KR20140056501A - Novel biomarker indicative of inflammatory arthritis and its uses - Google Patents
Novel biomarker indicative of inflammatory arthritis and its uses Download PDFInfo
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Abstract
Description
The present invention relates to a kit for the diagnosis or prognosis of inflammatory arthritis, a method for detecting an inflammatory arthritis marker, a method for screening a substance for preventing or treating inflammatory arthritis, and a pharmaceutical composition for preventing or treating inflammatory arthritis.
Inflammatory arthritis is an autoimmune disease that causes inflammation of the joints due to abnormalities in the human immune system due to various factors such as hereditary, environmental, and hormonal factors, including rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. On the other hand, degenerative arthritis such as osteoarthritis is classified as noninflammatory arthritis which is caused by aging and trauma, and there is a marked difference in symptoms as well as symptoms of inflammatory arthritis.
Rheumatoid Arthritis (RA) is a chronic disease that is estimated to be approximately 1% of the total population in Korea. The pathogenesis of rheumatoid arthritis is presumed to be extrinsic due to bacterial infection, viral infection, or
The treatment of rheumatoid arthritis is usually different from the treatment of choice depending on the course of the illness. Generally, nonsteroidal anti-inflammatory drugs (NSAIDs) are given at the early stage of diagnosis, and Disease-Modifying anti-rheumatic drugs (DMARD) are administered in addition to the NSAID if a definitive diagnosis is made. In particular, it is difficult to make a definite diagnosis at the initial stage of RA onset, and in the current situation, NSAID is administered and the progress is discriminated from other rheumatic diseases including collagen diseases while observing the course carefully. In some cases, a steroid drug is administered. In addition to drug therapy for pain, physical therapy and rehabilitation aids are performed for maintenance and recovery of joint function.
Currently, antibody drugs such as Enbrel, Humira, and Remicade, TNF blocking agents are used for the treatment of active rheumatoid arthritis and psoriatic arthritis in adults who do not respond properly to DMARD including methotrexate, but they are resistant to TNF There are disadvantages that can not be used in patients, and there are possibilities of serious side effects such as opportunistic infection or the development of lymphoma, so their use is limited. Therefore, other pathway inhibitors with different mechanisms can complement the existing antihypertensive medicines for treating arthritis, thus requiring a new therapeutic target of inflammatory arthritis.
To date, the diagnosis of rheumatoid arthritis is based on the American College of Rheumatology criteria, and the presence of rheumatoid factor as an objective index is only 33% within 3 months and 88% over 12 months. I have not been able to diagnose it. Although the above-mentioned treatment method for rheumatoid arthritis is capable of continuing the joint deformation despite its excellent effect, it may be difficult to appropriately treat due to the side effect of the drug, and since the drug cost is increased due to the development cost of the new drug, It is imperative to develop a method for quickly and accurately diagnosing and predicting prognosis and severity.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have sought to find a novel target for accurate diagnosis and effective treatment of inflammatory arthritis. As a result, the expression of TL1A (TNF-like ligand 1A) was significantly increased in inflammatory arthritis patients compared with those of non-inflammatory arthritis, and the production of IL-6, a major inflammatory factor in TL1A treatment, Thereby completing the present invention.
It is therefore an object of the present invention to provide a kit for the diagnosis or prognosis of inflammatory arthritis.
It is another object of the present invention to provide a method for detecting inflammatory arthritis markers in order to provide information necessary for diagnosis or prognosis analysis of inflammatory arthritis.
It is still another object of the present invention to provide a method for screening a substance for preventing or treating inflammatory arthritis.
It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammatory arthritis.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a kit for the diagnosis or prognosis of inflammatory arthritis, which comprises an antibody or a peptide aptamer that specifically binds to TL1A (TNF-like ligand 1A).
The present inventors have sought to find a novel target for accurate diagnosis and effective treatment of inflammatory arthritis. As a result, the expression of TL1A (TNF-like ligand 1A) was significantly increased in inflammatory arthritis patients compared with those of non-inflammatory arthritis, and the production of IL-6, a major inflammatory factor in TL1A treatment, Thereby completing the present invention.
The present invention is a novel inflammatory arthritis marker identified by identification of a protein specifically expressed in inflammatory arthritis, which is capable of accurately diagnosing and prognosing the disease.
As used herein, the expression "kit for the diagnosis or prognosis of inflammatory arthritis" refers to a kit containing a composition for the diagnosis or prognosis analysis of inflammatory arthritis. Therefore, the above expression can be used interchangeably or in combination with "composition for the diagnosis or prognosis analysis of inflammatory arthritis ".
