WO2018208108A1 - Dkk1 protein present specifically in regulatory t cells and use thereof - Google Patents

Abstract

The present invention relates to a Dickkopf-1 protein present specifically on the surface of regulatory T cells (Treg cells) among immune cells, an antibody specific therefor, and a use thereof. Being present specifically on the surface of immune cells, inter alia, regulatory T cells, the Dickkopt-1 (DKK1) protein of the present invention can be a new target of various therapeutic agents for immune-related diseases.

Description

DKK1 protein specific to regulatory T cells and uses thereof

The present invention relates to DKK1 (Dickkopf-1) proteins, antibodies specific thereto, and uses thereof, which are specifically present on the surface of regulatory T cells (Treg cells), among immune cells.

Among the immune cells, T cells are immune cells that are produced in the bone marrow and mature in the thymus, and function to regulate antibody production of B cells or to control the functions of innate immune cells. The T cells, also called T lymphocytes, act not only as a helper for other immune cells, but also destroy direct invading substances.

One of the most important functions of immune cells is that the immune response is suppressed against the antigenic substances that make up the self surrounding the immune cells, while the immune response is recognized and recognized for non-self antigenic substances. To induce. Immune cells induce no response to self antigens through developmental death, induction of mutations in receptors specific for self antigens, or inactivation of immune cells that recognize self antigens. This is called immunologic unresponsiveness or tolerance. Failure to self-tolerance induces an immune response to self antigens, which can lead to disease, which is called autoimmune disease.

In the early 1970s, inducing or maintaining the self-tolerance, the concept of a regulatory T cell that can control the effector function of conventional T cells was introduced (RK Gershon and K. Kondo, Immunology). , 1970, 18: 723-37) Research has been conducted to determine the immunological properties and functions of inhibitory T cells.

In normal individuals, the regulatory T cells control the function of conventional T cells to induce an excessive immune response or self tolerance. However, in autoimmune and chronic inflammatory diseases, the function and number of regulatory T cells are markedly reduced. Inability to perform their functions properly has been reported. Therefore, in patients with autoimmune diseases and chronic inflammatory diseases, returning regulatory T cells to normal levels of function and numbers may be one of the treatment methods.

As cell surface proteins that can target regulatory T cells, CD25, CTLA4, CD38, CD62L, GITR, CD45RB, etc. have been suggested through research, and animal and clinical studies have been conducted, but cell surface capable of targeting only regulatory T cells alone. Protein has not been revealed to date.

One object of the present invention is to provide a DKK1 (Dickkopf-1) protein or antigenic determinant thereof specifically present on the surface of regulatory T cells.

Another object of the present invention is to provide an antibody specific for the DKK1 protein specifically present on the surface of the regulatory T cells.

Still another object of the present invention is to provide regulatory T cells in which DKK1 protein is expressed on the cell surface.

Another object of the present invention is to provide a method for screening an immunosuppressant or immune activator.

It is another object of the present invention to provide a method for preventing or treating an immune related disease.

Another object of the present invention is to provide a method for diagnosing an immune related disease.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The inventors have discovered a DKK1 (Dickkopf-1) protein that is specifically present on the surface of immune cells, in particular regulatory T cells, selected epitopes of the protein, immune cells expressing the DKK1 protein, In particular, by selectively selecting regulatory T cells and co-culturing them with effector T cells, the present inventors have found that they can inhibit the activity of effector T cells.

In the present invention, the DKK1 is one of the DKK family genes DKK1, DKK2, DKK3, and DKK4, and encodes a secreted protein. The DKK protein generally consists of 255 to 350 amino acids, and in the case of DKK1, has a size of 24 KDa to 29 KDa, and the N-terminus is present in the form of glycosylated sugar. As an example of the present invention, the DKK1 may be represented by the amino acid sequence of SEQ ID NO: 1, and may be encoded by the nucleotide represented by SEQ ID NO: 2 (Table 1).

division	Sequence information
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 CARHFWSKIC 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 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 (SEQ ID NO: 2)

In the present invention, the DKK1 binds to a receptor stronger than a ligand such as Wnt3a in the Wnt signaling system involved in cell proliferation and wound recovery, thereby competitively inhibiting Wnt signaling, and thus, in the development of the heart, head, hands, etc. in embryonic development. It can play an important role. In addition, the increased secretion protein type DKK1 present in bone marrow plasma can identify osteolytic bone lesions in patients with multiple myeloma and determine the degree of cancer or bone disease. Can be used.

However, in the present invention, the DKK1 may be present on the surface of immune cells, particularly regulatory T cells, without being secreted out of cells as in a general case. This may help immune cells function as regulatory T cells for self tolerance.

According to one embodiment of the invention, it provides a regulatory T extracellular surface protein of DKK1 represented by SEQ ID NO: 3 (Table 2).

division	Sequence information
DKK1 extracellular domain	Column

 The regulatory T cell extracellular surface protein of DKK1 provided by the present invention may interact with a ligand present in effect T cells to reduce the activity of regulatory T cells.

According to another embodiment of the present invention, an antigenic epitope of DKK1, which is a surface protein of regulatory T cells, represented by any one of SEQ ID NOs: 4 to 36, is provided (Table 3).

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

In the present invention, the "antigen determinant" (Epitope) is a site that binds the antibody in the antigen molecule, and means a part that can be recognized by the antibody. In general, the antibody does not recognize the entire antigen molecule, but recognizes only a specific part, and even the same antigen molecule can recognize other antigenic determinant parts when the type of the antibody is different. For the purposes of the present invention, the antigenic determinant is an antigenic determinant of an antibody that can effectively bind to the remaining domains even when a portion of the DKK1 protein exposed to the outside of the cell is cleaved and secreted out of the cell It includes.

