KR101309910B1 - Syndecan-2 peptide fragment as a marker for diagnosing colorectal cancer and as a therapeutic agent thereof - Google Patents

Abstract

The present invention relates to colorectal cancer diagnostic markers, therapies and their applications, including synthecan protein fragments.

Description

Syndecan-2 peptide fragment as a marker for diagnosing colorectal cancer and as a therapeutic agent

The present invention relates to colorectal cancer diagnostic markers, therapies and their applications, including synthecan protein fragments.

Colorectal cancer is most commonly adenocarcinoma, and it is divided into colon cancer and rectal cancer. The incidence of each site is highest in the lower colon, or rectum, about 50%. Recent studies have shown that the incidence and mortality rates of colorectal cancer are increasing in Korea due to changes in dietary habits, and the incidence of colorectal cancer has increased by 420% from 1995 to 2002, (2003 Annual Report on Health Insurance Statistics, National Health Insurance Corporation). It is also the second leading cause of cancer-related deaths in the United States and Europe (American Cancer Society statics for 2009).

The cause of colorectal cancer is still unclear, but genetic factors, dietary habits associated with eating high-fat and low-fiber foods, and inflammatory bowel disease are considered. Colorectal cancer can occur in all age groups, but as the age increases, the incidence increases and occurs frequently in the 50s and 60s. The incidence ratios of men and women are higher in women with colon cancer and men with rectal cancer.

The treatment of colorectal cancer is based on surgical resection combined with chemotherapy and radiotherapy. Despite advances in surgical therapies, chemotherapy, and radiation therapy, mortality is known to be high if patients miss the point of operation after metastasizing to other organs due to the characteristics of colorectal cancer that progress without specific symptoms. The 5-year average survival rate is above 90% in stage 1, above 70% in stage 2, above 50% in stage 3 and below 5% in stage 4 (2004 Cancer Information, published by the National Cancer Center). Early detection and treatment of colorectal cancer shows a significant increase in survival. Therefore, there is an urgent need for a method for early diagnosis of colorectal cancer.

The diagnosis of colorectal cancer is performed by rectal digital examination, fecal occult blood test and colonography in patients with colon-related symptoms. Is done. The earliest detection methods currently available involve testing or endoscopic procedures for fecal blood.

However, significant tumor size must typically be present before fecal blood can be detected. The susceptibility of guaiacbased fecal testing is 26%, which means that 74% of patients with malignant lesions remain undetected (Ahlquist et al., Gastroenterol. Clin. North Am. 26: 41-55 , 1997). Visualization of precancerous and cancerous lesions is the best approach to early detection, but there is a problem that colonoscopy can cause pain and risk to the patient as well as significant costs (Silvis et al., JAMA 235: 928-930, 1976). Geenen et al., Am. J. Dig. Dis. 20: 231-235, 1975; Anderson et al., J. Natl. Cancer Institute 94: 1126-1133, 2002).

Conventional methods as described above are not only low in accuracy of diagnosis, but also impossible for early diagnosis of patients before colorectal cancer develops, and are inconvenient to the subjects. In order to make up for the shortcomings of these conventional diagnostic methods, there have been attempts to diagnose colorectal cancer by measuring the concentration of tumor markers in the patient's plasma (Clinton et al., Biomed. Sci. Instrum. 39: 408-). 414, 2003; Rui et al., Proteomics 3: 433-439, 2003; Soltormos et al., Dan. Med. Bull. 48: 229-255, 2001).

Currently, only diagnostic plasma tests based on fetal cancer antigens (CEA), tumor-associated glycoproteins are available for diagnostic assistance in the field of colorectal cancer. CEA is increased in 95% of tissues obtained from patients with colorectal cancer, gastric cancer, pancreatic cancer and the majority of breast carcinomas, lung carcinomas and head and neck carcinomas (Goldenberg et al., J. Natl. Cancer Inst. (Bethesda) 57: 11-22,1976). Elevated CEA levels have also been reported in patients with non-malignant diseases, with the majority of patients with colorectal cancer showing normal CEA levels in the serum, especially during the early stages of the disease (Carriquiry et al., Dis. Colon Rectum 42 : 921-929, 1999; Herrera et al., Ann. Surg. 183: 5-9, 1976; Wanebo et al., N. Engl. J. Med. 299: 48-451, 1978). The use of CEA measured from serum or plasma in detection relapse is controversial in reporting and not yet widely applied (Martell et al., Int. J. Biol. Markers 13: 145-149, 1998; Moertelet al., JAMA 270: 943-947, 1993).

Against this background, the present inventors have made diligent efforts to develop markers for early diagnosis of colorectal cancer, confirming that Syndecan-2 peptide fragments are detected in samples such as serum of colorectal cancer patients. Syndecan-2 peptide fragment was promoted to promote colorectal cancer activity, confirming the possibility of diagnosing colorectal cancer using Syndecan-2 fragment, and syndecan-2 fragment in blood of colorectal cancer patients. When removed, it was confirmed that there is no effect of promoting cancer activity in the blood of colorectal cancer patients, and when it was used, it was confirmed that the treatment effect of colorectal cancer was completed and the present invention was completed.

One object of the present invention is to provide a composition for diagnosing colorectal cancer, which comprises an agent for measuring the expression level of a syndecan-2 peptide fragment, which is a colorectal cancer marker.

Another object of the present invention to provide a colorectal cancer kit comprising the composition for diagnosing colorectal cancer.

It is still another object of the present invention to provide a method for detecting a colorectal cancer marker syndecan-2 peptide fragment in order to provide information for diagnosing colorectal cancer.

It is another object of the present invention to provide a method for screening a colorectal cancer therapeutic agent using a colorectal cancer marker syndecane-2 peptide fragment.

Still another object of the present invention is to provide a pharmaceutical composition for treating colorectal cancer, comprising an antibody that specifically binds to a Sindecan-2 peptide fragment.

As one embodiment for achieving the above object, the present invention provides a composition for diagnosing colorectal cancer comprising an agent for measuring the expression level of the Syndecan-2 peptide fragment.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the development of colorectal cancer.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" refers to a substance which can diagnose colon cancer cells separately from normal cells, and increases or decreases in cells with colorectal cancer as compared to normal cells. Organic biomolecules such as polypeptides or nucleic acids (eg, mRNAs), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, and the like) that exhibit a decrease. For the purposes of the present invention, the colorectal cancer diagnostic markers of the present invention are Syndecan-2 peptide fragments that exhibit a particularly high level of expression in colorectal cancer cells as compared to cells of normal colon tissue.

As used herein, the term "Syndecan-2 peptide fragment" is not limited thereto, but is a fragment of the Syndecan-2 protein that can distinguish between normal and colorectal cancer individuals. Syndecan-2 is a cell-bound receptor on the cell surface that is a transmembrane heparan-sulfate proteoglycan (Carey, DJ (1997). Syndecans: multifunctional cell-surface co-receptors. Biochem. J. Pt 1 , 1-16). The gene and protein information is registered in the National Center for Biotechnology Information (NCBI) (NM_002998, NP_002989). The present inventors confirmed that MMP-7 cleaved the extracellular domain of syndecane-2, and thus, a large number of syndecane-2 peptide fragments existed in serum in colon cancer, thereby enabling specific diagnosis of colon cancer. In addition, it was confirmed that the cutincan-2 peptide fragment, which is the cut off decane-2, plays an important role in the migration of colorectal cancer.

Preferably the syndecan-2 peptide fragment may be an amino acid sequence set forth in SEQ ID NO: 1 or 3. The present inventors have found that leucine, which is the 139th amino acid of the N-terminal extracellular domain of the human syndecan-2 protein of SEQ ID NO: 2 or the 149th amino acid of the N-terminal extracellular domain of the rat syndecan-2 protein, is MMP-7 It was first identified that the cleavage position of, and when the fragment is measured in the serum it was confirmed that it is possible to diagnose colon cancer quickly by non-invasive method. The part which can be used as such a colorectal cancer marker of Cindecan-2 is shown in FIG.

