KR20110076830A - Complement c9 as markers for the diagnosis of small cell lung cancer and non-small cell lung cancer - Google Patents

Abstract

PURPOSE: A cancer-specific biomarker, complement C9, is provided to determine metastasis and prognosis of non-small cell lung cancer. CONSTITUTION: A composition for diagnosing small cell lung cancer or non-small cell lung cancer contains an agent for measuring C9 mRNA or protein expression level. The agent for measuring the mRNA expression level contains a primer or probe which specifically binds to the complement C9 gene. The agent for measuring protein expression level contains an antibody which is specific to the complement C9 protein. A kit for diagnosing small cell lung cancer or non-small cell lung cancer contains the composition having the agent. The kit is a RT-PCR kit, DNA chip kit, or protein chip kit.

Description

Complement C9 as markers for the diagnosis of small cell lung cancer and non-small cell lung cancer}

The present invention relates to a cancer-specific biomarker complement C9 (Complement Component 9), comprising a composition for measuring the expression level of complement C9 small cell lung cancer or non-small cell lung cancer diagnostic composition, kit comprising the composition, the marker It relates to a method for detecting and screening for small cell lung cancer or non-small cell lung cancer therapeutic agent using the marker.

The number of cancer (malignant neoplasm) deaths in Korea was 62,887, which was 25.5% (29.6% of male deaths and 20.5% of female deaths) out of 246,515 deaths in Korea (512 deaths per 100,000 population). Death (130.7 deaths per 100,000 population) is the number one cause of death. The cancer death rankings are lung cancer, stomach cancer, liver cancer, colon cancer, and pancreatic cancer, with the deaths of these five cancers accounting for about 70% of all cancer deaths. The main cause of cancer deaths in men is lung cancer, stomach cancer, liver cancer, and colon cancer. The number of deaths from these four cancers (28,147) accounts for 70% of the total number of deaths from men (40,177). The main causes of cancer deaths are gastric cancer, lung cancer, liver cancer, colon cancer and pancreatic cancer. The number of deaths from these five cancers (13,630) accounted for 60% of all female cancer deaths (22,710).

There are dozens of these types of cancer, which have been identified to date, and are mainly classified by the location of the diseased tissue. Cancer grows very fast and metastasis occurs as it invades surrounding tissues, threatening life. These types of cancer include cerebrospinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer , Endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer. It may also be classified by other classification systems, depending on the mechanism or form of the cancer.

In particular, lung cancer was a rare disease until the 19th century. However, since the twentieth century, smoking is becoming more common, the incidence of lung cancer is increasing rapidly in Korea. In addition, lung cancer is poorly treated compared to other cancers, so the incidence is not the first, but deaths are known to be the most cancer patients.

It is not yet clear how and how cancer cells are produced. However, it is a general view that cancer is generated due to the generation of cells that are not controlled for proliferation due to the modification of a gene having a function of normal cell proliferation regulation. to be. These cancers are classified as early cancers in which the cancer cells are confined in the mucous membrane according to the degree of progression. In various cancers, the prognosis of treatment of patients found in the early cancer states appears to be relatively good. Therefore, early diagnosis and treatment of cancer are expected to contribute to lower cancer mortality rate and lower cancer treatment cost. However, in many cases, most of the early symptoms are asymptomatic, and even if it is mild enough to feel a slight indigestion or discomfort in the upper abdomen, most people overlook this, causing the cancer death rate.

Until now, most cancer testing means are physical. Examples include gastrointestinal X-rays, dual imaging, compression, or mucosal imaging. By using an endoscope to visually identify internal organs, you can find very small lesions that do not appear on X-rays. Biopsy can be performed directly at this suspicious place, increasing the diagnosis rate. However, this method has the disadvantages of hygiene problems and patient suffering during the examination.

In addition, most of the cancer treatments that have been advanced to date are surgically resected by surgery, and surgical resection is the only method particularly aimed at cure. In this surgical resection, the principle of resection is to include the widest possible range of surgery aimed at cure, but the extent of resection may be defined in consideration of the sequelae after extensive resection. However, even in this case, if the cancer has spread to other organs, radical surgery is impossible. Therefore, other methods such as chemotherapy are taken. At this time, the anti-cancer drugs that are currently on the market can be temporarily relieved or suppressed recurrence after survival. There is only a temporary effect of prolonging the duration, there is a limit to the treatment of the underlying cancer, and the patient suffers a double pain due to the side effects and economic burden of the anticancer drug.

Therefore, in order to treat cancer, the development of a diagnostic method of cancer with high sensitivity and specificity in the pre-treatment stage is of paramount importance, and this diagnosis must be one that can be found early in cancer. The development of a biomarker screening and diagnostic agent for the development of a diagnostic agent for the diagnosis of cancer and a surgical resection or an anti-cancer drug that enables accurate determination of the onset and progression of the cancer is intractable disease. It is an indispensable means to conquer human cancer.