As used herein, the term "diagnosing" is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an affected entity (e.g., determining the stage of inflammatory arthritis or determining the responsiveness of the disease to treatment), or evaluating therametrics (e.g., to provide information about therapeutic efficacy) And monitoring the status of the object).
According to a preferred embodiment of the present invention, the inflammatory arthritis is rheumatoid arthritis, psoriatic arthritis or ankylosing spondylitis, preferably rheumatoid arthritis.
As used herein, the term "rheumatoid arthritis" is a chronic inflammatory disease of unknown origin characterized by multiple arthritis. Initially, inflammation of the synovial membrane surrounding the joint gradually develops into inflammation of the surrounding cartilage and bones, And deformation. It is a disease that can invade whole body including anemia, dry syndrome, subcutaneous nodule, pulmonary fibrosis, vasculitis and skin ulcer due to joints as well as external joint symptoms.
As used herein, the term "psoriatic arthritis" is a disease in which arthritis is associated with psoriasis and is known to originate in the joints of the fingers or in the toes and various joint lesions. It may cause bone destruction of the joints, absorptions of the peaks, and may indicate a state of destructive arthritis. This type of joint may be accompanied by arthritis or spondylitis.
As used herein, the term " Ankylosing Spondylitis (AS) "is an inflammatory arthritis with major lesions of the osteoid stiffness of the vertebrae and the cephalad joint, and the HLA-B27 gene is known to play the most important role. There may be other causes. Intestinal bacteria, chlamydia and the like caused by infection, but the cause of rheumatoid arthritis, as well as the cause of the disease is still unclear.
The inventors of the present invention confirmed that the use of the marker of the present invention can provide high sensitivity and reliability for the incidence of inflammatory arthritis from an individual.
As used herein, the term "diagnostic marker, marker for diagnostic or diagnostic marker" refers to a substance capable of distinguishing inflammatory arthritis from steady-state or noninflammatory arthritis, and exhibits an increased expression pattern compared to steady- Organic biomolecules such as visible polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides and the like), and the like. For purposes of the present invention, the inflammatory arthritic diagnostic marker is TL1A. Such markers include not only proteins, but also DNA or RNA encoding proteins.
The expression "measurement of protein expression level" used in the diagnosis of inflammatory arthritis in the present invention is a process for confirming the presence and the degree of expression of the marker protein in a biological sample. Preferably, the antibody specifically binds to the marker protein Or peptide aptamer to identify the amount of the protein. More preferably, the amount of the protein is determined by using an antibody that specifically binds to the marker protein. Methods for analysis include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis (Immunoprecipitation Assay), Complement Fixation Assay (FACS), Fluorescence Activated Cell Sorter (FACS), and Protein Chip, and the like. The method of analysis is not limited.
In the present invention, "antibody" means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the new inflammatory arthritis marker protein has been identified as described above, the production of the antibody using the novel inflammatory arthritis marker protein can be easily performed using techniques well known in the art.
Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injecting the marker protein antigen described above into an animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.
Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519) or the phage antibody library (Clackson et al, Nature , 352: 624- 628, 1991; Marks et al . , J. Mol. Biol. , 222: 58, 1-597, 1991). The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with 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 an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.
In the present invention, the aptamer binding to TL1A is a peptide molecule, and the general contents of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).
According to a preferred embodiment of the present invention, the kit of the present invention is a kit for immunoassay, and preferably the immunoassay kit is a luminex assay kit, a protein microarray kit or an ELISA kit.
The luminex assay kit, protein microarray kit and ELISA kit of the present invention include a polyclonal antibody and a monoclonal antibody against the marker protein and a secondary antibody against the polyclonal antibody and the monoclonal antibody to which the labeling substance is bound .
The kit of the present invention may further include essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the marker protein. An antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the marker protein and little cross-reactivity to other proteins. In addition, ELISA kits can contain antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.
The kit of the present invention may further include essential elements necessary for performing a protein microarray to simultaneously analyze various samples. Microarray kits contain antibodies specific for the marker protein bound to the solid phase. Protein microarray kits may comprise reagents capable of detecting bound antibodies, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., fused with an antibody) and other substrates capable of binding to the substrate or antibody, . A method of analyzing a sample using a protein microarray is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It can provide the information needed to diagnose inflammatory arthritis.
