KR20130099131A - Antigen peptide for suppressing metastasis, anti-metastatic vaccine including same, composition for suppressing and treating metastasis, and method for screening metastasis-related target materials and metastasis suppressant - Google Patents

Abstract

PURPOSE: An antigen peptide which suppresses cancer metastasis and an anti-metastatic vaccine containing the same are provided to identify correlation between the expression level of epithin and transendothelial migration using various cancer cell lines, thereby controlling cancer progression and metastasis. CONSTITUTION: An antigen peptide which suppresses cancer metastasis is an oligopeptide selected among an oligopeptide with an amino acid sequence (KQARVVGGTNADEGEWPWQ) of sequence number 1, an oligopeptide with an amino acid sequence (CGDGSDEEG) of sequence number 2, an oligopeptide with an amino acid sequence (CSDGSDEKN) of sequence number 3, an oligopeptide with an amino acid sequence (RQARVVGGTDADEGEWPWQ) of sequence number 7, an oligopeptide with an amino acid sequence (CGDNSDEQG) of sequence number 8, an oligopeptide with an amino acid sequence (CSDGSDEKD) of sequence number 9, an oligopeptide with an amino acid sequence (RQARVVGGTDADEGEWPWQ) of sequence number 10, and an oligopeptide with an amino acid sequence (KQARVVGGTNADEGEWPWQ) of sequence number 11. An anti-metastatic vaccine contains the antigen peptide as an active ingredient.

Description

Antigen peptides that inhibit cancer metastasis, cancer metastasis vaccines comprising the same, compositions for inhibiting and treating cancer metastasis, and screening methods for cancer metastasis-related target substances and cancer metastasis inhibitors {ANTIGEN PEPTIDE FOR SUPPRESSING METASTASIS, ANTI-METASTATIC VACCINE INCLUDING SAME, COMPOSITION FOR SUPPRESSING AND TREATING METASTASIS, AND METHOD FOR SCREENING METASTASIS-RELATED TARGET MATERIALS AND METASTASIS SUPPRESSANT}

The present invention relates to an antigen peptide that inhibits cancer metastasis, a cancer metastasis vaccine comprising the same, and a composition for inhibiting and treating cancer metastasis, wherein the antigen peptide that inhibits cancer metastasis by migration and invasion of cancer cells, a cancer metastasis vaccine comprising the same It relates to a composition for inhibiting and treating cancer metastasis.

In addition, the present invention reveals that epithelial cell migration target epithelial cells and transendothelial migration (TEM) of cancer cells expressing it have a strong correlation with cancer metastasis, and use such endothelial cell permeation migration assays. The present invention relates to providing a technology for screening cancer metastasis-related target substances and cancer metastasis inhibitors.

It is reported that cancer is the largest refractory disease and various genes and proteins are involved, and cancer occurrence, inhibition and metastasis are controlled by a complex signaling system. Until now, research has been conducted to develop various diagnostic or anticancer agents for early diagnosis or treatment of cancer. Recently, studies on selective diagnostic agents or target anticancer agents using specific molecular targets have been actively conducted.

As a target anticancer agent using a target protein associated with cancer, currently commercially available target anticancer agents such as Avastin and Herceptin can predict the therapeutic effect and have an excellent therapeutic effect with fewer side effects. In order to develop an effective target anticancer agent, it is necessary to study the identification and function of cancer specific target protein along with general molecular understanding of cancer. That is, once the target protein related to cancer development is identified, it is easy to analyze the mechanism of cancer development using the target protein, and it is also possible to study the structure-activity association that develops various derivatives of anticancer drug candidates and performs optimal anticancer function.

In addition, cancer has a good prognosis in combination with surgical surgery and chemotherapy at the initial onset, whereas when the cancer has progressed considerably, even with a targeted anticancer agent, it is difficult to cure and is likely to recur within 5 years after being cured. In addition, when cancer recurs, cancer cells often metastasize to other organs in addition to the site of the first onset, causing cancer patients to die. In general, the need for early screening and recurrence screening for blood cancers such as leukemia, malignant lymphoma, gastric cancer, colon cancer, lung cancer, breast cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer is recognized. However, early screening is technically difficult due to the nature of the cancerous processes that start with very few cells. In particular, cancer metastasis, which is difficult to treat in case of cancer recurrence, has a poor prognosis and decreases the survival rate of patients, needs to be screened early in patients with cancer history, and the mechanism of cancer metastasis and cancer metastasis in patients with cancer history. The social demands on how to prevent this in advance are very high.

