 아자티오프린(Azathioprine), 마이코페놀레이트 모페틸(mycophenolate mofetil), 미조리빈(Mizoribin), 메토트렉세이트(Methotrexate), 레플루노미드(Leflunomid), 및 브레퀴나(Brequina)로 이루어진 군에서 선택된 1종 이상을 사용할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the immunosuppressant may be at least one selected from the group consisting of glucocorticoids, cyclophosphamide, cyclosporin, tacrolimus, rapamycin, type Ⅳ PDE inhibitors, p38 kinase inhibitors, azathioprine, mycophenolate mofetil, mizoribin, methotrexate, leflunomide, and brequina, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 치료를 필요로 하는 대상에게 본 발명의 상기 DKK-1 세포 외 표면 단백질 또는 상기 항원 결정기에 특이적인 항체를 투여하는 단계를 포함하는, 면역 관련 질환의 예방 또는 치료 방법에 관한 것이다. According to another embodiment of the present invention, there is provided a method for preventing or treating an immune-related disease, comprising administering to a subject in need of treatment an antibody specific for the DKK-1 extracellular surface protein of the present invention or an antigenic determinant.

본 발명에서 상기 치료를 필요로 하는 대상은 면역 관련 질환이 있거나, 그 증상이 의심되는 개체로, 여기서, 상기 면역 관련 질환의 일 예시로는 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등의 암일 수 있고, 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the subject requiring the treatment is an individual having an immune-related disease or suspected of having symptoms thereof, wherein, as an example of the immune-related disease, the cancer may be gastric cancer, liver cancer, glioma, 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, and as another example, the fibrosis may be selected from the group consisting of, but not limited to, fibrosis occurring in at least one organ selected from the group consisting of the kidney, liver, lung, heart, bone or bone marrow, and skin.

본 발명의 일 예시로, 상기 항체는 조절 T 세포의 표면에 발현되는 DKK-1 단백질 또는 이의 항원 결정기에 특이적으로 결합하여 조절 T 세포의 활성을 억제해 면역 관련 질환으로, 예를 들어 섬유증 또는 암을 효과적으로 예방, 개선 또는 치료할 수 있다. As an example of the present invention, the antibody specifically binds to the DKK-1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells, thereby effectively preventing, improving or treating immune-related diseases, such as fibrosis or cancer.

본 발명의 또 다른 구현 예에 따르면, 치료를 필요로 하는 대상에게 본 발명의 상기 DKK-1 세포 외 표면 단백질 또는 상기 항원 결정기를 코딩하는 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 마이크로RNA으로 이루어진 군에서 선택되는 어느 하나를 투여하는 단계를 포함하는, 면역 관련 질환의 예방 또는 치료 방법에 관한 것이다. According to another embodiment of the present invention, there is provided a method for preventing or treating an immune-related disease, comprising administering to a subject in need of treatment any one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the DKK-1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.

본 발명에서 상기 치료를 필요로 하는 대상은 면역 관련 질환이 있거나, 그 증상이 의심되는 개체로, 여기서, 상기 면역 관련 질환의 일 예시로는 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등의 암일 수 있고, 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the subject requiring the treatment is an individual having an immune-related disease or suspected of having symptoms thereof, wherein, as an example of the immune-related disease, the cancer may be gastric cancer, liver cancer, glioma, 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, and as another example, the fibrosis may be selected from the group consisting of, but not limited to, fibrosis occurring in at least one organ selected from the group consisting of the kidney, liver, lung, heart, bone or bone marrow, and skin.

본 발명의 일 예시로, 상기 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA는 조절 T 세포의 표면에 발현되는 DKK1 단백질 또는 이의 항원 결정기를 코딩하는 유전자에 특이적으로 결합하여 이들의 발현을 억제해 면역 관련 질환으로, 예를 들어 암을 효과적으로 예방, 개선 또는 치료할 수 있다. As an example of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA can specifically bind to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and inhibit their expression, thereby effectively preventing, improving or treating immune-related diseases, such as cancer.

본 발명의 다른 예시로, 상기 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA는 조절 T 세포의 표면에 발현되는 DKK1 단백질 또는 이의 항원 결정기를 코딩하는 유전자에 특이적으로 결합하여 이들의 발현을 증가시킴에 따라 면역 관련 질환을 효과적으로 예방 또는 치료할 수 있다. 상기 면역 관련 질환의 일 예시로는 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등의 암일 수 있고, 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA can effectively prevent or treat an immune-related disease by specifically binding to a gene encoding DKK1 protein or an antigenic determinant thereof expressed on the surface of regulatory T cells and increasing their expression. An example of the immune-related disease may be cancer such as gastric cancer, liver cancer, glioma, 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, and as another example, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 일 구체예에서 투여란, 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 경구 투여, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 비내 투여, 폐내 투여, 직장내 투여, 강내 투여, 복강 내 투여, 경막 내 투여가 이루어질 수 있으나, 이에 제한되지는 않는다. 본 발명의 치료 방법은 상기 항체, 안티센스 올리고뉴클레오타이드, siRNA, shRNA 또는 마이크로RNA를 약제학적 유효량으로 투여하는 것을 포함할 수 있다. 본 발명에서 유효량은 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 성인의 경우, 상기 유전자의 발현 억제제 또는 단백질의 활성 억제제를 1일 1회 내지 수회 투여시, siRNA일 경우 0.01ng/kg-10㎎/kg, 상기 유전자의 mRNA에 대한 안티센스 올리고뉴클레오타이드인 경우 0.01ng/kg-10㎎/kg, 화합물일 경우 0.1ng/kg-10㎎/kg, 상기 단백질에 대한 모노클로날 항체일 경우 0.1ng/kg-10㎎/kg의 용량으로 투여될 수 있다.In one specific embodiment of the present invention, administration means introducing the composition of the present invention into a patient by any appropriate method, and the route of administration of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intracavity administration, intraperitoneal administration, and intrathecal administration may be performed, but are not limited thereto. The treatment method of the present invention may include administering the antibody, antisense oligonucleotide, siRNA, shRNA, or microRNA in a pharmaceutically effective amount. In the present invention, the effective amount may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the effective ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, administration time, administration route, and secretion rate of the composition, treatment period, and concurrently used drugs. In adults, the expression inhibitor of the gene or the activity inhibitor of the protein may be administered once or several times a day at a dose of 0.01 ng/kg-10 mg/kg for siRNA, 0.01 ng/kg-10 mg/kg for antisense oligonucleotide to mRNA of the gene, 0.1 ng/kg-10 mg/kg for compound, and 0.1 ng/kg-10 mg/kg for monoclonal antibody to the protein.

본 발명에서 상기 투여 시 추가로 다른 항암제 또는 다른 면역 억제제를 병용하여 투여할 수 있다.In the present invention, additional administration may be performed in combination with another anticancer agent or another immunosuppressant.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기에 특이적인 항체를 유효 성분으로 포함하는, 섬유증 또는 암의 진단용 조성물을 제공한다. According to another embodiment of the present invention, a composition for diagnosing fibrosis or cancer is provided, comprising an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient.

본 발명에서 상기 "진단"이란, 병리 상태의 존재 또는 특징을 확인하는 것을 의미하며, 본 발명의 목적상 진단은 위암의 발병 유무 및 전이 여부를 확인하는 것이다.In the present invention, the term “diagnosis” means confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, diagnosis is confirming the presence or absence of gastric cancer and metastasis.

또한, 본 발명에서 상기 DKK1 세포 외 표면 단백질은 조절 T 세포 표면에 존재하는 것일 수 있다. Additionally, in the present invention, the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.

본 발명에서는 상기 항체를 이용하여 조절 T 세포의 표면에 존재하는 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기의 발현 수준을 측정함으로써 섬유증 또는 암을 진단할 수 있다. In the present invention, fibrosis or cancer can be diagnosed by measuring the expression level of DKK1 extracellular surface protein or the antigen determinant present on the surface of regulatory T cells using the antibody.

본 발명의 일 예시로, 상기 면역 관련 질환은 암일 수 있고, 보다 상세하게는 암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.As an example of the present invention, the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기를 코딩하는 유전자에 특이적인 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택되는 어느 하나를 포함하는, 섬유증 또는 암의 진단용 조성물을 제공한다. According to another embodiment of the present invention, a composition for diagnosing fibrosis or cancer is provided, comprising any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for the DKK1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant.

또한, 본 발명에서 상기 DKK1 세포 외 표면 단백질은 조절 T 세포 표면에 존재하는 것일 수 있다. Additionally, in the present invention, the DKK1 extracellular surface protein may be present on the surface of regulatory T cells.

본 발명에서는 상기 항체를 이용하여 조절 T 세포의 표면에 존재하는 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기를 코딩하는 유전자의 발현 수준을 측정함으로써 섬유증 또는 암을 진단할 수 있다. In the present invention, fibrosis or cancer can be diagnosed by measuring the expression level of the DKK1 extracellular surface protein present on the surface of regulatory T cells or the gene encoding the antigenic determinant using the antibody.

본 발명의 일 예시로, 상기 면역 관련 질환은 암일 수 있고, 보다 상세하게는 암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.As an example of the present invention, the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 본 발명의 진단용 조성물을 포함하는 섬유증 또는 암의 진단 키트를 제공한다. According to another embodiment of the present invention, a diagnostic kit for fibrosis or cancer comprising the diagnostic composition of the present invention is provided.