In the present invention, the term "agent for measuring the expression level of the Syndecan-2 peptide fragment" is a marker by confirming the expression level of the syndecan-2 peptide fragment, which is a marker for increasing expression in colorectal cancer cells as described above. Means a molecule that can be used for the detection of, preferably refers to an antibody, primer or probe specific for the marker. More preferably an antibody. This is a process of confirming the presence and expression of the protein expressed in the colorectal cancer marker gene in a biological sample in order to diagnose colorectal cancer, using an antibody that specifically binds to the protein of the gene. Check it. Analysis methods for this include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrophoresis. , Tissue immunity staining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS and protein chips (protein chip) and the like, but are not limited thereto.

Agents for measuring protein levels are preferably antibodies.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a Sindecan-2 peptide fragment, which is a marker of the present invention. Such an antibody may be obtained by cloning each gene into an expression vector according to a conventional method. The protein encoded by the gene can be obtained and prepared from conventional proteins by conventional methods. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

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

As another aspect, the present invention provides a kit for diagnosing colorectal cancer comprising the composition for diagnosing colorectal cancer.

The kit of the present invention can detect markers by confirming the expression level of the syndecane-2 peptide fragment, which is a colorectal cancer diagnostic marker. The kit for detecting markers of the present invention may include one or more other component compositions, solutions or devices suitable for analytical methods as well as antibodies that selectively recognize markers for measuring expression levels of colorectal cancer diagnostic markers.

As another specific example, the kit for measuring the expression level of the syndecan-2 peptide fragment in the present invention is a secondary antibody labeled with a substrate, a suitable buffer, a chromogenic enzyme or a fluorophore, for the immunological detection of the antibody. Substrates and the like. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The coloring enzyme may be peroxidase, Alkaline Phosphatase may be used as the fluorescent substance, FITC, RITC or the like may be used as the fluorescent substance, and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

The kit is preferably but not limited to i) a SPR (surface plasmon resonance) method or a SPRI (surface plasmon resonance imaging) method, or ii) fluorescence detection by fluorescent label attached to the antibody.

The antibody may be prepared by an antibody manufacturing method known to those skilled in the art for the epitope of the present invention.

The kits of the present invention can be used to measure Syndecan-2 peptide fragments, which are protein markers that differ in expression in individuals with cancer and in normal individuals. The kit not only makes it possible for the clinician to diagnose and screen the subject by discriminating whether the subject is cancerous or not, but also makes it possible to monitor the response of the subject to treatment and change the treatment according to the result.

The kit of the present invention may include a primary capture reagent that binds to the protein marker and a secondary capture reagent that does not bind to the primary capture reagent.

The primary acquisition reagent is an antibody or a metal chelate, preferably an antibody, and the secondary acquisition reagent is a secondary antibody conjugated with a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid. The chromogenic enzyme may be a peroxidase, an alkaline phosphatase or an acid phosphatase (e.g. horseradish peroxidase), and in the case of a fluorescent substance, a fluorescenecarboxylic acid ( FCA), fluorescein isothiocyanate (FITC), fluorescein thiourea (FTH), 7-acetoxycarin-3-yl, fluorescein-5-yl, 2 ', 7'-dichlorofluorescein-6-yl, dihydrotetramethyl rosamin-4-yl, tetramethylrhodamine-5-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl or 4,4-difluoro- 5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-ethyl.

When a sample obtained from an individual is contacted with an antibody that specifically binds to a first acquisition reagent, preferably a marker, the sample may be diluted to an appropriate degree before contact with the antibody and the antibody may be diluted to a suitable degree And can be fixed in a solid form to facilitate the step. The solid form can be glass or plastic, for example a microtiter plate, rod, bead or microbead. Antibodies can also be conjugated to probe substrates or protein chips. The sample can be placed at a constant temperature with the antibody and then washed to measure the antibody-marker complex. This is done by placing the washed mixture at a constant temperature with a secondary acquisition reagent, preferably a secondary antibody. Determination of the amount or presence of the antibody-marker complex can be accomplished through fluorescence, luminescence, chemiluminescence, absorbance, reflection or transmission. In addition to the above methods, protein markers in a sample can be detected by indirect assay methods, for example, competition or inhibition assay analysis with a monoclonal antibody that binds to another epitope of a protein marker.

In addition, the kit of the present invention can diagnose and screen colorectal cancer by treating the amount of antibody bound to the antibody after binding to the protein marker.

The high throughput screening (HTS) system is preferably used as a method for searching for the amount of the bound antibody. For example, a fluorescence method performed by detecting fluorescence by fluorescence labeling, a method of detecting the surface plasmon resonance It is preferable to use a surface plasmon resonance (SPR) method for measuring changes in real time or a surface plasmon resonance imaging (SPRI) method for imaging and verifying an SPR system. However, the present invention is not limited thereto.

The fluorescence method is a method of labeling an antibody binding to a specific antibody to a protein marker using a fluorescence scanner program and spotting the antibody to confirm the signal, and the degree of binding can be confirmed by applying this method. The fluorescent material is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (poly L-lysine-fluorescein

isothiocyanate (FITC), rhodamine-B-isothiocyanate (rhodamine-B-isothiocyanate, RITC), and any one selected from the group consisting of rhodamine (rhodamine), but is not limited thereto.

Unlike the fluorescence method, the SPR system does not need to label the sample with a fluorescent material, and it is possible to analyze the binding degree of the antibody in real time. In the case of SPRI, it is possible to perform simultaneous multiple sample analysis using microarray method.

In addition, the kit of the present invention may include a washing solution or an eluent which can remove the substrate and unbound protein and the like to develop a color reaction with an enzyme and retain only bound protein markers. Samples used for analysis include biological samples capable of identifying disease specific polypeptides that can be distinguished from normal conditions such as serum, urine, and tear saliva. Preferably from biological liquid samples such as blood, serum, plasma. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, serum samples obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, Can be pretreated using methods such as sequential extraction or gel electrophoresis.

In another aspect, the present invention provides a method for detecting colorectal cancer, comprising the steps of: measuring the expression level of a syndecan-2 peptide fragment from a sample of a suspected colorectal cancer; And comparing the expression level of the syndecane-2 peptide fragment with the expression level of the syndecane-2 peptide fragment of a normal control sample. .

In the present invention, a sample of an individual includes a sample such as tissue, cells, blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, in which the expression level of the syndecan-2 peptide fragment, which is a colorectal cancer marker, is different. It is not limited. Preferably, blood, serum or serum.

Through the above detection methods, it is possible to diagnose whether a colorectal cancer suspect is an actual cancer patient by comparing the expression level of the syndecane-2 peptide fragment in the normal control group with that of the colorectal cancer suspect. That is, the expression level of the marker of the present invention is measured from cells presumed to be colon cancer, the expression level of the marker of the present invention is measured from normal cells, and the expression level of the marker of the present invention is compared with that of normal cells Colon cancer is more likely to be expressed in a cell derived from the presumed colon cancer can be predicted to colon cancer cells.

Analytical methods for measuring syndecan-2 peptide fragment levels include Western blot, ELISA, radioimmunoassay, radioimmunoproliferation, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement Fixed assays, FACS, protein chips, and the like. Through the above assay methods, the amount of antigen-antibody complex formation in a normal control group and the amount of antigen-antibody complex formation in a suspected colorectal cancer subject can be compared, and the significance of the colorectal cancer marker gene to the synthecan-2 peptide fragment is significant. By determining whether the expression level is increased, it is possible to diagnose whether colorectal cancer is suspected to actually develop colorectal cancer.

In the present invention, the term antigen-antibody complex means a combination of a colorectal cancer marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formation can be quantitatively measured through the size of a signal of a detection label. .

Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] 2+ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotope includes include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re are not limited to .

Protein expression level measurement is preferably by using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is attached to a solid support, reacted with a sample, and then labeled with a labeled antibody that recognizes the antigen of the antigen-antibody complex, for enzymatic color development or for an antibody that recognizes the antigen of the antigen-antibody complex. Detected by sandwich ELISA method by attaching labeled secondary antibody and enzymatic color development. By checking the degree of complex formation of the colorectal cancer marker protein and antibody, it is possible to determine whether colorectal cancer is developed.

Also preferably, Western blot using at least one antibody against the colorectal cancer marker. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. By checking the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein produced by the expression of the gene can be confirmed to determine whether colorectal cancer is developed. The detection method comprises a method of examining the expression level of a marker gene in a control group and the expression level of a marker gene in a cell in which colon cancer has developed. The level of peptide fragments can be expressed as the absolute (eg μg / ml) or relative (eg relative intensity of signal) differences of the marker proteins described above.