As a target protein of interest in connection with such cancer metastasis, there is a protein called Matriptase or epithin. Epitin is the first protein identified in mouse thymic epithelial cells. Epitin has also been isolated from human epithelial cancer cells and may also be referred to as PRSS14, Matriptase, MT-SP1 or ST14. Epitin is markedly increased in most epithelial cell-derived cancer cells such as ovarian cancer, prostate cancer, cervical cancer and breast cancer, and recently, expression of activated macrophages has also been known.

Epitin is functionally a type II transmembrane serine protease, which is structurally an intracellular domain, a transmembrane region (TM), an SEA domain, a CUB domain, an LDLRA (or LDL) domain, and a serine. It consists of an extracellular region that contains a serine protease domain (see FIG. 1). Until now, epitin has been known to be involved in normal epithelial cell differentiation and cancer development. Recently, epitin interacts with several proteolytic enzymes in the initial cancer development, progression and metastasis, leading to the migration of cancer cells through degradation of extracellular matrix, invasion and angiogenesis of the basement membrane. It has been reported to play an important role in angiogenesis. Taken together, these results indicate that epitin is overexpressed in cancer cells and cancer tissues compared to normal cells, and thus is involved in all stages of cancer development, growth and metastasis. Thus, epitin is a new target protein for the diagnosis and treatment of cancer.

Therefore, the present inventors have studied intensively by paying attention to the fact that the mechanism of inhibiting cancer metastasis can be developed by elucidating the mechanism of cancer metastasis caused by epitin, and the cancer metastasis vaccine or monoclonal antibody treatment method can be developed based on this. As a result, it has been confirmed that meaningful studies have shown that polypeptides of water-soluble epitin motifs that are cut and shed out of cells are directly involved in angiogenesis. In addition, the present inventors promoted the vascular endothelial cell permeation migration of cancer cells while the structural stability between vascular cells disappeared by epitin acting on the Tie2 protein, which plays an important role in vascular stability and degrading the Tie2 moiety including a ligand binding domain. This led to the identification of the mechanism by which cancer metastasis was induced. Specifically, the present inventors in vitro ( in In vitro ) Reduced expression of epitin protein in 4T1 breast cancer cells resulted in a significant decrease in the number of vascular endothelial permeation migration (TEM) cells, and decreased 4T1 breast cancer in the blood vessels of mice in animal studies When the cells were injected, it was confirmed that tumor nodules metastasized to the lungs were reduced.

In other words, the present inventors have found a direct mechanism by which epitin plays a role in cancer metastasis, and by using a vaccine or antibody by identifying the correlation between epitin expression using various cancer cell lines and vascular endothelial cell permeation migration (TEM). It is now possible to provide a technological basis for controlling the progression and metastasis of Tin's cancer.

On the other hand, looking at the global trend of cancer vaccines and cancer metastasis vaccines, cancer vaccines are mostly used as a therapeutic vaccine to treat cancer with antibodies, and the human papilloma virus vaccine known to cause cancer is prevented. It has been developed and used as a vaccine, but side effects and ethical issues are being raised. Recently, prostate cancer vaccines and breast cancer vaccines using immune functions as clinical cancer vaccines are in clinical trials and are expected to be commercialized soon. Therefore, with the development of a target cancer therapy using monoclonal antibodies such as Herceptin, cancer vaccines that induce an immune response to a target protein associated with cancer without causing side effects in the human body have attracted great attention.