In the present invention, the polypeptide which is the antigenic determinant may include both a continuous or discontinuous sequence of a portion capable of binding an antibody to the DKK1 protein according to the present invention.

In addition, the polypeptide fragment which is the antigenic determinant of DKK1, which is the surface protein of the regulatory T cells provided by the present invention, may interact with a ligand present in the effect T cells to reduce the activity of the regulatory T cells.

According to another embodiment of the present invention, there is provided an extracellular surface protein of DKK1 represented by SEQ ID NO: 3 provided in the present invention, or a polynucleotide encoding the antigenic determinant of DKK1 of any one of SEQ ID NOs: 4 to 36. .

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

In the present invention, the "vector" is the 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 circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors are episomal mammalian vectors of bacterial replication origin). Other vectors (eg, non-episomal mammalian vectors) can enter the host cell and integrate into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can direct the expression of the genes to which they are linked in a working dimension. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably because the plasmid is the most commonly used form of the vector.

Specific examples of the expression vector in the present invention include commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; It can be selected from the group consisting of plant viruses, but is not limited thereto. All expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and the selection of the expression vector depends on the nature of the host cell of interest. The introduction of the vector into host cells can be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto. An introduction method suitable for the expression vector and the host cell can be selected and used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and may be selected depending on whether the product is produced using a vector that does not include the selection marker. The selection of the selection marker is selected by the host cell of interest, which uses methods already known to those skilled in the art and the present invention is not so limited.

In order to facilitate purification of the nucleic acid molecules of the present invention, tag sequences can be inserted and fused to an expression vector. The tag may include, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag that facilitates purification known to those skilled in the art may be used in the present invention.

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

The term "host cell" in the present invention includes individual cells or cell cultures that may or may have been recipients of the vector (s) for incorporation of the polypeptide insert. Host cells include the progeny of a single host cell, which may not necessarily be exactly the same (in morphological or genomic DNA complement) as the original parent cell because of natural, accidental or intentional mutations. Host cells include cells transfected in vivo with the polypeptide (s) herein.

In the present invention, the host cell may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; Fungal cells such as yeast cells and peach pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Chinese hamster ovary cells (CHO), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK (Baby hamster kidney cells, Baby Hamster Kidney cells), animal cells of HEK (Human Embryonic Kidney cells) or PERC.6 (human retinal cells); Or it may be a plant cell, but is not limited thereto, any cell that can be used as a host cell line known to those skilled in the art is available.

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

The regulatory T cells expressing the extracellular surface protein of DKK1 or antigenic determinants thereof on the surface of the present invention may inhibit the cellular activity through interaction with the effect T cells and suppress an immune response.

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

Regulatory T cells comprising the extracellular surface protein of DKK1 of the present invention may inhibit their cellular activity through interaction with effect T cells and inhibit an immune response.

In the present invention, regulatory T cells expressing DKK1 protein on the cell surface are immune-related diseases caused by hyperimmune reactions, for example, autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic, acute and chronic Inflammatory diseases and the like can be effectively prevented, improved or treated.

According to another embodiment of the present invention, the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient, provides a pharmaceutical composition for the prevention or treatment of immune-related diseases.

The DKK1 extracellular surface protein or antigenic determinant fragment provided by the present invention interacts with a ligand present in effect T cells to inhibit the activity of regulatory T cells, thereby effectively preventing immune related diseases, for example cancer. Can be improved or treated.

In the present invention, the term "cancer" refers to or indicates a physiological condition characterized by unregulated cell growth in mammals. In the present invention, the cancer to be prevented, improved or treated may be a solid tumor composed of agglomerates caused by abnormal growth of cells in a solid organ, and according to the site of the solid organ, stomach cancer, liver cancer, Glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer, and the like, and preferably, but not limited to melanoma.

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

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

In the present invention, the antibody may inhibit the activity of regulatory T cells to effectively prevent, ameliorate or treat cancer.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein of the present invention, which antibody can be cloned into an expression vector according to a conventional method to produce a protein encoded by the marker gene. Can be obtained and prepared from conventional proteins by conventional methods. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids.

The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

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

In one embodiment of the present invention, the antibody specifically binds to a DKK1 protein or antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells to effectively prevent immune-related diseases, for example cancer. Can be improved or treated.

In the present invention, the cancer may be a solid tumor composed of agglomerates generated by abnormal growth of cells in a solid organ, and according to the site of the solid organ, stomach cancer, liver cancer, glioblastoma, ovarian cancer, and colon cancer , Head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another embodiment of the present invention, the antibody binds specifically to a DKK1 protein or antigenic determinant thereof expressed on the surface of regulatory T cells, thereby increasing the activity of regulatory T cells. Disease, graft-versus-host disease, transplant rejection, asthma, atopy, acute and chronic inflammatory diseases can be effectively prevented, ameliorated or treated.

According to another embodiment of the present invention, any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA specific to the DKK1 extracellular surface protein of the present invention or the gene encoding the antigenic determinant is effective. Provided as an ingredient, a pharmaceutical composition for immune-related diseases.

In the present invention, the "antisense oligonucleotide" is a short length DNA synthesis strand (or DNA analog) that is antisense (or complementary) to a specific DNA or RNA target, and achieves gene-specific inhibition in vivo as well as in vitro. Used to Antisense oligonucleotides have been proposed to prevent 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 been successfully used in cell culture and animal models of disease. Other modifications of antisense oligonucleotides to make them more stable and resistant to degradation of oligonucleotides are known and understood by those skilled in the art. Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications to oligonucleotide backbones, modifications of sugar moieties or bases known in the art.