Also, preferably, at least one antibody against the colon cancer marker is arranged at a predetermined position on the substrate, and the protein chip is immobilized at a high density. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein, Colon cancer can be checked.

As a specific example, the measurement of the expression level of the syndecane-2 peptide fragment can be performed by electrophoresis, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (SALDI-TOF MS) The measurement is preferably performed using an enhanced laser desorption and ionization time-of-flight mass spectrometry method, but is not limited thereto.

As another aspect, the present invention provides a method for screening a colorectal cancer therapeutic agent, comprising measuring the expression level of a Syndecan-2 peptide fragment after treatment with a candidate substance for treating colorectal cancer.

Specifically, it can be usefully used for screening a colorectal cancer therapeutic agent by a method of comparing the increase or decrease of the expression of the syndecane-2 peptide fragment in the presence and absence of a candidate for treating colorectal cancer. Substances that directly or indirectly reduce the concentration of syndecane-2 peptide fragments may be selected as therapeutic agents for colorectal cancer. That is, the expression level of the marker syndecan-2 peptide fragment of the present invention in colorectal cancer cells in the absence of a candidate for colorectal cancer treatment is measured, and also the marker syndecan-2 peptide of the present invention in the presence of a candidate for colorectal cancer treatment After measuring the expression levels of the fragments and comparing the two, the agent for reducing the expression level of the marker of the present invention in the presence of the candidate for treating colorectal cancer is lower than the marker expression level in the absence of the candidate for treating colorectal cancer. It can be predicted as

Preferably, the method may further include measuring the expression level of MMP-7. The inventors confirmed that the expression level of the syndecane-2 peptide fragment was specifically increased in colorectal cancer, which was confirmed by MMP-7. Therefore, it can be seen that the decreased expression level of MMP-7 is a therapeutic agent for colorectal cancer.

In addition, the present inventors first identified that when mutant leucine, the 149th amino acid of the extracellular domain of SEQ ID NO: 4 of syndecane-2, is mutated to isoleucine, no synthecan-2 is cleaved by MMP-7. In addition, when mutant leucine, which is the 139th amino acid of the extracellular domain of SEQ ID NO: 2, is mutated to isoleucine, it was first identified that syndecane-2 was not cleaved by MMP-7.

Based on this, the therapeutic agent may be to mutate leucine, which is the 149th residue of the syndecane-2 extracellular domain of SEQ ID NO: 4, to isoleucine. Or an antibody that serves to protect MMP-7 from cleaving the 149th residue of the syndecane-2 extracellular domain of SEQ ID NO: 4. Alternatively, the therapeutic agent may be to mutate leucine, which is the 139th residue of the syndecan-2 extracellular domain of SEQ ID NO: 2, to isoleucine. Or the 139th residue of the syndecane-2 extracellular domain of SEQ ID NO: 2 may be an antibody that serves to protect MMP-7 from cleavage.

In another aspect, the present invention is to provide a pharmaceutical composition for treating colorectal cancer, comprising an antibody that specifically binds to a Sindecan-2 peptide fragment.

Such antibodies include polyclonal antibodies, monoclonal antibodies or antigen binding, some of which are included in the antibodies of the invention and all immunoglobulin antibodies. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies, and may include antibodies already known in the art in addition to novel antibodies. Such antibodies include functional fragments of antibody molecules, as well as complete forms having the full length of two heavy chains and two light chains, as long as they have the property of binding to specifically recognize synthecan-2 peptide fragments. The functional fragment of the molecule of an antibody means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.

The pharmaceutical composition for treating colorectal cancer of the present invention, using a synthecan-2 peptide fragment-specific antibody, to remove the synthecan-2 peptide fragment having the activity of increasing the movement of cancer in the serum of colorectal cancer patients It can be used in the method to treat colorectal cancer. The present inventors confirmed that synthecan-2 peptide fragment may be a target of colorectal cancer treatment by confirming that the effect of increasing the movement of colorectal cancer cells disappears when the decancetic-2 peptide fragment contained in the serum is removed using an antibody. It was.

Such a composition for treating colorectal cancer includes, without limitation, a substance capable of specifically binding to a synthecan-2 peptide fragment to promote cancer activity by the synthecan-2 peptide fragment, for example, a protein, an aptamer, a peptide, and the like. , Carbohydrates, compounds.

The composition of the present invention may be used alone, but radiation therapy or chemotherapy (cell arrest or cytotoxic substance, antibiotic-like substance, alkylating agent, anti-metabolic substance, hormone, immune agent, interferon type) may be used to increase the treatment efficiency. Substances, cyclooxygenase inhibitors, metallomatrix protease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor substances, anti-HER substances, anti-EGFR substances, anti-angiogenic substances, farnesyl transferase inhibitors , ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin conjugates, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.).

The composition of the present invention may be administered with a pharmaceutically acceptable carrier, and when used orally, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, and the like may be used. In the case of injections, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used. For topical administration, bases, excipients, lubricants, preservatives and the like can be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampoules or in multiple dosage forms.

As used herein, the term administration refers to introducing a composition of the invention to a patient in any suitable manner, and the route of administration of the composition of the invention may be administered via any general route as long as it can reach the desired tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but are not limited thereto. Do not.

The range of effective dosages of the compositions of the present invention may range from sex, body surface area, type and severity of disease, age, sensitivity to drugs, route of administration and rate of release, time of administration, duration of treatment, target cells, expression levels, etc. It may vary according to various well-known factors, and can be easily determined by those skilled in the art.

As can be seen from the present invention, the present invention can be used as a diagnostic marker because the cut decane-2 fragment plays an important role in the regulation of colorectal cancer cell migration and is detected in the serum of colorectal cancer patients. In addition, it can be used in the development of a new colorectal cancer therapeutic agent through a substance that removes or inhibits a truncated syndecane-2 fragment that promotes cancer activity in the blood of colorectal cancer patients.

