KR20190084879A - CTRP3 as biomarker for tendinopathy and Composition and method of treating tendinopathy targeting CTRP3 - Google Patents

Abstract

A composition according to the present invention can inhibit CTRP3, which is a main factor for developing tendonopathy, as identified in the present invention, thereby effectively suppressing the onset of tendonopathy and effectively treating tendonopathy. In addition, CTRP3, which is the major factor for developing tendonopathy, as identified in the present invention, can be usefully used as a diagnostic marker for tendonopathy.

Description

Technical Field [0001] The present invention relates to a use of CTRP3 as a symptom marker, and a composition and method for treating a tendinopathy targeting CTRP3 and a CTRP3 as biomarker for tendinopathy targeting CTRP3,

This is a technique related to the detection and treatment of troubles.

Guns (tendons) are fibrous soft tissues that attach the muscles to the bones, consisting of a parallel arrangement of collagen fibers. Tendinopathy is one of the most common disease-related diseases. Troubles are degenerative lesions that occur in dry tissues, characterized by gradual degeneration and irregular arrangement of dry tissues. Troubles are known to be caused by frequent use of exercise and lifestyle activities, especially in athletes and elderly people. In fact, in the case of rotator cuff tear, which is a type of problem, the number of patients treated in 2013 is 530,000, and 69% of all patients are in their 50s or older. As such, an aging society has been linked to a growing incidence of epidemics and the demand for treatment is expected to continue to increase.

Tumor disease has been relatively well studied for its causes and risks. However, the mechanism of the onset of the problem and the treatment method are relatively inadequate. In particular, there is no clearly defined treatment for the treatment of the problem. Currently, the most commonly used therapies for the treatment of trophic syndrome include inhibition of inflammatory response through steroidal anti-inflammatory drugs, platelet-rich plasma (PRP) injection, and surgical suturing of ruptured dry tissues. However, in the case of an anti-inflammatory agent or PRP injection, the effect is not consistent with the aim of alleviating symptoms without clearly recognizing the mechanism of development of the problem. In the case of suture surgery, it is merely a method of simply connecting the damaged tissue. Thus, it is necessary to develop a fundamental treatment that eliminates the cause and causes of the molecular pathogenesis of the disease.

Korean Patent Laid-Open Publication No. 2016-0105363 relates to autologous and allogeneic adipose-derived mesenchymal stem cell compositions for the treatment of tendon or ligament injuries and a method for producing the same.

Korean Patent Laid-Open Publication No. 2013-0000397 relates to a platelet-derived growth factor composition and method for the treatment of dry matter disease.

It is urgent to develop an antibody treatment method for treating the problem more effectively and fundamentally, which is directly related to the onset mechanism of the disease, rather than relieving the symptom of the disease.

This study aims to identify the factors associated with dry matter at the molecular level and to develop effective therapeutic strategies and treatments for it.

In one aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of a condition comprising a CTRP3 inhibitor.

In one embodiment, the inhibitor according to the present invention is an antibody capable of neutralizing the activity of the CTRP3 protein.

In another embodiment, the inhibitor according to the present invention is an siRNA or shRNA that inhibits expression of the CTRP3 gene into a protein.

In another aspect, the present invention provides a composition for the diagnosis of a condition, comprising a substance for CTRP3 detection.

In one embodiment, the detecting substance used in the diagnostic composition according to the present invention is a reagent capable of detecting the marker at the protein or nucleic acid level, for example, the protein level detecting reagent is Western blot, ELISA, But are not limited to, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, mass spectrometry, or protein microarray reagents. The nucleic acid level detection reagent may be a polymerase chain reaction But are not limited to, reagents used in the reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, RNase, S1 nuclease assay, in situ hybridization, nucleic acid microarrays or Northern blots.

In another embodiment, the detection reagent at the protein level in the detecting material used in the diagnostic composition according to the present invention is an antibody, antibody fragment, aptamer, or a fragment thereof that specifically recognizes the protein full- An avidity multimer or peptidomimetics, wherein the detection reagent of the nucleic acid level comprises a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a fragment of the nucleic acid sequence and a complementary sequence But are not limited to, a specific pair of primer pairs, or probes, or pairs of primers and probes.

In another aspect, the present invention provides a method for detecting a testicular disorder, comprising the steps of: detecting the level of nucleic acid and / or protein of a CTRP3 biomarker from a biological sample derived from a test subject to provide information necessary for diagnosis or prognosis; Comparing the nucleic acid and / or protein level detection results to a corresponding result of a corresponding marker of a control sample; And comparing the CTRP3 biomarker with the control sample, wherein the CTRP3 biomarker is associated with a diagnosis or prognosis of the subject when it is increased in the expression of the nucleic acid or protein of the subject-derived sample.

In another aspect, the present invention provides a method of treating cancer, comprising: providing cells that overexpress CTRP3; Treating the test substance expected to inhibit the expression or activity of CTRP3 in the cell; And a step of screening the test substance as a candidate for the treatment for tocopathy when the expression or activity of the CTRP3 is decreased in the cell as compared with a cell not treated with the test substance as a result of the treatment Or therapeutic agent screening method.

In one embodiment, the cells that may be used in the screening method include, but are not limited to, cells derived from the disease.

In another embodiment, the detecting in the screening method further comprises detecting AKT activation, wherein if the detection indicates that the AKT activation is reduced compared to a cell that has not been treated with the test substance, And a step of screening for candidate therapeutic agents for the disease.

In this study, we investigated whether CTRP3 protein overexpression was overexpressed in CTRP3 protein manifestation, which is a key factor in the onset of the disease, and relieved the trophic lesion using CTRP3 antibody, which inhibits CTRP3 protein function. Were confirmed in rats. Therefore, the CTRP3 protein identified in this study can be usefully used as a biomarker for the development of dryness and severity. Furthermore, CTRP3 acts as an important target for the treatment of dry symptom, and a composition containing a substance that inhibits the function, expression, etc. of CTRP3 can be effectively used for the treatment of dry symptom. CTRP3 protein can be usefully used for the development of a drug for the treatment of psychotic disease, which targets CTRP3 by identifying the effect of the protein on the molecular level.