In this regard, U.S. Patent Publication No. US2004 / 0105853 A1 discloses a pharmacological composition using a substance that binds to an epitin-like serine protease gene product. However, in the prior art, an epitope of an epitin-like protein antigen is disclosed. Induction of antibodies by the whole epitin-like protein consisting of 200 to 500 amino acids without elucidation of the epidermal or epithelial metastasis mechanism, and anti-antibody against polynucleotides encoding epitin-like protein It only discloses expression silencing of epitin-like proteins by antisense polynucleotides that are supposed to be sensed. However, since the antibody of the prior art is obtained by the immune response of a full length epitin-like protein, the specificity of the antibody is inferior and there is no data on activity in an animal model related to cancer progression and metastasis. Efficacy is difficult to be scientifically recognized and there is a problem that side effects are concerned. That is, it is difficult to generate antigen-specific monoclonal antibodies with long sequences of 200 to 500 amino acids described in the prior art, and is far from identifying tumor antigen oligopeptides directly related to cancer progression and metastasis. Was. In addition, the antisense polynucleotides of the prior art also have no proven efficacy, and the technology using antisense RNA can be implemented only at the cellular level and cannot be applied to individual units without the development of a drug delivery system. As in the prior art, there is no case of developing an antisense polynucleotide having a long base sequence as a therapeutic agent for an individual unit, and thus, the prior art is not applicable at all in relation to cancer development, progression, and metastasis.

In other words, the research on the conventional cancer metastasis as described above is still in its infancy, and many people are looking for genes or proteins that simply act on cancer metastasis without studying the mechanism, and target proteins based on precise cancer metastasis mechanism identification. Cancer metastasis vaccine research using the situation is still insignificant. In view of this, the development of cancer vaccines or monoclonal antibodies that inhibit cancer metastasis may be regarded as a breakthrough in medical technology that can prevent cancer recurrence in patients with cancer history and prevent cancer metastasis that is fatal to patients.

The present invention relates to an antigen peptide that inhibits cancer metastasis, a cancer metastasis vaccine comprising the same, and a composition for inhibiting and treating cancer metastasis, wherein the antigen peptide that inhibits cancer metastasis by migration and invasion of cancer cells, a cancer metastasis vaccine comprising the same To provide a composition for inhibiting and treating cancer metastasis.

In addition, an object of the present invention is to find that the epithelial cell migration target epithelial cell (TEM) and epithelial migration (TEM) of cancer cells expressing it has a deep correlation with the cancer metastasis, this endothelial cell permeation migration assay It is to provide a base technology for screening cancer metastasis related target substances and cancer metastasis inhibitor using.

The present inventors have intensively studied the direct mechanism that epitin acts on cancer metastasis. As a result, the present inventors have investigated the correlation between epithelial expression level and vascular endothelial cell permeation migration using various cancer cell lines. To provide a technical basis for controlling the progression and metastasis of the cancer of chitin, antigen peptides and cancer metastasis vaccines containing the same have been found to induce antibodies and inhibit cancer metastasis in vitro and in vivo. To develop.

Hereinafter, the term "epitin" as used in the specification of the present invention is used in a broad sense including epitopes derived from mouse as well as metlipases or other orthologs derived from humans. Thus, the antigenic peptides and cancer metastasis vaccines defined below may comprise oligopeptides of mouse origin, human origin, rat origin or rabbit origin.

Antigenic peptides that inhibit cancer metastasis of an embodiment of the present invention are oligopeptides having the amino acid sequence of K SEQARVVGGTNADEGEWPWQ, oligopeptides having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) ) Oligopeptide, an oligopeptide having the amino acid sequence of SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ), an oligopeptide having the amino acid sequence of SEQ ID NO: 8 (CGDNSDEQG), an oligopeptide having the amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), SEQ ID NO: At least one oligopeptide selected from the group consisting of an oligopeptide having an amino acid sequence of 10 (RQARVVGGTDADEGEWPWQ) and an oligopeptide having an amino acid sequence of SEQ ID NO: 11 (KQARVVGGTNADEGEWPWQ).

The cancer metastasis vaccine of one embodiment of the present invention is an oligopeptide having an amino acid sequence of KIDARVVGGTNADEGEWPWQ, an oligopeptide having an amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), an oligopeptide having an amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) Peptide, oligopeptide having the amino acid sequence of SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ), oligopeptide having the amino acid sequence of SEQ ID NO: 8 (CGDNSDEQG), oligopeptide having the amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), amino acid sequence of SEQ ID NO: 10 At least one oligopeptide selected from the group consisting of an oligopeptide having (RQARVVGGTDADEGEWPWQ) and an oligopeptide having the amino acid sequence of SEQ ID NO: 11 (KQARVVGGTNADEGEWPWQ) is included as an active ingredient.