Luminex Assay is a high-throughput quantitative assay that can simultaneously measure up to 100 different analyte without pretreatment small (10-20 μl) patient samples (Pg unit) and can be quantified in a short time (3-4 hours), which is an analytical method that can replace the existing ELISA or ELISPOT. The Luminex Assay is a multiplexed fluorescent microplate assay capable of simultaneously analyzing over 100 biological samples in each well in a 96-well plate using two types of laser detectors in real time Polystyrene beads of more than 100 different color groups are identified and quantified by progressing signal transduction. Each bead has an antibody attached to the protein to be analyzed, so that the protein can be quantitated by the immunoassay using the antibody. This sample is analyzed using two lasers, one laser detects the bead's unique number and the other laser detects the protein in the sample reacted with the antibody attached to the bead. Therefore, 100 in vivo protein analysis is possible simultaneously in one well. This assay has the advantage of being detectable with as little as 15 μl of sample. Luminex kits capable of carrying out the lumenx assays of the invention include antibodies specific for the marker protein. An antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the marker protein and little cross-reactivity to other proteins. In addition, luminex kits may contain antibodies specific for the control protein. Other luminex kits may also include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and other substrates capable of binding to the substrate or antibody . ≪ / RTI > The antibody may be a conjugated antibody conjugated with microparticles, and the microparticles may be colored latex or colloidal gold particles.
In the present invention, it is possible to compare the amount of antigen-antibody complex formed in the control group (for example, a sample of non-inflammatory arthritis patients) and the amount of antigen-antibody complex formed in the subject to be examined and whether or not the expression of TL1A protein is significantly increased It is possible to diagnose the actual inflammatory arthritis of patients with suspected inflammatory arthritis.
The term " antigen-antibody complex "as used herein refers to a combination of a marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the magnitude of the signal of the detection label. Such detection labels can be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules and radioisotopes, but is not limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructoketase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) [RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I and 186Re.
According to a preferred embodiment of the present invention, measurement of protein expression level is by ELISA. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. Identification of the complex formation of the marker protein and antibody can be used to confirm the onset of inflammatory arthritis.
According to another preferred embodiment of the present invention, the measurement of the protein expression level is performed using Western blot. The whole protein is separated from the sample, and the protein is separated according to the size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the marker protein can be confirmed by confirming the amount of the generated antigen-antibody complex using the labeled antibody, thereby confirming the onset of inflammatory arthritis.
According to another preferred embodiment of the present invention, measurement of protein expression level is performed by immunohistochemical staining. After collecting and fixing the tissue to be examined and the control tissue (for example, tissue derived from non-inflammatory arthritis patients), paraffin-embedded blocks are prepared by methods well known in the art. These are cut into pieces of several μm thick and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.
According to another preferred embodiment of the present invention, the measurement of the protein expression level is performed using a protein chip. An analysis method using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, and detecting the presence or the expression level of the protein to determine whether inflammatory arthritis .
According to a preferred embodiment of the present invention, the biological sample is tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine, preferably blood, serum, synovial tissue, synovial fluid or synovium It is synovial fluid.
According to another aspect of the present invention, the present invention provides a method for detecting the expression of TL1A (TNF-like ligand 1A) protein in a human biological sample to provide information necessary for diagnosis or prognosis analysis of inflammatory arthritis, A method for detecting a marker is provided.
The method of the present invention for detecting inflammatory arthritis markers is performed by measuring the expression level of the marker protein from a biological sample taken from a human and comparing the measured expression level with the expression level of the control sample.
When the method of detecting the inflammatory arthritic markers of the present invention is carried out with a biological sample of an inflammatory arthritis patient, it is possible to determine the prognosis of inflammatory arthritis in a patient sample.
According to a preferred embodiment of the present invention, the control sample includes a biological sample collected from a human who has not developed inflammatory arthritis or a biological sample collected from a patient with non-inflammatory arthritis. When the expression level of the marker protein in the control sample is compared with the expression level of the marker protein in the test sample and the expression level is higher than that of the control sample, inflammatory arthritis can be diagnosed.
According to another aspect of the present invention, the present invention provides a method for screening a substance for the prophylactic or therapeutic treatment of inflammatory arthritis comprising the steps of:
(a) contacting a sample to be analyzed with a cell or tissue expressing a TL1A (TNF-like ligand 1A) protein; And
(b) measuring the expression level of the TL1A protein, wherein the sample is judged to be a substance for the prophylaxis or treatment of inflammatory arthritis when the expression of TL1A is reduced.
According to the method of the present invention, a cell or tissue expressing the marker protein of the present invention is first brought into contact with a sample to be analyzed.
Preferably, the cell expressing the marker protein of the present invention is a synovial cell, and the tissue is a synovial tissue.