Figure 1 is a graph showing the increase in CTRP3 expression in human and mouse samples of behavioral disorders according to one embodiment of the present invention. (a) Volcano plot showing changes in gene expression of dry-damaged mouse models. (b) the percent expression of gene expression in normal and disease-prone mice. (c) Rotator cuff biceps tendon 1 to 3 stage histology results. Polarization microscopy was used for PSR staining. (d) Relative CTRP3 expression level of the first to third step tissues. (e) Box plot of lesion site and non-lesional CTRP3 expression in patients with trophic disease (GSE26051; n = 23).
FIG. 2 shows the results of a case in which an exogenous overexpression was induced in a mouse model of CTRP3 according to one embodiment of the present invention to induce symptoms. (a) shows the histology of normal mouse, empty vector, CTRP3 overexpressing vector electroporation mouse. (b) Bonn score was used to assess the severity of the problem of mouse tissue ((c)). (c) shows the sum of Bonar scores. (d) Exercise ability of colective vector electroporation mice and CTRP3 overexpressing vector electroporated mice evaluated as resistance in treadmill running. (e) Comparisons of the trophoblastic gene expression levels of the CTRP3 overexpressed vector electroporated mouse and the vector electroporated mouse. (f) histology results of Ad- eGFP injected mice and Ad- Ctrp3 injected mice. (g) Bonn score was used to evaluate the severity of the disease in mouse tissues ((f)). (h) Show the sum of Bonar score. (i) the athletic performance of Ad- eGFP injected mice and Ad- Ctrp3 injected mice evaluated in terms of resistance in treadmill running.
Figure 3 is a graph showing that the treatment of the alpha-CTRP3 antibody in a mouse disease model according to one embodiment of the present invention reduces the symptoms. (a) outline of dry and wound treatment and antibody treatment models, dry damage and antibody treatment models. (b) histology of mice treated with normal and control antibodies, mice treated with both control and control antibodies, and mouse treated with α-CTRP3 antibody. (c) Bonn score was used to assess the severity of the disease in the mouse tissue ((b)). (d) Bonar score. (e) Exercise ability of normal and control antibody-treated mice, vaccine and control antibody-treated mice, hepatocytes and α-CTRP3 antibody-treated mice as assessed by resistance in treadmill running. (f) histology of mice with dry damage and control antibody treatment, mouse injury, and α-CTRP3 antibody treated mice. (g) Bonn score was used to evaluate the severity of the disease in mouse tissues ((f)). (h) Show the sum of Bonar score.
FIG. 4 shows CTRP3-induced cartilage differentiation and differentiation inhibition according to one embodiment of the present invention. (a) The result of pellet culturing after transfection of a vector and a CTRP3 overexpressing vector into human mesenchymal stem cells. The histogram on the left is the results of the histochemistry, and the graph on the right is the Alcian blue staining range quantified by image-pro software. (b) Fibrin gel culture after transfection with CTRP3 overexpressing vector and human CTLs in human mesenchymal stem cells. The histogram on the left is the histogram, and the plot on the right is the thickness of the structure that is quantified by image-pro software. ERK1 / 2, p38, and JNK signaling proteins were treated with human CTLP3-overexpressed culture medium (c, d) in human mesenchymal stem cells (c) (Activation). (e, f) Human mesenchymal stem cells were transfected with overexpressing vector through GFP-overexpressing adenovirus and CTRP3-overexpressing adenovirus, followed by fibrin gel culture. The stomach is the result of quantification of the expression of the differentiation and cartilage differentiation-associated gene ((e)), and the histology results and the thickness of the dry structure (f) are shown below.
Figure 5 shows that treatment of the alpha-CTRP3 antibody in a rat manifestation model according to one embodiment of the present invention resulted in a reduction in symptoms. (a) Histology of normal and control antibody treated rats, dry and inactive antibody treated rats, dry and human α-CTRP3 antibody treated rats. The lesions were confirmed by hematoxylin & eosin staining and Alcian blue staining. The fluorescence immunohistochemistry showed the increase of CTRP3 and chondroitin sulfate (CS) in the lesion. (b) Bonn score was used to evaluate the degree of the trophoblastic disease in mouse tissue ((a)). (c) The ability of the normal and control antibody-treated rats, the ip-treated and control antibody-treated rats, the ip-treated and α-CTRP3 antibody treated rats to evaluate the strength of the forefoot with a grip strength test meter. (d) histology of dry injury and control antibody treated rats, dry injury and α-CTRP3 antibody treated rats. Fluorescence immunoassay confirmed the increase of MMP13, fibronectin (FN1) and type 3 collagen at lesion sites.
FIG. 6 is a schematic diagram of a treatment procedure using a CTRP3 target antibody in the case of dry-rupture and tendinitis.

Here, CTRP3 is overexpressed in trophic disease and is the essential regulator that causes the development / progression of behavioral problems, and is based on the discovery of its mechanism.

In one embodiment, the subject is related to the use of a biomarker for the detection of a trophic condition of CTRP3 (C1q / Tumor necrosis factor-Related Protein 3).

In one embodiment, the present invention provides a composition or kit for the diagnosis or prognosis of a condition for the diagnosis or prognosis of a condition comprising a substance for CTRP3 detection.

Here we show increased CTRP3 expression in human and mouse conjunctivitis (Fig. 1). In particular, overexpression of CTRP3 was commonly observed in human samples of tendon injured mice and rotator cuff tear. In addition, CTRP3 expression was increased with increasing degree of disease in rotavirus tear human samples. In addition, in this study, it was confirmed that the overexpression of CTRP3 caused the development of the disease (D, I, E in FIG. 2).

Therefore, CTRP3 according to the present invention can be used not only for the diagnosis of dryness but also for the determination of severity.

CTRP3 (C1q / Tumor necrosis factor-Related Protein 3) is a protein belonging to the C1q / TNF superfamily and is known to regulate metabolism, inflammation, and cell growth. CTRP3 has been actively studied mainly for metabolic syndrome and inflammation related diseases. However, there is no known association between it and its symptoms. CTRP3 protein and gene sequences are known, for example, in the case of humans, the gene and protein accession numbers of NCBI (National Center for Biotechnology Information) are NM_030945, NM_181435; NP_112207, NP_852100. In the case of mice, the gene and protein accession numbers of NCBI are NM_001204134, NM_030888; And NP_001191063, NP_112150.

Tendinopathy is the most common of the dry-related diseases. Guns (tendons) are fibrous soft tissues that attach the muscles to the bones, consisting of a parallel arrangement of collagen fibers. Troubles are degenerative lesions in these dry tissues, characterized by gradual degeneration and irregular arrangement of dry tissues. Troubles are caused by frequent use of tendons due to exercise and lifestyle activities, and especially in athletes and elderly people.