본 발명에서 상기 "키트"란, 특정한 목적을 위해 필요한 조성물 및 부속품들을 모아놓은 세트를 의미한다. 본 발명의 목적상, 본 발명의 키트는 암, 자가면역질환, 이식편대숙주 질환, 장기이식거부반응, 천식, 아토피, 급성 및 만성염증 질환 등의 면역 관련 질환의 진단 마커인 DKK1 세포 외 표면 단백질의 발현수준을 mRNA 또는 이의 단백질의 발현수준을 확인함으로써 상기 질환을 진단할 수 있다. 본 발명의 키트에는 면역 관련 질환의 진단 마커의 발현 수준을 측정하기 위한 프라이머, 프로브 또는 선택적으로 마커를 인지하는 항체뿐만 아니라 분석방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치가 포함될 수 있다.In the present invention, the "kit" refers to a set of compositions and accessories necessary for a specific purpose. For the purpose of the present invention, the kit of the present invention can diagnose the above diseases by confirming the expression level of the DKK1 extracellular surface protein, which is a diagnostic marker of immune-related diseases such as cancer, autoimmune disease, graft-versus-host disease, organ transplant rejection, asthma, atopy, and acute and chronic inflammatory diseases, as mRNA or the expression level of its protein. The kit of the present invention may include a primer, a probe, or optionally an antibody recognizing the marker for measuring the expression level of the diagnostic marker of the immune-related disease, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.

본 발명의 또 다른 구현 예에 따르면, 조절 T 세포에 존재하는 상기 DKK1의 세포 외 표면 단백질, 또는 상기 항원 결정기에 특이적인 항체를 이용하여 상기 세포 외 표면 단백질 또는 항원 결정기의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법을 제공한다. According to another embodiment of the present invention, a method is provided for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein or antigenic determinant of DKK1 present in regulatory T cells using an antibody specific for the extracellular surface protein or the antigenic determinant.

본 발명의 일 예시로, 상기 면역 관련 질환은 암일 수 있고, 보다 상세하게는 암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.As an example of the present invention, the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 조절 T 세포에서 본 발명의 상기 DKK1 세포 외 표면 단백질 또는 상기 항원 결정기를 코딩하는 유전자에 특이적인 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택되는 어느 하나를 이용하여 상기 유전자의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method is provided for providing information on fibrosis or cancer by measuring the expression level of a gene encoding the DKK1 extracellular surface protein of the present invention or the antigenic determinant in a regulatory T cell using any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for the gene.

본 발명의 일 예시로, 상기 면역 관련 질환은 암일 수 있고, 보다 상세하게는 암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 흑색종 또는 폐암 등일 수 있으며, 바람직하게는 흑색종일 수 있으나, 이에 제한되는 것은 아니다.As an example of the present invention, the immune-related disease may be cancer, and more specifically, may be cancer, liver cancer, glioma, 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, and preferably may be melanoma, but is not limited thereto.

본 발명의 다른 예시로, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.As another example of the present invention, the fibrosis may be selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin, but is not limited thereto.

본 발명의 또 다른 구현 예에 따르면, 섬유증 또는 암의 진단기기에 관한 것이다.According to another embodiment of the present invention, it relates to a diagnostic device for fibrosis or cancer.

본 발명의 상기 진단기기는 (a) 목적하는 개체로부터 얻어진 생물학적 시료에 대하여 DKK1; 또는 상기 단백질을 암호화하는 유전자의 발현 수준을 측정하는 측정부; 및 (b) 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 암의 유무를 출력하는 검출부; 를 포함하는 섬유증 또는 암의 진단기기에 관한 것이다.The diagnostic device of the present invention relates to a diagnostic device for fibrosis or cancer, comprising: (a) a measuring unit for measuring the expression level of DKK1 or a gene encoding the protein for a biological sample obtained from a target subject; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded thereby measured by the measuring unit.

본 발명에서 상기 "목적하는 개체"란 섬유증 또는 암이 발병하였거나 발병 가능성이 높은 개체로, 인간을 포함하는 포유 동물일 수 있고, 예를 들면, 인간, 래트, 마우스, 모르모트, 햄스터, 토끼, 원숭이, 개, 고양이, 소, 말, 돼지, 양 및 염소로 구성된 군으로부터 선택될 수 있고, 바람직하게는 인간일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the "target subject" refers to a subject that has developed or is likely to develop fibrosis or cancer, and may be a mammal including a human, and may be selected from the group consisting of, for example, a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep, and a goat, and is preferably a human, but is not limited thereto.

본 발명의 상기 방법에서 DKK-1, 섬유증, 암, 예후, 목적하는 개체, 생물학적 시료, 대조군 등에 관한 기재는 앞서 기재된 바와 중복되어 명세서의 과도한 복잡을 피하기 위하여 이하 그 자세한 기재를 생략한다. In the above method of the present invention, the description of DKK-1, fibrosis, cancer, prognosis, target entity, biological sample, control group, etc. is redundant with what has been described above, and thus, in order to avoid excessive complexity of the specification, a detailed description thereof is omitted below.

본 발명의 일 구현예에 따르면, DKK-1의 세포외 표면 단백질 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 포함하는, 섬유증 또는 암의 진단용 조성물을 제공한다.According to one embodiment of the present invention, a composition for diagnosing fibrosis or cancer is provided, comprising an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1 or a DKK-1 epitope represented by any one of SEQ ID NOS: 4 to 36.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.

본 발명의 또 다른 구현예에 따르면, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.

본 발명의 또 다른 구현예에 따르면, 상기 암은 고형암인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the cancer is a solid cancer.

본 발명의 또 다른 구현예에 따르면, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

본 발명의 일 구현예에 따르면, DKK-1 세포외 표면 단백질 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 프라이머, 프로브 또는 안티센스 뉴클레오티드를 포함하는, 섬유증 또는 암의 진단용 조성물을 제공한다.According to one embodiment of the present invention, a composition for diagnosing fibrosis or cancer is provided, comprising a primer, probe or antisense nucleotide specific for a gene encoding an epitope of DKK-1 extracellular surface protein or DKK-1 represented by any one of SEQ ID NOS: 4 to 36.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.

본 발명의 또 다른 구현예에 따르면, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.

본 발명의 또 다른 구현예에 따르면, 상기 암은 고형암인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, a diagnostic composition is provided, wherein the cancer is a solid cancer.

본 발명의 또 다른 구현예에 따르면, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단용 조성물을 제공한다.According to another embodiment of the present invention, the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

본 발명의 일 구현예에 따르면, 상기 조성물을 포함하는 섬유증 또는 암의 진단 키트를 제공한다.According to one embodiment of the present invention, a diagnostic kit for fibrosis or cancer comprising the composition is provided.

본 발명의 일 구현예에 따르면, DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 이용하여 상기 표면 단백질 또는 에피토프의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법을 제공한다.According to one embodiment of the present invention, a method is provided for providing information on fibrosis or cancer by measuring the expression level of the extracellular surface protein of DKK-1 or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36 using an antibody or antigen-binding fragment that specifically binds to the surface protein or epitope.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method for providing information is provided, wherein the DKK-1 is present on the surface of a regulatory T cell or is present in a dissolved state after being separated from the cell.

본 발명의 일 구현예에 따르면, DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택되는 어느 하나를 이용하여 상기 유전자의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법을 제공한다.According to one embodiment of the present invention, a method is provided for providing information on fibrosis or cancer by measuring the expression level of a gene using any one selected from the group consisting of a primer, a probe and an antisense nucleotide specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method for providing information is provided, wherein the DKK-1 is present on the surface of a regulatory T cell or is present in a dissolved state after being separated from the cell.

본 발명의 또 다른 구현예에 따르면, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method of providing information is provided, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.

본 발명의 또 다른 구현예에 따르면, 상기 암은 고형암인, 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method for providing information is provided, wherein the cancer is a solid cancer.

본 발명의 또 다른 구현예에 따르면, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 정보를 제공하는 방법을 제공한다.According to another embodiment of the present invention, a method for providing information is provided, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

본 발명의 일 구현예에 따르면, (a) 목적하는 개체로부터 얻어진 생물학적 시료에 대하여 DKK-1; 또는 상기 단백질을 암호화하는 유전자의 발현 수준을 측정하는 측정부; 및 (b) 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 암의 유무를 출력하는 검출부; 를 포함하는 섬유증 또는 암의 진단기기을 제공한다.According to one embodiment of the present invention, a diagnostic device for fibrosis or cancer is provided, comprising: (a) a measuring unit for measuring the expression level of DKK-1 or a gene encoding the protein for a biological sample obtained from a target individual; and (b) a detection unit for outputting the presence or absence of cancer from the expression level of the gene or the protein encoded thereby measured by the measuring unit.

본 발명의 다른 구현예에 따르면, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단기기를 제공한다.According to another embodiment of the present invention, a diagnostic device is provided, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.

본 발명의 또 다른 구현예에 따르면, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단기기를 제공한다.According to another embodiment of the present invention, the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

본 발명의 일 구현예에 따르면, DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising as an active ingredient an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.

본 발명의 일 구현예에 따르면, DKK-1 억제제를 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer comprising a DKK-1 inhibitor is provided.

본 발명의 다른 구현예에 따르면, 상기 DKK-1 억제제는 NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, endothelin-1 (ET-1)로 이루어진 군에서 선택되는 것인, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the DKK-1 inhibitor is selected from the group consisting of NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, and endothelin-1 (ET-1).

본 발명의 일 구현예에 따르면, DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 마이크로RNA으로 이루어진 군에서 선택되는 어느 하나를 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising as an active ingredient any one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA and microRNA specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.

본 발명의 또 다른 구현예에 따르면, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin.

본 발명의 또 다른 구현예에 따르면, 상기 암은 고형암인, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the cancer is a solid cancer.

본 발명의 또 다른 구현예에 따르면, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 약학 조성물을 제공한다.According to another embodiment of the present invention, the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

본 발명의 일 구현예에 따르면, 조절 T 세포에 존재하는 DKK-1의 세포 외 표면 단백질 및 면역글로불린 Fc 영역을 포함하는 융합 단백질을 유효성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising as an active ingredient a fusion protein comprising an extracellular surface protein of DKK-1 present in regulatory T cells and an immunoglobulin Fc region.