Accordingly, the present inventors have made efforts to develop a biomarker for effectively diagnosing cancer. As a result, the present inventors have found a biomarker that can be detected not only in cancer tissue but also in serum, and maintains a high level of sensitivity and specificity. Was completed.

One object of the present invention to provide a composition for diagnosing cancer comprising an agent for measuring the level of mRNA of the complement C9 gene or protein thereof.

Another object of the present invention to provide a cancer diagnostic kit comprising the cancer diagnostic composition.

It is another object of the present invention to provide a method for detecting cancer marker complement C9.

It is another object of the present invention to provide a method for screening a cancer therapeutic agent using cancer marker complement C9.

As one aspect for achieving the above object, the present invention relates to a composition for diagnosing cancer comprising an agent for measuring the expression level of Complement component 9 (Complement component 9).

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 determine whether the cancer has developed.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is a substance capable of diagnosing cancer cells from normal cells, and increases or decreases cancer cells in comparison with normal cells. Organic biomolecules such as visible polypeptides or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purposes of the present invention, the cancer diagnostic markers of the present invention are complement C9 genes and proteins encoded thereby that exhibit a specifically high level of expression in cancer cells as compared to cells of normal gastric tissue.

Complement is a protein complex consisting of 20 kinds of proteins involved in immune function in vivo, and is largely composed of subunits called C1 (component 1) to C9 (component 9). These complements, by complement fixation, bind to the cell membranes of the bacteria in which the antigen-antibody complexes are produced, resulting in cell lysis, and the complements bind to the antigen-antibody complexes to give phagocytes (neutral white blood cells or macrophages). It is known to perform opsonization which promotes the action of the back and the like). The present invention relates to a composition for markers capable of diagnosing cancer according to the expression pattern of C9, which is one of the components of the complement known to be involved in the immune response. Some of the components that make up complement have been known to increase in expression with the onset and progression of cancer, but not known for complement C9. Thus, the inventors found that complement C9 can act as a marker for various cancers, and in particular, it was confirmed that its expression is specifically increased in squamous cell carcinoma (FIG. 10). According to the experimental results of the present inventors as described above, preferably the complement C9 marker of the present invention can diagnose lung cancer, breast cancer, liver cancer, stomach cancer, kidney cancer and uterine cancer, and more preferably can be used as a diagnostic marker of lung cancer. More preferably, the marker of the present invention can be used as a diagnostic marker for small cell lung cancer and squamous cell carcinoma of the lung. In addition, the cancer marker of the present invention is increased in the expression of cancer tissues, but more preferably it was confirmed that the expression level in the serum significantly increased, the onset of cancer effectively without using an invasive diagnostic method in the individual Or it can be confirmed whether or not metastasis.

Lung cancer, a malignant tumor originating in the lung, is classified into small cell lung cancer and non-mall cell lung cancer according to its tissue type. Although small cell lung cancer is classified as a part of lung cancer by the location of the onset tissue, it is distinguished from other lung cancers because it has distinct characteristics in clinical course, treatment, and prognosis. Non-small cell lung cancer, on the other hand, is divided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma according to histologic type.

In particular, small cell lung cancer, unlike non-small cell cancer, most of the time, the surgical resection is difficult to progress at the time of diagnosis, it is rapidly growing and well-systemic metastasis, but is known to respond well to chemotherapy or radiation therapy. Small cell lung cancer has a strong malignancy and is often found in metastases to other organs, opposite lungs, and mediastinum through lymphatic vessels or blood circulation. It is known to first occur in the airway (bronchi or bronchi). In general, the mass is large, grayish white, and proliferates along the bronchial wall. The major metastasis organs are known to be in the order of brain, liver, bone, lung, adrenal gland and kidney.

Meanwhile, non-small cell lung cancer is divided into adenocarcinoma, squamous cell acrcinoma, adenocarcinoma and large-cell carcinoma as described above, and squamous cell carcinoma is mainly found in the center of lung. It is common in men and is known to be associated with smoking. As a clinical symptom, squamous cell carcinoma is mainly caused by the bronchial lumen growing and blocking the bronchus. Adenocarcinoma, on the other hand, occurs mainly in the periphery of the lungs, and occurs well in women or non-smokers, and even if the size is small, often metastases. Adenocarcinoma has recently been increasing in frequency. Finally, large cell cancer is a type of cancer that occurs in about 4 to 10% of lung cancers, and occurs mainly near the lung surface (peripheral lung), and about half occurs in large bronchus. Cell size is generally large, and some of them tend to proliferate and metastasize rapidly, which is known to have a poor prognosis compared to other non-small cell lung cancers.

Cancer diagnostic markers including complement C9 of the present invention showed high expression of the lung cancer as a whole, it was confirmed that it is possible to diagnose in most lung cancer, among them, especially for small cell lung cancer and squamous cell carcinoma of the sensitivity and specificity It was confirmed that the degree showed a high diagnostic result.

In more detail, the inventors found that complement C9 can be used as a marker for cancer diagnosis through the following verification.