Diagnosis herein refers to determining the susceptibility of a subject to a disease for a particular disease or disorder, determining whether a particular disease or disorder is presently present, the prognosis of a subject afflicted with the particular disease or disorder prognosis) or therametrics (e.g., monitoring the status of an object to provide information about the therapeutic efficacy).

The term biomarker or diagnosis marker used herein refers to a substance capable of diagnosing a disease-causing tissue or cell by distinguishing it from a normal cell or a tissue or cell subjected to appropriate treatment. Lt; RTI ID = 0.0 > or < / RTI > Herein, the diagnostic marker is a CTRP3 protein or a nucleic acid or gene encoding CTRP3 protein whose expression is increased in the tendon tissue in which the testicular disease occurs.

A biological sample herein refers to a substance or mixture of substances, including one or more components capable of detecting biomarkers, including, but not limited to, living organisms, particularly cells and tissues derived from humans. It also includes cells or tissues cultured in Invitro as well as those directly derived from the organism. In one embodiment, cell cultures may be used, including, but not limited to, tissues / cells or invitrocells obtained from, or suspected of developing, or suspected of developing trophic conditions. And also includes fractions or derivatives of said cells or tissues. When a cell or tissue is used, the cell itself or a fusion of cells or tissues may be used.

The CTRP3 protein and its nucleic acid sequence herein are well known and include, but are not limited to, the functional equivalents thereof as described above.

Detection herein includes quantitative and / or qualitative analysis, including detection of presence and absence and expression level detection, and such methods are well known in the art, and those skilled in the art will be able to select appropriate methods for the practice of the invention It will be possible.

The markers according to the present invention can be detected by quantitative or qualitative analysis at the level of detection of the presence of nucleic acids, in particular mRNA and / or protein and / or their expression levels themselves, changes in expression levels, and differences in expression levels.

In one embodiment, the diagnostic marker according to the present invention is based on its functional and / or antigenic characteristics. Can be detected using the activity, function of the protein, or a nucleic acid encoding the protein, particularly an agent that specifically interacts at the mRNA and / or protein level.

In this respect, the present application also includes a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to said nucleic acid sequence, a fragment of said nucleic acid sequence, or an antibody or aptamer that specifically recognizes a protein encoded by said nucleic acid sequence To a diagnostic marker.

As used herein, the term detecting substance is a substance which can detect the marker of the present invention for quantitative determination of the presence or absence of a protein or nucleic acid, for example, at an mRNA level.

Methods for analysis of the amount and presence expression pattern of a protein are well known and include, for example, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Immunoprecipitation assays, Complement Fixation Assays, FACS, protein chips, and the like can be given as examples of the immunoassay method. Reagents for protein or nucleic acid level detection are well known, for example the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker, Ligand or cofactor, or a mass spectrometer can be used. Reagents or substances that specifically interact or bind to the markers of the present invention may be used in conjunction with chip-based or nanoparticles.

The diagnostic compositions according to the present invention may comprise CTRP3 markers for the detection of proteins or nucleic acids, for example at the mRNA level. For example, reagents detectable at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands or cofactors, and the like. These reagents can be incorporated into nanoparticles or chips as needed. Proteins can also be detected using a mass spectrometer.

(RT-PCR) / polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assay, chip or Northern blot to detect the expression level, Methods can be used and these assays are well known and can also be performed using commercially available kits, and those skilled in the art will be able to select appropriate ones for the practice of the present application. For example, as a detection reagent in a method for measuring the presence and amount or pattern of the mRNA by RT-PCR, for example, a primer specific to the mRNA of the marker of the present invention. A primer refers to a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding with a template and allowing the reverse transcriptase or DNA polymerase to initiate replication of the template.

The detection reagent used in the present invention may be conjugated with a coloring material such as a fluorescent material for signal detection.

Detection herein includes quantitative and / or qualitative analysis, including detection of presence and absence and expression level detection, and such methods are well known in the art, and those skilled in the art will appreciate that methods suitable for the practice of the present application You will be able to choose. According to one embodiment of the invention, the detection reagent comprises an antibody, and the detection of the CTRP3 protein of the present invention is carried out using a monoclonal antibody that specifically binds thereto.

Antibodies that may be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) Or phage antibody library methods (Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with an antibody-producing lymphocyte, and the techniques required for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay, capture-ELISA, inhibition or competitive assay, sandwich assay, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984, which is incorporated herein by reference. By analyzing the intensity of the final signal by the above-described immunoassay process, that is, performing signal contrast with a normal sample, diagnosis of disease occurrence can be made.

According to another embodiment of the invention, the detection reagent is a reagent for RNA analysis, and the detection according to the present invention is carried out at the mRNA level. Detection of mRNA is usually performed through Northern blot or reverse transcription PCR (polymerase chain reaction). In the latter case, it is possible to detect a specific gene in a specimen by isolating RNA of the specimen, specifically mRNA, synthesizing cDNA therefrom, and then using a specific primer or a combination of a primer and a probe to detect the presence / Or the amount of expression can be determined. Such a method is described in, for example, Han, H., Bearss, DJ; Browne, LW; Calaluce, R .; Nagle, RB; Von Hoff, DD. Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002 , 62, (10), 2890-6).

Here we confirmed the effect of CTRP3 as a target for the treatment of goitosis. Specifically, the present inventors confirmed that CTRP3 antibody can inhibit insomnia due to damage due to overuse of tendons (FIG. 3). Specifically, the CTRP3 neutralizing antibody effect was verified using a mouse challenge model (Achilles tendon) and a rat manifestation model (rotator cuff). As a result, the histological characteristics (A, B and D in Fig. 5) of the trophoblast were reduced by the CTRP3 antibody and the physical ability (C in Fig. 5) was maintained.

In another aspect, the present invention is directed to a pharmaceutical composition for the treatment of a symptomatic disease comprising a CTRP3 inhibitory substance.

The CTRP3 inhibitory substance contained in the composition according to the present invention is a substance capable of lowering the concentration of the CTRP3 protein or inhibiting its activity.

Expression of CTRP3 involves both expression of the gene into mRNA, expression of the mRNA into the protein, and mRNA degradation or proteolysis. The activity of CTRP3 is the biological function / activity of the CTRP3 protein in cells or tissues.

Therefore, various substances exhibiting such effects can act as inhibitors of the present invention. Substances capable of inhibiting expression or activity at transcriptional and / or translation levels include, for example, proteins or RNAs comprising low molecular weight compounds, antibodies and polypeptides, and nucleic acid molecules including DNA, such as antisense oligonucleotides, siRNA, shRNA or miRNA, or any combination thereof.