In the present invention, "siRNA (small interfering RNA)" is a nucleic acid molecule that can mediate RNA interference or gene silencing, and because it can suppress the expression of target genes, it is an efficient gene knockdown method or gene therapy method. Used. When the siRNA molecule is used in the present invention, a double stranded structure or self-complementary (sense strand (sequence corresponding to the WLS mRNA sequence) and antisense strand (sequence complementary to the WLS mRNA sequence) is located opposite each other to form a double-chain -complementary) can have a single chain structure with sense and antisense strands. siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but can be paired by mismatch (the corresponding base is not complementary), expansion / protrusion (there is no base corresponding to one chain), and the like. Parts that do not achieve may be included. The siRNA terminal structure can be either a blunt end or a sticky end as long as the expression of the WLS gene can be inhibited by RNA interference (RNAi) effect. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures. The siRNA molecule is not limited thereto, but may have a total length of 15 to 30 bases, preferably 19 to 21 bases.

In the present invention, the "shRNA (short hairpin RNA)" is a single stranded RNA having a length of 45 to 70 nucleotides, and connects 3-10 base linkers between the sense strand of the target gene siRNA sequence and the complementary antisense strand. After synthesizing oligo DNA and cloning into a plasmid vector or inserting and expressing shRNA into the retrovirus lentivirus and adenovirus, a shuffle with a looped hairpin structure is produced and the siRNA is produced by intracellular dicer. Is converted to show RNAi effect.

In the present invention, the "microRNA" regulates various biological processes such as development, differentiation, proliferation, conservation and apoptosis. MicroRNAs generally regulate the expression of the gene encoding the target mRNA by destabilizing the target mRNA or disrupting translation.

Regulatory sequences useful in the expression constructs / vectors with the antisense oligonucleotides, siRNAs, shRNAs or microRNAs in the present invention (eg, constitutive promoters, inducible promoters, tissue specific promoters or combinations thereof) are also known in the art. It may be appropriately selected from the known contents in the following.

In one embodiment of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA specifically binds to a gene encoding a DKK1 protein or antigenic determinants thereof expressed on the surface of regulatory T cells to inhibit their expression and immunity. Related diseases can, for example, effectively prevent, ameliorate or treat cancer.

In the present invention, the cancer may be a solid tumor composed of agglomerates generated by abnormal growth of cells in a solid organ, and according to the site of the solid organ, stomach cancer, liver cancer, glioblastoma, ovarian cancer, and colon cancer , Head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another embodiment of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA specifically binds to the gene encoding the DKK1 protein or antigenic determinants expressed on the surface of regulatory T cells to increase their expression. Therefore, as an immune-related disease, for example, autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases can be effectively prevented, improved or treated.

In the present invention, "prophylaxis" may include without limitation any action that blocks, suppresses or delays the symptoms of the disease using the pharmaceutical composition of the present invention.

In addition, in the present invention, "treatment" may include without limitation any action that improves or benefits the symptoms of the disease using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in that the capsule, tablets, granules, injections, ointments, powder or beverage form, the pharmaceutical composition may be characterized in that it is intended for humans. Preferably, the pharmaceutical composition according to the present invention may be prepared in the form of an injection and injected directly to a site where cancer or an immune disease occurs, but is not limited thereto.

The pharmaceutical compositions of the present invention can be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. In the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or in multiple dosage forms. have. And other solutions, suspensions, tablets, capsules, sustained release preparations and the like.

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

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The pharmaceutical composition of the present invention may be further administered in combination with other anticancer agents, thereby effectively inhibiting general cancer cell proliferation and cancer metastasis, thereby being used for the treatment of cancer.

Wherein the anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, zefitinib, vandetanib, nirotinib, semasanib, conservinib, axitinib, cediranib , Restautinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, biscumalboom, asparaginase, tretinoin, hydroxycarba Amide, dasatinib, estramastine, gemtuzumab ozogamycin, ibritumab tucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate Chitosan, gemcitabine, doxyfluridin, pemetrexed, tegapur, capecitabine, gimerasin, oteracil, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enosi Tarbin, flutamide, Capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmoper, raltitrexed, docetaxel, paclitaxel, irinotecan, velotecan, topotecan, vinorelbine, etoposide, vinblastine, idarubi Sin, mitomycin, bleomycin, dactinomycin, pyrurubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, iphosphamide, cyclophosphamide, melfaran, alt Lettmin, dacarbazine, cheotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutesimid, anagrelide, olopalip, navelvin, padrazole , At least one selected from the group consisting of tamoxifen, toremifene, testosterone, anastrozole, letrozole, borosol, bicalutamide, lomustine, 5FU, vorinostet, entinostet, and carmustine However, the limitation is no.

The pharmaceutical composition of the present invention may be further administered in combination with other immunosuppressive agents, thereby effectively inhibiting diseases due to general hyperimmune reactions.

In the present invention, the immunosuppressive agents include glucocorticoids, cyclophosphamid, cyclosporin, tacrolimu, rapamycin, type IV PDE inhibitors (Type IV PDE inhibitors). , p38 kinase inhibitors, azathioprine, mycophenolate mofetil, mizoribin, methotrexate, leflunomid, and brequinamide Brequina) may be used one or more selected from the group consisting of, but is not limited thereto.

According to another embodiment of the present invention, a method for preventing or treating an immune related disease, comprising administering to a subject in need thereof an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention. It is about.

The subject in need of the treatment in the present invention is an individual with an immune related disease or suspected of the symptoms, wherein one example of the immune related disease is gastric cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, two Cancer, such as cervical cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer, and other examples include autoimmune diseases, graft versus host disease, organ transplant rejection, asthma, atopy or Acute and chronic inflammatory diseases.