Specifically, serum samples were collected to remove albumin (albumin) and IgG present in large amounts in serum, and to identify proteins that specifically increase expression in cancer among the remaining serum proteins. Fucosylated glycoproteins were then isolated, and the glycoproteins isolated by electrophoresis were cleaved into peptides using trypsin. After cleavage, peptide mass spectrometry and protein identification were performed, resulting in the identification of complement C9 of the present invention as a protein with increased expression specifically in cancer (FIG. 1).

As used herein, the term "agent for measuring the level of expression of complement C9" refers to a molecule that can be used for detection of a marker by confirming the level of expression of complement C9, a marker that increases expression in cancer cells, as described above. Preferably an antibody, primer or probe specific for the marker.

The expression level of complement C9 can be determined by confirming the expression level of the mRNA of the complement C9 gene or the protein encoded by the gene, and the expression level can be known by measuring one or more levels of one gene alone. In the present invention, "mRNA expression level measurement" refers to a process of confirming the presence and expression level of mRNA of a cancer marker gene in a biological sample in order to diagnose cancer, and can be known by measuring the amount of mRNA. Analytical methods for this include RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting (Northern) blotting) and DNA chips, but are not limited thereto.

The agent for measuring mRNA level of the gene is preferably a primer pair or probe, and the nucleic acid sequence of the complement C9 gene is known as the nucleic acid sequence of NC_000005.9 or the mRNA sequence of NM_001737.3 and the like. Primers or probes can be designed that specifically amplify specific regions of these genes.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. In the present invention, the PCR can be performed using the sense and antisense primers of the complement C9 polynucleotide to diagnose cancer through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.

In the present invention, the term "probe" refers to nucleic acid fragments such as RNA or DNA, which are short to several bases to hundreds of bases, which are capable of specific binding with mRNA, and are labeled to identify the presence of a specific mRNA. Can be. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization may be performed using a probe complementary to the complement C9 polynucleotide, and cancer may be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

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

According to a specific embodiment of the present invention, the cancer diagnostic marker detection composition comprises a primer pair specific for the complement C9 gene.

In the present invention, "protein expression level measurement" refers to a process of confirming the presence and expression level of a protein expressed in a cancer marker gene in a biological sample in order to diagnose cancer. An antibody that specifically binds to the protein of the gene. Check the amount of protein using. Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket ) Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip, 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 complement C9, a marker of the present invention, which is encoded by the marker gene by cloning each gene into an expression vector according to a conventional method. The resulting protein can be obtained and prepared by conventional methods from the obtained protein. Also included are partial peptides that may be made from such proteins, and the partial peptides of the present invention include at least seven amino acids, preferably nine amino acids, more preferably twelve or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

Antibodies used in the detection of 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.

In another aspect, the present invention relates to a cancer diagnostic kit comprising the cancer diagnostic composition.

The kit of the present invention can detect markers by confirming the expression level of mRNA or protein with the expression level of complement C9, a cancer diagnostic marker. The kit for detecting markers of the present invention includes primers, probes, or antibodies that selectively recognize markers for measuring the expression level of a cancer diagnostic marker, as well as one or more other component compositions, solutions, or devices suitable for analytical methods. Can be.

As a specific example, the kit for measuring the mRNA expression level of complement C9 in the present invention may be a kit containing the necessary elements required to perform RT-PCR. RT-PCR kits, in addition to each primer pair specific for the marker gene, RT-PCR kits can be used in test tubes or other appropriate containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymers. Enzymes such as lyase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as the quantitative control. Also preferably, the kit of the present invention may be a diagnostic kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent probe. In addition, the substrate may comprise cDNA corresponding to the quantitative control gene or fragment thereof.

As another specific example, the kit for measuring the protein expression level of Complement C9 in the present invention comprises a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody. It may include. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Fatase (Alkaline Phosphatase) can be used, the fluorescent material can be used FITC, RITC, etc., the color substrate substrate is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

In another aspect, the present invention relates to a method for detecting cancer marker complement C9 by comparing the expression level of complement C9 from a patient's sample to the expression level of normal cells in order to provide information necessary for cancer diagnosis.

More specifically, the expression level of the gene can be detected at the mRNA level or the protein level, and separation of the mRNA or protein from the biological sample can be performed using known processes.

In the present invention, the term patient's sample includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine, which differ in expression level of complement C9, a cancer marker gene.

Through the detection methods, it is possible to diagnose whether the cancer suspect patient is a real cancer patient by comparing the gene expression level in the normal control group with the gene expression level in the cancer suspect patient. That is, after measuring the expression level of the marker of the present invention from the cells suspected of cancer, comparing the two by measuring the expression level of the marker of the present invention from normal cells, the expression level of the marker of the present invention is that of normal cells. If more expression is derived from a cell suspected of being cancer, a cell suspected of being cancer can be predicted as cancer.

Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, and DNA chip. Through the above detection methods, it is possible to compare the mRNA expression level in the normal control group and the mRNA expression level in the suspected cancer patient, and to determine whether the expression level of the cancer marker gene is significantly increased in the mRNA, and whether the cancer suspected patient actually develops cancer. Can be diagnosed.

mRNA expression level measurement is preferably by using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific for the gene used as a cancer marker.

The reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band by checking the mRNA expression and degree of the gene used as a cancer diagnostic marker and compare it with the control group, it is easy to determine whether cancer Diagnosis can be made.

On the other hand, the DNA chip uses a DNA chip in which the nucleic acid corresponding to the cancer marker gene or a fragment thereof is attached to a glass-like substrate with high density, and isolates the mRNA from the sample, and cDNA labeled at the end or the inside with a fluorescent substance Probes can be prepared, hybridized to DNA chips, and read for cancer.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto. Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in cancer suspected patients can be compared, and whether the significant expression level of the cancer marker gene to the protein is increased. By judging, it is possible to diagnose whether the cancer suspected patient actually develops cancer.

Preferably, the protein marker identification through the antibody-antigen reaction may be performed after preferentially separating and selecting the glycated protein in the previous step. Proteins undergo glycosylation according to protein in post-transcriptional modification, depending on the inherent characteristics of the protein, and in some cancers, this may occur because the glycosylation pattern is different from that of normal individuals. Therefore, the present inventors focused on the relationship between cancer and glycation of proteins, and preferentially separated glycated proteins in the step before performing the antibody-antigen reaction, and in particular, fucosylation proceeded according to whether fucosylation was performed during glycosylation. Cancer markers were identified for the proteins. Protein identification method according to the progress of the glycosylation can be used without limitation the conventional methods performed in the art, in the preferred embodiment of the present invention performed a lectin blot to separate the fucosylated glycoprotein (Example 3 ).

In the present invention, the term antigen-antibody complex means a combination of a cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed 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 Therapase, 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. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, 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. Radioisotopes include, but are not limited to, 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, and the like. .

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 enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. The degree of complex formation of the cancer marker protein and antibody can be checked to determine whether cancer has developed.

Also preferably, Western blot using at least one antibody against the cancer marker. The whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody. By checking the amount of the generated antigen-antibody complexes using labeled antibodies, the amount of protein produced by the expression of genes can be confirmed to determine whether cancer is developed. The detection method consists of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the cancer-causing cells. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

Also preferably, immunohistostaining is performed using at least one antibody against the cancer marker. After collecting and fixing normal tissue and suspected cancer, paraffin embedding blocks are prepared by methods well known in the art. These are cut into slices of several μm thickness, adhered to glass slides, and reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

In addition, preferably, one or more antibodies against the cancer marker are arranged at a predetermined position on the substrate to use a protein chip 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, You can check for cancer.

As another aspect, the present invention relates to a method for screening a cancer therapeutic agent, comprising measuring the expression level of the complement C9 gene or the level of a protein encoded by administration of a substance expected to be able to treat cancer. will be.

Specifically, it can be usefully used for screening cancer therapeutic agents by a method of comparing the increase or decrease in the expression of complement C9 in the presence and absence of cancer treatment candidates. Substances that indirectly or directly reduce the concentration of complement C9 can be selected as therapeutic agents for cancer. That is, by measuring the expression level of the marker complement C9 of the present invention in cancer cells in the absence of a cancer treatment candidate, and also measuring the expression level of the marker complement C9 of the present invention in the presence of a cancer treatment candidate, Subsequently, it is possible to predict an agent for treating cancer in which the expression level of the marker of the present invention when the cancer treatment candidate is present is lower than the marker expression level in the absence of the cancer treatment candidate.

The present invention is expected to find an effect that can be used as an indicator of data useful for the treatment and prognosis of cancer by discovering and providing a diagnostic marker that can determine cancer metastasis and prognosis. Using the cancer marker complement C9 according to the present invention can accurately and easily determine the presence or absence of cancer, can be used as a specific target in cancer-specific anti-cancer drug development research, furthermore it can be used in the study of cancer tumor formation In addition, it is expected to contribute to the early diagnosis of cancer.