As used herein, the term " expression " includes all steps of the process in which a gene is made into a protein in vitro or in a cell, for example, transcription from a gene to mRNA, and translation from mRNA to protein.

As used herein, "active" means a biological action that allows an expressed protein to function intrinsically in a cell.

In other embodiments of the invention, the inhibitor is an antibody or antigen-binding fragment capable of specifically recognizing CTRP3 and interfering with, inhibiting, interfering with or inhibiting its activity and / or mechanism of action. The term antibody as used herein includes complete antibodies, antigen-binding fragments of antibody molecules, and antibodies or fragments thereof functionally equivalent thereto. The antibodies of the invention may also be of the IgG, IgM, IgD, IgE, IgA or IgY type and may be of the IgGl, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, short chain, heterologous, humanized and humanized antibodies and active fragments thereof and antibody mimetics.

Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) Or phage antibody library methods (Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with an antibody-producing lymphocyte, and the techniques required for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

The antigen-binding fragment comprises a portion of an antibody that lacks some amino acids but is capable of specifically binding to the antigen, as compared to the full-length chain consisting of two heavy and two light chains. Such fragments may be specifically biologically active in terms of being capable of specifically binding to the target antigen or competing with another antibody or antigen binding fragment for binding to a particular epitope. In one embodiment, such a fragment comprises at least one CDR present in a full-length light chain or heavy chain, and in some embodiments, a short chain heavy and / or light chain, or a portion thereof. Such biologically active fragments can be produced by recombinant DNA technology or can be produced, for example, by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include but are not limited to Fab, Fab ', F (ab') 2, scFab, dsFv, Fv, scFV, scFV-Fc, diabody, minibody, scAb, and dAb But not limited to, any mammal, including a human, mouse, rat, camelid or rabbit. The "Fc" region herein encompasses two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. These two heavy chain fragments are linked to each other by hydrophobic interactions of two or more disulfide bonds and the CH3 domain. A "Fab fragment" herein is composed of one light chain and one heavy chain comprising only the variable region and CH1. The heavy chain of a Fab molecule can not form a disulfide bond with another heavy chain molecule. The scFab is a two-molecule Fab linked by a flexible linker. As used herein, the term "Fab 'fragment" is intended to include a region between the CH1 and CH2 domains of the heavy chain in addition to the Fab fragment, such that a chain interdisulfide bond is formed between the two heavy chains of the two Fab' ) to form a _second molecule. As used herein, the term "F (ab ') ₂ fragment" includes two light chains, including two heavy chains comprising two light chains and a variable region, CH1 and a portion of the constant region between the CH1 and CH2 domains, Inter-chain disulfide bonds are formed between the heavy chains. Thus, the F (ab ') ₂ fragment is composed of two Fab' fragments, and the two Fab 'fragments are associated with each other by a disulfide bond therebetween. As used herein, "Fv region" is a fragment of an antibody that comprises variable regions of heavy and light chains but not constant regions. sdFV is a heavy chain and light chain linked by a disulfide bond. The scFv is linked by a flexible linker with Fv. scFv-Fc is an Fc linked to scFv. The mini body is connected to scFV with CH3. The diabody contains two scFVs. As used herein, a "single chain antibody" (scAb) is a single polypeptide chain comprising a constant region or a light chain constant region of the heavy chain to which the heavy and light chain variable regions are linked by a flexible linker. As used herein, "domain antibody" (dAb) is an immunologically functional immunoglobulin fragment comprising only the variable region of the heavy chain or the variable region of the light chain.

Materials / methods that can inhibit the function and activity of a protein are well known in the art, and those skilled in the art will readily be able to select appropriate ones. For example, an antibody against which a neutralizing antibody can be used is a kind of protein that binds to a specific epitope of CTRP3, and the binding effect is different depending on the binding ability, the binding site, and the type of the antibody. Antibodies that neutralize the function or activity of the CTRP3 are used herein. Such antibodies are known in the art and can be selected, for example, with reference to those described in the Examples herein.

Antibodies that may be used herein are polyclonal or monoclonal antibodies or antigen binding fragments thereof. The antibody may be derived from any mammal, including a human, mouse, rat, camelid or rabbit.

In other embodiments of the disclosure, the inhibitor is a polypeptide capable of interfering with, interfering with, or inhibiting CTRP3 expression, activity and / or action / mechanism. As used herein, the term polypeptide refers to a polymer of a natural or synthetic amino acid and does not refer to a specific length as long as it has such an action, and includes peptides and oligopeptides. Such polypeptides include those modified by natural or artificial modifications such as glycosylation, acetylation, phosphorylation, and the like.

In another embodiment according to the present application, the inhibitor which may be included in the composition according to the present invention is a nucleotide such as small interfering RNA (siRNA) or small hairpin RNA or miRNA (microRNA). siRNAs, shRNAs and miRNAs, through RNA interference, for example, short-length interfering RNAs (siRNAs) bind to the transcript in a sequence-specific manner to form an RNA Induced Silencing Complex (RISC) RNA is silenced so that it can not be expressed as a protein in cells. The siRNA, shRNA or miRNA has a sequence that is highly complementary to its target sequence. A highly complementary sequence means at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with at least 15 consecutive bases in length of the target gene. Antisense oligonucleotides, siRNAs, shRNAs and / or miRNAs targeting a CTRP3 gene according to the present invention may be used as long as they bind to the target nucleic acid and function as silencing, and their biological equivalents, derivatives and analogues . Antisense oligonucleotides are as known in the art and are, for example, short synthetic nucleic acids that bind to any coding sequence of the target protein and inhibit / reduce the expression of the target protein. The antisense RNA may have an appropriate length depending on the target gene and the delivery method, for example, about 6, 8 or 10 to 40, 60 or 100 nucleotides. In one embodiment, the siRNA is used to inhibit CTRP3 gene expression. Such siRNAs may include various sequences as long as they achieve the desired effect by reducing the expression of the gene of interest.

The term "treatment ", as used herein, refers to any act that improves or alleviates symptoms of a condition or disease caused by administration of a composition according to the invention. Those skilled in the art will be able to ascertain the precise criteria of the disease by referring to the data presented by the Korean Medical Association, and to judge the degree of improvement, improvement, and cure.

In addition to the above-mentioned active ingredients, the composition of the present invention may further comprise at least one pharmaceutically or physiologically acceptable carrier.