상기 융합 단백질은 DKK-1의 리간드인 LRP-5, 6와 경쟁적으로 결합하는 바, 궁극적으로 DKK-1의 기능을 억제함으로써, 섬유증 또는 암을 예방 또는 치료할 수 있다.The above fusion protein competitively binds to LRP-5, 6, which are ligands of DKK-1, and ultimately inhibits the function of DKK-1, thereby preventing or treating fibrosis or cancer.

면역글로불린(immunoglobulin)은 4개의 폴리펩타이드 쇄, 즉 쇄간 디술피드 결합을 통해 회합된 2개의 중쇄 및 2개의 경쇄를 포함한다. 각각의 경쇄는 2개의 도메인, 즉 가변 경쇄 도메인 (VL) 및 불변 경쇄 도메인 (CL)을 갖고, 각각의 중쇄는 2개의 영역, 즉 가변 중쇄 영역 (VH) 및 불변 중쇄 영역 (CH)을 갖는다. 불변 중쇄 영역 (CH)은 숫자에 의해 지정된 불변 중쇄 영역 (예를 들어, CH1, CH2, CH3 등)으로 이루어진다. 면역글로불린은 그의 생물학적 특성, 유기체 내의 위치 및 상이한 항원을 처리하는 능력에 기초하여 상이한 이소형(즉, IgG, IgM, IgA, IgD 및 IgE)으로 분류된다. 이뮤노글로불린 이소형에 따라, 불변 중쇄 영역 (CH)은 3개 또는 4개의 CH 도메인을 가질 수 있다. 또한, 일부 이소형 (IgA, IgD 및 IgG)에서, 중쇄는 분자에 유연성을 부가하는 힌지 영역을 포함한다.Immunoglobulins comprise four polypeptide chains, two heavy chains and two light chains, which are joined by interchain disulfide bonds. Each light chain has two domains, a variable light domain (VL) and a constant light domain (CL), and each heavy chain has two domains, a variable heavy domain (VH) and a constant heavy domain (CH). The constant heavy domain (CH) is made up of constant heavy domains, which are designated by numbers (e.g., CH1, CH2, CH3, etc.). Immunoglobulins are classified into different isotypes (i.e., IgG, IgM, IgA, IgD, and IgE) based on their biological properties, location in an organism, and ability to process different antigens. Depending on the immunoglobulin isotype, the constant heavy domain (CH) may have three or four CH domains. Additionally, in some isotypes (IgA, IgD, and IgG), the heavy chain contains a hinge region that adds flexibility to the molecule.

인간에는 4개의 IgG 하위클래스 (IgG1, 2, 3, 4)가 있고, 이들은 혈청에 풍부한 순서에 따라 명명된다 (IgG1이 가장 풍부하다). IgG 이소형은 2개의 경쇄 및 2개의 중쇄로 이루어지고, 각각의 중쇄는 3개의 불변 중쇄 도메인 (CH1, CH2, CH3)을 포함한다. IgG의 2개의 중쇄는 서로에 대해 및 경쇄에 각각 디술피드 결합 (-S-S-)에 의해 연결되어 있다. IgG의 항원 결합 부위는 가변 경쇄 (VL) 및 가변 중쇄 (VH) 도메인뿐만 아니라 불변 경쇄 (CL) 및 불변 중쇄 (CH1) 도메인을 포함하는 단편 항원 결합 영역 (Fab 영역)에 위치한다. IgG의 단편 결정화가능 영역 (Fc 영역)은 신생아 Fc 수용체 (FcRn)를 포함하는 특정 세포의 표면에서 발견되는 Fc 수용체에 결합하는 CH2 및 CH3 도메인을 함유하는 중쇄의 일부이다. IgG의 중쇄는 또한 Fab 영역을 Fc 영역으로부터 분리하고 2개의 중쇄를 디술피드 결합을 통해 함께 연결할 때 참여하는, CH1과 CH2 사이의 힌지 영역 (힌지)을 갖는다. 힌지 영역의 구조는 4개의 IgG 하위클래스 각각의 특유한 생물학적 특성에 기여한다.There are four IgG subclasses in humans (IgG1, 2, 3, 4), named according to their order of abundance in serum (IgG1 being the most abundant). An IgG isotype consists of two light chains and two heavy chains, each heavy chain containing three constant heavy domains (CH1, CH2, CH3). The two heavy chains of IgG are linked to each other and to the light chain by disulfide bonds (-S-S-). The antigen-binding site of IgG is located in the fragment antigen-binding region (Fab region), which includes the variable light (VL) and variable heavy (VH) domains, as well as the constant light (CL) and constant heavy (CH1) domains. The fragment crystallizable region (Fc region) of IgG is the part of the heavy chain that contains the CH2 and CH3 domains that bind to Fc receptors found on the surface of certain cells, including the neonatal Fc receptor (FcRn). The heavy chain of IgG also has a hinge region (hinge) between CH1 and CH2 that separates the Fab region from the Fc region and participates in linking the two heavy chains together via a disulfide bond. The structure of the hinge region contributes to the unique biological properties of each of the four IgG subclasses.

IgG는 쉽게 조직을 관류할 수 있도록 허용하는 크기가 작은 단량체로 분비된다. 이것은 자궁 내의 태아를 보호하기 위해 인간 태반을 통과하는 것을 촉진하는 수용체 (신생아 Fc 수용체 (FcRn))를 갖는 유일한 이소형이다. 태반을 통해 흡수된 IgG는 그 자체 면역계가 발달하기 전에 체액성 면역을 신생아에게 제공한다.IgG is secreted as a small monomer that allows it to easily perfuse tissues. It is the only isotype that has a receptor (neonatal Fc receptor (FcRn)) that facilitates its passage across the human placenta to protect the fetus in the womb. IgG absorbed through the placenta provides humoral immunity to the newborn before its own immune system develops.

IgG 신생아 Fc 수용체 (FcRn) 결합 부위는 항체의 Fc 영역에 위치한다. FcRn은 일반적으로 인간의 태반 및 상피 세포에서 발현되고, IgG의 분해를 막는 세포내이입 샐비지 경로에 참여한다. 이 샐비지 경로는 산성 pH에서 FcRn에 대한 IgG의 높은 pH 의존성 결합 친화도에 의해 매개된다. 산성 pH에서 FcRn에 대한 IgG의 높은 친화도는 산성 엔도솜 내로의 흡수 후에 내재화된 IgG의 FcRn에 대한 결합을 유발하는 것으로 생각된다. 대부분의 가용성 단백질은 내재화 후에 리소솜으로 향하지만, 내재화된 FcRn-결합 IgG는 형질 막으로 되돌아가고, 기본 분해 경로로부터 효과적으로 구출된다. 세포외 공간의 중성 pH에 노출시, IgG는 FcRn으로부터 해리되어 순환계로 되돌아갈 수 있다. 따라서, 항체의 연장된 혈청 반감기 특성은 Fc 단편에서 유지된다.The IgG neonatal Fc receptor (FcRn) binding site is located in the Fc region of the antibody. FcRn is commonly expressed on human placental and epithelial cells and participates in the endocytic salvage pathway that protects IgG from degradation. This salvage pathway is mediated by the high pH-dependent binding affinity of IgG for FcRn at acidic pH. The high affinity of IgG for FcRn at acidic pH is thought to drive binding of internalized IgG to FcRn after uptake into acidic endosomes. While most soluble proteins are directed to lysosomes after internalization, internalized FcRn-bound IgG are returned to the plasma membrane and are effectively rescued from the canonical degradation pathway. Upon exposure to the neutral pH of the extracellular space, IgG can dissociate from FcRn and return to the circulation. Thus, the prolonged serum half-life properties of antibodies are maintained in the Fc fragment.

본 발명에서, "면역글로불린 Fc 영역"은, 면역글로불린의 중쇄와 경쇄 가변영역을 제외한, 중쇄 불변영역 2(CH2) 및/또는 중쇄 불변영역 3(CH3) 부분을 포함하는 부위를 의미한다. 상기 면역글로불린 Fc 영역은 본 발명의 단백질 결합체의 모이어티를 이루는 일 구성일 수 있다.In the present invention, the "immunoglobulin Fc region" means a region including a heavy chain constant region 2 (CH2) and/or a heavy chain constant region 3 (CH3) portion, excluding the heavy chain and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component forming a moiety of the protein complex of the present invention.

본 발명에서 상기 면역글로불린 Fc 영역이 최종 제조될 융합 단백질의 구조적 유연성(flexibility)에 영향을 줄 수 있고, 융합 단백질의 생산성 및 안정성을 보다 높일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the immunoglobulin Fc region can affect the structural flexibility of the fusion protein to be finally manufactured, and can further increase the productivity and stability of the fusion protein, but is not limited thereto.

본 발명에서 상기 면역글로불린 Fc 영역은 중쇄 불변영역에 힌지(hinge) 부분을 더 포함함으로써 최종 제조될 융합 단백질의 구조적 유연성(flexibility)에 영향을 줄 수 있고, 융합 단백질의 생산성 및 안정성을 보다 높일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the immunoglobulin Fc region may further include a hinge portion in the heavy chain constant region, thereby affecting the structural flexibility of the final fusion protein to be manufactured, and may further increase the productivity and stability of the fusion protein, but is not limited thereto.