1 is a schematic diagram showing the overall scheme for detecting the marker protein of the present invention.
2 is a diagram showing the results of analyzing the protein of serum (lane 1), albumin and IgG in the serum (lane 2) and serum samples (lane 3) from which albumin and IgG are removed from normal and small cell lung cancer patients.
3 is a conceptual diagram showing fucosylation, which is a type of glycosylation.
FIG. 4 shows the identification of fucosylated AFP of L3 fragments through lectin-electrophoresis.
5 is a schematic diagram showing the mechanism of occurrence of abnormal aberrant fucosylation (J. Biochem. 2008. 143. 725-729) in Hepatocellular carcinoma.
Figure 6 is a schematic diagram showing the process of separating the fucosylated glycoproteins bound to AAL lectin.
7 is a schematic diagram showing a process for confirming the isolation and expression of the fucosylated glycoprotein.
Figure 8 is a photograph showing the results of the lectin blot analysis to identify the fucosylated glycoproteins present in the sample.
FIG. 9 shows the results of Western blot expression of complement C9 protein in serum of small cell lung cancer patients and healthy subjects.
Figure 10 shows the results confirmed by Western blot the complement C9 expression pattern of patients with squamous cell carcinoma of lung cancer.
Figure 11 shows the results confirmed by Western blot the complement C9 expression in the sample of adenocarcinoma patients in lung cancer.
12 is a graph showing the amount of C9 complement expressed in normal, adenocarcinoma patients, small cell lung cancer patients and squamous cell carcinoma patients.
Figure 13 shows the results of adding samples of normal and squamous cell carcinoma patients to the protein chip array.
14 shows the results of Western blot expression of complement C9 complement from samples of normal, small cell lung cancer patients, squamous cell carcinoma patients, breast cancer patients, liver cancer patients, gastric cancer patients, kidney cancer patients and ovarian cancer patients.
FIG. 15 is a schematic diagram illustrating a process of performing a hybrid lectin eliza experiment using a sandwich ELISA technique. FIG.
FIG. 16 is a graph showing the degree of fucosylation of C9 complement included in each sample of normal (HEC), adenocarcinoma patients (ADC), small cell lung cancer patients (SCLC), and squamous cell carcinoma patients (SQLC).
FIG. 17 is a graph showing the degree of fucosylation of C9 complement included in each sample of normal (HEC), breast cancer patients (BC), liver cancer patients (HCC), gastric cancer patients (STC), and squamous cell carcinoma patients (SQLC). .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Collection of Serum Samples

Serum samples of 13 normal and 13 lung cancer patients were provided from Seoul Samsung Hospital. The provided sample was stored at -70 ° C until use.

Example  2: albumin ( albumin ) And IgG  remove

There are many kinds of proteins in the blood and they exist in various concentration distributions. For example, albumin, the highest concentration protein, is present at a high concentration of about 30-40 mg / ml, which accounts for half of the protein in the blood, and IgG (Immunoglobulin G) is also contained in the serum at high concentrations. On the other hand, the concentration of substances such as cytokines, especially interleukin, which directly reflects the situation in the body due to increased expression by special circumstances in the body, such as the development or progression of cancer and certain diseases, Since only about 10 ng / ml, in the study of cancer-specific marker proteins generated by the cytokine-like substance or the cancer surrounding environment, the removal of proteins such as albumin and IgG in large quantities in the blood There was a problem that should be prioritized.

Thus, the present inventors removed the albumin and IgG in the protein in the serum using a protein removal kit (ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit, Sigma, USA) in order to overcome the above problems. Specifically, when each of the obtained serum samples in a column included in the kit, only albumin and IgG are bound to the resin of the column, and the remaining proteins pass through the column. Therefore, a sample passed through the column was collected to obtain a serum sample from which albumin and IgG were removed (FIG. 2).

Figure 2 is an electrophoresis picture showing the result of removing albumin and IgG from the sample of normal people and patients, lane 1 of Healthy represents the mixed serum of the normal sample before removing albumin and IgG, lane 2 of Healthy The albumin and IgG derived from the mixed serum of the normal sample bound to the column of the kit for healthy, and lane 3 of Healthy represents the mixed serum of the normal sample from which albumin and IgG were removed. In addition, lane 1 of small cell lung cancer represents the mixed serum of the patient sample before the albumin and IgG removed, lane 2 of small cell lung cancer is derived from the mixed serum of the patient sample bound to the column of the protein removal kit Albumin and IgG, and lane 3 of small cell lung cancer shows the mixed serum of albumin and IgG samples. As shown in Figure 2, it was confirmed that the majority of albumin and IgG in the serum of normal people and patients are removed.

Example  3: Fucosylated ( Fucosylated Glycoprotein Isolation

Glycoprotein refers to a protein in which a sugar chain is bonded to a side chain of a peptide by a covalent bond. The glycoprotein is a post-transcriptional modification called glycosylation. Attached to the protein. Glycoproteins produced in vivo by such glycosylation mechanisms are present as extracellular secreted proteins or proteins attached to the membrane, and may be used for immune reactions or cell-cell actions ( It is known to be responsible for many functional parts such as cell-cell interaction. Fucosylation refers to the glycosylation process in which fucose binds to protein as a sugar chain, which is the most common glycosylation process in vivo, and the action of various fucosyltransferases. , GDP-fucose synthesis and GDP-fucose transpoter are known to be involved (FIG. 3).

This fucosylation has been reported as one of the major glycosylation processes in cancer since the relationship with cancer was first identified in 1979. For example, AFP (alpha-fetoprotein), a well-known marker of liver cancer, is known to cause abnormal aberrant fucosylation by microenvironment of liver cancer (FIGS. 4 and 5).

Lectin is a sugar-binding protein that specifically binds to different sugar moieties according to its type, and is used to recognize cells or proteins in vivo. It is known to be involved. There are many kinds of these lectins, and depending on the type has the ability to specifically bind each sugar chain. Therefore, when using the lectin, not only can confirm specific glycosylation, but also can be utilized for the enrichment of the sample and the diagnosis using a biomarker, the inventors of the present invention is a kind of lectin AAL ( Aleuria Aurantia Lectin ) was used to select fucosylated glycoproteins from serum samples of albumin- and IgG-depleted patients.