"Carrier" as used herein is intended to mean a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. Examples of such carriers include buffers such as saline, Ringer's solution, buffered saline, phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, Or immunoglobulins; Other hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, For example, mannitol or sorbitol, salt forming counter ions such as sodium, and / or nonionic surfactants such as tweens, polyethylene glycol (PEG) and PLURONICS.

If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administering the composition of the present invention is not particularly limited thereto, and the known method of administering the inhibitor may be applied. The parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical application) Or parenteral administration. In the case of parenteral administration, it can be administered through a patch type nasal / respiratory patch attached to the skin. In order to obtain a rapid therapeutic effect, administration by intravenous injection is preferable.

The dosage ranges vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of disease, and doses of known inhibitors can be applied. For protein preparations, including siRNAs, miRNAs, antisense oligonucleotides, shRNA preparations and polypeptides, parenteral administration may be preferred but not excluding other routes and means. In the case of typical medicaments, the dosage unit comprises, for example, from about 0.01 mg to about 100 mg, but does not exclude the following ranges and above. The daily dose may be about 1 μg to 10 g, and may be administered once a day or divided into several times a day.

The present invention is based on the finding that CTRP3 is important for development and initiation of dry symptom, and can be developed as a therapeutic agent for the disease by screening substances that inhibit the activity of CTRP3.

In this regard, the present invention is directed to a method for screening for a condition for treating a condition for treating a condition comprising the following steps.

In one embodiment, the method comprises: providing a cell that overexpresses CTRP3; Treating the cell with a test substance expected to inhibit the expression or activity of CTRP3 in the cell; And when the expression or activity of the CTRP3 is decreased in the cell as compared with a cell not treated with the test substance as a result of the treatment, the test substance is selected as a candidate substance for the treatment of the disease.

The CTRP3 used in the present invention can be used in the present method in a variety of ways depending on the specific method for screening. For example, mammals, especially those derived from humans or mice, can be used. In addition, even the same host, for example, a human, may have sequence variation depending on a specific individual, region, environment, and the like. Of course, this sequence may be modified (deletion, substitution, addition) Can be used in the present invention.

CTRP3, which may be used in the methods herein, may be provided in isolated form or in the form of cells expressing it. In the case of a cell, it may be a cell strain expressing or overexpressing the protein employed internally in the method of the present invention or introducing the plasmid expressing it (transient or stable delivery).

In one embodiment, the cell line that may be used in the method according to the present invention includes, but is not limited to, a dry cell line, and one skilled in the art will be able to select the appropriate one with reference to the examples herein.

The amount of the protein used, the type of the cell, and the amount and type of the test substance used in the present method will depend on the specific experimental method used and the kind of the test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance which inhibits or reduces the expression or activity of CTRP3 in the presence of the test substance as compared with the control group not in contact with the test substance is selected as a candidate substance. Decrease by about 95%, decrease by about 90%, decrease by about 85%, decrease by about 80%, decrease by about 75%, decrease by about 70%, decrease by about 65% Less than 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 30%, less than about 20% no.

Refers to a substance that is expected to inhibit the expression or activity of CTRP3 and may be a low molecular weight compound, a high molecular weight compound, a mixture of compounds (e.g., a natural extract or a cell or tissue culture), or a biologic But are not limited to, proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes and DNAzymes) or sugars and lipids. The test substance may be a polypeptide having two or more amino acid residues, such as six, ten, twelve, twenty or fewer or more than twenty such as 50 amino acid residues. The test materials can be obtained from libraries of synthetic or natural compounds, and methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich Available from MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, "1-bead 1-compound" library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like.

For example, for the purpose of screening a drug that inhibits the expression or activity of CTRP3, the compound may be used that has a therapeutic effect of low molecular weight. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such library of compounds includes peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

For example, biologics can also be used for screening. Biologics refers to a cell or a biomolecule, which refers to a substance produced using biomolecules, proteins, nucleic acids, carbohydrates, lipids, or in vivo and in vitro cell systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organic materials found in polynucleotides, peptides, antibodies, or other plasma.

Here we demonstrate that activation of CTRP3-mediated Akt (also known as protein kinase B, PKB) signaling is essential for the function of CTRP3 in the development of trophic conditions. Here, activation means that the phosphorylation of a specific amino acid in Akt protein is finally increased. According to the present invention, the phosphorylation of the serine residue (Ser473), which is the 473rd amino acid of the Akt protein, was found to increase, and activation of Akt signal was activated when the phosphorylation of Ser473 was increased.

Therefore, the method according to the present invention further comprises the step of detecting Akt activation, and when the test substance is reduced as compared to the untreated cell, the step of screening the test substance as a candidate for to treat disease drug do.

AKT is a serine / threonine kinase 1 protein, also known as Protein Kinase B, PKB, whose sequence is known in the art. For example, the NCBI accession numbers of human genes and proteins are NM_001014431, NM_001014432, NM_005163; And NP_001014431, NP_001014432, NP_005154, and the NCBI accession numbers of mouse genes and proteins are known as NM_001165894, NM_001331107, NM_009652; And NP_001159366, NP_001318036, NP_033782.

Accordingly, in another aspect, the present invention provides a method for diagnosing or diagnosing a symptomatic disease, comprising the steps of: providing a biological sample derived from the subject, Detecting CTRP3 in said biological sample; And detecting CTRP3 in Invitro when the CTRP3 is increased in the biological sample as a result of the detection as compared with a control.

In one embodiment, the detection according to the invention can be detected at the mRNA or protein level as an expression level of CTRP3, which method is well known in the art, for example the nucleic acid comprises a nucleic acid amplification method such as PCR, northern blot, The protein includes, but is not limited to, an immunoassay using an antibody capable of specifically recognizing CTRP3, and may also refer to those described herein.

Biological samples that may be used in the method according to the present invention described above are tissues, e.g., cancer cells or tissues, in which the expression of CTRP3 can be detected.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Human samples. Human samples were obtained at Boramae Hospital, Seoul. Nineteen human rotator cuff tendons were obtained from 14 rotator cuff tear patients and four cruciate ligaments samples were obtained from four knee joint degenerative arthritis patients. Written consent was provided to all participants, and the study passed IRB deliberations. Evaluation of histology was done through a modified Bonar scoring system. All histological and immunohistochemical samples were independently evaluated by two pathologists with the relevant expertise. The evaluators were unaware of any of the above-categorized features of the sample.