In addition, the cancer metastasis vaccine of one embodiment of the present invention may contain a pharmaceutically acceptable carrier in addition to the oligopeptide administered as an active ingredient. Cancer metastasis vaccines of one embodiment of the present invention can be used, for example, with an adjuvant or immune enhancer (or "immune adjuvant") (adjuvnat), such as alum. The cancer metastasis vaccine of one embodiment of the present invention is a mineral such as aluminum sulfate, aluminum hydroxide, magnesium hydroxide, or aluminum phosphate, surfactant such as phosphorus lecithin and Pluronic polyol, polyanion, Oily emulsions and the like. In addition, the cancer metastasis vaccine of one embodiment of the present invention may be mixed in liposomes or may contain various sugars.

Another embodiment of the cancer transfer vaccine of the present invention is an oligopeptide having an amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), an oligopeptide having an amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), an amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) Oligopeptides, oligopeptides having the amino acid sequence of SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ), oligopeptides having the amino acid sequence of SEQ ID NO: 8 (CGDNSDEQG), oligopeptides having the amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), amino acids of SEQ ID NO: 10 A DNA encoding at least one oligopeptide selected from the group consisting of an oligopeptide having a sequence (RQARVVGGTDADEGEWPWQ) and an oligopeptide having an amino acid sequence of SEQ ID NO: 11 (KQARVVGGTNADEGEWPWQ) is included as an active ingredient.

In another cancer metastasis vaccine of the present invention, a nucleic acid encoding an antigen peptide that inhibits cancer metastasis, preferably DNA, is inserted into an expression vector, and then the vector is administered to the animal to produce cancer immunity. can do. Such DNA vaccines are described in WO2000 / 6602 or J. Immunol., 160, p. 1717, 1998 and the like can be technically implemented.

In addition, the cancer metastasis inhibiting and therapeutic composition of an embodiment of the present invention is an oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), an oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), the amino acid sequence of Oligopeptide with (CSDGSDEKN), oligopeptide with amino acid sequence of SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ), oligopeptide with amino acid sequence of SEQ ID NO: 8 (CGDNSDEQG), oligopeptide with amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), Containing an antibody or an antibody induced by an immune response against at least one antigenic peptide selected from the group consisting of an oligopeptide having the amino acid sequence of SEQ ID NO: 10 (RQARVVGGTDADEGEWPWQ) and an oligopeptide having the amino acid sequence of SEQ ID NO: 11 (KQARVVGGTNADEGEWPWQ) It contains serum. Preferably, the antibody is a monoclonal antibody.

On the other hand, in an antigen peptide which inhibits cancer metastasis of one embodiment of the present invention, a cancer metastasis vaccine comprising the same, or a composition for inhibiting and treating cancer metastasis, "administration" means introducing a predetermined substance into a patient by any suitable method. Means, the administration route of the antigen peptide, cancer metastasis vaccine comprising the same, or a composition for inhibiting and treating cancer metastasis may be administered through any general route as long as they can reach the target tissue. For example, it may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, and the like. In addition, an effective amount of the antigenic peptide which inhibits cancer metastasis of one embodiment of the present invention, a cancer metastasis vaccine comprising the same, or a composition for inhibiting and treating cancer metastasis is required for administration in order to expect cancer metastasis suppression, prophylactic and therapeutic effect. Means quantity. Therefore, it can be adjusted according to the cancer type of the patient, the progress and metastasis state of the cancer, the type of formulation, the age, sex, weight, health condition, diet, administration time and administration method of the patient. For example, when administering an antigen peptide that inhibits cancer metastasis, an cancer metastasis vaccine comprising the same, or a composition for inhibiting and treating cancer metastasis, 0.1 mg / kg to 100 mg / kg once daily It may be administered in a dose.