Specifically, AAL was charged to the washed empty spin column, stabilized, and then serum samples of patients with albumin and IgG removed were added, reacted for 12 hours, and proteins bound to the column. By eluting, fucosylated glycoproteins bound to AAL were obtained (FIG. 6).

Then, to confirm the obtained fucosylated glycoproteins, the sample was electrophoresed and the AAL-biotin complex (ALL-biotin complex) capable of binding with the fucosylated glycoprotein to the electrophoretic sample and the The presence of fucosylated glycoproteins was sequentially added to the biotin-binding streptavidin-HRP complex, followed by confirmation of the activity of HRP using ECL (Electrochemiluminescence) technique (FIG. 7). It was confirmed (Fig. 8).

Figure 8 is a photograph showing the results of the lectin blot analysis to identify the fucosylated glycoproteins present in the sample, lane 1 shows the mixed serum of the normal sample before removing albumin and IgG, lane 2 is albumin and IgG Mixed serum of the patient sample prior to removal of lanes, lane 3 represents the mixed serum of the normal sample with albumin and IgG removed, lane 4 represents the mixed serum of the patient sample with albumin and IgG removed, lane 5 Mixed serum of normal sample not bound to AAL column is shown, lane 6 represents mixed serum of patient sample not bound to AAL column, lane 7 represents mixed serum of normal sample bound to AAL column, lane 8 is AAL Mixed serum of normal samples bound to the column is shown. As shown in Figure 8, it was confirmed that the sample bound to the AAL-column contained a relatively high content of the fucosylated glycoprotein.

Example  4: protein mass spectrometry and candidate discovery

After obtaining a fucosylated glycoprotein sample using the AAL lectin technique, trypsin digestion was performed, and the sample was analyzed using an LC-MS / MS mass spectrometer based on a proteomics method. Glycoprotein was analyzed. Then, we performed a method of identifying proteins based on database search and various software. Later, we compared the results of the glycoproteins of the lung cancer group and the normal group to find the complement C9 (complemnet component 9 (C9)), a glycoprotein specific to the lung cancer group.

Example  5: normal people In lung cancer patients  Manifested C9 Complementary  compare

In order to confirm whether the C9 discovered in Example 4 can be used as a diagnostic marker for lung cancer, a number of small cell lung cancer, squamous cell acrcinoma or adenocarcinoma patients The amount of C9 protein expressed was compared to that of normal persons using Western blot method.

Example  5-1: Normal Person and Small Cell Lung Cancer In the patient  Manifested C9 Complementary  compare

Serum samples were obtained from 38 normal individuals and small cell lung cancer patients, respectively, and albumin and IgG were removed from each sample by the method of Example 2, and then subjected to Western blot using anti-C9 antibody. The amount of C9 complement expressed in normal and small cell lung cancer patients was compared (FIGS. 9A and 9B).

Figure 9a is a photograph showing the results of Western blots performed on a part of the normal (HE) serum samples and a part of the small cell lung cancer patients (SCC) serum samples, Figure 9b is a densito the concentration of the band color developed by Western blot It is a graph showing the result of measuring in meters (densitometry).

As shown in Figure 9a and 9b, it was confirmed that C9 is expressed at a significantly higher level in patients with small cell lung cancer than normal people, it can be seen that the C9 can be used as a diagnostic marker for small cell lung cancer.

Example  5-2: normal people Squamous cell carcinoma In the patient  Manifested C9 Complementary  compare

Serum samples were obtained from 121 normal subjects and 120 squamous cell carcinoma patients, and albumin and IgG were removed from each sample by the method of Example 2, followed by Western blot using anti-C9 antibody. Was performed to compare the amount of C9 complement expressed in normal and squamous cell carcinoma patients (FIGS. 10A and 10B).

Figure 10a is a photograph showing the results of Western blot performed on a portion of the serum samples of normal (HE) samples and a portion of the squamous cell carcinoma patients (SQLC) serum, Figure 10b is a concentration of the bands developed by Western blot It is a graph showing the results measured with a densitometer (densitometry).

As shown in Figure 10a and 10b, it was confirmed that C9 is expressed at a significantly higher level in patients with squamous cell carcinoma than normal, it can be seen that the C9 can be used as a diagnostic marker for squamous cell carcinoma.

Example  5-3: normal people Adenocarcinoma In the patient  Manifested C9 Complementary  compare

Serum samples were obtained from 121 normal individuals and 120 adenocarcinoma patients, and albumin and IgG were removed from each sample by the method of Example 2, and then subjected to Western blot using anti-C9 antibody. The amount of C9 complement expressed in normal and adenocarcinoma patients was compared (FIGS. 11A and 11B).

FIG. 11A is a photograph showing Western blot results of a part of a normal (HE) serum sample and a part of adenocarcinoma patient (LAC) serum sample, respectively, and FIG. 11B is a densitometer of concentration of bands colored by Western blot. It is a graph showing the result of measuring (densitometry).