Mouse transduction and transfection. For mouse transduction, the right hind limb of the mouse was epilated and 40 micrograms of plasmid DNA was injected into the muscle around the hind limb tendon. Thereafter, a conductive gel was applied and electric shock was applied to the caliper electrode of 5 mm gap. The stabbing was performed with BTX Gemini X2 (Harvard Bioscience, Inc.) at 225 V / cm, 25 ms, 5 times. The Ad- eGFP transgenic mice or Ad- Ctrp3 for injection was injected every 3 weeks a total of seven days in the hind leg muscles. For the experiment, 8th week C57BL / 6J male mice were used and were used for the experiment after 5 weeks of electric shock or first virus injection.

Induction of murine manifestation by overuse. 8th week C57BL / 6J male mice were purchased from Daehan-Biolink Co.). The mouse treadmill running consisted of 1 week (15 m / min, 15 min / day, 6 days / week, 10 ° ramp), 2 weeks (15 m / min, 1 hour / day, 6 days / ), 3 weeks (15 m / min, 2 hour / day, 6 days / week, 10 ° slope) for 6 weeks. Air puffs attached to the treadmill were used for treadmill running induction.

Induction of rheumatoid arthritis by overuse. 12 Sprague Dawley male rats were purchased from Daehan-Biolink Co.). Rat treadmill running was 1 week (15 m / min, 30 min / day, 6 days / week, 10 ° downward slope), 2 weeks (gradually increasing 15-20 m / min, 1 hour / day, 6 days / week , 10 ° downward slope), 2 weeks (20m / min, 2 hour / day, 6 days / week, 10 ° downward slope) for 5 weeks. Air puffs attached to the treadmill were used for treadmill running induction.

Partial transection Achilles tendon injury model. An 8-week C57BL / 6J male mouse was anesthetized, and skin and synovial sheath were exposed with a scalpel blade. Using a 1.5mm ear punch, a hole of 0.75mm semicircularity was drilled in the Achilles tendon of the right hind leg. The skin was sutured and raised in a cage for 3 weeks and then treated for 2 weeks.

Behavioral experiments. Treadmill running (15 m / min, 30 min, 10 ° uphill slope) was performed to evaluate the motor ability of the mice. An electric foot shock was used behind the treadmill to induce the mouse running. The resting behavior in treadmill running was measured by the number of electric shocks. In order to evaluate the exercise capacity of the rats, the maximum force that the rats could hold the bar with the front foot was measured using a grip strength test meter.

Neutralization antibody therapy. Neutralization antibody treatment was performed after anesthesia. One microgram of anti-CTRP3 antibody (Abcam) was injected twice weekly for a total of 2 weeks into the peripheries of the tendons.

Histology and immunohistochemistry. Human rotator cuff samples and cruciate ligament samples and mouse Achilles tendon were fixed in PBS for 4 days in paraformaldehyde for 7 days and 3 days, respectively. After paraffin embedding, sections were 7 mm thick. For histological staining, the sections were deparaffinized with xylene and hydrated with ethanol. Samples were stained with H & E, Alcian Blue, Nuclear Fast Red, and Picrosirius Red. The histological evaluation of the samples used a modified Bonar scoring system. For immunohistochemical staining, the sections were incubated in 70 占 폚 citrate buffer (pH 6.0) for 1 hour to recover the antigen. The sample was blocked with PBS containing 1% BSA and the primary antibody was treated at 4 ° C overnight, and the secondary antibody was treated at room temperature for 1 hour.

In vitro chonrogenesis. Human mesenchymal stem cells are mixed with the DNA of 30 micro-grams of the back was resuspended in 2.5 X 10 ^5/200 ul using Opti-MEM (Gibco) was electric shock to 2mm gap cuvette. Electrical shock was performed using BTX Gemini X2 (Harvard Bioscience, Inc.) at 500 V, 0.1 ms, and 1 time. After 3 days, the cells were transferred to a 15-ml tube and centrifuged at 400 g for 4 minutes. Cells were then seeded in 500 μl of con- dible medium (DMEM / F-12 supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 250 ng / ml amphotericin B, 1.25 mg / ml BSA, 1% insulin-transferrin-selenium, 1 mM sodium pyruvate, 50 μM L-aspartic acid, 50 μM L-proline and 100 nM dexamethasone). The tube lid was opened briefly and incubated for 3 days at 37 ° C in 5% CO ₂ and replaced with new conrogenic media containing 10 ng / ml TGF - β1. The media change was conducted for a total of 28 days every three days. The pellet was then fixed in 4% PFA and then cryo-sectioned to 5 micrometers and stained with Alcian blue / Nuclear fast red. Measurements of dyeing were performed with Image Pro Premier software.

In vitro neo-tendon construction. 6 well plates were coated with SYLGARD (Dow Corning) and incubated overnight at 55 < 0 > C. After sterilization with 70% ethanol and UV treatment. Human mesenchymal stem cells using Opti-MEM (Gibco) 2.0 X 10 5/200 back was resuspended in 30 ul of a mixture of DNA micro grams had electrical shock to 2mm gap cuvette. Electrical shock was performed using BTX Gemini X2 (Harvard Bioscience, Inc.) at 500 V, 0.1 ms, and 1 time. The cells were then seeded in a 60 mm dish, incubated for 3 days, and seeded in a 6 well dish with 2 mg of fibrinogen and 1 U of thrombin. Culture media containing 100 micrograms of L-ascorbic acid 2-phosphate was treated every 2 days for 2 weeks, after which the dry constituents were fixed in 4% PFA, embedded in paraffin, sectioned at 7 micrometer and then stained with H & E . The thickness of the gun construction was performed with Image Pro Premier software.

Adenovirus. Adctrp3 (ADV-254235) was purchased from Vector Biolabs. Ad- eGFP was obtained from Prof. Kim Jae-beom (Department of Life Science, Seoul National University). Adenovirus titer measurements were performed according to the protocol of the Rapid Titer Kit (Applied Biological Materials Inc.).

Production of CTRP3 overdose culture. The HEK293T cell line was transfected with the CTRP3 overexpression vector using PEI chemistry to allow the HEK293T culture to contain excess CTRP3 secreted from the cells. The CTRP3 over-content medium was sterilized with a 0.22 micrometer filter. It was confirmed by immunoblotting that CTRP3 was excessively contained in the CTRP3-over-culture medium.

CTRP3 overexpressing plasmid vector production. Mouse Ctrp3 mRNA was extracted from Achilles tendon of C57BL / 6 mice. Ctrp3 cDNA was synthesized from mRNA by reverse transcriptase reaction. Ctrp3 cDNA was inserted into p3xFLAG-CMV14 plasmid to construct a plasmid capable of overexpressing Ctrp3 in mammalian cells.