1 is a diagram showing that MMP-7 regulates cleavage of the extracellular domain of syndecane-2. (A) Slotted blotting was performed with conditioned media (CM) obtained from indicated colon cancer cells using monoclonal-(# 197) or polyclonal (M-140) anti-syndecan-2 antibodies. Serum-free medium (SFM) was used as negative control. (B) Water soluble decane-2 in conditioned medium of HT-29 cells was isolated by DEAE-Sepharose column chromatography. Each DEAE fraction was used for slot blotting (left panel). The proteoglycan containing elution fraction was digested with heparinase and western blotting was performed with anti-synthecan-2 antibody (right panel). (C) APMA (1 mM) was treated for the time indicated on HT-29 cells, and the conditioned medium was subjected to slot blotting with anti-synthecan-2 antibody. APMA-activated (+) and non-activated (-) recombinant-pro-MMP-7 were separated by SDS-PAGE gel, transferred to PVDF membrane, and then the latent form (28kDa) or activated form of MMP-7 (19 KDa) was labeled with an anti-MMP-7 antibody. (D) HCT116 cells were transfected with 1.5 μg control (VEC) or MMP-7 coding expression vector (left panel) and transfected with pSuper-only (VEC) or MMP-7 shRNA plasmid (right panel) ). Expression levels of MMP-7 were measured by RT-PCR (top panel) and western blotting (middle panel). Conditioned medium obtained from HCT116 cells was subjected to slot blotting with the indicated anti-syndecan-2 antibody. Ponceau S staining was used as a loading control (bottom panel). (E) HT-29 cells were treated with pro-MMP-7 at the indicated times and concentrations. The conditioned medium was subjected to slot blotting with the indicated anti-syndecan-2 antibody. (F) HT-29 cells were treated with the indicated pro-MMPs (1 mg / ml). The conditioned medium was subjected to slot blotting with the indicated anti-syndecan-2 antibody.
2 shows MMP-7 cleaving an N-terminal Leu residue in the extracellular domain of syndecane-2. (A) Purified GST-syndecan-2 (GST-SDC2) was incubated with indicated amounts of pro-MMP-7 at 37 ° C. for 1 hour, separated by 8% SDS-PAGE, followed by CBB R-250 ( Coomassie Brilliant Blue R-250) (left panel) and immunoblotting with anti-syndecan-2 antibody (right panel). Arrows indicate dimers (D) and monomers (M) of syndecane-2, and arrowheads indicate truncated fragments of syndecane-2 (S). (B) The same amount of GST-SDC2 and syndecane-4 (SDC4) were incubated with pro-MMP-7 at 37 ° C. for 1 hour and immunoblotting was performed with anti-GST antibody. (C) An equal amount of purified GST or GST-SDC2 was incubated with purified pro-MMP-7 at 37 ° C. for 1 hour, separated by SDS-PAGE, stained with silver, or immunosorbed with anti-synthecan-2 antibody. Lot. (D) The truncated SDC2 polypeptide was isolated on a silver-stained gel and a protein-free gel plug was used as a cleavage control. MassLynx ^Tm Trypsin Autologous Proteolysis Product As an internal standard for calibration of MS spectra using (Waters Co., UK).
FIG. 3 shows MMP-7 cleaving the endogenous glycated extracellular domain of syndecan-2. FIG. (A) S2E-Fc was prepared by connecting the extracellular domain of syndecane- _{2 to} the Fc region of human IgG ₁ (top panel). HEK293T cells were transfected with VEC or S2E-Fc cDNA and mRNA expression of syndecane-2 was analyzed by RT-PCR using the indicated primers: β-actin was amplified as a control. Slot blotting was performed with anti-synthecan-2 or HRP conjugated anti-human IgG antibodies in conditioned medium (top panel). (B) HEK293T cells transfected with S2E-Fc were incubated with pro-MMP-7 at 37 ° C. for the indicated times. After the reaction mixture was incubated with protein A-Sepharose, the bound material was separated by 15% SDS-PAGE, and the gel was performed by silver staining or immunoblotting with HRP conjugated anti-human IgG antibody. (C) Conditioned media obtained from VEC or S2E-Fc transfected HEK293T cells were incubated with pro-MMP-7 in the presence or absence of GM6001 for 1 hour at 37 ° C. After the culture was reacted with protein A-Sepharose, the bound material was separated by 15% SDS-PAGE and transferred to PVDF membrane. Proteins on PVDF membranes were stained with CBB R-250. (D) Proteins on PVDF membranes stained with CBB R-250 were sent to Tufts Core Facility (USA) for N-terminal protein sequencing analysis (3 cycles).
4 is a diagram showing the resistance of the mutants of the syndecan-2 extracellular domain cleaved by MMP-7. (A) Representative schematic of the extracellular domain of wild type syndecan-2 (SDC2-WT, GenBank accession number NM_013082.3) and the following SDC2 mutants are shown: Leucine (L ¹⁴⁹ ) → Serine (S) (SDC2-NS) ), Asparagine (N ¹⁴⁸ ) / Leucine (L ¹⁴⁹ ) → Serine (S) (SDC2-SS), and Leucine (L ¹⁴⁹ ) → Isoleucine (I) (SDC2-NI). Cleavage sites are indicated by arrow heads (upper panel). Equal amounts of purified GST-SDC2-WT, -SDC2-NS, SDC2-SS, and -SDC2-NI were incubated with purified pro-MMP-7 at 37 ° C for 1 hour, separated by SDS-PAGE, CBB Stained with R-250 (bottom panel). (B) HCT116 cells were transfected with SDC2-WT or indicated SDC2 mutants. MRNA expression of syndecane-2 was analyzed by RT-PCR using the indicated primers: β-actin was amplified as a control (top panel). Conditioned media was slot blotted with anti-synthecan-2 antibody (bottom panel). (C) HT-29 (left panel) and HCT116 (right panel) cells were transfected with 1.5 μg of VEC, SDC2-WT or SDC2-NI mutants (N148 / L149I; NI) and synthesized from syndecan-2. mRNA expression was analyzed by RT-PCR using the indicated primers: β-actin was amplified as a control (top panel). Conditioned media isolated from indicated cells were slot blotted with anti-synthecan-2 antibody. Ponceau S staining was used as a loading control (bottom panel).
5 is a diagram showing that the cut off decande-2 is important for the movement of cancer cells. (A) HT-29 and HCT116 colorectal cancer cells were treated with 0.2 μg / ml of purified truncated syndecane-2, and transwell migration assays were performed by the method of Example. Error bar = 95% confidence interval (HT-29, P = .05, HCT116, P <.01 versus control). (B) HT-29 (left panel) and HCT116 (right panel) cells transfected with 1.5 μg of VEC, SDC2-WT or SDC2-NI mutants (N148 / L149I; NI) and transfected cells (1 × 10 ⁶ ) was moved as described in (A). The experiment was performed three times. Error bar = 95% confidence interval (HT-29, SDC2-WT: P <.01; SDC2-NI mutant: P <.05, HCT116, SDC2-WT or -NI mutant: P <.01 versus VEC control ). (C) B16F10 melanoma cells were transfected with 1 μg vector control (VEC), SDC2-WT, or SDC2-NI mutant (N148 / L149I; NI) and mRNA expression of syndecane-2 was expressed in primers RT-PCR was analyzed using: β-actin was amplified as a control (left, top panel). Conditioned media was slot blotted with anti-synthecan-2 antibody (left , middle panel). Transfected cells (1 × 10 ⁵ ) were moved in a gelatin-coated Transwell apparatus (left, bottom panel, SDC-2 WT, -NI mutant, P <.01 versus VEC control). The experiment was performed three times. Error bar = 95% confidence interval. B16F10 cells were transfected alone with siRNA targeting 225 pmol of mouse syndecane-2, or with the indicated 1 μg let synthecan-2 construct and injected into C57BL / 6 mice (n = 10). Two weeks later, mice were sacrificed and lungs were removed and examined. Two independent experiments were performed (n = 3 mice per group). The front and back of each lung were photographed. The bar graph shows the number of lung metastatic nodules (right panel). Bars represent median values of pulmonary metastasis nodules (n = 3). Error bar = 95% confidence interval.
6 shows that cleaved syndecane-2 increases cancer cell migration and does not regulate proliferation and adhesion. (A) Representative schematic diagram of the production of Fc receptor-cut truncated synthecan-2 chimera (sS2E-Fc) by linking the extracellular domain of syndecan-2 (Met ¹ -Asn ¹⁴⁸ ) to the Fc region of human IgG ₁ (top) panel). HCT116 cells were transfected with VEC or sS2E-Fc, mRNA expression of syndecane-2 was analyzed by RT-PCR using the indicated primers, and conditioned media isolated from the indicated transfected cells was obtained to obtain anti- Slot blotting with syndecane-2 antibody (bottom, left panel). And a migration analysis was performed (bottom, right panel). HCT116 cells were treated with conditioned media of cells transfected with VEC- and sS2E-Fc and the migration was analyzed (bottom, right panel). The experiment was performed three times. Error bar = 95% confidence interval, P <.05 versus control. (B) A mixture of conditioned medium (final 10% v / v ) and HCT116 cells (1 × 10 ⁵ cells / well) of VEC- and sS2E-Fc-transfected cells was added to the previously hardened bottom agar and colonies were added. Counted after 2 weeks. Clonogenicity was calculated as percentage by colony formation relative to the VEC control (n = 3; p <0.05). (C) Human embryonic kidney (HEK) 293T cells were transfected with VEC or sS2E-Fc and subjected to slot blotting with anti-synthecan-2 antibody with conditioned media obtained from the indicated transfected cells. Ponceau S staining was used as a loading control. CI (Cell index) was performed by the method of Example. CI = slope * time + intercept. (D) HCT116 cells were treated with conditioned medium (final 10% v / v ) obtained from VEC- and sS2E-Fc-transfected HEK293 cells for the indicated times and the cell number was treated with MTT (3- (4, 5) -dimethylthiazol-2-yl) -2 and 5-diphenyltetrazolium bromide) were counted. The experiment was performed three times. Error bar = 95% confidence interval. (E) Vector or sS2E-Fc expression conditioned medium (final 10% v / v) and HCT116 cell mixture (5 × 10 ⁴ cells / well) were added to each RTCA E-Plate wells (Roche Diagnostics GmbH). Plates were incubated at 37 ° C. for 5% CO ₂ , 6 hours and the results analyzed by RTCA software.
7 is a diagram showing the cancer active site of cleaved syndecane-2. (A) Partial removal of the extracellular domains up to syndecan-2 (Met ¹ -Asn ¹⁴⁸ ) followed by concatenation of the Fc region of human IgG ₁ to the Fc receptor-cut synthecan-2 chimera (sS2E-Fc-I, II , III, IV) is a representative schematic. (B) Transfecting HEV293 cells with each VEC or sS2E-Fc-I-IV, obtaining conditioned media isolated from the indicated transfected cells and slot blotting with anti-human IgG antibody (left) Western blotting with anti-synthecan-2 antibody (right). (C) Conditioned medium (final 10% v / v) and HCT116 cell mixture (5 × 10 ⁴ cells / well) obtained in (B) were added to each RTCA CIM-Plate wells (Roche Diagnostics GmbH). Plates were incubated at 37 ° C. for 5% CO ₂ , 20 hours and the results analyzed by RTCA software. (D) The conditioned medium (final 10% v / v) and HCT116 cell mixture (5 × 10 ⁴ cells / well) obtained in (B) were transferred in a gelatin-coated Transwell apparatus. The experiment was performed three times. Error bar = 95% confidence interval.
8 is a diagram showing an increase in the amount of syndecane-2 cut in the serum of colorectal cancer patients. (A) Serum samples obtained from normal donors (n) or indicated cancer patients (Ade: adenoma (adenoma), CIS: carcinoma in situ (carcinoma), AC: advanced carcinoma (advanced colorectal cancer)) Slot blotting was performed using specific antibodies. (B) Serum samples obtained from normal donors (n) or advanced carcinoma patients were incubated with protein A sepharose containing PBS buffer for 1 hour and then eluted with PBS buffer containing 1M NaCl. The eluted samples and binding supernatant proteins were subjected to slot blotting with syndecane-2 antibodies. (C) Serum samples from normal donors (n) and advanced cancer patients were subjected to slot blotting as in B above with or without pretreatment with nitrite. (D) Serum samples obtained from normal donors (n) or indicated cancer patients were pretreated with nitrous acid and slot blotting was performed with synthecan-2 specific antibodies. (E) Serum samples pretreated with nitrous acid were analyzed by SDS-PAGE and Western blotting was performed using syndecan-2 specific antibodies. (F) Slot blotting was performed using mouse monoclonal (# 197) or rabbit polyclonal (M-140) syndecan-2 antibodies on serum samples obtained from normal donors (n) and colon or gastric advanced cancer patients. It was. (G) Serum samples (40 μl each) from normal (N) or Apc / Min mice were treated with nitrous acid and slot blotting with synthecan-2 antibody. Serums from patients with advanced cancer (AC) were used as controls. * Indicates that the image has taken longer exposure.
9 is a diagram showing that truncated syndecane-2 in serum increases in animal models of colorectal cancer. Tissues of normal colon mucosa (control) and AOM tumor mouse model (AOM-derived as described in the Examples) were immunostained with syndecan-2 antibody.
FIG. 10 shows that the level of truncated syndecane-2 increased in serum correlates with increased mobility of colorectal cancer cells. (A) Serum samples from normal donors (n) and truncated syndecane-2 fragments from patients with high serum (+) and low serum (-) adenoma (Ade) or advanced cancer (AC) patients were identified. Slot blotting was performed with decan-2 antibody (top panel). Transwell migration assays of HCT116 cells were performed using the serum of the cancer patients described. The cells were allowed to migrate for 20 hours in (1 × 10 ⁵ ) gelatin encoded transwell plates. After fixing the cells and staining with 0.6% hematoxylin and 0.5% eosin, the number of migrated cells were counted (*, P <0.05; **, compared to P <0.01 control). (B) Serum samples obtained from patients with advanced cancer (AC) with high serum (+) and low serum (-) with severely cut sindecan-2 fragments were incubated with protein A sepharose at room temperature for 30 minutes and then unbound. The material was reincubated with protein G Sepharose to which an IgG- or syndecane-2 antibody was attached. Supernatants were blotted onto nitrocellulose membranes and slot blotting with specific syndecane-2 antibodies (top panel). In addition, supernatants were used for Transwell migration assays using HCT116 cells as described in A (left panel, P <0.05 control). The error range is expressed as mean ± standard deviation (number of trials = 3). (C) Serine samples of advanced cancer (AC) patients with high serum (+) and low serum (-) concentrations of syndecan-2 fragments were treated with the described cancer cells, followed by gelatin coated (10 μg / ml) Transwell plates. Shifted in phase. After incubation, cells were stained and counted with hematoxylin-eosin as described in A (**, P <0.01 control). (D) Total RNA was extracted from the biopsy tissue of the indicated colorectal cancer patients to assess the expression level of MMP-7 by RT-PCR (left, top panel). Serum samples obtained from indicated cancer patients were subjected to slot blotting with specific syndecane-2 antibodies (left, bottom panel). Transwell migration assays were performed on HCT116 cells treated with the indicated serum samples as described in A (right, top panel) (*, P <0.05, **, P <0.01 control). Serum samples (final 10% v / v ) and HCT116 cells (1 × 10 ⁵ cells / well) mixture from AC patients were added to the previously hardened bottom agar and colonies were counted after 2 weeks. Clonogenicity was calculated as percent colony formation relative to the VEC control (right, bottom panel) (n = 3; p <0.05).
FIG. 11 shows the sequence of the decane-2 sequence and the sequence of the decane-2 peptide fragment of human and rat, respectively. The part indicated in yellow represents the syndecan-2 peptide fragment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1: cell culture and Transfection

