KR102179375B1 - CTRP3 as biomarker for tendinopathy and Composition and method of treating tendinopathy targeting CTRP3 - Google Patents

Abstract

The composition according to the present application inhibits CTRP3, which is a major factor causing tendonosis as identified herein, effectively inhibits the onset of tendonosis, and can effectively treat tendonosis. In addition, CTRP3, which is a major factor in generating tendonopathy identified herein, can be usefully used as a diagnostic marker for tendonosis.

Description

[CTRP3 as biomarker for tendinopathy and Composition and method of treating tendinopathy targeting CTRP3}

This application is a technology related to the detection and treatment of tendonosis.

The tendon (tendon) is a fibrous soft tissue that attaches muscles to bones, consisting of parallel arrangements of collagen fibers. Tendinopathy is the most common tendon-related disease. Tennopathy is a degenerative lesion of tendon tissue, characterized by gradual degeneration and irregular arrangement of tendon tissue. It is known that tendonpathy is caused by frequent use of the suggestion due to exercise and life activities, and the incidence rate is very high, especially in athletes and the elderly population. In fact, in the case of rotator cuff tear, a type of tendonopathy, the number of patients treated as of 2013 was 530,000, and 69% of all patients were in their 50s or older. The incidence of tendinopathy, which is linked with such an aging society, has been increasing every year, and it is believed that the demand for treatment will continue to increase.

Tennopathy has been studied relatively much for its causes and risk factors. However, the pathogenesis and treatment of tendonpathy are relatively poorly studied. In particular, there is no well-established treatment for tendonosis. Currently, the most commonly used treatments for tendonopathy include steroid anti-inflammatory drugs to suppress inflammatory reactions, platelet-rich plasma (PRP) injection, and surgical suture of ruptured tendon tissue. However, in the case of anti-inflammatory drugs or PRP injection, the effect is inconsistent as a treatment aimed at relieving symptoms without clearly understanding the mechanism of occurrence of tendonpathy, and in the case of suture surgery, it is simply a method of connecting damaged tendon tissues. As such, it is necessary to identify the molecular biological pathogenesis mechanism of tendonosis and to develop a fundamental treatment that eliminates the cause.

Korean Patent Application Publication No. 2016-0105363 relates to an autologous and allogeneic adipose-derived mesenchymal stem cell composition for healing tendon or ligament damage, and a method for producing the same.

Korean Patent Application Publication No. 2013-0000397 relates to a platelet-derived growth factor composition and method for the treatment of tendon diseases.

There is an urgent need to develop an antibody therapy for treating tendonosis more fundamentally and effectively as it is directly related to the pathogenesis of tendonpathy, not to relieve symptoms of tendonpathy.

The present application is to identify factors related to tendinopathy at the molecular level, and to develop effective strategies and therapeutic agents for tendonpathy targeting.

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

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

In another embodiment, the inhibitor according to the present application is siRNA or shRNA that inhibits the expression of the CTRP3 gene into a protein, a pharmaceutical composition for the treatment or prevention of tendonosis.

In another aspect, the present application provides a composition for diagnosis of tendonosis, including a substance for detecting CTRP3.

In one embodiment, the detection material used in the diagnostic composition according to the present application is a reagent capable of detecting the marker at the protein or nucleic acid level, for example, the protein level detection reagent is Western blot, ELISA, radioimmunoassay, Reagents for immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray may be mentioned, but are not limited thereto, and the nucleic acid level detection reagent is polymerase chain Reagents used in reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, Nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization method, nucleic acid microarray or Northern blot, but are not limited thereto.

In another embodiment, the detection reagent of the protein level in the detection material used in the diagnostic composition according to the present application is an antibody, antibody fragment, aptamer, which specifically recognizes the entire protein length of the marker or a fragment thereof, Avimer (avidity multimer) or peptidomimetic (peptidomimetics) is included, the nucleic acid level detection reagent is the nucleic acid sequence of the marker, the nucleic acid sequence complementary to the nucleic acid sequence, the nucleic acid sequence and a fragment of the complementary sequence A primer pair or probe that is specifically recognized, or a primer pair and probe, but is not limited thereto.

In another aspect, the present disclosure provides information necessary for diagnosis or prognosis of tendonopathy, comprising: detecting the level of nucleic acid and/or protein of the CTRP3 biomarker from a biological sample derived from a test subject; Comparing the detection result of the nucleic acid and/or protein level with the corresponding result of the corresponding marker of the control sample; And compared with the control sample, when the expression of the nucleic acid or protein of the sample derived from the subject is increased, it provides a method of detecting a CTRP3 biomarker comprising the step of correlating with the diagnosis or prognosis of the subject's tendonosis.

In another aspect, the present application provides a cell overexpressing CTRP3; Treating the cells with a test substance expected to inhibit the expression or activity of CTRP3; And when the expression or activity of the CTRP3 is decreased in the cells as a result of the treatment, compared with the cells not treated with the test substance, selecting the test substance as a candidate substance for treating tendonosis. Or, it provides a therapeutic agent screening method.

In one embodiment, the cells that can be used in the screening method are not limited thereto, but the cells include tendonopathy-derived cells.

In another embodiment, the detecting step in the screening method further comprises detecting AKT activation, and as a result of the detection, when the AKT activation decreases compared to cells not treated with the test substance, the test substance is And selecting a candidate substance for treating tendonosis.

The present study recognizes that the CTRP3 protein is overexpressed in tendonopathy and is a key factor in the onset of tendonosis, alleviates tendonopathy lesions using CTRP3 antibody that inhibits the function of the CTRP3 protein, and restores structural and functional functions of the Achilles tendon and rotator cuff tendon. Was confirmed in rats. Accordingly, the CTRP3 protein identified herein can be usefully used as a biomarker of the occurrence and severity of tendonosis. Furthermore, CTRP3 acts as an important target for the treatment of tendonosis, and a composition containing a substance that inhibits the function and expression of CTRP3 can be effectively used for the treatment of tendonosis. The adverse effects of CTRP3 protein in the occurrence of tendonosis can be identified up to the molecular level, and can be usefully used in the development of drugs for treating tendonosis targeting CTRP3.