또한 본 발명의 면역글로불린 Fc 영역은 천연형과 실질적으로 동등하거나 향상된 효과를 갖는 한, 면역 글로불린의 중쇄와 경쇄 가변영역만을 제외하고, 일부 또는 전체 중쇄 불변영역 1(CH1) 및/또는 경쇄 불변영역 1(CL1)을 포함하는 확장된 Fc영역일 수 있다. 또한, CH2 및/또는 CH3에 해당하는 상당히 긴 일부 아미노산 서열이 제거된 영역일 수도 있다. In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region including part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1), excluding only the heavy and light chain variable regions of the immunoglobulin, as long as it has substantially the same or improved effect as the native type. In addition, it may also be a region in which a relatively long part of the amino acid sequence corresponding to CH2 and/or CH3 is deleted.

예컨대, 본 발명의 면역글로불린 Fc 영역은 1) CH1 도메인, CH2 도메인, CH3 도메인 및 CH4 도메인, 2) CH1 도메인 및 CH2 도메인, 3) CH1 도메인 및 CH3 도메인, 4) CH2 도메인 및 CH3 도메인, 5) CH1 도메인, CH2 도메인, CH3 도메인 및 CH4 도메인 중 1개 또는 2개의 이상의 도메인과 면역글로불린 힌지 영역(또는 힌지 영역의 일부)와의 조합, 6) 중쇄 불변 영역 각 도메인과 경쇄 불변영역의 이량체일 수 있다. 그러나, 이에 제한되는 것은 아니다. For example, the immunoglobulin Fc region of the present invention may be a dimer of 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), 6) each domain of a heavy chain constant region and a light chain constant region. However, it is not limited thereto.

또한, 본 발명의 면역글로불린 Fc 영역은 천연형 아미노산 서열뿐만 아니라 이의 서열 유도체를 포함한다. 아미노산 서열 유도체란 천연 아미노산 서열 중의 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 것을 의미한다. In addition, the immunoglobulin Fc region of the present invention includes not only a natural amino acid sequence but also a sequence derivative thereof. An amino acid sequence derivative means a sequence having a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in a natural amino acid sequence.

예를 들면, IgG Fc의 경우 결합에 중요하다고 알려진 214 내지 238, 297 내지 299, 318 내지 322 또는 327 내지 331번째 아미노산 잔기들이 변형을 위해 적당한 부위로서 이용될 수 있다.For example, for IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

또한, 이황화 결합을 형성할 수 있는 부위가 제거되거나, 천연형 Fc에서 N-말단의 몇몇 아미노산이 제거되거나 또는 천연형 Fc의 N-말단에 메티오닌 잔기가 부가될 수도 있는 등 다양한 종류의 유도체가 가능하다. 또한, 이펙터 기능을 없애기 위해 보체결합부위, 예로 C1q 결합부위가 제거될 수도 있고, ADCC (antibody dependent cell mediated cytotoxicity) 부위가 제거될 수도 있다. 이러한 면역글로불린 Fc 영역의 서열 유도체를 제조하는 기술은 국제특허공개 제WO 97/34631호, 국제특허공개 제96/32478호 등에 개시되어 있다.In addition, various types of derivatives are possible, such as those in which a site capable of forming a disulfide bond is removed, in which several amino acids at the N-terminus of a native Fc are removed, or in which a methionine residue is added to the N-terminus of a native Fc. In addition, a complement binding site, e.g., a C1q binding site, may be removed to eliminate effector function, or an ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for producing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication No. WO 97/34631, International Patent Publication No. 96/32478, etc.

분자의 활성을 전체적으로 변경시키지 않는 단백질 및 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다(H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation), 아세틸화(acetylation) 및 아미드화(amidation) 등으로 수식(modification)될 수도 있다.Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. Modifications may also occur, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

상기 기술한 Fc 유도체는 본 발명의 Fc 영역과 동등한 생물학적 활성을 나타내며 Fc 영역의 열, pH 등에 대한 구조적 안정성을 증대시킨 것일 수 있다. The Fc derivative described above exhibits biological activity equivalent to the Fc region of the present invention and may have enhanced structural stability of the Fc region with respect to heat, pH, etc.

또한, 이러한 Fc 영역은 인간, 소, 염소, 돼지, 마우스, 래빗, 햄스터, 랫트 또는 기니아 픽 등의 동물의 생체 내에서 분리한 천연형으로부터 얻어질 수도 있고, 형질 전환된 동물세포 또는 미생물로부터 얻어진 재조합형 또는 이의 유도체일 수 있다. 여기서, 천연형으로부터 획득하는 방법은 전체 면역글로불린을 인간 또는 동물의 생체로부터 분리한 후, 단백질 분해효소를 처리하여 획득하는 방법일 수 있다. 파파인으로 처리할 경우에는 Fab 및 Fc로 절단되고, 펩신으로 처리할 경우에는 pF'c 및 F(ab)2로 절단된다. 크기 배제 크로마토그래피(size-exclusion chromatography) 등을 이용하여 Fc 또는 pF'c를 분리할 수 있다. 더 구체적인 실시 형태에서는 인간 또는 마우스 유래의 Fc 영역을 미생물로부터 수득한 재조합형 면역글로불린 Fc 영역이다.In addition, the Fc region may be obtained from a natural type isolated from an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or may be a recombinant or a derivative thereof obtained from a transformed animal cell or microorganism. Here, the method for obtaining from a natural type may be a method of isolating the entire immunoglobulin from a human or animal body and then treating it with a protease to obtain it. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography, etc. In a more specific embodiment, the Fc region derived from a human or mouse is a recombinant immunoglobulin Fc region obtained from a microorganism.

또한, 면역글로불린 Fc 영역은 천연형 당쇄, 천연형에 비해 증가된 당쇄, 천연형에 비해 감소한 당쇄 또는 당쇄가 제거된 형태일 수 있다. 이러한 면역글로불린 Fc 당쇄의 증감 또는 제거에는 화학적 방법, 효소학적 방법 및 미생물을 이용한 유전 공학적 방법과 같은 통상적인 방법이 이용될 수 있다. 여기서, Fc에서 당쇄가 제거된 면역글로불린 Fc 영역은 보체(c1q)와의 결합력이 현저히 저하되고, 항체-의존성 세포독성 또는 보체-의존성 세포 독성이 감소 또는 제거되므로, 생체 내에서 불필요한 면역 반응을 유발하지 않는다. 이런 점에서 약물의 캐리어로서의 본래의 목적에 보다 부합하는 형태는 당쇄가 제거되거나 비당쇄화된 면역글로불린 Fc 영역이라 할 것이다.In addition, the immunoglobulin Fc region may be a native sugar chain, a sugar chain with an increased amount compared to the native type, a sugar chain with a decreased amount compared to the native type, or a form in which the sugar chain is removed. Conventional methods such as a chemical method, an enzymatic method, and a genetic engineering method using microorganisms can be used for the increase or removal of the immunoglobulin Fc sugar chain. Here, the immunoglobulin Fc region in which the sugar chain is removed from the Fc has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so that it does not induce unnecessary immune responses in vivo. In this respect, the form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region in which the sugar chain is removed or is non-glycosylated.

본 발명에서 "당쇄의 제거(Deglycosylation)"는 효소로 당을 제거한 Fc 영역을 말하며, 비당쇄화(Aglycosylation)는 원핵동물, 더 구체적인 실시 형태에서는 대장균에서 생산하여 당쇄화되지 않은 Fc 영역을 의미한다.In the present invention, “deglycosylation” refers to an Fc region from which a sugar has been removed by an enzyme, and aglycosylation refers to an Fc region that has not been glycosylated and is produced in a prokaryotic animal, or in a more specific embodiment, in E. coli.

한편, 면역글로불린 Fc 영역은 인간 또는 소, 염소, 돼지, 마우스, 래빗, 햄스터, 랫트, 기니아 픽 등의 동물 유래일 수 있으며, 바람직한 일 예시로 인간 또는 마우스 유래일 수 있다.Meanwhile, the immunoglobulin Fc region may be of animal origin, such as human or cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, and may be of human or mouse origin as a preferred example.

또한, 본 발명의 면역글로불린 Fc 영역은 IgG, IgA, IgD, IgE, IgM 유래 Fc 영역, 중쇄 불변영역 2(CH2), 중쇄 불변영역 3(CH3), 힌지(hinge), 이의 단편, 또는 이들의 조합(combination), 또는 이들의 조합을 포함하는 하이브리드 Fc(hybrid Fc)일 수 있다. In addition, the immunoglobulin Fc region of the present invention may be a hybrid Fc comprising an Fc region derived from IgG, IgA, IgD, IgE, IgM, heavy chain constant region 2 (CH2), heavy chain constant region 3 (CH3), a hinge, a fragment thereof, or a combination thereof, or a combination thereof.

한편, 본 발명에서 "조합(combination)"이란 이량체 또는 다량체를 형성할 때, 동일 기원 단쇄 면역글로불린 Fc 영역, 중쇄 불변영역 2(CH2) 또는 중쇄 불변영역 3(CH3)을 코딩하는 폴리펩타이드가 상이한 기원의 단쇄 폴리펩타이드와 결합을 형성하는 것을 의미한다. 즉, IgG, IgA, IgM, IgD 또는 IgE 유래의 Fc 영역, 중쇄 불변영역 2(CH2) 또는 중쇄 불변영역 3(CH3)으로부터 선택된 2개 이상의 단편으로부터 이량체 또는 다량체의 제조가 가능하다. Meanwhile, in the present invention, the term "combination" means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region, heavy chain constant region 2 (CH2) or heavy chain constant region 3 (CH3) of the same origin forms a bond with a single-chain polypeptide of different origin. That is, it is possible to produce a dimer or multimer from two or more fragments selected from the Fc region, heavy chain constant region 2 (CH2) or heavy chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD or IgE.