Human colorectal cancer cells HT-29 (K30038), Caco-2 (K30037.1), LoVo (K10229), and HCT116 (K10247) and mouse melanoma cells B16F10 (K80008) were described in the Korean Cell Bank (http: // cellbank. snu.ac.kr). The cells were 37 ° C., 5% CO ₂ MEM (Caco-2), McCoy's 5a (HT-29, HCT116) with gentamycin (50 μg / ml, Sigma) and 10% (v / v) or 20% (v / v) fetal calf serum in a humid environment Cultured with RPMI (LoVo), DMEM (B16F10). Transfection into HT-29 cells using LipofectAMINE LTX reagent (Invitrogen (Carlsbad, Calif.)) Or effectene reagent (Qiagen (Hilden, Germany)) using HCT116 and B16F10 using manufacturer's protocol.

Example  2: slot Blotting

Conditioned medium or patient's serum was slot-blotted onto nitrocellulose membrane using a Bio-Rad device and washed once with PBS. The membrane was then blocked under TBS (Tris-buffered saline: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl), 5% Tween-20 containing 5% fat free oil, washed and washed for 24 hours at 4 ° C. After labeling with an appropriate primary antibody, it was labeled with a species-specific horseradish peroxidase-bound secondary antibody purchased from Amersham Life Science (West Grove, Pennsylvania). The signals were detected by ECL (enhanced chemiluminescence; Anigen, Korea). In some experiments, nitrous acid consisting of 30 μl (final 43% v / v ) of 0.5 MH ₂ SO ₄ and 5.5 M NaNO ₂ in 40 μl of human serum in a microtube was treated at room temperature for 10 minutes.

Example  3: water soluble Sindecan Partial purification of -2

Water soluble syndecane-2 is described in Koda, JE, Rapraeger, A. and Bernfield, M. (1985). J. Biol. Chem. 13, 8157-8162 was isolated from the conditioned medium of HT-29 cells via a modification of the method described. The HT-29 cells were incubated in serum-free medium for 24 hours, culture medium was obtained, and after centrifugation (1,000 rpm, 10 minutes), the supernatant was buffered [50 mM Tris-HCl pH 8.0, 4 M Urea, 200]. mM NaCl, 0.1% Tween-20, 10 mM NEM, 25 mM EDTA and 2 mM PMSF] were applied to a DEAE-Sepharose column (Amersham Biosciences) equilibrated. The column was washed with 10 volumes of buffer 1 and then buffer 2 [30 mM Sodium acetate pH 4.0, 4M Urea, 200 mM NaCl, 0.1% Tween-20, 10 mM NEM, 25 mM EDTA and 2 mM PMSF pH 4.0] was applied. After washing the column, the bound protein was eluted with buffer 3 [150 mM Sodium acetate pH 4.0, 4M GuHCl, 0.1% Tween-20].

Example  4: Heparinase  process

A water-soluble synthecan-2 extracellular domain (ectodomain) was mixed with a mixture of 2 U / ml heparinase III and 20 U / ml chondroitinase ABC and heparinase reaction buffer (50 mM Hepes pH 6.5, 50 mM NaOAc, 150 mM NaCl, and 5 mM CaCl). ₂ ) was treated at 37 ° C for 8 hours.