1 is a result showing an increase in CTRP3 expression in a human sample and a mouse sample of tendonosis according to an embodiment of the present application. (a) It is a volcano plot showing changes in gene expression in a tendon injury mouse model. (b) It shows the gene expression in percentiles in normal mice and tendon injured mice. (c) Histological results of the 1st to 3rd stages of the human rotator cuff biceps tendon. PSR staining was performed using a polarization microscope. (d) It shows the relative CTRP3 expression level in the tissues of steps 1 to 3. (e) The lesion site and non-lesion CTRP3 expression level of the tendonopathy patient (GSE26051; n = 23) are box plots.
FIG. 2 is a result showing that tendonopathy is induced when exogenous overexpression is performed in a CTRP3 mouse model according to an embodiment of the present application. (a) Histology of normal mice, empty vectors, and CTRP3 overexpression vectors electroporated mice. (b) The degree of tendonosis of mouse tissue ((c)) was evaluated by Bonar score. (c) It shows the sum of Bonar scores. (d) The exercise capacity of the ball vector electroporated mice and the CTRP3 overexpression vector electroporated mice evaluated by the degree of resistance in treadmill running. (e) Comparison of the expression levels of tendonpathy genes between empty vector electroporated mice and CTRP3 overexpression vector electroporated mice. (f) Histological results of Ad- eGFP injected mice and Ad- Ctrp3 injected mice. (g) The degree of tendonopathy of mouse tissue ((f)) was evaluated by Bonar score. (h) It shows the sum of Bonar scores. (i) The exercise capacity of Ad- eGFP- injected mice and Ad- Ctrp3- injected mice evaluated by the degree of resistance in treadmill running.
3 is a result showing that tendonpathy is reduced when α-CTRP3 antibody is treated in a mouse tendonopathy model according to an embodiment of the present application. (a) It is an overview of tendon overuse and antibody treatment model, tendon injury and antibody treatment model. (b) Histology of normal and control antibody-treated mice, tendon overuse and control antibody-treated mice, tendon overuse and α-CTRP3 antibody-treated mice. (c) The degree of tendonopathy of mouse tissue ((b)) was evaluated by Bonar score. (d) It shows the sum of Bonar scores. (e) It is the exercise capacity of normal and control antibody-treated mice, overuse and control antibody-treated mice, and over-use and α-CTRP3 antibody-treated mice, evaluated for the degree of resistance in treadmill running. (f) Histology of tendon injury and control antibody-treated mice, tendon injury and α-CTRP3 antibody-treated mice. (g) The degree of tendonopathy of mouse tissue ((f)) was evaluated by Bonar score. (h) It shows the sum of Bonar scores.
4 is a result of promoting cartilage differentiation and inhibiting tendon differentiation by CTRP3 according to an embodiment of the present application. (a) The result of pellet culture after transfecting human mesenchymal stem cells with an empty vector and a CTRP3 overexpression vector. The left is histological results, and the right graph is the range of Alcian Blue staining quantified with Image-Pro software. (b) The result of fibrin gel culture after transfecting human mesenchymal stem cells with an empty vector and a CTRP3 overexpression vector. The left is histological results, and the right graph is the thickness of the key structure as quantified with Image-Pro software. (c, d) Human mesenchymal stem cells ((c)) and mouse tendon cells ((d)) were treated with a culture medium containing an excess of CTRP3, and phosphorylation of Akt, ERK1/2, p38, and JNK signaling proteins by immunoblot This is the result of checking the degree of (activation). (e, f) The result of fibrin gel culture after transfecting human mesenchymal stem cells with an overexpression vector through GFP overexpressing adenovirus and CTRP3 overexpressing adenovirus. The above is the result of quantifying the expression change ((e)) of genes related to dry differentiation and cartilage differentiation, and the bottom is the histology result and the thickness of the quantified tendon structure ((f)).
5 is a result showing that tendonpathy is reduced when α-CTRP3 antibody is treated in a rat tendonpathy model according to an embodiment of the present application. (a) Histology of normal and control antibody-treated rats, tendon overuse and control antibody-treated rats, tendon overuse, and α-CTRP3 antibody-treated rats. The lesion was confirmed by hematoxylin & eosin staining and Alcian blue staining in the upper part, and the increase in CTRP3 and chondroitin sulfate (CS) at the lesion site was confirmed by fluorescence immunostaining. (b) The degree of tendonopathy of mouse tissue ((a)) was evaluated by Bonar score. (c) Exercise capacity of normal and control antibody-treated rats, overuse and control antibody-treated rats, and over-use and α-CTRP3 antibody-treated rats whose strength of the forearm was evaluated with a grip strength test meter. (d) Histology of tendon injury and control antibody treated rats, tendon injury and α-CTRP3 antibody treated rats. The increase in MMP13, fibronectin (FN1), and type 3 collagen at the lesion site was confirmed by fluorescence immunostaining.
6 is a schematic diagram of a treatment procedure using a CTRP3 target antibody in patients with tendon rupture and tendonpathy.

Here, CTRP3 is overexpressed in tendinopathy, is an essential regulator of the development/progression of tendonopathy, and is based on the discovery of its mechanism.

In one aspect, the present application relates to the use of a tenopathy detection biomarker of CTRP3 (C1q/Tumor necrosis factor-Related Protein 3).

In one embodiment, the present application provides a composition or kit for diagnosing tendonopathy or measuring prognosis, including a substance for detecting CTRP3.

In the present application, it was confirmed that CTRP3 expression was increased in human and mouse tendonopathy (Fig. 1). In particular, CTRP3 overexpression was confirmed in common in tendon-injured mice and rotator cuff tear human samples. In addition, it was confirmed that CTRP3 expression increased as the severity of disease increased in human samples of rotator cuff tear. In addition, in the present application, it was confirmed that tendonopathy caused by overexpression of CTRP3 (Fig. 2D, I, E).

Accordingly, CTRP3 according to the present application can be usefully used not only for diagnosis of tendonosis, but also for determining 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, it is not known about its association with tendonopathy. CTRP3 protein and gene sequences are known, for example, in the case of humans, the NCBI (National Center for Biotechnology Information) gene and protein accession numbers are NM_030945, NM_181435, respectively; It is known as NP_112207, NP_852100. In the case of mice, the gene and protein accession numbers of NCBI are NM_001204134, NM_030888, respectively; And NP_001191063, NP_112150.