본 발명에서 상기 "하이브리드 Fc"는 인간 IgG 서브클래스의 조합 또는 인간 IgD 및 IgG의 조합으로부터 유도될 수 있다. 하나의 실시예에서, 상기 하이브리드 Fc는 예를 들어, IgD 힌지 영역 및 CH2 N-말단 영역 + IgG4 CH2 및 CH3 영역을 포함할 수 있으며, 예를 들어, 한국등록특허 제0897938호에 개시된 하이브리드 Fc 형태를 동일하게 차용하여 사용할 수 있고, 본 명세서에 참조로서 도입된다. 본 발명에서 상기 하이브리드 Fc는 생물학적 활성 분자, 폴리펩타이드 등에 결합하는 경우, 생물학적 활성 분자의 혈청 반감기를 증가시킬 뿐만 아니라 Fc-폴리펩타이드 융합 단백질을 코딩하는 뉴클레오티드가 발현될 때 폴리펩타이드의 발현 수준을 높이는 효과가 있다. In the present invention, the "hybrid Fc" can be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. In one embodiment, the hybrid Fc can include, for example, an IgD hinge region and a CH2 N-terminal region + an IgG4 CH2 and CH3 region, and can be used by borrowing the same hybrid Fc form disclosed in, for example, Korean Patent No. 0897938, which is incorporated herein by reference. In the present invention, when the hybrid Fc binds to a biologically active molecule, a polypeptide, etc., it not only increases the serum half-life of the biologically active molecule, but also has the effect of increasing the expression level of the polypeptide when a nucleotide encoding an Fc-polypeptide fusion protein is expressed.

본 발명의 일 예시로 상기 면역글로불린 Fc 영역은 IgG, IgA, IgM, IgD 또는 IgE 유래의 Fc 영역이거나, 혹은 상기 IgG, IgA, IgM, IgD 또는 IgE 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있으나, 이에 제한되는 것은 아니다. As an example of the present invention, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgM, IgD or IgE, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG, IgA, IgM, IgD or IgE, but is not limited thereto.

본 발명의 일 예시로 상기 면역글로불린 Fc 영역은 인간 혈액에 가장 풍부한 IgG 또는 IgM 유래 Fc 영역이거나, 혹은 상기 IgG 또는 IgM 유래 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있고, 다른 예시로 리간드 결합 단백질의 반감기를 향상시키는 것으로 공지된 IgG 유래 Fc 영역이거나, 혹은 상기 IgG 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있고, 또 다른 예시로 IgG1, IgG2, IgG3 또는 IgG4 유래 Fc 영역이거나, 혹은 상기 IgG1, IgG2, IgG3 또는 IgG4 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있으며, 또 다른 예시로 IgG1 또는 IgG2 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있다.As an example of the present invention, the immunoglobulin Fc region may be an Fc region derived from IgG or IgM which is most abundant in human blood, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG or IgM, or as another example, an Fc region derived from IgG known to enhance the half-life of a ligand binding protein, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG, or as another example, an Fc region derived from IgG1, IgG2, IgG3 or IgG4, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from IgG1 or IgG2.

본 발명에서 바람직한 예시로서 상기 면역글로불린 Fc 영역은 서열번호 37로 표시되는 인간 IgG1 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함하거나, 서열번호 38으로 표시되는 마우스 IgG2 유래의 중쇄 불변영역 2(CH2) 및 중쇄 불변영역 3(CH3)을 포함할 수 있으나, 이에 제한되는 것은 아니다.As a preferred example in the present invention, the immunoglobulin Fc region may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from human IgG1 represented by SEQ ID NO: 37, or may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) derived from mouse IgG2 represented by SEQ ID NO: 38, but is not limited thereto.

본 발명의 일 예시로, 상기 면역글로불린 Fc 영역은 IgG, IgA, IgM, IgD, IgE, 또는 아바타셉트(Abatacept) 유래의 힌지 영역을 포함할 수 있고, 또 다른 예시로 IgG, IgD 또는 아바타셉트(Abatacept) 유래의 힌지 영역을 포함할 수 있으며, 혹은 IgG1, IgG2, IgG3, IgG4, IgD 또는 아바타셉트(Abatacept) 유래의 힌지 영역을 포함할 수 있으나, 이에 제한되는 것은 아니다. As an example of the present invention, the immunoglobulin Fc region may include a hinge region derived from IgG, IgA, IgM, IgD, IgE, or Abatacept, or as another example, may include a hinge region derived from IgG, IgD or Abatacept, or may include a hinge region derived from IgG1, IgG2, IgG3, IgG4, IgD or Abatacept, but is not limited thereto.

본 발명에서 바람직한 예시로서 상기 면역글로불린 Fc 영역은 서열번호 39로 표시되는 인간 IgG1 유래의 힌지 영역; 서열번호 40로 표시되는 마우스 IgG2 유래의 힌지 영역; 서열번호 41로 표시되는 인간 IgD 유래의 힌지 영역; 및 서열번호 42으로 표시되는 아바타셉트(Abatacept)의 힌지 영역;으로 이루어진 군에서 선택된 1종 이상을 포함함으로써 최종 제조될 융합 단백질의 구조적 유연성(flexibility)을 높이고, 융합 단백질의 생산성 및 안정성을 현저히 향상시킬 수 있으나, 이에 제한되는 것은 아니다.As a preferred example in the present invention, the immunoglobulin Fc region may include at least one selected from the group consisting of a hinge region derived from human IgG1 represented by SEQ ID NO: 39; a hinge region derived from mouse IgG2 represented by SEQ ID NO: 40; a hinge region derived from human IgD represented by SEQ ID NO: 41; and a hinge region of Abatacept represented by SEQ ID NO: 42; thereby increasing the structural flexibility of the fusion protein to be finally manufactured and significantly improving the productivity and stability of the fusion protein, but is not limited thereto.

본 발명에서 상기 DKK-1 단백질의 세포 외 도메인과 Fc 영역이 링커를 통해 연결되는 경우, 상기 링커는 Fc 단편의 N-말단, C-말단 또는 유리기 (free radical)에 연결될 수 있고, DKK-1 단백질의 세포 외 도메인의 N-말단, C-말단 또는 유리기에 연결될 수 있다. 링커가 펩타이드 링커인 경우, 연결은 임의의 부위에서 일어날 수 있다. 예를 들면, 상기 링커는 상기 DKK-1 단백질의 세포 외 도메인의 C-말단 및 상기 면역글로불린의 Fc 영역의 N-말단에 연결될 수 있고, 혹은 상기 면역글로불린의 Fc 영역의 C-말단 및 상기 DKK-1 단백질의 세포 외 도메인의 N-말단에 연결될 수 있다. In the present invention, when the extracellular domain and the Fc region of the DKK-1 protein are connected via a linker, the linker can be connected to the N-terminus, the C-terminus or a free radical of the Fc fragment, and can be connected to the N-terminus, the C-terminus or a free radical of the extracellular domain of the DKK-1 protein. When the linker is a peptide linker, the connection can occur at any site. For example, the linker can be connected to the C-terminus of the extracellular domain of the DKK-1 protein and the N-terminus of the Fc region of the immunoglobulin, or can be connected to the C-terminus of the Fc region of the immunoglobulin and the N-terminus of the extracellular domain of the DKK-1 protein.

본 발명에서 상기 "링커(linker)"는 융합 단백질 내 DKK-1 단백질의 세포 외 도메인과 면역글로불린 Fc 영역 사이의 간섭 효과를 줄여 타겟 세포에서 상기 DKK-1 단백질의 세포 외 도메인의 목적하는 활성을 높일 수 있다. 또한, 본 발명에서 상기 링커는 목적하는 질환의 조직 또는 세포 내에서 과발현되는 효소에 의해 절단될 수 있는 서열을 포함할 수 있다. 상기와 같이 과발현되는 효소에 의해 절단될 수 있는 경우에는 Fc 부분으로 인하여 폴리펩타이드의 활성이 저하되는 것을 효과적으로 방지할 수 있다.In the present invention, the "linker" can reduce the interference effect between the extracellular domain of the DKK-1 protein and the immunoglobulin Fc region in the fusion protein, thereby increasing the desired activity of the extracellular domain of the DKK-1 protein in the target cell. In addition, the linker in the present invention can include a sequence that can be cleaved by an enzyme that is overexpressed in the tissue or cell of the desired disease. In the case where it can be cleaved by the overexpressed enzyme as described above, the activity of the polypeptide can be effectively prevented from being reduced due to the Fc portion.

또한, 본 발명에서 상기 링커의 일 예시로, 혈액 내에 가장 많이 존재하는 인간 알부민의 282번 내지 314번째 부분에 위치한 33개의 아미노산으로 이루어진 펩타이드 링커, 보다 바람직하게는 292번 내지 304번째 부분에 위치한 13개의 아미노산으로 이루어진 펩타이드 링커일 수 있으며, 이러한 부분은 3차원적인 구조상 대부분 외부에 노출된 부분으로서 체내에서 면역반응을 유도할 가능성이 최소화된 부분이다. 단, 이에 제한되는 것은 아니다. In addition, as an example of the linker in the present invention, a peptide linker composed of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in blood, more preferably a peptide linker composed of 13 amino acids located at positions 292 to 304, is a portion that is mostly exposed to the outside in the three-dimensional structure and thus has a minimal possibility of inducing an immune response in the body. However, it is not limited thereto.