Example  5: Sindecan Construction of Site-Specific Mutants of -2

Site-specific mutation of the proteolytic cleavage site of the syndecan-2 extracellular domain was performed using a QuikChange site-directed mutagenesis kit purchased from Clontech (Palo Alto, Calif.). Asn ¹⁴⁸ and Leu ¹⁴⁹ residues of the syndecan-2 extracellular domain were replaced with Ser and / or Ile. The primer sequence is as follows:

Synthecan-2 NS forward 5'-GTACACCGAGAAACATTCAGACAATTCAAGCGGACGG-3 '(SEQ ID NO: 5)

Syndecane-2 SS forward 5'-ACGTGTACACCGAGAAACATTCAGACTCGTCGTTCAAGCGG-3 '(SEQ ID NO: 6)

Syndecan-2 NI forward 5'-ACGTGTACACCGAGAAACATTCAGACAATATCTTCAAGCGG-3 '(SEQ ID NO: 7) and

reverse 5'-CCGTCCGCTTGAACGAATTGTCTGAATGTTTCTCGGTGTAC-3 '(SEQ ID NO: 8).

Syndecane-2 site-specific mutant products were inserted into pcDNA 3 vectors using EcoRI / XhoI cloning sites.

Example  6: GST In vitro cleavage and mass spectrometry of fusion proteins

Oh, ES, Woods, A. and Couchman, JR (1997). J. Biol. Chem. GST fusion syndecan-2 (GST-SDC2) and -4 (GST-SDS4) purified with glutathione-sepharose 4B as described in 18, 11805-11811 were converted into 1 mM APMA (Sigma, St. Louis, MO). Incubated for 1 hour at 37 ° C. with pro-MMP-7 in cleavage buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl ₂ ) in the presence or absence of conditions. The cleavage was separated by SDS-PAGE and analyzed by silver staining. The masses of peptides and cleavage products were determined by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) using an Autoflex II mass spectrometer (Brucker Daltonics) from Ewha Womans University for Proteolytic Pathways.

Example  7: move ( Migration ) analysis

Gelatin (10 μg / ml in PBS) was added to each well of a transwell plate purchased from Corning costar (Cambridge, Mass.); 8-um pore size, the membrane was dried at 25 for 1 hour. Connect the transwell to a 24-well plate and fill the lower chamber with DMEM containing 10% FBS and 100 ng / ml bFGF (B16F10) or Mccoy5A containing 10% FBS and 0.1% BSA (HT29, HCT116). It was. Cells were added to each upper chamber and the plates were incubated at 37 ° C., 5% CO ₂ for 8 or 20 hours. Cells migrated to the bottom surface of the filter were stained with 0.6% hematoxylin and 0.5% eosin and counted.

Example  8: Tumor Metastasis Assay

For in vivo lung metastasis analysis, B16F10 melanoma cells (1 × 10 ⁵ cells / mouse) were injected into the tail vein of C57BL / 6 mice ( n = 10) on day 0. On day 13, the lungs were excised and photographed for counting metastatic nodules.

Example  9: SDC Antibody Production Against A-2

Syndecane-2 monoclonal antibody was produced by AdipoGen Inc. The extracellular domain of rat syndecan-2 was amplified by PCR and cloned into a certain region (Fc), a human immunoglobulin heavy chain, pAGCF, a leader sequence present in nature, and a plasminogen activity inhibition-1 leader peptide, pAGPCF2. It was. The Fc fusions were transfected into HEK293 cells, and then cultured for 2 days in serum-free medium conditions in which the cells were full to obtain a cell medium containing the rat syndecane-2-Fc fusion protein. After collecting the cell medium, the cells were removed by centrifugation, and the pH was adjusted to 8.0 using 1M Tris. The medium was filtered using a 0.4-㎛ filter, and then hung on a Protein A column and eluted under 100 mM glycine, pH 3.0. And immediately neutralized with 1 M Tris, pH 8.0. The BALB / c mice generated an immune response with the S2E-Fc fusion protein to contain polyclonal sera through a strong immune response. After spleens were removed from the mice and fused to mouse myeloma Sp2 / 0 cells, screening and single cell cloning were performed according to a defined protocol. After obtaining ascites from BALB / c mice that do not have an immune response, a stable hybridoma clone (clone 197) was obtained that produced an immunoglobulin fragment via Protein G to secrete monoclonal antibodies against syndecan-2. Produced.

Example  10: AOM Establishment of an Induced Colorectal Cancer Model

Four-week-old male FVB / J mice were intraperitoneally injected once weekly with 10 mg / kg AOM (azoxymethane) for six weeks. Mice were sacrificed 30 weeks after the AOM final injection. All animals were maintained in the SPF barrier area of the experimental animal facility at the veterinary college (Seoul National University, Korea). All animals were housed in plastic cages under limited humidity (50 ± 5%), light (12/12 h light / dark cycle) and temperature (23 ± 2 ° C.). All courses were approved by IACUC (Institutional Animal Care and Use Committee) in accordance with Seoul National University's Experimental Animal Breeding Management Committee.

Example 11: Preparation of Fc Receptor-Soluble Cindecan-2 Chimera

The extracellular domain (Met ¹ -Asn ^148, SEQ ID NO: 3) and cleavage (sS2E-Fc-I, II, III) water soluble syndecan-2 chimera (sS2E-Fc) were linked to the Fc region of human IgG ₁ . A 444 base FcR-SDC2 cDNA was inserted by cleaving the EcoRI / XhoI enzyme site of the pcDNA 3.1 expression vector comprising the Fc region.

Example  12: Sindecan -2- Fc  Receptor Chimeric  In vitro cleavage and N-terminal sequencing

HETK293T cells were transfected with the vector or plasmid S2E-Fc and the conditioned medium was conditioned for 1 hour at 37 ° C. with pro-MMP-7 on cleavage buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl ₂ ). Incubated. Protein A-Sepharose slurry was added, and further incubated for 1 hour at 25 ℃ for 1 hour. Bound material was added to 15% (w / v) SDS-PAGE and blotted onto PDVF membrane (GE Healthcare Life Sciences). Membrane-binding proteins were stained with CBB R-250 (Coomassie Brilliant Blue R-250), and N-terminal sequencing of the cleaved S2E-Fc fragment on the membrane was performed by Edman cleavage at Tufts Core Facility (Tufts University, MA, USA). It was performed by the method.

Example  13: Cell proliferation assay

Proliferation of HCT116 cells was confirmed by Park, H., et al., (2002) in the presence of conditioned medium containing sS2E-Fc (final 10% v / v ). J. Biol. Chem. 33, 29730-29736. MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) method according to the method described in.

Example  14: soft Agarose  ( soft agarose Adhesion-independent on) Anchorage - independent A) growth analysis

Each well of a 6 well plate was coated with 3 ml bottom agar mixture (Mccoy's 5A / 10% FBS / 0.6% agar). After the bottom agar membrane solidified, conditioned medium containing VEC (control) or sS2E-Fc or normal or cancer patient serum was mixed with HCT116 cells and Mccoy's 5A / 10% FBS / 0.3% to create a top agar mixture and added to each well. The culture was incubated at 37 ° C., 5% CO ₂ . Colony formation was monitored daily with light microscopy, and colonies were photographed with a camera after 14 days of incubation.

result

One. MMP -7 is Extracellular Sindecan Adjust the cleavage of -2.

To analyze the syndecan-2 shedding mechanism, we first identified the presence of syndecan-2 soluble forms in cancer cell conditioned media.

As a result, soluble forms of syndecane-2 were detected in conditioned media of various human colorectal cancer cells, including HT-29, Caco-2, LOVO, and HCT116 cells ( FIG. 1A ). The water-soluble truncated syndecane-2 was partially separated from the medium of the slot HT-29 cells and confirmed its presence by slot blotting (FIG. 1B left) or western blotting (FIG. 1B right). Interestingly, the level of truncated syndecane-2 increased after treatment with the well-known MMP activator, 4-aminophenylmercuric acetate (APMA) (FIG. 1C), suggesting that MMP is involved in the regulation of cleavage.

Next we examined whether syndecane-2 cleavage was caused by MMP-7.