In one embodiment of the present invention, the antibody specifically binds to a DKK1 protein or antigenic determinant thereof expressed on the surface of regulatory T cells to inhibit the activity of regulatory T cells to effectively prevent immune-related diseases, for example cancer. Can be improved or treated.

In another embodiment of the present invention, the antibody binds specifically to a DKK1 protein or antigenic determinant thereof expressed on the surface of regulatory T cells, thereby increasing the activity of regulatory T cells. Disease, graft-versus-host disease, transplant rejection, asthma, atopy, acute and chronic inflammatory diseases can be effectively prevented, ameliorated or treated.

According to another embodiment of the present invention, a group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the DKK1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant to a subject in need thereof It relates to a method for preventing or treating an immune related disease, comprising the step of administering any one selected from.

The subject in need of the treatment in the present invention is an individual with an immune related disease or suspected of the symptoms, wherein one example of the immune related disease is gastric cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, two Cancer, such as cervical cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer, and other examples include autoimmune diseases, graft versus host disease, organ transplant rejection, asthma, atopy or Acute and chronic inflammatory diseases.

In one embodiment of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA specifically binds to a gene encoding a DKK1 protein or antigenic determinants thereof expressed on the surface of regulatory T cells to inhibit their expression and immunity. Related diseases can, for example, effectively prevent, ameliorate or treat cancer.

In another embodiment of the present invention, the antisense oligonucleotide, siRNA, shRNA or microRNA specifically binds to the gene encoding the DKK1 protein or antigenic determinants expressed on the surface of regulatory T cells to increase their expression. Therefore, as an immune-related disease, for example, autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases can be effectively prevented, improved or treated.

Administration in one embodiment of the invention means introducing the composition of the invention to the patient in any suitable manner, the route of administration of the composition of the invention being administered via any general route as long as it can reach the desired tissue. Can be. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not. The method of treatment of the present invention may comprise administering the antibody, antisense oligonucleotide, siRNA, shRNA or microRNA in a pharmaceutically effective amount. In the present invention, the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently. In adults, when the expression inhibitor of the gene or the activity of the protein is administered once or several times, 0.01ng / kg-10mg / kg for siRNA, 0.01ng for the antisense oligonucleotide for mRNA of the gene / kg-10mg / kg, 0.1ng / kg-10mg / kg for the compound, 0.1ng / kg-10mg / kg for the monoclonal antibody to the protein can be administered.

In the present invention, the above-mentioned administration may be administered in combination with another anticancer agent or another immunosuppressant.

According to another embodiment of the present invention, (a) contacting a sample to be assayed with an extracellular surface protein of DKK1 or a regulatory T cell comprising the antigenic determinant; (b) measuring the expression level of said extracellular surface protein or said antigenic determinant; And (c) determining that the sample is an immune activator or immunosuppressant when the level of expression of the extracellular surface protein or antigenic determinant is measured to be down or upregulated. A method for screening an agent or an immunosuppressant.

In the present invention, the "screening" is to select a substance having a specific characteristic of interest from a candidate group consisting of various substances by a specific manipulation or evaluation method. For the purposes of the present invention, the screening of the present invention is directed to immunosuppressive or immunosuppressive agents when the expression of a protein of the present invention is reduced or increased after administration of a candidate agent for the treatment of such a disease to a subject suspected of an immune related disease. It is a screening method to discriminate with an activator. Means such as molecular biological assays, digital imaging, cytological and histological examinations can be used to measure the activity of the genes and proteins of the invention on disease pathological or suspicious tissue.

In the present invention, the expression level of the extracellular surface protein of the DKK1 or the antigenic determinant, the amount or activity may be carried out using an antibody capable of specifically binding to the extracellular surface protein of the DKK1 or the antigenic determinant. . In the present invention, the antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the same is used.

In the present invention, the "antigen-antibody complex" means a combination of a protein antigen for identifying the presence or absence of a gene of interest in a biological sample and an antibody which recognizes the same. The detection of the antigen-antibody complex can be detected using methods as known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.

In the present invention, a method for measuring or comparing the expression level of the protein may include protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and SELDI-TOF. (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbent assay (ELISA). no.

According to another embodiment of the present invention, (a) contacting a sample to be analyzed with a regulatory T cell comprising the extracellular surface protein of DKK1 or a gene encoding the antigenic determinant; (b) measuring the expression level of the gene; And (c) determining that the sample is an immune activator or immunosuppressant when it is determined that the expression level of the gene is reduced or upregulated. It is about.

In the present invention, the expression level of the extracellular surface protein of DKK1 or the gene encoding the antigenic determinant may be performed using primers, probes or antisense nucleotides capable of specifically binding to the gene. Since the information of the extracellular surface protein of the DKK1 or the antigenic determinant and the gene encoding the same according to the present invention is known, those skilled in the art based on the primer, probe or antisense nucleotide to specifically bind to the gene encoding the protein It will be easy to design.

In the present invention, the primer refers to a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3-terminal hydroxyl group and serving as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, the PCR can be performed using the sense and antisense primers of genes encoding the extracellular surface protein of DKK1 to diagnose cancer through the generation of desired products. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

In addition, in the present invention, the probe refers to nucleic acid fragments such as RNA or DNA corresponding to short bases to hundreds of bases capable of specific binding with mRNA, and are labeled to confirm the presence or absence of specific mRNAs. have. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, hybridization is performed using a probe complementary to a gene encoding an extracellular surface protein of DKK1, and cancer can be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, "capsulation", substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, Phosphoramidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

In the present invention, as a method for measuring or comparing the expression level of the gene, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. may be used. May be, but is not limited thereto.