As shown in FIGS. 11a and 11b, it was confirmed that C9 is significantly expressed in adenocarcinoma patients rather than normal patients, and thus, C9 may be used as a diagnostic marker for adenocarcinoma.

Example 5-4 Comparison by Western Blot of C9 Complement Expressed in Normal Subjects and Various Lung Cancer Patients

As shown in the results of Examples 5-1 to 5-3, since C9 of the present invention is expressed at a significantly high level in patients with small cell lung cancer, squamous cell carcinoma and adenocarcinoma, The expression level of C9 complement was directly compared.

Specifically, each serum sample was obtained from 20 normal patients, adenocarcinoma patients, small cell lung cancer patients and squamous cell carcinoma patients, and albumin and IgG were removed from each sample by the method of Example 2, and then each sample was subjected to Western blot using anti-C9 antibody was performed to compare the amounts of C9 complement expressed in normal, adenocarcinoma patients, small cell lung cancer patients, and squamous cell carcinoma patients (FIGS. 12A and 12B).

FIG. 12A is a photograph showing Western blot results of a portion of each serum sample obtained from a normal person (HEC), adenocarcinoma patient (ADC), small cell lung cancer patient (SCLC) and squamous cell carcinoma patient (SQLC). , FIG. 12B is a graph showing the result of measuring the density of the bands colored by Western blot (densitometry).

As shown in Figure 12a and 12b, it was confirmed that C9 is expressed at a significantly higher level in all lung cancer patients than normal people. In particular, in patients with squamous cell carcinoma, it was confirmed that C9 was expressed at a higher level than those of other lung cancer patients as well as normal people. Therefore, it was found that C9 can be used as a diagnostic marker for lung cancer.

Example 5-5 Comparison of Protein Chip Arrays of C9 Complement Expressed in Normal and Various Lung Cancer Patients

As shown in the results of Examples 5-4, C9 of the present invention is expressed at the highest level in patients with squamous cell carcinoma, so in order to confirm again, the squamous cell carcinoma patients and normal patients with the protein chip array method The expression level of C9 complement was directly compared to the sample of.

That is, each serum sample was obtained from 100 normal and squamous cell carcinoma patients, and albumin and IgG were removed from each sample by the method of Example 2, and then each sample was subjected to anti-C9 antibody. Western blots were performed to compare the amount of C9 complement expressed in normal and small cell lung cancer patients (FIGS. 13A, 13B and 13C).

Figure 13a is a photograph showing the results of applying a sample of normal and squamous cell carcinoma patients to the protein chip array, Figure 13b is a graph showing the result (densitometry) of measuring the degree of color development on the protein chip, Figure 13c is a graph showing the Receiver-operator characteristic (ROC) curve for C9. The degree of detection is the detection rate of squamous cell carcinoma of the normal human (HEC), the sensitivity is 53%, the specificity is about 89%. The area-under-the-curve (AUC) value was 0.708.

As shown in Figure 13a to 13c, it was confirmed once again that C9 is expressed at a significantly higher level in patients with squamous cell carcinoma than normal.

Example  6: with various cancer patients From normal people  Manifested C9 Complementary  compare

From Example 5, it was confirmed that the C9 of the present invention can be used as a diagnostic marker for most lung cancers, and therefore, it was intended to confirm whether the C9 can be used for diagnosis of cancers other than lung cancer.

Specifically, from 12 normal, 12 small cell lung cancer patients, 12 squamous cell carcinoma patients, 9 breast cancer patients, 9 liver cancer patients, 9 gastric cancer patients, 6 kidney cancer patients and 3 ovarian cancer patients, respectively A serum sample was obtained, and albumin and IgG were removed from each sample by the method of Example 2, and then Western blot using an anti-C9 antibody was performed on each sample. The amounts of C9 complement expressed in cancer patients, breast cancer patients, liver cancer patients, gastric cancer patients, kidney cancer patients and ovarian cancer patients were compared (Figs. 14A and 14B).

14A shows normal people (HE), small cell lung cancer patients (SCC), squamous cell carcinoma patients (SQC), breast cancer patients (BC), liver cancer patients (HCC), gastric cancer patients (SC), kidney cancer patients (RCC) and ovaries Part of each serum sample obtained from cancer patients (OC) is a photograph showing the results of the Western blot, respectively, Figure 14b is a densitometry of the concentration of the band developed by Western blot (densitometry) It is a graph.

As shown in Figure 14a and 14b, it was confirmed that C9 is expressed at a significantly higher level in all cancer patients than normal people. In particular, the ovarian cancer, squamous cell carcinoma and small cell lung cancer patients showed higher levels of C9 expression than normal and other cancer patients, and renal, gastric, breast and liver cancer patients were significantly higher than normal. Since it was confirmed that the expression, it was found that the C9 can be used as a diagnostic marker for various cancers as well as lung cancer.