Example 1. Upregulation of CTRP3 expression in human and mouse disease

RNA sequencing was performed using samples of sham and damaged mouse Achilles tendons to identify causative factors for specifically proliferating tendinopathy in disease states. Further, in view of its potential advantages in terms of pharmacogenetics by antibody-based therapies, the present inventors have investigated candidate genes classified as 'cytokines' in the database of Ingenuity Pathway Analysis (IPA) (1740 gene) and UniProt (1662 gene) Respectively. CTRP3 was identified as the most significantly up-regulated cytokine under the impaired condition (multiple change = 68.204; P value = 1.83 x 10 ^-7 ) (Fig. 1A). When arranged according to the percentile order, CTRP3 was found to be one of the most expressed genes in the damaged transcriptome (Fig. 1B) and cytokine transcript in the damaged tendon.

CTRP3 expression in both mRNA and protein levels in patients with rotator cuff tendon injury correlated with the severity of symptoms (C and D in Fig. 1). Consistently, when cross validated with the public data set of human problem (GSE26051) (39), CTRP3 expression was increased in injured lesions of the tendinitis lesion tendons compared to non-lesioned controls (FIG. The tendons and ligaments are closely related connective tissue and represent tightly arranged collagen fibers that resist tensile stress (40, 41). Ligaments undergo similar degenerative changes during injury or abuse (40, 42). In fact, collagen fiber alignment and cartilage layer disassembly were observed in ligaments with high posterior cruciate ligament injuries (Fig. 1F). CTRP3 was significantly upregulated in high grade ligament injuries (Fig. 1F). As a rodent model of trophoblastic disease, a long-running treadmill run method (43) was used to cause symptoms of trophic symptoms in the Achilles tendon of mice (G and H in Figure 1). Both CTRP3 mRNA and protein levels were significantly increased in the Achilles tendon of rats that received 5 weeks of overproduction (G and I in Figure 1).

Example 2. Onset of testicular hyperplasia by overexpression of CTRP3 in mouse

The increased expression of CTRP3 in the tendon of the disease indicates the relevance to the pathogenesis of the disease. The role of CTRP3 in tendons was examined by overexpression of CTRP3 protein in mouse Achilles tendon. First, it was confirmed that exogenously expressed CTRP3 was secreted into the medium by primary mouse cultured mouse tendon cells. Ctrp3 expression vector was injected into the tendon and then overexpression of CTRP3 in the tendon cells of the Achilles tendon was confirmed by electroporation (Fig. 2, A). In particular, overexpression of CTRP3 alone resulted in major manifestations of the disease, including disintegration and denaturation of collagen fibers, abnormal accumulation of proteoglycan between irregular cells and fibers (Fig. 2 A and B). CTRP3 overexpression resulted in a significant increase in the severity of the behavioral condition evaluated by the Bonar scoring system (B and C in Figure 2). In addition, exercise evaluation by treadmill running experiments indicates that overexpression of CTRP3 significantly impairs exercise capacity in mice (FIG. 2D), indicating a malfunction-related dysfunction. At the molecular level, CTRP3 overexpression induced upregulation of the disease-associated collagen type, Col3a1 and cartilage matrix genes Col2a1 and Acan (FIG. 2E). Indeed, changes in ECM composition during development of the disease, including the accumulation of type III collagen and cartilage specific matrix proteins, are major factors in deteriorating tendon properties by weakening tensile strength.

The role of CTRP3 in trophoblastic disease was investigated by injecting adenovirus expressing Ctrp3 mice (Ad-Ctrp3) around the tendons in mice. Overexpression of CTRP3 in tendon-surrounding tissues surrounding the Achilles tendon induced manifestations of trophic symptoms in terms of histopathology and behavior (Figs. 2F-I). These results indicate that secreted CTRP3 into the bursa can induce degenerative changes in tendon cells in a non-autonomous manner.

Example 3. Prevention of development of tongue in mouse due to blocking of CTRP3

Next, we investigated the therapeutic efficacy of antibodies that block the function of CTRP3 in the development of trophic conditions. The selected -CTRP3 antibody binds effectively to the secreted native form of CTRP3, suggesting the possibility of neutralizing CTRP3 function. Antibody therapy was first tested in Achilles tendinopathic model mice induced by prolonged onset treadmill running (FIG. 3, A; top panel). Compared to control immunoglobulin G (IgG) therapy, anti-CTRP3 antibody therapy is associated with a reduction in the severity of symptoms associated with hypertension induced by overuse including sternal deformity, collagen fiber breakdown, and proteoglycan accumulation (FIGS. 3, B and C) Significantly reduced major symptoms. Expression of the trophoblast-associated gene in the tendon induced by transient disappeared by treatment with anti-CTRP3 antibody. Consistently, the interruption of CTRP3 restored the athletic performance of the disease-prone model mice (Figure 3E) as well as a substantial reduction in the severity of the severity of symptoms assessed by the Bonar scoring system (Figures 3, C and D) It shows the therapeutic effect of CTRP3 neutralization in pathology.

We also investigated whether blockade of CTRP3 improves the healing process of partially amputated mouse Achilles tendon (Fig. 3A, bottom panel). Damaged tendons treated with control IgG were restored to fibricartilaginous scar tissue as evidenced by acute cartilage tissue degeneration and proliferative (FIGS. 3F-H). In contrast, treatment of damaged tendons with anti-CTRP3 antibodies effectively inhibited fibrous cartilage features in the regenerated tissue and restored normal cellular and collagen fiber alignment (Fig. 3, F, H).

Example 4. Activation of cartilage gene program in tendons by CTRP3

To gain insight into the role of CTRP3 in the development of trophic conditions, we performed transcriptomic analysis using tendons isolated from various rodent disease manifestations. Transcripts of tendon transfected with the Ctrp3 overexpression vector were compared with transcripts transfected with control vector and naive tendon. Uncontrolled hierarchical clustering analysis grouped the differentially expressed genes into three clusters (Fig. 4A). Among them, 'Cluster 2' represents a set of genes specifically activated for CTRP3 overexpression (Table S2).

Transcriptome analysis was performed on the tendinitis tendon and naïve tendon due to transient use treated with control IgG or anti-CTRP3 antibody. Of the two clusters identified, 'Cluster 4' is a set of genes activated by the use of tendon motility, but mitigated by blocking CTRP3 function (FIG. 4B). In practice, cluster 4 is highly overlapped with cluster 2 (initial P = 1.55 × 10 ^-34 ) (FIG. 4C).