According to another embodiment of the present invention, the DKK1 extracellular surface protein of the present invention or an antibody specific for the antigenic determinant is provided as an active ingredient, a diagnostic composition for an immune related disease.

In the present invention, the "diagnosis" refers to confirming the presence or characteristic of a pathological state, and for the purpose of the present invention, the diagnosis is to confirm the onset and metastasis of gastric cancer.

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

In the present invention, the immune-related disease can be diagnosed by measuring the expression level of the DKK1 extracellular surface protein or the antigenic determinant present on the surface of regulatory T cells using the antibody.

In one embodiment of the present invention, the immune-related disease may be cancer, more specifically cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, Pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another example of the present invention, the immune-related diseases may be autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases, but are not limited thereto.

According to another embodiment of the present invention, immune-related, including any one selected from the group consisting of primers, probes and antisense nucleotides specific for the DKK1 extracellular surface protein of the present invention or the gene encoding the antigenic determinant Provided is a composition for diagnosing a disease.

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

In the present invention, immune-related diseases can be diagnosed by measuring the expression level of DKK1 extracellular surface protein or the gene encoding the antigenic determinant present on the surface of regulatory T cells using the antibody.

In one embodiment of the present invention, the immune-related disease may be cancer, more specifically cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, Pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another example of the present invention, the immune-related diseases may be autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases, but are not limited thereto.

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

In the present invention, the "kit" means a set of compositions and accessories necessary for a specific purpose. For the purposes of the present invention, the kit of the present invention is a DKK1 extracellular surface protein which is a diagnostic marker for immune related diseases such as cancer, autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases. The disease level can be diagnosed by checking the expression level of mRNA or a protein thereof. Kits of the invention include primers, probes or optionally antibodies that recognize the expression level of a diagnostic marker of an immune related disease, as well as one or more other component compositions, solutions or devices suitable for the assay. Can be.

According to another embodiment of the present invention, the expression level of the extracellular surface protein or antigenic determinant is measured by using an antibody specific for the extracellular surface protein of DKK1 or the antigenic determinant present in regulatory T cells. It provides a method of providing information about related diseases.

In one embodiment of the present invention, the immune-related disease may be cancer, more specifically cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, Pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another example of the present invention, the immune-related diseases may be autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases, but are not limited thereto.

When the diagnosis of the immune-related disease in the present invention may further comprise the step of administering a therapeutic agent for the immune-related disease.

According to still another embodiment of the present invention, any one selected from the group consisting of primers, probes and antisense nucleotides specific for the gene encoding the DKK1 extracellular surface protein or the antigenic determinant of the present invention in regulatory T cells By measuring the expression level of the gene to provide a method for providing information on immune-related diseases.

In one embodiment of the present invention, the immune-related disease may be cancer, more specifically cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, breast cancer, metastatic cancer, prostate cancer, Pancreatic cancer, melanoma or lung cancer, and the like, preferably may be melanoma, but is not limited thereto.

In another example of the present invention, the immune-related diseases may be autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, acute and chronic inflammatory diseases, but are not limited thereto.

When the diagnosis of the immune-related disease in the present invention may further comprise the step of administering a therapeutic agent for the immune-related disease.

In the present invention, since DKK1 protein is specifically present on the surface of immune cells, especially regulatory T cells, it can be used as a new target of therapeutic agents for various immune related diseases.

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

Figure 2 shows the result of predicting the epitope of the antigen of the DKK1 protein according to an embodiment of the present invention.

Figure 3 shows the results of predicting the epitope of the antigen of the DKK1 protein according to an embodiment of the present invention.

Figure 4 shows the results of predicting the epitope of the antigen of the DKK1 protein according to an embodiment of the present invention.

Figure 5 shows the results of predicting the epitope in the 3S8V chain X of the DKK1 protein according to an embodiment of the present invention.

Figure 6 shows the results of predicting the epitope in the 3SOQ chain Z of the DKK1 protein according to an embodiment of the present invention.

Figure 7 shows the results of predicting the epitope in the 5FWW chain B of the DKK1 protein according to an embodiment of the present invention.

Figure 8 shows the results of predicting the epitope in the 5FWW chain B of the DKK1 protein according to an embodiment of the present invention.

Figure 9 shows the expression level of mRNA according to an embodiment of the present invention.

Figure 10 shows the expression level of mRNA according to an embodiment of the present invention.

Figure 11 shows the expression of DKK1 protein on the surface of regulatory T cells according to an embodiment of the present invention.

According to an embodiment of the present invention, a regulatory T extracellular surface protein of DKK1 represented by SEQ ID NO: 3 is provided.

According to another embodiment of the present invention, an antigenic epitope of DKK1, which is a surface protein of regulatory T cells, represented by any one of SEQ ID NOs: 4 to 36, is provided.

According to another embodiment of the present invention, there is provided an extracellular surface protein of DKK1 represented by SEQ ID NO: 3 provided in the present invention, or a polynucleotide encoding the antigenic determinant of DKK1 of any one of SEQ ID NOs: 4 to 36. .

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

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

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

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

According to another embodiment of the present invention, the DKK1 extracellular surface protein or the antigenic determinant of the present invention as an active ingredient, provides a pharmaceutical composition for the prevention or treatment of immune-related diseases.

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

According to another embodiment of the present invention, any one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA specific to the DKK1 extracellular surface protein of the present invention or the gene encoding the antigenic determinant is effective. Provided as an ingredient, a pharmaceutical composition for immune-related diseases.

According to another embodiment of the present invention, a method for preventing or treating an immune related disease, comprising administering to a subject in need thereof an antibody specific for the DKK1 extracellular surface protein or the antigenic determinant of the present invention. It is about.