또한, 본 발명에서 상기 링커 및 Fc 영역이 별개로 발현된 후에 서로 결합될 때, 링커는 당업계에 알려진 가교제일 수 있다. 상기 가교제는, 예를 들어, 1,1-비스(디아조아세틸)-2-페닐에탄 (1,1-bis(diazoacetyl)-2-phenylethane), 글루타르알데하이드 (glutaraldehyde), 4-아지도살리실릭산 (4-azidosalicylic acid)과 같은 N-하이드로옥시석신이미드 에스테르 (N-hydroxysuccinimide ester), 3,3'-디사이오비스(석신이미딜프로피오네이트) (3,3'-dithiobis(succinimidylpropionate))와 같은 디석신이미딜에스테르 (disuccinimidyl esters)를 포함하는 이미도에스테르 (imidoesters), 및 비스-N-말레이미도-1,8-옥테인과 같은 이중 기능적 말레이미드 (bifunctional maleimides)일 수 있으나, 이에 제한되는 것은 아니다.Additionally, when the linker and Fc region in the present invention are expressed separately and then linked to each other, the linker may be a cross-linking agent known in the art. The crosslinking agent may be, but is not limited to, imidoesters including, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as 4-azidosalicylic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.

본 발명의 일 구현예에 따르면, LRP-5, 6 및 면역글로불린 Fc 영역을 포함하는 융합 단백질을 유효성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis or cancer is provided, comprising a fusion protein comprising LRP-5, 6 and an immunoglobulin Fc region as an active ingredient.

상기 융합 단백질은 DKK-1과 결합하는 리간드인 LRP-5, 6을 포함하고 있으므로, LRP-5, 6과 상기 융합 단백질은 경쟁적으로 DKK-1에 결합하는 바, DKK-1을 경쟁적으로 억제함으로써 섬유증 또는 암을 치료할 수 있다.Since the above fusion protein contains LRP-5, 6, which is a ligand that binds to DKK-1, LRP-5, 6 and the above fusion protein competitively bind to DKK-1, and thus fibrosis or cancer can be treated by competitively inhibiting DKK-1.

상기 융합 단백질에 대해서는 앞서 살펴본 DKK-1과 면역글로불린 Fc 영역을 포함하는 융합 단백질에서 자세히 서술하였는바, 중복을 피하기 위하여 생략한다.The above fusion protein has been described in detail in the fusion protein comprising DKK-1 and the immunoglobulin Fc region discussed above, and is omitted to avoid duplication.

본 발명의 다른 구현예에 따르면, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 약학 조성물을 제공한다.According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells.

도 1는 DKK-1 발현 수준이 인간 특발성 폐 섬유증 환자에게서 상승한 것을 나타낸 것이다.Figure 1 shows that DKK-1 expression levels are elevated in patients with human idiopathic pulmonary fibrosis.

도 2은 BLM으로 자극된 폐에서 DKK-1의 발현이 증가된 것을 나타낸 것이다.Figure 2 shows that the expression of DKK-1 was increased in the lungs stimulated with BLM.

도 3은 DKK-1 저형 (hypomorphic) Dkk-1d/d 마우스는 BLM 매개 폐 손상으로부터 보호됨을 나타낸 것이다.Figure 3 shows that DKK-1 hypomorphic Dkk-1 d/d mice are protected from BLM-mediated lung injury.

도 4은 혈소판은 DKK-1 조직학적 관찰의 주요 소스임을 나타낸 것이다.Figure 4 shows that platelets are the main source of DKK-1 histological observations.

도 5은 골수유래 대식세포(Bone marrow-derived macrophage, BMDM)에서 DKK-1 매개 대안 활성화 대식세포 (Alternatively activated macrophage, AAM) 마커 발현을 나타낸 것이다.Figure 5 shows the expression of alternatively activated macrophage (AAM) markers mediated by DKK-1 in bone marrow-derived macrophages (BMDM).

도 6은 DKK1 수용체 LRP6의 제거는 BLM 유발 손상으로부터 마우스를 보호함을 나타낸 것이다.Figure 6 shows that deletion of the DKK1 receptor LRP6 protects mice from BLM-induced injury.

도 7은 지연된 DKK1 항체 치료는 BLM 유발 폐 손상 모델에서 콜라겐 침착을 억제함 나타낸 것이다.Figure 7 shows that delayed DKK1 antibody treatment inhibits collagen deposition in a BLM-induced lung injury model.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, 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.

실시예Example

[실시예 1] DKK-1 단백질 발현 수준이 인간 특발성 폐 섬유증 (Idiopathic Pulmonary Fibrosis, IPB) 환자 폐 조직에서 상승함을 확인.[Example 1] It was confirmed that the level of DKK-1 protein expression was increased in lung tissue of human idiopathic pulmonary fibrosis (IPB) patients.

폐섬유화 조직에서의 DKK-1이 발현을 인간 조직에서 확인하기 위해 파라핀 블록 고정화 되어 있는 인간 폐 조직 array kit를 구매하였다 (Pulmonary interstitial fibrosis tissue array, LC561; Biomax). 각 폐 조직은 Histoclear로 탈 파라핀화 (deparaffinize) 한 뒤 탈수하였다. Sodium citrate buffer (10 mM, pH 6,0)로 antigen을 제거하였다. Endogeneous peroxidase 활성을 저해하기 위해 3% H2O2로 블락킹 하였다. 각 section은 5% BSA에서 블락킹 한 뒤, 1차 항체를 (Goat polyclonal anti-m/hDKK1, R&D systems; AF1096) 처리하였고, 5 ug/mL농도로 2차항체를 염색하였다. DAB substrate kit (SK-4100; Vector Laboratories)을 이용하여 항체 발색을 하였고, 더불어 H&E 염색을 동시에 진행하여 이미지를 관찰하였다. 그 결과를 도 1에 나타내었다.To confirm the expression of DKK-1 in pulmonary fibrosis tissues in human tissues, a human lung tissue array kit fixed in paraffin blocks was purchased (Pulmonary interstitial fibrosis tissue array, LC561; Biomax). Each lung tissue was deparaffinized with Histoclear and dehydrated. Antigens were removed with sodium citrate buffer (10 mM, pH 6.0). To inhibit endogenous peroxidase activity, the sections were blocked with 3% H2O2. Each section was blocked in 5% BSA, treated with primary antibody (Goat polyclonal anti-m/hDKK1, R&D systems; AF1096), and stained with secondary antibody at a concentration of 5 ug/mL. Antibody color development was performed using a DAB substrate kit (SK-4100; Vector Laboratories), and H&E staining was performed simultaneously to observe the images. The results are shown in Figure 1.

도 1에서 보는 바와 같이, 건강한 사람보다 인간 특발성 폐 섬유증 (Idiopathic Pulmonary Fibrosis, IPB) 환자에서 DKK1의 발현이 매우 높음을 확인할 수 있었다.As shown in Figure 1, it was confirmed that the expression of DKK1 was much higher in patients with human idiopathic pulmonary fibrosis (IPB) than in healthy people.

[실시예 2] DKK-1은 블레오마이신 (Bleomycin, BLM)을 처리한 경우 발현이 증가함을 확인.[Example 2] It was confirmed that the expression of DKK-1 increased when treated with bleomycin (BLM).

C57BL/6 마우스는 BLM 4U/kg을 40 uL PBS에 용해시켜 각 마우스에 비강 내 (intranasal)로 처리하였다. 8~10 주령 마우스에 BLM을 처리하고 2주 뒤 마우스를 희생하여 폐를 적출한다. 적출한 폐는 폐 관류 (lung perfusion)을 거쳐 10% 포르말린으로 최소 48시간 고정하고 조직학 분석을 위해 파라핀 블록화 하였다. 이것을 microtome으로 절개 (section) 하였다. 파라핀 블록화 된 폐조직은 [실시예 1]에 기재한 방법으로 동일하게 면역염색법을 실시하였다. 그 결과를 도 2에 나타내었다.C57BL/6 mice were treated intranasally with 4 U/kg of BLM dissolved in 40 μL PBS. Mice aged 8 to 10 weeks were treated with BLM, and the mice were sacrificed 2 weeks later and their lungs were removed. The removed lungs were fixed in 10% formalin for at least 48 hours after lung perfusion and embedded in paraffin blocks for histological analysis. These were sectioned using a microtome. The paraffin-blocked lung tissues were subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Fig. 2.

도 2에서 보는 바와 같이, 블레오마이신 (Bleomycin, BLM)으로 섬유증이 유발된 경우 DKK-1의 발현이 증가함을 알 수 있었다.As shown in Figure 2, it was found that the expression of DKK-1 increased when fibrosis was induced by bleomycin (BLM).

[실시예 3] DKK-1 저형 (hypomorphic) Dkk-1[Example 3] DKK-1 hypomorphic Dkk-1 d/dd/d 마우스는 BLM 매개 폐 손상으로부터 보호됨을 확인.Mice were found to be protected from BLM-mediated lung injury.

WT 및 DKK-1d/d 마우스(8-10주령)를 BLM 유발 폐 섬유증 마우스 그룹 또는 식염수 그룹으로 무작위 배정했다. BLM으로 유도된 폐 섬유증 마우스 그룹을 1% 펜토바르비탈 나트륨으로 마취한 다음 보고된 바와 같이 기관내 경로를 통해 40μl 생리식염수 중 4U/kg BLM (BLM, S121415; Selleckchem, Houston, TX)을 투여했다. 동일한 양의 생리 식염수를 투여한 마우스를 대조군으로 사용하고 폐 섬유증 분석을 위해 BLM 투여 후 14일 후에 마우스를 희생시켰다. 마우스로부터 폐를 적출하여, 폐 관류을 거쳐 10% 포르말린으로 최소 48시간 고정하고 조직학 분석을 위해 파라핀 블록화 하였다. 이것을 microtome으로 절개하였다. 파라핀 블록화 된 폐조직은 [실시예 1]에 기재한 방법으로 동일하게 면역염색법을 실시하였다. 그 결과를 도 3에 나타내었다.WT and DKK-1 d/d mice (8-10 weeks old) were randomly assigned to BLM-induced lung fibrosis mice group or saline group. BLM-induced lung fibrosis mice group was anesthetized with 1% sodium pentobarbital and administered 4 U/kg BLM (BLM, S121415; Selleckchem, Houston, TX) in 40 μl saline via the intratracheal route as reported. Mice administered the same amount of saline were used as controls, and mice were sacrificed 14 days after BLM administration for lung fibrosis analysis. Lungs were removed from mice, fixed in 10% formalin for at least 48 h after lung perfusion, and embedded in paraffin blocks for histological analysis. These were sectioned with a microtome. The paraffin-blocked lung tissues were subjected to immunostaining in the same manner as described in [Example 1]. The results are shown in Fig. 3.