Herein, tendinopathy is the most common quality among tendon-related diseases. The tendon (tendon) is a fibrous soft tissue that attaches muscles to bones, consisting of parallel arrangements of collagen fibers. Tennopathy is a degenerative lesion of the tendon tissue, characterized by gradual degeneration and irregular arrangement of tendon tissue. Tennopathy is caused by frequent use of a suggestion due to exercise and life activities, and is a disease with a very high incidence rate, especially in athletes and elderly populations.

Diagnosis herein is to determine the susceptibility of a test subject to a disease for a specific disease or disease, to determine whether or not to have a specific disease or disease, the prognosis of a subject suffering from a specific disease or disease ( prognosis) or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).

As used herein, the term diagnostic biomarker or diagnostic marker is a substance capable of diagnosing a diseased tissue or cell by distinguishing it from a normal cell or a tissue or cell that has received appropriate treatment. It includes a protein or nucleic acid exhibiting an increase or decrease pattern at the site. The diagnostic marker herein is a CTRP3 protein whose expression is increased in tendon tissue in which tendonosis has occurred, or a nucleic acid or gene encoding the same.

As used herein, a biological sample refers to a substance or a mixture of substances containing one or more components capable of detecting a biomarker, and includes, but is not limited to, cells and tissues derived from living organisms, particularly humans. It also includes cells or tissues cultured in vitro as well as directly derived from an organism. In one embodiment, a tissue/cell or in vitro cell culture obtained from an organism in which tendonosis has occurred or is suspected of or likely to occur may be used, but is not limited thereto. It also includes a fraction or derivative of the cell or tissue. When using cells or tissues, cells themselves or lysates of cells or tissues may be used.

In the present application, the CTRP3 protein and its nucleic acid sequence are known, and are as mentioned above, but are not limited thereto, and include functional equivalents thereof.

As used herein, detection includes quantitative and/or qualitative analysis, and includes detection of presence and absence and detection of expression levels, and such methods are known in the art, and those skilled in the art may select an appropriate method for the practice of the present application. I will be able to.

The marker according to the present application can be detected at the level of detection of the presence or absence of nucleic acids, in particular mRNA and/or proteins, and/or the level of expression thereof, changes in expression levels, and differences in expression levels through quantitative or qualitative analysis.

In one embodiment, the diagnostic marker according to the present application is based on its functional characteristics and/or antigenic characteristics. The activity, function or nucleic acid encoding the protein can be detected using agents that specifically interact at the mRNA and/or protein level.

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

As used herein, the term detection substance is a substance capable of detecting the marker of the present application quantitatively at the level of a protein or nucleic acid, such as an mRNA, or to determine the presence or absence.

Methods for analyzing the expression pattern of the amount and presence of protein are known, for example, Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, and Ouchterloni ( Ouchterlony) immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS, protein chip (protein chip), and the like. Reagents for detecting protein or nucleic acid levels are known, for example, the former is 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, or It can be detected through reaction with a ligand or cofactor, or a mass spectrometer can be used. The reagent or material that specifically interacts or binds to the marker of the present application may be used in a chip manner or in combination with nanoparticles.

The diagnostic composition according to the present disclosure may contain reagents necessary for detection of the CTRP3 marker at the protein or nucleic acid, for example, mRNA level. For example, reagents that can be detected at the protein level may include monoclonal antibodies, polyclonal antibodies, substrates, aptamers, receptors, ligands or cofactors. These reagents can be incorporated into nanoparticles or chips if necessary. Proteins can also be detected using mass spectrometry.

Transcription polymerase chain reaction (RT-PCR)/polymerase chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assay, chip or northern blot for detection at the RNA level, expression level or pattern Modes can be used, such assays are known, and can also be performed using commercially available kits, and those skilled in the art will be able to select one suitable for practice herein. For example, as a detection reagent in a method for measuring the presence or absence of the mRNA and its amount or pattern by RT-PCR, for example, a primer specific for the mRNA of the present marker is included. The primer refers to a nucleic acid sequence having a free 3'hydroxyl group capable of binding complementarily to the template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template.

The detection reagent used herein may be conjugated with a color developing material such as a fluorescent material for signal detection.

As used herein, detection includes quantitative and/or qualitative analysis, and includes detection of presence and absence, and detection of expression levels, and such methods are known in the art, and those skilled in the art will use a method suitable for the practice of the present application. You will be able to choose. According to an embodiment of the circle, the detection reagent includes an antibody, and the CTRP3 protein detection of the present application is performed using a monoclonal antibody that specifically binds thereto.

Antibodies that can be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies are methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56). Alternatively, it can be prepared by the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production 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, which documents are incorporated herein by reference. For example, the production of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be easily performed. Polyclonal antibodies can be obtained by injecting protein antigens into suitable animals, collecting antisera from these animals, and then isolating the antibodies from antisera using known affinity techniques.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format includes radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbant assay), capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, and immunity. Including, but not limited to, fluorescent staining and immunoaffinity purification. The immunoassay or immunostaining method is 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, etc., the document 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, it is possible to diagnose whether a disease has occurred.

According to another embodiment of the present application, the detection reagent is a reagent for RNA analysis, and the detection according to the present application is performed at the mRNA level. mRNA detection is usually performed through Northern blot or reverse transcription PCR (polymerase chain reaction). In the latter case, a specific gene in the sample is detected using a specific primer or a combination of primers and probes after separating the RNA from the sample, particularly mRNA, and then synthesizing cDNA from it. Or it is a method that can determine the expression level. Such methods are described 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).

In the present application, the effect of CTRP3 as a target for the treatment of tendonosis was confirmed. Specifically, in the present application, it was confirmed that the CTRP3 antibody can suppress tendonosis caused by the overuse of tendons (FIG. 3). Specifically, the effect of the CTRP3 neutralizing antibody was verified using a mouse tendinopathy model (Achilles tendon overuse) and a rat tendonpathy model (rotator cuff overuse). As a result, the histological characteristics (A, B, and D of Fig. 5) of tendonopathy were decreased by CTRP3 antibody and the physical ability (C of Fig. 5) was maintained.