According to another embodiment of the present invention, a group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the DKK1 extracellular surface protein of the present invention or a gene encoding the antigenic determinant to a subject in need thereof It relates to a method for preventing or treating an immune related disease, comprising the step of administering any one selected from.

According to another embodiment of the present invention, (a) contacting a sample to be assayed with an extracellular surface protein of DKK1 or a regulatory T cell comprising the antigenic determinant; (b) measuring the expression level of said extracellular surface protein or said antigenic determinant; And (c) determining that the sample is an immune activator or immunosuppressant when the level of expression of the extracellular surface protein or antigenic determinant is measured to be down or upregulated. A method for screening an agent or an immunosuppressant.

According to another embodiment of the present invention, (a) contacting a sample to be analyzed with a regulatory T cell comprising the extracellular surface protein of DKK1 or a gene encoding the antigenic determinant; (b) measuring the expression level of the gene; And (c) determining that the sample is an immune activator or immunosuppressant when it is determined that the expression level of the gene is reduced or upregulated. It is about.

According to another embodiment of the present invention, the expression level of the extracellular surface protein or antigenic determinant is measured by using an antibody specific for the extracellular surface protein of DKK1 or the antigenic determinant present in regulatory T cells. It provides a method of providing information about related diseases.

According to still another embodiment of the present invention, any one selected from the group consisting of primers, probes and antisense nucleotides specific for the gene encoding the DKK1 extracellular surface protein or the antigenic determinant of the present invention in regulatory T cells By measuring the expression level of the gene to provide a method for providing information on immune-related diseases.

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

Example

Example 1 Confirmation of DKK1 C-terminal Structure

DKK1 is known to bind to LRP6, a ligand specific for it, in the form of a secreted protein, so that the amino acid structure of No. 182-266 of the C-terminal portion is important for DKK1 to bind to the ligand, thereby high DKK1 and LRP6 Because of its ability to bind, its binding plays an important role in Wnt cell signaling. The structure of the C-terminal protein of the DKK1 is shown in FIG.

[Example 2] DKK1 epitope amino acid sequence prediction

Prediction of the sequences is ABCpred (FIG. 2), BCEpred (FIG. 3), Discotope and Ellipro servers (http://tools.iedb.org), which are epitope prediction software based on the structure of the DKK1 protein. / ellipro /) (FIGS. 4-8). The Ellipro and Discotope search engines used the search engines known to be the most reliable among the algorithms for predicting existing antigenic determinants.

After entering the extracellular domain analyzed in Example 1 into the antigenic determinant prediction software, the predicted contiguous or discontinuous amino acid sequences of the predicted antigenic determinants are shown in Figures 2-8.

As shown in FIGS. 2 to 8, a total of 19 consecutive antigenic determinant amino acid sequences were predicted with SEQ ID NOs: 4 to 22, and a total of 14 discontinuous antigenic determinant amino acid sequences with SEQ ID NOs: 23 to 36 were predicted.

Example 3 Confirmation of Specific Expression of DKK1 mRNA in Regulatory T Cells

We verified that DKK1 protein can act as a biomarker specific for regulatory T cells.

For this validation, CD4 + T cells were isolated using magnet-activated cell sorting (MACS) through CD4 beads from the spleen of mice to which C57BL / 6 or Foxp3 and FRP were commonly expressed.

Thereafter, regulatory T (CD4 + CD25 + T) cells and non-regulated T (CD4 + CD25− T) cells were isolated using a fluorescent activated cell sorter (FACS) using a CD25 antibody. Each cell and the cells differentiated in Preparation Example 1 extracted mRNA using Trizol, and genomic RNA removed gDNA by a protocol provided by a company using gDNA extraction kit (Qiagen). . gDNA removed mRNA was synthesized into cDNA through the BDsprint cDNA Synthesis Kit (Clonetech).

Real time PCR was performed to quantitatively confirm the expression level of DKK1 mRNA in the cDRNA. The real-time polymerase chain reaction was performed by SYBR Green (Molecular Probes) using a protocol provided by the manufacturer for 3 minutes at 95 ℃, 15 seconds at 61 ℃, 40 cycles of 30 seconds at 72 ℃, Table 4 It was performed using a primer, relative gene expression was calculated using the ΔCT method, normalized using HPRT, the results are shown in FIG.

division		order
DKK1	Forward	5′-GCG GCA AGA CCT ACA CCA AGA G-3 ′
	Reverse	5′-CTT TCG GTA GTG GCG GGT AAG C-3 ′
Gapp	Forward	5′-GCC TTC CGT GTT CCT ACC-3 ′
	Reverse	5′-GCC TGC TTC ACC ACC TTC-3 ′
DKK2	Forward	5′-TGG ATC ATG ACT TCC GTGACG CTT-3 ′
	Reverse	5′-GCC ATC TTG CCA TGG TCT TGG TTT-3 ′
DKK3	Forward	5′-AGA ACT CCA AGA GGC ACA GAG CAA-3 ′
	Reverse	5′-TGT TGT GAG GGA TCT GACAAG CCA-3 ′
DKK4	Forward	5′-TGG TGG TGG CTC TCC TTG-3 ′
	Reverse	5′-AGT TCC GCA CAT CCT TCT TG-3 ′
Wnt3a	Forward	5′-TCG GAG ATG GTG GTA GAG AAA CAC-3 '
	Reverse	5'-AAG TTG GGT GAG GCC TCG TAG TAG-3 '
Wnt5a	Forward	5'-ACA ACC TGG CAG ATG TAG CCT GTA-3 '
	Reverse	5'-CGC GCT ATC ATA CTT CTC CTT GAG-3 '
Wnt7b	Forward	5'-CCC AAT GGA GAC AAA TCC CTT TAC-3 '
	Reverse	5'- GAA ACC GTT TTG AGT GTG ACT GGT-3 '
Wnt10b	Forward	5'-GTT TCC GTG AGA GTG CTT TCT CCT-3 '
	Reverse	5'-TCT TGC TCA CCA CTA CCC TTC CAT-3 '
Wnt11	Forward	5'- GGG CTT CAA AGG AAA CTG ATA GGA-3 '
	Reverse	5'-CCT TGA AAG GTC AAA TGC ACA GTC-3 '

As shown in FIG. 9, the expression of the DKK family gene and the Wnt gene in the regulatory T cells was hardly confirmed, but the expression of the DKK1 gene was remarkably high. It can be seen that it is very likely to be used.