Screening method of the cancer metastasis inhibitor of an embodiment of the present invention, (a) culturing the endothelial cells to prepare an endothelial cell layer, (b) culturing the cells expressing epitin, and ( c) adding the epitin-expressing cells onto the endothelial cell layer, (d) counting the number of cells that have migrated through the endothelial cell layer to determine cancer metastasis reference values, and (e) Adding the cancer metastasis inhibiting candidate and performing the steps (a) to (c), counting the number of cells moved through the endothelial cell layer and comparing the cancer metastasis reference value obtained in the step (d). It includes. If the number of epitin-expressing cells permeated through the endothelial cell layer was decreased by the addition of a cancer metastasis suppression candidate, the candidate material inhibited the expression or activity of epitin, indicating that the endothelial cell layer permeation migration was reduced. This suggests that the candidate is an effective substance that inhibits cancer metastasis.

Screening method of a cancer metastasis-related target material of an embodiment of the present invention, (a) culturing the endothelial cells to prepare an endothelial cell layer, (b) culturing cells expressing cancer metastasis-related candidate target material is prepared (C) adding cells expressing the cancer metastasis-related candidate target material on the endothelial cell layer, and (d) counting the number of cells that have migrated through the endothelial cell layer and counting cancer metastasis reference values. And (e) preparing a cell having reduced expression or activity of a cancer metastasis-related candidate target material and adding it to the endothelial cell layer and counting the number of cells that have migrated through the endothelial cell layer. and comparing the cancer metastasis reference value obtained in step (d). If the number of cells that permeate the endothelial cell layer is decreased in the case of cells which decrease the expression or activity of the cancer metastasis-related candidate target material, the cancer metastasis-related candidate target material may be involved in endothelial cell permeation migration. This suggests that the cancer metastasis-related candidate target material is a substance related to cancer metastasis.

In the method of the present invention, the endothelial cell layer may be an MS1 cell layer which is a mouse endothelial cell, and the cells added to the endothelial cell layer are 427.1.86 cells which are thymic epithelial cancer cells or 4T1 which are breast cancer cells. May be a cell. However, the present invention is not limited thereto, and it goes without saying that a cancer cell strain derived from various cancer tissues can be used. In addition, the cells added to the endothelial cell layer may be labeled with a label such as fluorescent protein to facilitate counting the number of cells that have migrated through the endothelial cell layer. For example, as a label, a fluorescent material such as rhodamine, FITC, or CFSE, and a fluorescent protein such as GFP, EGFP, YFP, CFP, or RFP may be used. However, the present invention is not limited thereto, and various labeling materials such as radioactive materials or luminescent materials known in the art can be used.

According to the present invention, it is possible to control epithelial cancer progression and metastasis by using a vaccine or an antibody by identifying the correlation between epitin expression level and vascular endothelial permeation migration using various cancer cell lines.

Specifically, the present invention reveals that epithelial cell permeation target of epithelial cancer and epithelial cell permeation of cancer cells expressing it has a strong correlation with cancer metastasis. It is possible to provide a base technology for screening substances and cancer metastasis inhibitors.

In addition, the antigen peptides that inhibit cancer metastasis of the present invention and cancer metastasis vaccines comprising the same may function as cancer metastasis antigens to induce an immune response to produce antibodies, and inhibit metastasis of cancer cells, thereby preventing and treating cancer metastasis. Very useful for