In another aspect, the present application relates to a pharmaceutical composition for treating tendonopathy comprising a substance for inhibiting CTRP3.

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

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

Therefore, various substances exhibiting such effects can act as inhibitors of the present application. Substances capable of inhibiting expression or activity at the level of transcription and/or translation are, for example, small molecule compounds, proteins including antibodies and polypeptides, or nucleic acid molecules including RNA and DNA, such as antisense oligonucleotides, siRNAs, Includes, but is not limited to, shRNA or miRNA or any combination thereof.

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

As used herein, "active" refers to a biological action that allows the expressed protein to function intact within a cell.

In another embodiment of the present application, 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 is intended to include intact antibodies, antigen-binding fragments of antibody molecules, and antibodies or fragments functionally equivalent thereto. In addition, the antibody of the present invention may be of the IgG, IgM, IgD, IgE, IgA or IgY type, and may be of the IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 class or a subclass thereof. Antibodies of the present invention include monoclonal, polyclonal, chimeric, single chain, bispecific, ape and humanized antibodies and active fragments and antibody mimetics thereof.

Antibodies are methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56). Alternatively, it can be prepared by the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production 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, which documents are incorporated herein by reference. For example, the production of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortal cell line with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be easily performed. Polyclonal antibodies can be obtained by injecting protein antigens into suitable animals, collecting antisera from these animals, and then isolating the antibodies from antisera using known affinity techniques.

The antigen-binding fragment lacks some amino acids compared to a full-length chain consisting of two heavy chains and two light chains, but contains a portion of an antibody capable of specifically binding to an antigen. Such fragments can be said to be biologically active in that they can specifically bind to a target antigen, or compete with other antibodies or antigen-binding fragments to bind to a specific epitope. In one aspect, such fragments comprise at least one CDR present within the full length light or heavy chain, and in some embodiments, comprise a short chain heavy and/or light chain, or a portion thereof. Such biologically active fragments can be produced by recombinant DNA technology or, 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. It can be derived from any mammal including, but not limited to, humans, mice, rats, camelids or rabbits. The “Fc” region herein includes two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. These two heavy chain fragments are bound to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The term "Fab fragment" herein consists of one light chain and one heavy chain comprising only the variable region and CH1. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. In scFab, two molecules of Fab are connected by a flexible linker. As used herein, "Fab'fragment" includes a region between the CH1 and CH2 domains of the heavy chain in addition to the Fab fragment, and an interchain disulfide bond is formed between the two heavy chains of the Fab' fragment of two molecules, resulting in F(ab' ) ₂ molecules can be formed. The term "F(ab') ₂ fragment" herein includes two light chains, and two heavy chains comprising a variable region, a portion of a constant region between the CH1 and CH1 and CH2 domains, as described above, Interchain disulfide bonds are formed between heavy chains. Thus, the F(ab') ₂ fragment consists of two Fab' fragments, and the two Fab' fragments are associated with each other by a disulfide bond between them. A "Fv region" as used herein is a fragment of an antibody that includes each variable region of the heavy and light chain, but not the constant region. In sdFV, heavy and light chains are linked by disulfide bonds. The scFv is Fv linked by a flexible linker. scFv-Fc is an Fc linked to scFV. The minibody is the scFV with CH3 connected. Diabodies contain two molecules of scFV. As used herein, a "single-chain antibody" (scAb) is a single polypeptide chain including one constant region or a light chain constant region of a heavy chain in which the heavy and light chain variable regions are linked by a flexible linker. A "domain antibody" (dAb) herein is an immunologically functional immunoglobulin fragment comprising only the variable region of the heavy chain or the variable region of the light chain.

Substances/methods capable of inhibiting the function and activity of a protein are known in the art, and those skilled in the art can easily select an appropriate one. For example, an antibody to which a neutralizing antibody can be used is a type of protein that binds to a specific epitope of CTRP3, and the effect of the binding is different depending on the binding ability, binding site, and type of antibody. Among these antibodies, an antibody that neutralizes the function or activity of CTRP3 is 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 can be used herein are polyclonal or monoclonal antibodies or antigen binding fragments thereof. Antibodies can be derived from any mammal including human, mouse, rat, camelid or rabbit.

In other embodiments herein, the inhibitor is a polypeptide capable of interfering, interfering with and or inhibiting CTRP3 expression, activity and/or action/mechanism. As used herein, the term polypeptide refers to a polymer of natural or synthetic amino acids, 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 naturally or artificially, such as those modified by glycosylation, acetylation, phosphorylation, and the like.

In another embodiment according to the present application, the inhibitor that can be included in the composition according to the present application is a nucleotide such as siRNA (small interfering RNA) or shRNA (small hairpin RNA) or miRNA (microRNA). siRNA, shRNA and miRNA are transcribed through RNA interference, for example, short-length interfering RNA (siRNA) sequence-specifically binds to the transcript to form an RNA Induced Silencing Complex (RISC). It makes it impossible for RNA to be expressed as a protein in the cell (silencing). siRNA, shRNA or miRNA has a sequence that is highly complementary to its target sequence. By highly complementary sequence is meant at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with a sequence of at least 15 contiguous bases in length of the target gene. Antisense oligonucleotides, siRNAs, shRNAs and/or miRNAs of various origins targeting the CTRP3 gene according to the present application may be used as long as it binds to the target nucleic acid and functions as a silencing, and biological equivalents, derivatives and analogs thereof It also includes. Antisense oligonucleotides are as known in the art and are short synthetic nucleic acids that, for example, bind to any coding sequence of the target protein and inhibit/reduce the expression of the target protein. Antisense RNA may have an appropriate length depending on the target gene and delivery method, for example about 6, 8 or 10 to 40, 60 or 100 nucleotides. In one embodiment, siRNA is used to suppress CTRP3 gene expression. Such siRNA may contain various sequences as long as it achieves the desired effect by reducing the expression of the desired gene.