The term "sample" used in reference to the screening method of the present invention means an unknown substance used in screening to check whether the expression level of the marker of the present invention is affected. The sample includes a chemical substance, an antibody, a peptide, a small molecule substance, and an aptamer, and is preferably an antibody.
Next, the expression level of the marker of the present invention is measured in the sample-treated cells. Measurement of the expression level can be carried out as described above. When the result of measurement shows that the high-specificity of the marker protein of the present invention is inhibited, the sample can be judged to be a substance for preventing or treating inflammatory arthritis. As demonstrated in the following examples, the production of inflammatory cytokines is markedly increased by treatment with TL1A, so that a substance that inhibits high expression of TL1A can be used as a prophylactic and therapeutic agent for inflammatory arthritis.
According to another aspect of the present invention, there is provided a kit for the treatment of a disease comprising: (a) a population consisting of (i) an antibody, a peptide aptamer, a compound and DcR3 (Decoy Receptor 3) specifically binding to a TL1A (Ii) a TL1A expression inhibitor selected from the group consisting of antisense oligonucleotides, nucleic acid aptamers, small interference RNA (siRNA) and shRNA (short hairpin RNA) comprising a sequence complementary to a TL1A protein coding nucleotide ≪ / RTI > And
(b) a pharmaceutical composition for the prophylaxis or treatment of inflammatory arthritis comprising a pharmaceutically acceptable carrier.
The TL1A activity inhibitor used in the present invention, DcR3, is a water-soluble receptor encoded by the TNFRSF6B gene and binds to TL1A to neutralize TL1A.
The TL1A expression inhibitor used in the present invention includes antisense oligonucleotides, nucleic acid aptamers, siRNA and shRNA.
As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and refers to a complementary sequence in mRNA, It acts to inhibit translation. Antisense sequence refers to a DNA or RNA sequence that is complementary to TL1A mRNA and capable of binding to TL1A mRNA and has an essential activity for TL1A mRNA translation, translocation into the cytoplasm, maturation, or any other overall biological function . ≪ / RTI > The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.
As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method. The siRNA molecule of the present invention may have a structure in which the sense strand and the antisense strand are located on opposite sides to form a double strand. Also, according to another embodiment, the siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands. The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. SiRNA molecules of the present invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence The hairpin structure is formed by hybridization, and the stem-and-loop structure as a whole is formed.
As used herein, the term "shRNA" refers to a short hairpin RNA in which the sense and antisense sequences of the siRNA target sequence are located between loops of 5-9 bases. shRNA is used to overcome the disadvantages of high cost biosynthesis cost of siRNA, short time maintenance of RNA interference effect due to low cell transfection efficiency, and the like by using adenovirus, lentivirus and plasmid expression vector system from RNA polymerase III promoter It is widely known that siRNA is converted into an siRNA having a correct structure by siRNA processing enzyme (Dicer or Rnase III) present in the cell to induce silencing of the target gene.
As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve efficacy or activity of the activity inhibitor or expression inhibitor of the present invention.
The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, have.
The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.0001-1000 mg / kg (body weight) per day.
The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
The features and advantages of the present invention are summarized as follows:
(i) The present invention provides novel molecular markers for inflammatory arthritis and uses thereof (kits, marker detection methods, screening methods and pharmaceutical compositions).
(ii) The marker TL1A of the present invention has an increased expression in patients with inflammatory arthritis such as rheumatoid arthritis, and the expression of TL1A increases the production of inflammatory cytokines, resulting in the development, progression and worsening of inflammatory arthritis.
(iii) The TL1A delivery pathway blocking agent of the present invention is expected to block the pathway prior to tumor angiogenic factor secretion, thereby reducing the likelihood of side effects such as infection or malignant tumors appearing in a tumor angiogenesis inhibitor (e.g., Enbrel). In addition, the new pathway can be used for the treatment of patients who have not responded to existing tumor angiogenesis inhibitors, and a synergistic effect can be expected in the combination therapy with tumor angiogenesis inhibitors.
(iv) Thus, the present invention provides a diagnostic and prognostic analysis of inflammatory arthritis; Screening of inflammatory arthritis remedies; And for the treatment of inflammatory arthritis.
1 is a photograph of immunochemical staining showing the level of TL1A expression in synovial tissues of rheumatoid arthritis patient (a) and degenerative arthritis patient (b).
Figure 2 is a graph showing the levels of TL1A expression measured in serum and synovial fluid (SF) of rheumatoid arthritis patient (a) and degenerative arthritis patient (b).