도 3에서 보는 바와 같이, DKK-1 저형인 경우 블레오마이신 (Bleomycin, BLM)으로부터 유발되는 폐 손상으로부터 상대적으로 보호받음을 알 수 있었다.As shown in Figure 3, it was found that DKK-1 low-formation mice were relatively protected from lung damage induced by bleomycin (BLM).

[실시예 4] 혈소판은 DKK-1 조직학적 관찰의 주요 소스임을 확인.[Example 4] Platelets were confirmed to be the main source of DKK-1 histological observations.

혈소판은 이전에 설명된 대로 일부 변형하여 마우스에서 분리했다. 심장 천자를 통해 혈액을 3.8% 구연산나트륨, pH 7.4 (2:1, 부피)로 수집하고 250g에서 10분 동안 원심분리하여 혈소판이 풍부한 혈장 (platelet rich plasma, PRP)을 얻었다. 혈소판 자극을 위해 PRP를 100 μM thrombin receptor activating peptide (SFLLRN; Sigma-Aldrich)로 표시된 시간 동안 처리한 후 657g에서 7분 동안 원심분리하였다. 상청액을 혈장으로 수집하고 혈소판 펠렛도 수집했다. 혈소판 방출물 수집을 위해 PRP를 657g에서 7분 동안 원심분리하여 혈소판을 펠릿화했다. 그런 다음 혈소판을 Tyrode-HEPES (N-2-하이드록시에틸피페라진-N'-2-에탄설폰산) 완충액에 재현탁하고 0.1U/mL 트롬빈 (Sigma-Aldrich)으로 90분 동안 자극했다. 이후 혈소판 방출물을 4°C에서 10분 동안 2500g에서 원심분리하여 수집했다.Platelets were isolated from mice as previously described with some modifications. Blood was collected by cardiac puncture into 3.8% sodium citrate, pH 7.4 (2:1, by volume) and centrifuged at 250 g for 10 min to obtain platelet-rich plasma (PRP). For platelet stimulation, PRP was treated with 100 μM thrombin receptor activating peptide (SFLLRN; Sigma-Aldrich) for the indicated times and then centrifuged at 657 g for 7 min. The supernatant was collected as plasma, and the platelet pellet was also collected. For platelet release, PRP was centrifuged at 657 g for 7 min to pellet platelets. Platelets were then resuspended in Tyrode-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer and stimulated with 0.1 U/mL thrombin (Sigma-Aldrich) for 90 min. Platelet release was then collected by centrifugation at 2500 g for 10 min at 4°C.

그 결과, 도 4에서 보는 바와 같이, 혈소판은 DKK-1 조직학적 관찰의 주요 소스가 됨을 알 수 있었다.As a result, as shown in Fig. 4, it was found that platelets were the main source of DKK-1 histological observation.

[실시예 5] 골수유래 대식세포(Bone marrow-derived macrophage, BMDM)에서 DKK-1 매개 대안 활성화 대식세포 (Alternatively activated macrophage, AAM) 마커 발현 확인[Example 5] Confirmation of expression of alternatively activated macrophage (AAM) marker mediated by DKK-1 in bone marrow-derived macrophages (BMDM)

DKK-1이 대안 활성화 대식세포의 발현을 조절하는지 확인하기 위해 골수유래 대식세포(Bone marrow-derived macrophage, BMDM)에서 DKK-1 처리 유무에 따른 Arg1과 Cd206의 발현양 변화를 유세포 분석으로 관찰하였다. BMDM 분화를 위해 마우스를 희생하고 day 0에 골수 세포를 추출하고, 20% FBS, GlutaMAX, MEM-NEAA, sodium pyruvate, 1x penicillin & streptomycin, 20 ng/mL의 MCSF (macrophage colony-stimulating factor) 환경의 DMEM 배지에서 배양하였다. 2일마다 배지를 교체해주고, day 6에 IL-4와 IL-13을 처리하여 M2-like 대식세포로의 분화를 유도하였다. To determine whether DKK-1 regulates the expression of alternatively activated macrophages, the expression levels of Arg1 and Cd206 in bone marrow-derived macrophages (BMDM) treated with or without DKK-1 were observed by flow cytometry. For BMDM differentiation, mice were sacrificed, bone marrow cells were extracted on day 0, and cultured in DMEM medium containing 20% FBS, GlutaMAX, MEM-NEAA, sodium pyruvate, 1x penicillin & streptomycin, and 20 ng/mL MCSF (macrophage colony-stimulating factor). The medium was changed every two days, and IL-4 and IL-13 were treated on day 6 to induce differentiation into M2-like macrophages.

도 5에서 보는 바와 같이, Arg+ 와 CD206+ 대식세포에서 DKK-1의 발현이 높음을 알 수 있었다.As shown in Figure 5, high expression of DKK-1 was found in Arg + and CD206 + macrophages.

[실시예 6] DKK1 수용체 LRP6의 제거는 골수유래 대식세포(Bone marrow-derived macrophage, BMDM) 유발 손상으로부터 마우스를 보호함을 확인.[Example 6] Deletion of the DKK1 receptor LRP6 protects mice from bone marrow-derived macrophage (BMDM)-induced damage.

선행연구를 통해 DKK-1의 결합분자로 알려진 LRP6가 DKK-1 매게 BLM-induced lung injury에 미치는 영향을 알아보았다. LRP6fl/fl 과, LysM-Cre LRP6fl/fl conditional KO 마우스를 BLM으로 폐손상을 유도하였고, 콜라겐 (collagen) mRNA 과 면역조직학적염색을 통해 폐 손상정도를 비교하였다. We investigated the effect of LRP6, known as a binding molecule of DKK-1 through previous studies, on DKK-1-mediated BLM-induced lung injury. LRP6 fl/fl and LysM-Cre LRP6 fl/fl conditional KO mice were induced with BLM, and the degree of lung damage was compared through collagen mRNA and immunohistochemical staining.

도 6에서 보는 바와 같이, DKK1 수용체 LRP6의 제거는 BLM 유발 손상으로부터 마우스를 보호함을 알 수 있었다.As shown in Figure 6, deletion of the DKK1 receptor LRP6 was found to protect mice from BLM-induced damage.

[실시예 7] 지연된 DKK1 항체 치료는 BLM 유발 폐 손상 모델에서 콜라겐 침착을 억제함을 확인.[Example 7] Delayed DKK1 antibody treatment was confirmed to inhibit collagen deposition in a BLM-induced lung injury model.

C57BL6 마우스에 BLM을 day 0에 처리하였고, DKK-1항체의 치료효과를 확인하기 위해 투여방법을 두가지로 나누어 150 μg/mouse DKK-1 항체를 도입하였다. 첫번째 투여군은 day -1,1,3,7,9 11에 항체를 처리하였고, 두번째는 day 7,9,11에 처리하였다. C57BL6 mice were treated with BLM on day 0, and 150 μg/mouse DKK-1 antibody was introduced in two administration methods to confirm the therapeutic effect of DKK-1 antibody. The first administration group was treated with antibody on days -1, 1, 3, 7, 9, and 11, and the second was treated on days 7, 9, and 11.

도 7에서 보는 바와 같이 두 투여군 모두 DKK-1의 발현양을 감소시켰고, 그에 따라 폐의 콜라겐 침착을 저해하였다. 지연된 DKK1 항체 치료도 미리 투여한 군과 비슷한 정도의 콜라겐 침착을 자해하는 충분한 치료효과를 나타내었다. As shown in Figure 7, both administration groups reduced the expression level of DKK-1, thereby inhibiting collagen deposition in the lungs. Delayed DKK1 antibody treatment also showed sufficient therapeutic effects, inhibiting collagen deposition to a similar degree as the pre-administered group.

[결론][conclusion]

DKK-1 저형 더블릿지(DKK-1 hypomorphic doubleridge) 마우스에서 DKK-1 발현 감소는 BLM을 통하여 유발된 염증 및 섬유증으로부터 보호된다. 또한 DKK-1PKO 마우스에서 DKK-1의 혈소판 특이 결실은 Arg1+ 및 CD206+와 같은 AAM 마커를 감소시키고 DKK-1은 2형 사이토카인 없이 여러 염증 유발 및 섬유화 촉진 마커를 유도한다. 더불어 DKK-1은 IL-13 매개 AAM 표현형을 강화하고 STAT6과 JNK의 공동 억제는 과도한 AAM 유사 대식세포 유전자 발현을 제거하기에 충분하다. 또한, Lrp6MKO 마우스의 골수 계통 세포에서 DKK-1 수용체 제거는 BLM을 통하여 유발된 염증 및 섬유증으로부터 보호된다. DKK-1 항체의 지연 치료는 BLM 유발 염증 및 섬유증을 억제한다.In DKK-1 hypomorphic doubleridge mice, reduction in DKK-1 expression protects against BLM-induced inflammation and fibrosis. Furthermore, platelet-specific deletion of DKK-1 in DKK-1 PKO mice reduces AAM markers such as Arg1 + and CD206 + , and DKK-1 induces several proinflammatory and profibrotic markers in the absence of type 2 cytokines. Furthermore, DKK-1 enhances IL-13-mediated AAM phenotype, and co-inhibition of STAT6 and JNK is sufficient to eliminate excessive AAM-like macrophage gene expression. Furthermore, deletion of the DKK-1 receptor in myeloid lineage cells of Lrp6 MKO mice protects against BLM-induced inflammation and fibrosis. Delayed treatment with DKK-1 antibodies suppresses BLM-induced inflammation and fibrosis.