As used herein, the term "treatment" refers to any act of improving or beneficially altering the symptoms of tendonopathy or a disease caused by administration of the composition according to the present application. Those of ordinary skill in the art to which the present application pertains will be able to grasp the exact criteria of the disease, and determine the degree of improvement, improvement, and treatment by referring to data presented by the Korean Medical Association.

The composition of the present application may be prepared by including one or more pharmaceutically or physiologically acceptable carriers in addition to the above-mentioned active ingredients.

As used herein, "carrier" refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to the dosage and concentration used. Examples of such carriers include saline, Ringer's solution, buffered saline, buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin. Or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars Alcohols such as mannitol or sorbitol, salt forming counter ions such as sodium, and/or nonionic surfactants such as tween, polyethylene glycol (PEG) and PLURONICS.

If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets, and can act specifically on target organs. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. have.

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

The dosage range is very diverse depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and a known dosage of an inhibitor can be applied. For protein preparations including siRNA, miRNA, antisense oligonucleotides, shRNA preparations, and polypeptides, parenteral administration may be preferred, but other routes and means are not excluded. In the case of a typical drug, the dosage unit includes, for example, about 0.01 mg to 100 mg, but the range below and above the above range is not excluded. The daily dosage may be about 1 μg to 10 g, and may be administered once to several times a day.

The present application is based on finding out that CTRP3 is important for the development and initiation of tendonosis, and it can be developed as a therapeutic agent for tendonosis by screening for substances that inhibit the activity of CTRP3.

In this respect, the present application relates to a screening method for treating tendonopathy comprising the following steps.

In one embodiment, the method comprises the steps of providing a cell overexpressing CTRP3; Treating cells with a test substance expected to inhibit the expression or activity of CTRP3 in the cells; And when the expression or activity of CTRP3 in the cells is decreased as a result of the treatment, compared with cells not treated with the test substance, selecting the test substance as a candidate substance for treating tendonosis.

CTRP3 used herein can be used in the present method according to the specific method of screening, for example, mammals, particularly those derived from humans or mice may be used. In addition, even those derived from the same host, for example, humans, may have sequence variations depending on a specific individual, region, environment, etc., as well as some sequence modifications (deletion, substitution, addition), but all functionally equivalent variants It can be used in the present invention.

CTRP3 that can be used in the methods herein can be provided in an isolated form or in the form of cells expressing the same. In the case of a cell, it may be a cell line that expresses or overexpresses the protein employed in the method of the present application endogenously, or by introduction of a plasmid expressing it (transient or stable transduction).

In one embodiment, a cell line that can be used in the method according to the present application includes, but is not limited to, a tendon cell line, and those skilled in the art will be able to select an appropriate one by referring to Examples herein.

The amount of protein used in this method, the type of cell, and the amount and type of the test substance depend on the specific experimental method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance that suppresses or decreases the expression or activity of CTRP3 in the presence of the test substance is selected as a candidate substance compared to the control group not in contact with the test substance. In the case of reduction, compared to the control, about 99% or less, about 95% or less, about 90%, about 85%, about 80%, about 75%, about 70%, about 65% or less, about 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%, but excluding ranges outside this no.

"Test substance" herein refers to a substance expected to inhibit the expression or activity of CTRP3, a low molecular weight compound, a high molecular weight compound, a mixture of compounds (eg, natural extract or cell or tissue culture), or a biopharmaceutical (E.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme), or sugars and lipids and the like, but are not limited thereto. The test substance may be a polypeptide having two or more amino acid residues, such as 6, 10, 12, 20 or less, or more than 20, such as 50 amino acid residues. The test substance can be obtained from a library of synthetic or natural compounds, and methods of obtaining a library 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 (USA), and libraries of natural compounds are available from Pan Laboratories (USA) and Available from MycoSearch (USA). Test substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution. It can be obtained by the required synthetic library method, the "1-bead 1-compound" library method, and the synthetic library method using affinity chromatography selection. For the synthesis method of molecular library, 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, etc.

For example, for the purpose of screening a drug that inhibits the expression or activity of CTRP3, a compound having a low molecular weight therapeutic effect may be used. For example, a compound having a weight of about 1000 Da such as 400 Da, 600 Da, or 800 Da may be used. Depending on the purpose, these compounds may constitute a part of the compound library, and the number of compounds constituting the library varies from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on templates, such as benzodiazepines, hydantoin, biaryl, carbocycle and polycycle compounds (such as naphthalene, phenothiol). Azine, acridine, steroids, etc.), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is only exemplary. It is not limited to this.

Also, for example, Biologics can be used for screening. Biologics refers to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced in vivo and in vitro using cellular systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organic substances found in plasma.

In this application, it was found that activation of CTRP3-mediated Akt (Protein Kinase B, also known as PKB) signaling is essential for the function of CTRP3 in tendonopathy development. Here, activation means that the phosphorylation of a specific amino acid in the Akt protein is finally increased. According to the present application, it was confirmed that phosphorylation of the serine residue (Ser473), which is the 473th amino acid of the Akt protein, is increased, and that when the phosphorylation of Ser473 is increased, Akt signaling is activated.

Therefore, the method according to the present application further includes the step of detecting Akt activation, and when the test substance is reduced compared to untreated cells, the method according to the present application includes the step of selecting the test substance as a candidate substance for treating tendonosis. do.

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

Thus, in another aspect, the present application provides information on the diagnosis or prognosis of the subject's tendonosis, providing a biological sample derived from the subject; Detecting CTRP3 in the biological sample; And as a result of the detection, when the CTRP3 is increased in the biological sample compared to the control, it provides a method for detecting CTRP3 in vitro for diagnosing the subject as tendonopathy.

In one embodiment, the detection according to the present application can be detected at the mRNA or protein level by the expression level of CTRP3, and such a method is known in the art, for example, the nucleic acid includes a nucleic acid amplification method such as PCR, Northern blot, Proteins include, but are not limited to, immunoassay methods using antibodies capable of specifically recognizing CTRP3, and reference may be made to those described herein.

The biological sample that can be used in the above-described method according to the present application is a tissue in which the expression of CTRP3 can be detected, such as tendon cells or tissues.