Example  7: C9 Complementary Fucosylation ( Fucosylated Degree of comparison

As described above in Example 3, fucosylation is known to be one of the most important glycosylation processes in cancer, and thus, the fucosylation of C9 complement detected in samples of three lung cancer patients is compared and mutually compared. The fucosylation of the C9 complement detected in the sample was compared with each other.

Example  7-1: Depending on the type of lung cancer C9 Complementary Fucosylation ( Fucosylated Degree of comparison

Hybrid lectin ELISA experiment using the sandwich ELISA technique was measured and compared with the degree of fucosylation of C9 complement contained in the sample of each lung cancer patient (Fig. 15).

That is, AAL-biotin capable of binding 20 samples of adenocarcinoma patients, small cell lung cancer patients and squamous cell carcinoma patients to the plate coated with the anti-C9 antibody, and binding to the fucosylated glycoprotein, respectively. Allo-biotin complex and streptavidin-HRP complex capable of binding to the biotin were sequentially added, and then the level of fucosylation of C9 complement was confirmed by confirming HRP activity with an ELISA detector. Was measured (FIG. 16).

FIG. 16 is a graph showing the degree of fucosylation of C9 complement included in each sample of normal (HEC), adenocarcinoma (ADC), small cell lung cancer (SCLC) and squamous cell carcinoma patients (SQLC). As shown in FIG. 16, the C9 complement contained in the sample of each lung cancer patient showed a high level of fucosylation compared to the C9 complement included in the sample of a normal person, but the Cucco complement of the C9 complement was different between the samples of different types of lung cancer patients. It was confirmed that the degree of misfire did not show a significant difference.

Example  7-2: Depending on the type of cancer C9 Complementary Fucosylation ( Fucosylated Degree of comparison

From the results of Example 7-1, it was confirmed that the degree of fucosylation of C9 complement did not show a significant difference between samples of different types of lung cancer patients. We attempted to determine whether the degree of fucosylation showed a significant difference.

Specifically, samples of 15 normal individuals, breast cancer patients, liver cancer patients, gastric cancer patients, and squamous cell carcinoma patients, were added to the plate coated with the anti-C9 antibody, respectively, and reacted with the fucosylated glycoprotein. AAL-biotin complex (ALL-biotin complex) and streptavidin-HRP complex that can bind to the biotin was added sequentially, and then confirmed the activity of HRP by the Elisa detector, C9 complement of The degree of fucosylation was measured (FIG. 17).

FIG. 17 is a graph showing the degree of fucosylation of C9 complement included in each sample of normal (HEC), breast cancer patients (BC), liver cancer patients (HCC), gastric cancer patients (STC), and squamous cell carcinoma patients (SQLC). . As shown in FIG. 17, the C9 complement contained in all cancer patients compared to the C9 complement included in a normal human sample showed a high level of fucosylation. In particular, gastric cancer patients (STC) and squamous cell carcinoma patients It was confirmed that the degree of fucosylation of the C9 complement included in the sample of (SQLC) shows a relatively high level.

Claims (15)

Composition for diagnosing small cell lung cancer or non-small cell lung cancer comprising an agent for measuring the expression level of the mRNA of the complement C9 gene or protein thereof.
According to claim 1, wherein the agent for measuring the expression level of the gene mRNA composition for diagnosing small cell lung cancer or non-small cell lung cancer that comprises a primer specifically binding to the complement C9 gene.
The composition for diagnosing small cell lung cancer or non-small cell lung cancer according to claim 1, wherein the agent for measuring the expression level of the gene mRNA comprises a probe specifically binding to the complement C9 gene.
The composition for diagnosing small cell lung cancer or non-small cell lung cancer of claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody specific for complement C9 protein.
The cancer diagnostic composition of claim 1, wherein the non-small cell lung cancer is squamous cell carcinoma.
Small-cell lung cancer or non-small cell lung cancer diagnostic kit comprising the composition according to any one of claims 1 to 5.
The kit for diagnosing small cell lung cancer or non-small cell lung cancer according to claim 6, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit.
Measuring the expression level of the complement C9 gene or the protein encoded by the gene from a sample of the suspected cancer to provide information necessary for diagnosing small cell lung cancer or non-small cell lung cancer; And comparing the expression level of the gene or the level of the protein encoded by the gene with the expression level or protein level of the gene in the normal control sample.
The method of claim 8, wherein the expression level of the gene measures the mRNA expression level of the gene.
The method of claim 9, wherein the method of measuring mRNA levels is by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip.
The method of claim 10, wherein the mRNA expression level is measured by reverse transcriptase polymerization using a primer.
The method of claim 8, wherein the protein expression level uses an antibody specific for that protein.
The method of claim 8, wherein the method for measuring protein expression level is Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement Any one of a fixed assay, FACS or protein chip method.
The method of claim 8, wherein the non-small cell lung cancer is squamous cell carcinoma.
A method for screening for a small cell lung cancer or non-small cell lung cancer therapeutic agent, comprising measuring the expression level of the complement C9 gene or the protein encoded by the gene after administration of a substance expected to be able to treat cancer.

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