Figure 3 is a graph showing the amount of IL-6 produced in TL1A-treated rheumatoid arthritis synovial cells.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1. Comparison of TL1A expression levels in synovial tissues of inflammatory arthritis and noninflammatory arthritis
The present inventors performed immunochemical staining to determine whether there is a difference in expression of TL1A in a synovial tissue of a representative inflammatory arthritis, ie, rheumatoid arthritis and a representative non-inflammatory arthritis, ie, degenerative arthritis patients. Immunochemical staining was performed using Abcam (USA) reagent according to the manufacturer's instructions. The experimental results are shown in Fig.
As shown in Fig. 1, the expression of TL1A was significantly increased in synovial proliferation sites in patients with rheumatoid arthritis, compared with synovial growth sites in patients with degenerative arthritis (Fig. 1).
Example 2. Analysis of TL1A expression level in blood and synovial fluid of rheumatoid arthritis patients
Serum and synovial fluid were sampled simultaneously in 37 patients with rheumatoid arthritis and 27 patients with degenerative arthritis, and the amount of TL1A expressed was quantitated by ELISA. TL1A assays for serum and synovial fluid were performed using primary and secondary anti-TL1A antibodies purchased from Peprotech (UK). The values of each sample were determined using a standard curve obtained through three step dilutions. The experimental results are shown in Fig.
As shown in FIG. 2, the amount of TLIA protein in patients with inflammatory arthritis, rheumatoid arthritis, was increased by about 3-fold in sera and 4-5-fold in synovial fluid compared with patients with degenerative arthritis, which is a non-inflammatory arthritis. In addition, TL1A protein expression was increased in synovial fluid rather than in serum (Fig. 2).
Example 3: TL1A induced induction of inflammatory response in synovial cells
To investigate the changes of TL1A-associated inflammatory cytokines in the synovial membrane of patients with true inflammatory arthritis, TL1A stimulation was induced in rheumatoid arthritis synoviocytes (RA-FLS) at concentrations of 0, 40, 200 ng / And 72 hours, and the amount of IL-6 was measured by obtaining a cell culture supernatant. Pro-inflammatory cytokines were measured using the milliplex human cytokine magnetic bead panel; HCYTOMAG-60K (Millipore Corp., USA). The test method was carried out in compliance with the test method of the kit manufacturer. All the samples were performed without dilution, and all the procedures were performed at room temperature where light was blocked. The reaction results were measured using a Luminex 200 system. Median Fluorescent Intensity (MFI) was calculated using the MasterPlex QT ™ software programs (MiraiBio, Canada) and the optimal standard curve for each cytokine was determined. Respectively. The experimental results are shown in Fig.
As shown in FIG. 3, IL-6 production was increased in proportion to the treatment concentration of TL1A in rheumatoid arthritis synoviocytes (FIG. 3). These results indicate that TL1A is a diagnostic and therapeutic target for inflammatory arthritis, including rheumatoid arthritis.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (9)
(a) contacting a sample to be analyzed with a cell or tissue expressing a TL1A (TNF-like ligand 1A) protein; And
(b) measuring the expression level of the TL1A protein, wherein the sample is judged to be a substance for the prophylaxis or treatment of inflammatory arthritis when the expression of TL1A is reduced.
(b) a pharmaceutical composition for the prophylaxis or treatment of inflammatory arthritis comprising a pharmaceutically acceptable carrier.
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| KR1020120119904A KR20140056501A (en) | 2012-10-26 | 2012-10-26 | Novel biomarker indicative of inflammatory arthritis and its uses |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016010397A1 (en) * | 2014-07-18 | 2016-01-21 | 고려대학교 산학협력단 | Method for screening anti-inflammatory drug or anti-cancer drug |
| CN111435137A (en) * | 2019-01-14 | 2020-07-21 | 深圳先进技术研究院 | Quantitative detection kit for DcR3 |
| CN112094846A (en) * | 2020-05-20 | 2020-12-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
-
2012
- 2012-10-26 KR KR1020120119904A patent/KR20140056501A/en not_active Application Discontinuation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016010397A1 (en) * | 2014-07-18 | 2016-01-21 | 고려대학교 산학협력단 | Method for screening anti-inflammatory drug or anti-cancer drug |
| CN111435137A (en) * | 2019-01-14 | 2020-07-21 | 深圳先进技术研究院 | Quantitative detection kit for DcR3 |
| CN112094846A (en) * | 2020-05-20 | 2020-12-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
| CN112094846B (en) * | 2020-05-20 | 2022-02-18 | 中山大学孙逸仙纪念医院 | Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof |
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