Hereinafter, examples are presented to aid in understanding the present invention. However, the following examples are provided for easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Human sample. Human samples were obtained at Boramae Hospital, Seoul. 19 samples of human rotator cuff tendons were obtained from 14 patients with rotator cuff tear, and 4 samples of cruciate ligaments were obtained from 4 patients with knee joint degenerative arthritis. Written informed consent was provided to all participants, and the study passed IRB review. Histology evaluation was made through a modified Bonar scoring system. All histological and immunohistochemical samples were independently evaluated by two pathologists with appropriate expertise. The evaluators were completely unaware of the above classified features of the sample.

Mouse transduction and transfection. For mouse transduction, the right hind limb of the mouse was depilated and 40 micrograms of plasmid DNA was injected into the muscles around the tendon of the hind limb. Thereafter, a conductive gel was applied and electric shock was applied with a caliper electrode with a 5 mm gap. The electric shock was performed under the conditions of 225 V/cm, 25 ms, 5 times using a BTX Gemini X2 (Harvard Bioscience, Inc.). For mouse transfection, Ad- eGFP or Ad- Ctrp3 was injected into the hind limb muscle every 7 days for a total of 3 weeks. C57BL/6J male mice at 8 weeks were used for the experiment, and 5 weeks after electric shock or the first virus injection were used in the experiment.

Induction of mouse tendonosis by overuse. Week 8 C57BL/6J male mice were purchased from Biolink Company (Daehan-Biolink Co.). Mouse treadmill running is 1 week (15 m/min, 15 min/day, 6 days/week, 10° uphill slope), 2 weeks (15 m/min, 1 hour/day, 6 days/week, 10° uphill slope) ), 3 weeks (15 m/min, 2 hour/day, 6 days/week, 10° uphill slope) for a total of 6 weeks. An air puff attached to the treadmill was used to induce treadmill running.

Rat tendonopathy induction by overuse. Week 12 Sprague Dawley male rats were purchased from the Biolink Company (Daehan-Biolink Co.). Rat treadmill running is 1 week (15 m/min, 30 min/day, 6 days/week, 10° downhill slope), 2 weeks (gradually increased 15-20 m/min, 1 hour/day, 6 days/week) , 10° downhill slope), 2 weeks (20 m/min, 2 hour/day, 6 days/week, 10° downhill slope) for a total of 5 weeks. An air puff attached to the treadmill was used to induce treadmill running.

Partial transection Achilles tendon injury model. After anesthetizing C57BL/6J male mice at 8 weeks, the skin and synovial sheath were exposed with a scalpel blade. Using a 1.5mm ear punch, a 0.75mm semicircular hole was drilled in the right hind limb Achilles tendon. The skin was sutured, grown in a cage for 3 weeks, and then treated with antibodies for 2 weeks.

Behavioral experiment. Treadmill running (15 m/min, 30 min, 10° uphill slope) was performed to evaluate the tendonpathy-related motor ability of the mouse. An electric foot shock at the back of the treadmill was used to induce the mouse to run. Resting behavior in treadmill running was measured by the number of electric shocks. In order to evaluate the rat's tendonpathy-related motor ability, a grip strength test meter was used to measure the maximum force that the rat could hold by holding the iron bar with its forefoot.

Neutralization antibody therapy. Neutralizing antibody treatment was performed after anesthesia. 1 microgram of anti-CTRP3 antibody (Abcam) was injected into the periphery of the tendon twice a week for a total of 2 weeks.

Histology and immunohistochemistry. Human rotator cuff sample, cruciate ligament sample, and mouse Achilles tendon were washed with PBS and fixed in 4% paraformaldehyde for 7 days and 3 days, respectively. After paraffin embedding, it was sectioned to a thickness of 7 mm. 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 was performed using a modified Bonar scoring system. For immunohistochemical staining, the sections were incubated in a citric acid buffer (pH 6.0) at 70° C. for 1 hour to recover antigens. After blocking the sample with PBS containing 1% BSA, 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. Electric shock was performed using a BTX Gemini X2 (Harvard Bioscience, Inc.) under conditions of 500 V, 0.1 ms, and once. Thereafter, the cells were seeded in a 60 mm dish, and after 3 days, transferred to a 15 ml tube, centrifuged at 400 g for 4 minutes, and 500 ul of chondrogenic media (DMEM/F-12 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ ml amphotericin B, 1.25mg/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 slightly opened and incubated for 3 days at 37° C. 5% CO ₂ , and then replaced with new chondrogenic media containing 10 ng/ml TGF-β1. The media change took place every 3 days for a total of 28 days. After that, the pellet was fixed in 4% PFA and then cryosectioned with 5 micrometers, followed by Alcian blue/Nuclear fast red staining. Measurement of staining was performed with Image Pro Premier software.

In vitro neo-tendon construction. A 6-well plate was coated with SYLGARD (Dow Corning) and incubated overnight at 55°C. After sterilization with 70% ethanol, UV treatment was performed. 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. Electric shock was performed using a BTX Gemini X2 (Harvard Bioscience, Inc.) under conditions of 500 V, 0.1 ms, and once. Thereafter, the cells were seeded in a 60 mm dish, cultured 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 were treated every 2 days for a total of 2 weeks, after which the dried composition was fixed in 4% PFA, embedded in paraffin, sectioned into 7 micrometers, and stained with H&E. . The thickness of the building construction was performed with Image Pro Premier software.

Adenovirus. Ad- ctrp3 (ADV-254235) was purchased from Vector Biolabs. Ad- eGFP was obtained from Professor Jaebeom Kim's team (Seoul National University Department of Life Science). Adenovirus titer measurement was performed according to the protocol of Rapid Titer Kit (Applied Biological Materials Inc.).

Production of cultures containing excess CTRP3. The HEK293T cell line was transfected with a CTRP3 overexpression vector using PEI chemical so that the HEK293T culture medium contained an excess of CTRP3 secreted from the cells. The culture medium containing an excess of CTRP3 was sterilized with a 0.22 micrometer filter, and it was confirmed that the culture medium containing an excess of CTRP3 contained an excess of CTRP3 by immunoblot method.