Example 4 Confirmation of DKK1 mRNA Expression in Immune Cells

We verified that DKK1 protein can act as a biomarker specific for regulatory T cells.

Among the CD4 + T cells isolated as in Example 3, Treg (CD4 + RFP +) cells, naïve T cells, and effector T cells were classified into CD62L antibody (ebioscience) and fluorescent activated cell sorter. And CD8 + T cells, B cells are separated using an antibody against CD8 β chain, an antibody against CD19, and then with or without antibodies (ebioscience) to CD3 and CD28, or CD4 + T After culturing in a condition capable of differentiating into cell subtypes, extracting mRNA in the same manner as in Example 3, confirming the expression of DKK1 mRNA, and the results are shown in FIG. 10.

As shown in FIG. 10, the DKK1 gene according to the present invention was higher in regulatory T cells, and in the same regulatory T cells, the group treated with antibodies to CD3 and CD28 had higher expression of DKK1, resulting in differentiation signals. However, the expression of DKK1 was higher in thymus-produced natural Tregs than induced Tregs differentiated into experimental T-cells experimentally.

Example 5 Confirmation of Specific Expression of DKK1 Protein in Regulatory T Cells

Since DKK1 protein must be expressed on the surface of regulatory T cells in order to be a target of cell therapy, the target therapy can be more effectively performed. Therefore, it was confirmed whether DKK1 protein is expressed on the surface.

Each differentiated T cell subtype of Example 4 above was stained with anti-CD4-APC and anti DKK1-PE antibodies and DKK1 at each cell surface using a Fluorescence-Activated Cell Sorter (FACS). The expression amount of was measured and the result is shown in FIG.

As shown in FIG. 11, when T cell receptor signal was activated, regulatory T cells (CD4 + Foxp +) expressed higher DKK1 than other effect T cells (Effector T cells) that did not express Foxp3.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims. It will be self-evident to those who have knowledge of.

The present invention relates to DKK1 (Dickkopf-1) proteins, antibodies specific thereto, and uses thereof, which are specifically present on the surface of regulatory T cells (Treg cells), among immune cells.

SEQ ID NO: 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 CARHFWSKIC KPVLKEGQVC TKHRRKGSHG LEIFQRCYCG EGLSCRIQKD HHQASNSSRL HTCQRH

SEQ ID NO: dickkopf-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

SEQ ID NO: DKK1 extracellular domain

TLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTRSKQEGCKVCRKGSRGS

SEQ ID NO: 3S2K chain C Linear epitope

RIQKRL

SEQ ID NO: 3S2K chain C Linear epitope

HRRKGSHGLE IF

SEQ ID NO: 3S2K chain C Linear epitope

KGQEGSVCLR SSDCASG

SEQ ID NO: 3S8V chain X Linear epitope

RIQSRL

SEQ ID NO: 3S8V chain X Linear epitope

HRRKGSHGLE IF

SEQ ID NO: 3S8V chain X Linear epitope

GEGL

SEQ ID NO: 10 3S8V chain X Linear epitope

QEGSVCLRSS DCASG

SEQ ID NO: 3S8V chain X Linear epitope

KEGQ

SEQ ID NO: 12 3S8V chain X Linear epitope

NSNAIKN

SEQ ID NO: 13 5FWW chain B Linear epitope

RVPGASHIH

SEQ ID NO: 14 5FWW chain B Linear epitope

GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGE

SEQ ID NO: 15 5FWW chain B Linear epitope

KDHGNPPPLT GTSKTSNKL

SEQ ID NO: 16: 5FWW chain B Linear epitope

FSDRINQ

SEQ ID NO: 17 5FWW chain B Linear epitope

CYVGVYWK

SEQ ID NO: 18 5FWW chain B Linear epitope

IRDSADMVEL LDGYTHRVLA RFHGRSRPPL SFNVSLDFVI

SEQ ID NO: 19: 5FWW chain B Linear epitope

LGEHNYC

SEQ ID NO: 20: 5FWW chain B Linear epitope

FCGNNPDYWK YGE

SEQ ID NO: 21 5FWW chain B Linear epitope

SQRFK

SEQ ID NO: 22 5FWW chain B Linear epitope

ATGRV

SEQ ID NO: 23 3S2K chain C Discontinuous epitope

HKGSGL

SEQ ID NO: 24 3S2K chain C Discontinuous epitope

IQL

SEQ ID NO: 25 3S2K chain C Discontinuous epitope

FWIF

SEQ ID NO: 26 3S2K chain C Discontinuous epitope

EGQGEGRH

SEQ ID NO: 27 3S2K chain C Discontinuous epitope

KGQEGSVCLR SDASG

SEQ ID NO: 28 3S8V chain X Discontinuous epitope

IQSRL

SEQ ID NO: 29 3S8V chain X Discontinuous epitope

HKGSGL

SEQ ID NO: 30 3S8V chain X Discontinuous epitope

QGSVCRSD

SEQ ID NO: 31 3S8V chain X Discontinuous epitope

FWIF

SEQ ID NO: 32 3S8V chain X Discontinuous epitope

EGQGGLR

SEQ ID NO: 33 3S8V chain X Discontinuous epitope

ASG

SEQ ID NO: 34 5FWW chain B Discontinuous epitope

GTQNWTALQG GKPCLFWNET FQHPYNTLKY PNGELGEHNY CPCYVGVYWK C

SEQ ID NO: 35 5FWW chain B Discontinuous epitope

AMYATGRVRV PGASHIHIRD SADMELDGYT HRVLARHGRS PPLSFNVSLD FVIFSDRINQ AQAVK

SEQ ID NO: 36: 5FWW chain B Discontinuous epitope

KDHGNPPPLT GTSKTSNKLF SQRFKSGYFC GNNPDYWKYG ECFGGDG

Claims (26)