그러므로 DKK-1은 섬유증 (fibrosis) 발생을 유도하는 중요한 타겟 단백질이므로, DKK-1의 기능이나 활성을 억제하는 저분자 물질 또는 단일클론항체가 섬유증 치료에 대한 새로운 치료제가 될 수 있고, 섬유증의 제어에 대하여 DKK-1+ Treg 세포가 중요하므로 DKK-1+ Treg 세포를 세포치료제로 이용할 수 있다. 또한 섬유증을 매개로 하는 고형암에서 DKK-1이 중요한 역할을 담당하는 바, DKK-1 또는 DKK-1+ Treg 세포 또는 DKK-1에 의하여 유도되는 TAM의 기능저해가 이들 섬유증 관련 고형암 (fibrosis-associated solid cancer)의 예방 및 치료에 중요한 역할을 담당할 수 있다.Therefore, since DKK-1 is an important target protein that induces the occurrence of fibrosis, small molecule substances or monoclonal antibodies that inhibit the function or activity of DKK-1 may be new therapeutic agents for the treatment of fibrosis, and since DKK-1 + Treg cells are important for the control of fibrosis, DKK-1 + Treg cells can be used as cell therapy agents. In addition, since DKK-1 plays an important role in solid cancer mediated by fibrosis, inhibition of the function of DKK-1 or DKK-1 + Treg cells or TAM induced by DKK-1 may play an important role in the prevention and treatment of these fibrosis-associated solid cancers.

이상에서 본 발명에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical idea of the present invention described in the claims.

Claims (31)

DKK-1의 세포외 표면 단백질 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 포함하는, 섬유증 또는 암의 진단용 조성물.A composition for diagnosing fibrosis or cancer, comprising an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1 or a DKK-1 epitope represented by any one of SEQ ID NOS: 4 to 36. 제1항에 있어서, In the first paragraph, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 진단용 조성물.A diagnostic composition wherein the above DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells. 제1항에 있어서, In the first paragraph, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단용 조성물.A diagnostic composition, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin. 제1항에 있어서,In the first paragraph, 상기 암은 고형암인, 진단용 조성물.A diagnostic composition wherein the above cancer is a solid cancer. 제4항에 있어서, In paragraph 4, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단용 조성물.A diagnostic composition, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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 muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. DKK-1 세포외 표면 단백질 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 프라이머, 프로브 또는 안티센스 뉴클레오티드를 포함하는, 섬유증 또는 암의 진단용 조성물.A composition for diagnosing fibrosis or cancer, comprising a primer, probe or antisense nucleotide specific for a gene encoding an epitope of DKK-1 as represented by any one of SEQ ID NOS: 4 to 36 or an extracellular surface protein of DKK-1. 제2항에 있어서, In the second paragraph, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 진단용 조성물.A diagnostic composition wherein the above DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells. 제6항에 있어서, In Article 6, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단용 조성물.A diagnostic composition, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin. 제6항에 있어서,In Article 6, 상기 암은 고형암인, 진단용 조성물.A diagnostic composition wherein the above cancer is a solid cancer. 제6항에 있어서, In Article 6, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단용 조성물.A diagnostic composition, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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 muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. 제1항 내지 제10항 중 어느 한 항의 조성물을 포함하는 섬유증 또는 암의 진단 키트. A diagnostic kit for fibrosis or cancer comprising a composition according to any one of claims 1 to 10. DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 이용하여 상기 표면 단백질 또는 에피토프의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법.A method for providing information about fibrosis or cancer by measuring the expression level of the extracellular surface protein of DKK-1 or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36 using an antibody or antigen-binding fragment that specifically binds to the surface protein or epitope. 제12항에 있어서, In Article 12, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 정보를 제공하는 방법.A method for providing information, wherein the above DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells. DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택되는 어느 하나를 이용하여 상기 유전자의 발현 수준을 측정하여 섬유증 또는 암에 관한 정보를 제공하는 방법.A method for providing information about fibrosis or cancer by measuring the expression level of a gene using any one selected from the group consisting of primers, probes and antisense nucleotides specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36. 제14항에 있어서, In Article 14, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 정보를 제공하는 방법.A method for providing information, wherein the above DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells. 제12항 내지 제15항 중 어느 한 항에 있어서, In any one of Articles 12 to 15, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 정보를 제공하는 방법.A method of providing information, wherein said fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin. 제12항 내지 제15항 중 어느 한 항에 있어서, In any one of Articles 12 to 15, 상기 암은 고형암인, 정보를 제공하는 방법.A method of providing information, wherein the cancer is a solid cancer. 제12항 내지 제15항 중 어느 한 항에 있어서, In any one of Articles 12 to 15, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 정보를 제공하는 방법.A method for providing information, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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 muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. (a) 목적하는 개체로부터 얻어진 생물학적 시료에 대하여 DKK-1; 또는 상기 단백질을 암호화하는 유전자의 발현 수준을 측정하는 측정부; 및 (a) a measuring unit for measuring the expression level of DKK-1 or a gene encoding the protein in a biological sample obtained from a target individual; and (b) 상기 측정부에서 측정된 유전자 또는 이에 의해 코딩되는 단백질의 발현 수준으로부터 암의 유무를 출력하는 검출부; 를 포함하는 섬유증 또는 암의 진단기기.(b) A diagnostic device for fibrosis or cancer, comprising a detection unit that outputs the presence or absence of cancer from the expression level of a gene or a protein encoded by the gene measured in the above measurement unit. 제19항에 있어서, In Article 19, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 진단기기.A diagnostic device, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin. 제19항에 있어서,In Article 19, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 진단기기.A diagnostic device, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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 muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. DKK-1의 세포 외 표면 단백질 및 면역글로불린 Fc 영역을 포함하는 융합 단백질을 유효성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating fibrosis or cancer, comprising as an active ingredient a fusion protein comprising the extracellular surface protein of DKK-1 and an immunoglobulin Fc region. LRP-5, 6 및 면역글로불린 Fc 영역을 포함하는 융합 단백질을 유효성분으로 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prevention or treatment of fibrosis or cancer, comprising as an active ingredient a fusion protein comprising LRP-5, 6 and an immunoglobulin Fc region. DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프에 특이적으로 결합하는 항체 또는 항원 결합 단편을 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating fibrosis or cancer, comprising as an active ingredient an antibody or antigen-binding fragment that specifically binds to an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36. DKK-1 억제제를 포함하는 섬유증 또는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating fibrosis or cancer comprising a DKK-1 inhibitor. 제25항에 있어서,In Article 25, 상기 DKK-1 억제제는 NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, endothelin-1 (ET-1)로 이루어진 군에서 선택되는 것인, 약학 조성물.A pharmaceutical composition, wherein the above DKK-1 inhibitor is selected from the group consisting of NCI8642 (Gallocyanine), BHQ880, DKN-01, RH2-18, Hetero-DS, Cyclized Oligopeptide, and endothelin-1 (ET-1). DKK-1의 세포 외 표면 단백질, 또는 서열번호 4 내지 36 중 어느 하나로 표시되는 DKK-1의 에피토프를 코딩하는 유전자에 특이적인 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 마이크로RNA으로 이루어진 군에서 선택되는 어느 하나를 유효 성분으로 포함하는, 섬유증 또는 암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating fibrosis or cancer, comprising as an active ingredient any one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA and microRNA specific for a gene encoding an extracellular surface protein of DKK-1, or an epitope of DKK-1 represented by any one of SEQ ID NOS: 4 to 36. 제22항 내지 제27항 중 어느 한 항에 있어서, In any one of Articles 22 to 27, 상기 DKK-1은 조절 T 세포 표면에 존재하거나, 세포로부터 분리되어 용해된 상태로 존재하는 것인, 약학 조성물.A pharmaceutical composition wherein the above DKK-1 is present on the surface of regulatory T cells or is present in a dissolved state after being separated from the cells. 제28항에 있어서, In Article 28, 상기 섬유증은 신장, 간, 폐, 심장, 뼈 또는 골수 및 피부로 구성된 군으로부터 선택되는 적어도 하나의 기관에 발생된 섬유증으로 이루어진 군에서 선택되는, 약학 조성물.A pharmaceutical composition, wherein the fibrosis is selected from the group consisting of fibrosis occurring in at least one organ selected from the group consisting of kidney, liver, lung, heart, bone or bone marrow and skin. 제28항에 있어서, In Article 28, 상기 암은 고형암인, 약학 조성물.A pharmaceutical composition wherein the cancer is a solid cancer. 제28항에 있어서, In Article 28, 상기 암은 위암, 갑상선암, 부갑상선암, 난소암, 대장암, 췌장암, 간암, 유방암, 자궁경부암, 폐암, 비소세포성폐암, 전립선암, 담낭암, 담도암, 비호지킨 림프종, 호지킨 림프종, 혈액암, 방광암, 신장암, 흑색종, 결장암, 골암, 피부암, 두부암, 자궁암, 직장암, 뇌종양, 항문부근암, 나팔관암종, 자궁내막암종, 질암, 음문암종, 식도암, 소장암, 내분비선암, 부신암, 연조직 육종, 요도암, 음경암, 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS central nervoussystem) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 적어도 하나인, 약학 조성물.A pharmaceutical composition, wherein the cancer is at least one selected from the group consisting of gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical 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 muscle cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.
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