As a result, overexpression of MMP-7 in HCT116 cells increased the level of soluble syndecan-2 in conditioned media, whereas knocking down MMP-7 using the pSuper-MMP-7 shRNA plasmid had the opposite effect. It was confirmed (FIG. 1D). When pre-MMP-7 was pretreated in HT-29 cells, MMP-7 increased syndecane-2 cleavage time- and concentration-dependently (FIG. 1E). In addition, MMP-7-mediated syndecane-2 cleavage was more effective than MMP-2 or MMP-9 (FIG. 1F).

These results support that syndecane-2 cleavage is mediated, in particular, by MMP-7.

2. MMP -7 is Sindecan -2 Extracellular  N-terminal of the domain Leu The residue  Cut.

To identify cleavage sites for syndecane-2, GST-recombinant syndecane-2 (GST-SDC2) was incubated with purified pro-MMP-7 enzyme in vitro.

As a result, MMP-7 cleaved syndecane-2 dose-dependently as expected (FIG. 2A). However, MMP-7 did not effectively cleave recombinant syndecane-4, which had the closest homology to syndecane-2 (FIG. 2B).

To identify the cleavage site of syndecane-2, purified GST-SDC2 was incubated with MMP-7 in vitro and analyzed by SDS-PAGE.

As a result, MMP-7 cleaved the GST-recombinant syndecane-2, resulting in a cleavage product of about 45 kDa (FIG. 2C). Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed that syndecane-2 was cleaved at the N-terminal Leu ¹⁴⁹ residue of the extracellular domain (FIG. 2D).

Syndecane-2 has a number of glycosaminoglycan chains so that core proteins can be protected from proteolytic cleavage. To address this problem, the present inventors have identified a syndecan-2 extracellular domain-Fc receptor chimera (syndecan-2 extracellular) that linked the syndecan-2 extracellular domain (Met ¹ -Glu ¹⁵⁴ ) to the Fc region of human IgG ₁ . A domain-Fc receptor chimera (S2E-Fc) construct was constructed and the expression vector encoding the construct was transfected into HEK293T cells to obtain an endogenously glycosylated protein.

As a result, the soluble form of S2E-Fc was confirmed in conditioned medium in which HEK293T cells were grown (FIG. 3A). Purified S2E-Fc was smeared on SDS-PAGE, which is a common form of glycosylation. MMP-7 cleaved S2E-Fc to yield a cleavage product of about 36 kDa size, indicating that cleavage occurs in the extracellular domain of syndecane-2 (FIG. 3B). GM6001, a diverse range of MMP inhibitors, completely blocked S2E-Fc cleavage (FIG. 3C). N-terminal sequencing results showed that MMP-7 cleaves Leu ¹⁴⁹ at the N-terminus of glycated S2E-Fc (FIG. 3D). These results support that MMP-7 mediates the cleavage of syndecane-2.

In order to further analyze the function of MMP-7 in cleaving syndecan-2, we constructed various syndecan-2 mutants (cleavage site mutants) in which Asn ¹⁴⁸ and Leu ¹⁴⁹ residues were replaced with Ser or Ile. (FIG. 4A). Recombinant synthecan-2 and syndecan-2 cleavage site mutants were expressed as GST-recombinant proteins, purified using glutathione-agarose beads and incubated with MMP-7 in vitro. 0.05 units of MMP-7 cleaved 50% of the recombinant wild-type syndecane-2, whereas the replacement of Leu ¹⁴⁹ with Ile ¹⁴⁹ (Asn ¹⁴⁸ -Ile ¹⁴⁹ ) reduced the cleavage by a factor of five. Asn ¹⁴⁸ -Leu ¹⁴⁹ a mutant replaced by Ser (Ser ¹⁴⁸ -Ser ¹⁴⁹⁾ is decreased significantly by cutting MMP-7. In contrast, Ser ¹⁴⁸ -Ser ¹⁴⁹ The mutants were five times more resistant to cleavage by MMP-7 (FIG. 4A). Interestingly, the amino acid sequence of the Cindecan-2 Asn ¹⁴⁸ -Ile ¹⁴⁹ mutant is similar to its closest homologue, Cindecan-4, which supports the concept that it is not effectively cleaved by pro-MMP-7 (Fig. 4A).

Level as expected, a truncated Shin decane -2 wild type cells than were transfected with control ^{vector, Asn 148 -Ser 149, Ser 148} -Ser 149 Increased in HCT116 cells transfected with a vector expressing syndecan-2 mutant. However, Asn ^148- Ile ¹⁴⁹ This was not the case in cells transfected with the vector expressing the mutant (FIG. 4B). Similar results were obtained in HT-29 cells (FIG. 4C).

Next we examined whether the cut decane-2 mutant regulates colorectal cancer migration. Migration of HCT116 cells transfected with syndecane-2 was significantly increased compared to cells transfected with vectors. However, cell migration by syndecan-2 cleavage site mutants was reduced in all (FIG. 4B; P <0.05). In particular, Ser ¹⁴⁸ -Ser ¹⁴⁹ mutants with minimal cleavage resulted in the greatest reduction of colon cancer cell migration (the most effective inhibition result). This effect was similar in both HT-29 and HCT116 cells (FIG. 4C; P <0.05). These results indicate that cleavage of syndecane-2 is involved in the regulation of migration of colorectal cancer cells.

3. Clipped Sindecan -2 external domain ( ectodomain ) Promotes cancer cell migration.

We investigated whether truncated syndecane-2 promotes cancer cell migration through self-secretion.

As shown in FIG. 5A, treatment of truncated syndecane-2 purified from the culture promoted cell migration in both HT-29 and HCT116 cells (* P <0.05, ** P <0.01).

We next sought to investigate whether syndecan-2 mutants could regulate colon cancer cell migration. Although cell migration was markedly increased in HT29 and HCT116 cells transfected with a vector expressing wild type Sindecan-2 than vector-transfected cells, it expresses the Sindecan-2 Asn ¹⁴⁸ -Ile ¹⁴⁹ mutant. It was confirmed that syndecane-2-mediated cell migration was significantly reduced in the cells to be treated, which means that cleavage was minimized (FIG. 5B. HT-29, vector = 1.0-fold, wild-type = 2.59-fold, Asn ¹⁴⁸ -Ile ¹⁴⁹ = 1.54- fold, difference, vector vs. wild type = 1.59- fold, 95% CI = 1.54- to 1.64- fold, P <.01; difference, vector vs. Asn ¹⁴⁸ -Ile ¹⁴⁹ = 0.54- fold, 95% CI = 0.53- to 0.56- fold, P <.01; HCT116, vector = 1.0- fold, wild type = 2.12- fold, Asn ¹⁴⁸ -Ile ¹⁴⁹ = 1.26- fold, difference, vector vs. wild type = 1.12- fold, 95 % CI = 1.07- to 1.16-fold, P <.01; vector vs. Asn ¹⁴⁸ -Ile ¹⁴⁹ = 0.26-fold, 95% CI = 0.24- to 0.27-fold, P <.01).

We have previously reported that dedecane-2 regulates metastatic potential in B16F10 mouse melanoma cells. Similar to colorectal cancer cells, we found that Asn ¹⁴⁸ -Ile ¹⁴⁹ increased levels of cutincan-2, which was truncated in conditioned media from B16F10 cells expressing wild type syndecan-2. In mutants it was confirmed that it is not (Fig. 5C, above). Similarly, B16F10 cells overexpressed with dedecane-2 increased cell migration but decreased cell migration capacity in cells expressing mutants (FIG. 5C, below, vector = 1.0-fold, wild-type = 2.53-fold, Asn ^148). -Ile ¹⁴⁹ = 1.48-fold).

We analyzed whether cutindecan-2 can induce cell migration even in vivo. Knock down mouse synthecan-2 (mSDC2) to mouse specific siRNA in B16F10 cells and re-express with a vector encoding rat syndecan-2 (rSDC2) cDNA (wild-type or cleavage mutant), and Lung metastasis model was constructed by injecting B16F10 cells into the 6-week-old C57BL / 6 mouse tail vein.

As a result, lung samples isolated from mice injected with B16F10 cells transfected with mutants had reduced lung metastasis compared to mice injected with cells transfected with wild type (FIG. 5D). These data support that metastatic activity induced by syndecane-2 is associated with cleavage of the extracellular domain.