1. (a) contacting a sample to be assayed with regulatory T cells comprising an extracellular surface protein of DKK1 (Dickkopf-1) or an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36;

   (b) measuring the expression level of said extracellular surface protein or said antigenic determinant; And

   (c) determining that the sample is an immune activator or immunosuppressant when the level of expression of the extracellular surface protein or antigenic determinant is determined to be down or upregulated. Or a method of screening an immunosuppressant.
2. The method of claim 1,

   The expression level of the extracellular surface protein or the antigenic determinant is performed using an antibody capable of specifically binding to the extracellular surface protein or the antigenic determinant.
3. The method of claim 1,

   The extracellular surface protein of DKK1 is represented by SEQ ID NO: 3, the screening method.
4. The method of claim 1,

   The expression level of the extracellular surface protein or the antigenic determinant is determined by protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF), and Surface Enhanced Laser (SELDI-TOF). Desorption / Ionization Time of Flight Mass Spectrometry, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass Carried out by a method selected from the group consisting of liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), western blot and enzyme linked immunosorbent assay (ELISA). , Screening method.
5. (a) contacting a sample to be assayed with a regulatory T cell comprising an extracellular surface protein of DKK1 (Dickkopf-1) or a gene encoding an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36;

   (b) measuring the expression level of the gene; And

   (c) determining that the sample is an immune activator or immunosuppressant when the level of expression of the gene is determined to be reduced or upregulated.
6. The method of claim 5,

   The expression level of the gene is performed using primers, probes or antisense nucleotides capable of specifically binding to the gene.
7. The method of claim 5,

   The extracellular surface protein of DKK1 is represented by SEQ ID NO: 3, the screening method.
8. The method of claim 5,

   The expression level of the gene is performed by a method selected from the group consisting of reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting and DNA chip. .
9. An antigenic determinant of DKK1 (Dickkopf-1) represented by any one of SEQ ID NOs: 4 to 36.
10. A pharmaceutical composition for preventing or treating an immune related disease comprising a regulatory T cell (Treg cell) comprising an extracellular surface protein of DKK1 (Dickkopf-1) represented by SEQ ID NO: 3 as an active ingredient.
11. The method of claim 10,

    The immune-related disease is selected from the group consisting of autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopic and acute and chronic inflammatory diseases.
12. Pharmaceutical composition for the prevention or treatment of immune-related diseases comprising a regulatory T cell (Treg cell) comprising an antigenic determinant of DKK1 (Dickkopf-1) represented by any one of SEQ ID NOs: 4 to 36 as an active ingredient.
13. The method of claim 12,

    The immune-related disease is selected from the group consisting of autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopic and acute and chronic inflammatory diseases.
14. An extracellular surface protein of DKK1 or an antibody specific for an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36 as an active ingredient, a pharmaceutical composition for preventing or treating an immune related disease.
15. The method of claim 14,

    The immune related disease is cancer, pharmaceutical composition.
16. The method of claim 14,

    The immune-related disease is selected from the group consisting of autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopic and acute and chronic inflammatory diseases.
17. As an active ingredient, an extracellular surface protein of DKK1, or any one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs, and microRNAs specific for a gene encoding an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36 as an active ingredient Pharmaceutical composition for immune-related diseases.
18. The method of claim 17,

    The immune related disease is cancer, pharmaceutical composition.
19. The method of claim 17,

    The immune-related disease is selected from the group consisting of autoimmune disease, graft versus host disease, organ transplant rejection, asthma, atopic and acute and chronic inflammatory diseases.
20. A composition for diagnosing an immune related disease, comprising an antibody specific for a DKK1 extracellular surface protein or a protein represented by any one of SEQ ID NOs: 4 to 36.
21. A composition for diagnosing an immune related disease, comprising a primer, a probe, or an antisense nucleotide specific for a DKK1 extracellular surface protein or a gene encoding an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36.
22. 23. A diagnostic kit for an immune related disease comprising the composition of claim 20 or 21.
23. The expression level of the surface protein or antigenic determinant is measured by using an antibody specific for an extracellular surface protein of DKK1 present in regulatory T cells or an antigenic determinant represented by any one of SEQ ID NOs: 4 to 36. How to Provide Information.
24. Using an extracellular surface protein of DKK1 present in regulatory T cells, or any one selected from the group consisting of primers, probes and antisense nucleotides specific for genes encoding antigenic determinants represented by any one of SEQ ID NOs: 4-36. Measuring the expression level of said gene to provide information about an immune related disease.
25. A method for preventing or treating an immune related disease, comprising administering to a subject in need thereof an antibody specific for said DKK1 extracellular surface protein or said antigenic determinant.
26. Administering to the subject in need thereof any one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs and microRNAs specific for the DKK1 extracellular surface protein of the invention or the gene encoding the antigenic determinant A method of preventing or treating an immune related disease, comprising.

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