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

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

RNA sequencing was performed using samples of sham and injured mouse Achilles tendons in order to identify the causative factor of tendonopathy that was specifically proliferated in the disease state. Furthermore, we investigated candidate genes classified as'cytokines' in the databases of Ingenuity Pathway Analysis (IPA) (1740 gene) and UniProt (1662 gene), taking into account its potential advantage from the viewpoint of drugization potential by antibody-based therapy. I did. CTRP3 was identified as the most significantly upregulated cytokine under the damage condition (fold change = 68.204; P value = 1.83×10 ^-7 ) (Fig. 1A). When arranged according to the percentile rank, CTRP3 was found to be one of the genes most frequently expressed in damaged tendons among the total transcriptome (FIG. 1B) and cytokine transcripts.

In patients with rotator cuff tendon injury, CTRP3 expression at both the mRNA and protein levels was correlated with the severity of tendonosis (FIG. 1C and D). Consistently, when cross-validating with the public dataset of human tendonosis (GSE26051) (39), it was found that CTRP3 expression was increased in the damaged area of tendon lesions tendon compared to the non-lesion control group (Fig. 1E). The tendons and ligaments are closely related connective tissues and represent densely arranged collagen fibers that resist tensile stress (40, 41). Ligaments similarly undergo degenerative changes during injury or abuse (40, 42). Indeed, collagen fiber alignment and dissociation of the cartilage-forming layer were observed in ligaments with high posterior cruciate ligament injury (FIG. 1F). CTRP3 was significantly upregulated in high grade ligament injury (FIG. 1F ). As a rodent model of tendonosis, a long-term treadmill execution method 43 that causes tendonpathy symptoms in the Achilles tendon of a mouse was used (Fig. 1G and H). Both CTRP3 mRNA and protein levels were significantly increased in the Achilles tendon of mice that received 5 weeks of overuse experiments (G and I in Fig. 1).

Example 2. Onset of tendonopathy by overexpression of CTRP3 in mice

Increased expression of CTRP3 in the diseased tendon indicates an association with the pathogenesis of tendonosis. The role of CTRP3 in the tendon was examined by overexpressing the CTRP3 protein in the mouse Achilles tendon. First, it was confirmed that the exogenously expressed CTRP3 was secreted into the medium by the primary cultured mouse tendon cells. After injection of the Ctrp3 expression vector into the tendon, overexpression of CTRP3 (Fig. 2A) was confirmed in tendon cells of the Achilles tendon by electroporation. In particular, only overexpression of CTRP3 showed major symptoms of tendonosis, including dissociation and degeneration of collagen fibers, irregular cells, and abnormal accumulation of proteoglycans between fibers (Figs. 2A and 2B). CTRP3 overexpression resulted in a significant increase in tendonopathy grades assessed by the Bonar scoring system (Figure 2B and C). In addition, exercise evaluation by a treadmill running experiment indicates that CTRP3 overexpression significantly impairs motor performance in mice (FIG. 2D), which indicates tennopathy-related dysfunction. At the molecular level, CTRP3 overexpression caused upregulation of the tendonopathy-related collagen type, Col3a1, and cartilage matrix genes Col2a1 and Acan (FIG. 2E). Indeed, changes in ECM composition during tendonopathy, including accumulation of type III collagen and cartilage-specific matrix proteins, are a major factor deteriorating tendon properties by weakening tensile strength.

The role of CTRP3 in tendonopathy was investigated by injecting adenovirus (Ad-Ctrp3) expressing Ctrp3 mice around the tendon into mice. Overexpression of CTRP3 in the tendon surrounding tissues surrounding the Achilles tendon caused tendonpathy symptoms in terms of histopathology and behavior (FIGS. 2, F to I). These results indicate that CTRP3 secreted into the synovial sac can induce degenerative changes in tendon cells in a non-irritating manner.

Example 3. Prevention of occurrence of tendonosis in mice due to blockade of CTRP3

Next, the therapeutic utility of an antibody that blocks the function of CTRP3 in the occurrence of tendonosis was investigated. The selected -CTRP3 antibody effectively binds to the secreted natural form of CTRP3, suggesting the potential to neutralize CTRP3 function. Antibody therapy was first tested in Achilles tendonosis model mice induced by long-term uphill treadmill running (Figure 3A; top panel). Compared to the control immunoglobulin G (IgG) treatment, anti-CTRP3 antibody treatment was used to treat tendonpathies caused by excessive use including alteration of sternum morphology, destruction of collagen fibers, and accumulation of proteoglycans (Figure 3, B and C). Major symptoms were significantly reduced. Expression of tennopathy-related genes in tendons caused by excessive use was eliminated by treatment with anti-CTRP3 antibodies. Consistently, blockade of CTRP3 restored the motor capacity of the tendonosis model mice (FIG. 3E) as well as a substantial reduction in the severity of tendonosis as assessed by the Bonar scoring system (FIGS. 3, C and D ). It shows the therapeutic effect of CTRP3 neutralization in the disease.

In addition, we investigated whether blockade of CTRP3 enhances the healing process of partially severed 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 deformity and proliferative properties (FIG. 3, F to H). In contrast, when the damaged tendon was treated with an anti-CTRP3 antibody, fibrous cartilage characteristics were effectively suppressed in the regenerated tissue, and normal cellularity and collagen fiber alignment were restored (Fig. 3, F to H).

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

In order to gain insight into the role of CTRP3 in tendonosis development, transcriptomic analysis was performed here using tendons isolated from various rodent tendonosis models. Transcripts of tendons transfected with Ctrp3 overexpression vector were compared with transcripts of tendons and naive tendons transfected with a control vector. Genes differentially generated by unsupervised hierarchical cluster analysis were grouped 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 tendonpathic tendons and naive tendons resulting from overuse treated with control IgG or anti-CTRP3 antibodies. Among the two confirmed clusters,'Cluster 4'was a set of genes activated by the overuse of tendons, but was alleviated by blocking CTRP3 function (FIG. 4B). In fact, cluster 4 is highly overlapped with cluster 2 (initial P = 1.55×10 ⁻³⁴ ) (Fig. 4C).