To further test the effect of truncated synthecan-2 on cancer cell migration, we linked the Fc portion of human IgG ₁ to the truncated extracellular domain (amino acids Met ¹ -Asn ¹⁴⁸ ) to the Fc receptor. -Sindecan-2 chimera (sS2E-Fc) was constructed and the vector encoding this construct was transfected into HCT116 cells. Cell migration was significantly increased in HCT116 cells transfected with sS2E-Fc compared to vector transfected cells (P <0.05), and conditioned media derived from HCT116 cells expressing sS2E-Fc migrated to HCT116 cells. (FIG. 6A; P <0.05).

Similarly, sS2E-Fc overexpression increased adhesion-dependent growth of HCT116 cells on soft agar (FIG. 6B). The xCELLigence system was used to support that sS2E-Fc increased the migration of HCT116 cells (FIG. 6C, Vector = 1.0-fold, sS2E-Fc = 3.16-fold). However, sS2E-Fc had no effect on cell proliferation (FIG. 6D) or adhesion of HCT116 cells (FIG. 6E). These results indicate that truncated syndecane-2 promotes colon cancer cell migration and colony formation.

Furthermore, to determine the cancerous active site of the truncated syndecane-2 fragment, the Fc portion of human IgG ₁ partially eliminated the extracellular domain of the truncated syndecane-2 (sS2E-Fc-I). : Met ¹ -Asp ⁵⁸ , sS2E-Fc-II: Met ¹ -Ser ⁸⁸ , sS2E-Fc-III: Met ¹ -Asp ¹¹⁸ ) to construct various Fc receptor-syndecan-2 chimeras (FIG. 7A) , The constructs were transfected into HEK293T cells and cultured to confirm that they were properly expressed (FIG. 7B). This was performed at the sS2E-Fc-II site using the xCELLigence system (FIG. 7C) and trasnewell (FIG. 7D) (Asp ^58). - Ser ⁸⁸ ) to increase the migration of HCT116 cells. This is interestingly consistent with the site where MMP-7 is expected to bind.

4. In serum Clipped Sindecan Amount of -2 colon cancer In patients  Increases.

To investigate the function of syndecane-2 as a biomarker for colorectal cancer, we collected serum from different stages of colorectal cancer patients and analyzed the cutincan-2 level cut in serum using slot blotting. Truncated syndecane-2 is 2/16 (12.5%) ade, 4/16 (25%) carcinoma in While shed sindecan-2 was not detected in situ (CIS) and 11/16 (69%) advanced colorectal cancer (AC) patients (0/16, 8A), indicating that colorectal cancer patients produce truncated syndecane-2. Interestingly, the amount of cut off can be increased in the development of colorectal cancer (FIG. 8A). To avoid nonspecific interactions between syndecan-2 antibodies and serum immunoglobulins, colon cancer patient serum was preincubated with protein A-beads. After prior removal of serum immunoglobulin, truncated syndecane-2 detection was greatly improved (FIG. 8B), indicating specific binding between syndecane-2 and syndecane-2 antibodies away from serum. To further improve detection of cutincan-2 cut in serum, colon cancer patient serum was treated with nitrite (HNO ₂ ). As can be seen in FIG. 8C, removal of nonspecific proteins in serum further enhanced truncated syndecane-2 detection. Fallen Cindecan-2 has 70% adenomas and 80% carcinomas in It was detected in situ and 90% of advanced colorectal cancer patients (FIG. 8D). Similar results were obtained with Western blotting using mouse monoclonal syndecane-2 antibody (FIG. 8E). On the other hand, the cut decane-2 was not detected in the serum of gastric cancer, another gastrointestinal cancer (FIG. 8F). These data indicate that syndecane-2 truncation occurs during colorectal cancer development and that syndecane-2 truncation is correlated with colorectal cancer development.

Consistent with this, we found that syndecane-2 expression increased in the colon of AOM (azoxymethane) -induced mice (FIG. 9). Thirty weeks after AOM injection, all AOM-injected FVB / J mice showed significant colorectal cancer lesions. Significant syndecane-2 expression was observed in the proliferative epithelium of the colon lesions of these mice, but no such occurrence was observed in normal control FVB / J mice. In the Apc / min mouse model of another colon cancer animal model, familial adenomatous polyposis, cutinc-2 was detected in serum (FIG. 9). These findings suggest that truncated syndecane-2 is secreted into the serum during colorectal carcinogenesis in both human and animal models, supporting the fact that syndecane-2 peptide fragments can be used as specific markers for colorectal cancer. .

5. In the Serum of Colorectal Cancer Patients Clipped Sindecan The increase of -2 correlates with the increased mobility of colorectal cancer cells.

We analyzed whether syndecane-2, cut from the serum of colorectal cancer patients, is associated with colorectal cancer activity. The extent of cutincan-2 was examined using slot blotting with syndecan-2 antibodies, and each serum of colon cancer patients was used as a chemoattractant for cancer cell migration assays. Serum containing a lot of truncated syndecane-2 from adenomas, advanced carcinomas significantly reduced cell migration of HCT116 colorectal cancer cells compared to cells treated with a relatively small amount of truncated syndecane-2. (FIG. 10A; * P <0.05, ** P <0.01). In addition, when the truncated syndecane-2 contained in the serum was removed, the effect of increasing the migration of colorectal cancer cells by the serum disappeared (FIG. 10B; P <0.05).

Interestingly, testing of truncated syndecane-2-containing serum derived from advanced carcinoma resulted in unchanged cell proliferation (data not shown) and increased mobility of most colorectal cancer cells, whereas breast, gastric, liver and lung cancers It was not effective in the same other cancer cells (FIG. 10C). This means that they have specificity for the migration of colon cancer cells. In addition, when the expression of MMP-7 in the tissues was high, the cutincan-2 level cut in the serum was also high and the mobility was increased (* P <0.05, ** P <0.01). In addition, adhesion-dependent colony formation also increased (FIG. 10D).

All these data strongly imply that truncated syndecane-2 present in serum increases the mobility of colon cancer cells.

Attach an electronic file to a sequence list

Claims (16)

delete A composition for diagnosing colon cancer, comprising an agent for measuring the expression level of a Syndecan-2 peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
The composition for diagnosing colorectal cancer according to claim 2, wherein the agent for measuring the expression level of the peptide fragment comprises an antibody specific for a Syndecan-2 peptide fragment.
A kit for diagnosing colorectal cancer, comprising the composition of claim 2.
The kit for diagnosing colorectal cancer according to claim 4, wherein the kit is a protein chip kit.
5. The colon of claim 4, wherein the kit is measured in quantity by i) detecting fluorescence by a surface plasmon resonance (SPR) method or surface plasmon resonance imaging (SPRI) method, or ii) a fluorescent label attached to an antibody. Cancer diagnostic kit.
To provide information necessary for diagnosing colorectal cancer, the expression level of a syndecan-2 peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 from an isolated sample of a suspected colon cancer Measuring; And comparing the expression level of the syndecane-2 peptide fragment with the expression level of the syndecane-2 peptide fragment of the normal control sample.
8. The method of claim 7, wherein the sample is any one selected from blood, serum or plasma.
delete The method of claim 7, wherein the expression level of the Syndecan-2 peptide fragment is electrophoresis, Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (SELD-TOF) or SELDI-TOF The method is measured using the surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (MS) method.
The method of claim 7, wherein the expression level of the Syndecan-2 peptide fragment is measured using an antibody that specifically binds to the Syndecane-2 peptide fragment.
A candidate for treating colorectal cancer is treated with a sample expressing a syndecan-2 peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, and then the expression level of the Syndecan-2 peptide fragment is adjusted. A method for screening a colorectal cancer therapeutic agent, comprising the step of measuring.
The method of claim 12, further comprising measuring the expression level of MMP-7 (Matrix metalloprotease-7).
The method of claim 12, wherein the therapeutic agent mutates leucine, which is the 139th residue of the sindecan-2 extracellular domain of SEQ ID NO: 2 to isoleucine, or the 149th residue of the sindecan-2 extracellular domain of SEQ ID NO: The leucine is mutated to isoleucine.
A pharmaceutical composition for treating colorectal cancer, comprising an antibody that specifically binds to a Sindecan-2 peptide fragment consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
The composition of claim 2, wherein the composition is for early colon cancer diagnosis.

Images