To evaluate the role of CTRP3 in the development of atherosclerosis, Biological Network Gene Ontology tool (BiNGO) (44) was used to analyze biological processes represented by cluster 2. Histologically structured tin associated with cartilage development and proteoglycan metabolism appeared to be very abundant in cluster 2 (FIG. 4D). Indeed, gene set inhibition assay (GSA) revealed that the 'proteoglycan synthesis' gene set and 'cartilage development' of IPA were positively enriched in CTRP3 overexpressing total transcripts (FIG. 4E). Similarly, the genes listed in the two annotations were up-regulated in comparison with naïve mice in the over-inducible challenge model. CTRP3 antibody treatment effectively inhibited this set of genes in mice with behavioral problems (FIG. 4E). Finally, we examined how gene clusters activated by CTRP3 overexpression are regulated in human disease. The cluster 2 gene set was positively enriched in tendinous tendinitis as compared to the non-lesioned control (Fig. 4F). Taken together, CTRP3 indicates activating the tendon cartilage transcriptional program to account for important molecular and morphological changes associated with the disease.

Example 5. Inhibition of tendon development by CTRP3 mediated signal transduction

In tendons with fever, cartilage deformation is easily observed simultaneously in the accumulation of proteoglycan and in the lesion area (18-22). Indeed, the trophic features observed in rodent models are due to abnormal differentiation into non-vascular cell types such as the cartilage of endogenous mesenchymal stem cells (MSCs). Because the results of this study indicate the role of CTRP3 in activating cartilage-related transcriptional programs, we investigated how CTRP3 affects cartilage formation and the determination of MSC fate in the tendon. As a result, CTRP3 overexpression effectively promoted cartilage differentiation of human MSCs grown in pelleted cultures (FIG. 5A). In contrast, CTRP3 inhibited the formation of new tendons in MSCs grown in fixed fibrin gels, resulting in a marked reduction in the thickness of the new tendon constructions (Fig. 5B).

Next, the signal path affected by CTRP3 was examined. CTRP3 induces signaling through various subtypes of mitogen-activated protein kinase (MAPK) cascade (31,45) and phosphatidylinositol-3-kinase (PI3K) -Akt pathway, depending on cell type and cell status (26, 31 ). CTRP3-conditioned medium in primary cultured MSCs and tendon cells activates PI3K-Akt signaling, but not MAPK pathway driven by extracellular signal-regulated kinases (ERKs), c-Jun N terminal kinases (JNKs) and p38 protein kinase (C and D in Fig. 5). Notably, the inhibition of the PI3K-Akt pathway essentially abolished the false expression of CTRP3-mediated COL2A1 and ACAN, restoring the strong expression of COL1A1 (FIG. 5E). Consistently, the inhibitory effect of MSRs of CTRP3 on the formation of new tendon was alleviated by inhibiting PI3K-Akt signaling (Fig. 5F). Taken together, activation of CTRP3-mediated PI3K-Akt signaling is essential for the function of CTRP3 in the development of symptoms.

Example 6. Treatment of rotator cuff tendinopathy in mice using CTRP3 antibody

Rotator cuff tendinopathy It is a major cause of shoulder disability (46). Rotational mastopathophilia occurs when the rotator cuff tendons are exposed to overload or repeated stress without adequate recovery time (47-49). In this study, long-term downhill running was used to induce rotavirus troubles in rats by (50, 51), and the effect of antibody therapy targeting CTRP3 was tested (Fig. 5A). CTRP3 neutralizing antibodies were effectively injected around the tendons to effectively inhibit collagen fiber disruption and abnormal basal deposition (e.g., chondroitin sulfate) by transient transient use (FIG. 6B). Consistently, the strength of the forelimb mice that had problems with troubles was restored to CTRP3 antibody therapy (Fig. 6C). At the molecular level, the biomarkers associated with symptoms such as MMP13, fibronectin, and COL3A1 were detected at a minimum level in the rotator cuff tendons treated with CTRP3 antibody, indicating that the tendons were totally healed due to the blockage of CTRP3.

Claims (10)

A pharmaceutical composition for the treatment or prophylaxis of a disease associated with a CTRP3 inhibitor.
The method according to claim 1,
Wherein said inhibitor is an antibody capable of neutralizing the activity of CTRP3 protein.
The method according to claim 1,
Wherein the inhibitor is siRNA or shRNA that inhibits expression of the CTRP3 gene into a protein.
A composition for CTRP3 detection.
5. The method of claim 4,
The detection substance is a reagent capable of detecting the marker at a protein or nucleic acid level,
The protein level detection reagent may be a reagent for Western blotting, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry,
The nucleic acid level detecting reagent may be a reagent used in a polymerase chain reaction, a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, an Nuclease protection assay (RNase, S1 nuclease assay), an in situ hybridization method, a nucleic acid microarray or a Northern blot , A composition for diagnosing a condition.
6. The method of claim 5,
The detection reagent at the protein level includes an antibody, an antibody fragment, an aptamer, an avidity multimer or peptidomimetics that specifically recognizes the protein full length of the marker or a fragment thereof,
The detection reagent for the nucleic acid level may comprise a nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a pair of primers or a probe or a pair of primers that specifically recognize the nucleic acid sequence and a fragment of the complementary sequence, , A composition for diagnosing a condition.
In order to provide information necessary for the diagnosis or prognosis of the disease,
Detecting the level of nucleic acid and / or protein of the CTRP3 biomarker from the biological sample derived from the test subject;
Comparing the nucleic acid and / or protein level detection results to a corresponding result of a corresponding marker of a control sample; And
Wherein the increase in the expression of the nucleic acid or protein of the subject-derived sample, as compared to the control sample, is associated with a diagnosis or prognosis of the symptomatic diagnosis of the subject.
Providing cells that overexpress CTRP3;
Treating the test substance expected to inhibit the expression or activity of CTRP3 in the cell; And
As a result of the above-mentioned treatment, it was found that compared with the cells not treated with the test substance,
And screening the test substance as a candidate for a therapeutic agent for oncopathy when the expression or activity of the CTRP3 is decreased.
9. The method of claim 8,
Wherein said cell is a testicular disease-derived cell.
11. The method according to any one of claims 8 to 10,
Wherein the detecting further comprises detecting AKT activation,
And when the AKT activation is reduced as compared with the cells that have not been treated with the test substance, selecting the test substance as a candidate substance for the disease-causing treatment.

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