To evaluate the role of CTRP3 in the induction of tendonosis, the Biological Network Gene Ontology tool (BiNGO) (44) was used to analyze the biological process represented by cluster 2. Hierarchically structured annotations related to cartilage development and proteoglycan metabolism processes were found to be very abundant in cluster 2 (Fig. 4D). In fact, gene set enrichment analysis (GSA) revealed that the'proteoglycan synthesis' gene set and'cartilage development' of IPA were positively enriched in the entire transcript of the tendon overexpressing CTRP3 (Fig. 4E). Similarly, the genes listed in the two annotations were upregulated compared to naive mice in an overuse-induced tendonpathy mouse model. CTRP3 antibody treatment effectively inhibited this gene set in mice with tendonopathy (Fig. 4E). Finally, we investigated how the gene cluster activated by CTRP3 overexpression is regulated in human tendonpathy. The cluster 2 gene set was positively enriched in tendon suffered from tendonopathy compared to the non-lesion control group (FIG. 4F). Taken together, CTRP3 has been shown to activate the tendon cartilage transcription program that accounts for important molecular and morphological changes associated with tendonopathy.

Example 5. Interference of tendon generation by CTRP3-mediated signal transmission

In tendonological tendons, cartilage deformity is easily observed simultaneously in the accumulation of proteoglycans and in the lesion area (18-22). In fact, the tendonological features observed in rodent models are due to the abnormal differentiation of endogenous mesenchymal stem cells (MSCs) into non-tendonous cell types such as cartilage. Since the results according to the present application indicate the role of CTRP3 in the activation of cartilage-related transcription programs, we investigated how CTRP3 influences cartilage formation and the fate determination of MSCs that differentiate into tendons. As a result, CTRP3 overexpression effectively promoted cartilage differentiation of human MSCs grown in pellet culture (Fig. 5A). In contrast, CTRP3 inhibited the formation of new tendons of MSCs grown on immobilized fibrin gels, resulting in a significant reduction in the thickness of the new tendon constructs (FIG. 5B).

Next, the signaling pathways affected by CTRP3 were investigated. CTRP3 induces signals through various subtypes of the mitogen-activated protein kinase (MAPK) cascade (31,45) and the phosphatidyl inositol-3-kinase (PI3K)-Akt pathway, depending on the cell type and cell context (26, 31 ). In primary cultured MSCs and tendon cells, CTRP3-conditioned medium activates PI3K-Akt signaling, but the MAPK pathway is bound by extracellular signaling kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 protein kinases. Did not activate (FIG. 5C and D). Notably, inhibition of the PI3K-Akt pathway essentially abolished the false expression of CTRP3-mediated COL2A1 and ACAN, and restored strong expression of COL1A1 (Fig. 5E). Consistently, the inhibitory effect of CTRP3 on MSC formation of new tendons 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 tendonopathy development.

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

Rotator cuff tendinopathy It is a major cause of shoulder disorders (46). Rotator cuff tendonosis occurs when the tendon tendon is exposed to overload or repeated stress without a sufficient recovery period (47-49). In the present application, a long-term downhill running was used to induce rotator cuff tendonopathy in rats by (50, 51), and the effect of antibody therapy targeting CTRP3 was tested (Fig. 5A). The CTRP3 neutralizing antibody was injected around the tendon to effectively inhibit collagen fiber dissociation and abnormal basal material deposition (eg, chondroitin sulfate) due to excessive use of the tendon (FIG. 6B ). Consistently, the strength of the forelimb rats caused by tendonopathy was also recovered by CTRP3 antibody treatment (FIG. 6C). At the molecular level, biomarkers associated with tendonosis such as MMP13, fibronectin and COL3A1 were detected at a minimal level in the rotator cuff tendons of rats treated with CTRP3 antibody, indicating that the tendon was healed as a whole due to blockade of CTRP3.

Claims (10)

A pharmaceutical composition for the treatment or prevention of tendonosis comprising a CTRP3 inhibitor, wherein the inhibitor is an antibody capable of neutralizing the activity of CTRP3 protein, or siRNA or shRNA that inhibits the expression of the CTRP3 gene into a protein. Or a pharmaceutical composition for prevention.
delete delete Comprising a substance for detecting CTRP3 marker, a composition for diagnosis of tendonosis.
The method of claim 4,
The detection material is a reagent capable of detecting the marker at the protein or nucleic acid level,
The protein level detection reagent is a reagent for Western blot, ELISA, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray,
The nucleic acid level detection reagent is a polymerase chain reaction, a reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, a Nuclease protection assay (RNase, S1 nuclease assay), an in situ hybridization method, a nucleic acid microarray or a reagent used in Northern blot. , A composition for diagnosis of tendonosis.
The method of claim 5,
The protein-level detection reagent includes an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full length of the protein of the marker or a fragment thereof,
The nucleic acid-level detection reagent is a primer pair or probe that specifically recognizes the nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, the nucleic acid sequence and a fragment of the complementary sequence, or a primer pair and a probe. , A composition for diagnosis of tendonosis.
To provide information necessary for the diagnosis or prognosis of tendonopathy,
Detecting the level of nucleic acid and/or protein of the CTRP3 biomarker from the biological sample derived from the test subject;
Comparing the detection result of the nucleic acid and/or protein level with the corresponding result of the corresponding marker of the control sample; And
Compared with the control sample, when the expression of the nucleic acid or protein of the sample derived from the subject is increased, the method of detecting a CTRP3 biomarker comprising the step of correlating with the diagnosis or prognosis of the subject's tendonosis.
Providing a cell overexpressing CTRP3;
Treating the cells with a test substance expected to inhibit the expression or activity of CTRP3; And
As a result of the treatment, compared with the cells not treated with the test substance, in the cells
When the expression or activity of CTRP3 is decreased, screening method for suppressing the onset of tendonosis or therapeutic agent comprising the step of selecting the test substance as a candidate material for treating tendonosis.
The method of claim 8,
The cell is a tendonpathy-derived cell, a method for screening the onset of tendonosis or therapeutic agent.
The method of claim 8 or 9,
The detecting step further comprises detecting AKT activation,
When the detection result of the AKT activation decreases compared to cells not treated with the test substance, comprising the step of selecting the test substance as a candidate substance for treating tendonosis, screening method for suppressing the onset of tendonosis or therapeutic agent.

Images