The above and other technical problems and features of the present invention will be apparent to those skilled in the art through the description of the embodiments of the present invention made with reference to the accompanying drawings.
1 is a schematic diagram showing the protein structure of epitin.
2 is a diagram illustrating step-by-step endothelial cell permeation migration assay of the present invention.
Figure 3 is a micrograph showing the shape and location of cells labeled with fluorescent proteins that migrated the endothelial cell layer in the endothelial cell permeation transfer assay of the present invention.
Figure 4 is a graph comparing the number of GFP-427 cells and GFP-427-Epi-KD cells that permeate the MS1 endothelial cell layer using the endothelial cell permeation transfer assay of the present invention.
5 is a graph comparing the number of 427 cells and 427-Epi-KD cells that permeate the MS1 endothelial cell layer using the endothelial cell permeation migration assay of the present invention.
FIG. 6 shows epitope gene knockdown from 4T1 cell line. Electrophoresis showing the results of Western blotting using mAb5, an anti-epitin antibody against clones and control clones (A7, D5, B3, C5, G3).
7 shows that in the endothelial cell permeation migration assay of the present invention, cells of the A7 and D5 clones expressing the epitin effectively penetrate the endothelial cell layer while the epitope expression is knocked down B3, C5 and G3 The cells of the clones are graphs showing the inability to penetrate the endothelial cell layer easily.
FIG. 8 shows that in the endothelial cell permeation migration assay of the present invention, TEM ability was restored in cells of AB-B3 clone obtained by transfecting B3 clone knocked down with epitin expression with pcDNA3.1 / metlipase. It is a graph showing that.
9 is a photograph of tumor nodules metastasized to the lungs of mice injected with clones derived from the 4T1 cell line.
FIG. 10 is a graph counting the number of tumor nodules in FIG. 9 and quantifying each clone. FIG.
FIG. 11 is a graph showing that endothelial cell migration of 4T1 cell lines is inhibited by mouse antibodies AG22-1, AG22-3, and AG22-5 immuno-induced by the antigen oligopeptide of the present invention.
12 is a graph showing the percentage of cancer cell migration when the antibody of FIG. 11 is administered.
Figure 13 shows the immune enhancers, "immune enhancer + Epi-2LDL", "immune enhancer + Epi-4LDL" in the mouse Adj, Adj + Epi-2LDL, Adj + Epi-4LDL and Adj + Epi-SP groups, respectively, in the first animal experiment. And survival curves measured for each group after immunization with immune enhancer + Epi-SP and injection of cancer cells.
FIG. 14 is a photograph taken when the lungs of the mouse are removed and placed in a dish on the 19th day of the experiment of FIG. 13.
FIG. 15 is a graph showing numerical values of lung weight and nodule number of Adj, Adj + Epi-2LDL, Adj + Epi-4LDL, and Adj + EpiSP groups after the experiment of FIG. 13.
FIG. 16 is a graph showing the percentages obtained by dividing all individuals by their average values based on the average value of the nodule number of the Adj group after the experiment of FIG. 13.
Figure 17 shows the immune enhancers, "immune enhancer + Epi-2LDL", "immune enhancer + Epi-4LDL" and "immune enhancer + in the mouse Adj, Adj + 2LDL, Adj + 4LDL, Adj + SP groups, respectively, in the second animal experiment After immunization with Epi-SP and injection of cancer cells, each group was photographed on the surface of the nodular lung.
18 is a graph quantifying the number of nodules in the groups Adj, Adj + 2LDL, Adj + 4LDL, and Adj + SP after the experiment of FIG. It is a graph showing each group after obtaining.
Figure 19 after immunization with "immune enhancer + Epi-SP" and "immune enhancer + Epi-SP-sc" in each of the third animal experiment and injected 4T1 cancer cells (4T1 Con-D5 or 4T1 EpiKD-B3) This is a picture of the surface of the nodule in each group.
20 is a graph showing the number of nodules numerically for each group after the experiment of FIG. 19.
21 is a graph showing the percentages obtained by dividing all individuals by their average value based on the average value of the nodule number of the Adj + 4T1 group after the experiment of FIG. 19.
22 is a photograph of the electrophoresis results of Western blotting on MCF7 with serum obtained from Adj + SP + 4T1 group.
Figure 23 is a photograph showing that the antibody formed of the antigen oligopeptide of the present invention inhibits the fibrillation of 427.1.86 cells induced by epitin.
24 is a photograph showing that the antibody formed with the antigen oligopeptide of the present invention inhibits the fibrillation of MS1 cells induced by soluble epitin.

Hereinafter, the present invention will be described in detail according to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. Therefore, what can be easily inferred by the expert in the technical field to which this invention belongs from the detailed description and the Example of this invention is interpreted as belonging to the scope of the present invention. The references cited in the present invention are incorporated herein by reference.

Materials and methods

Reagents, antibodies and laboratory animals used

Most of the reagents used in the following examples of the present invention were purchased from Invitrogen Corporation, USA. Anti-epitin (anti-metlipase) antibody (H-270), anti-Tie2 antibody, control shRNA lentivirus and epitin shRNA lentivirus were purchased from Santa Cruz Biotechnology, INC, USA. , Anti-tubulin was purchased from Sigma, USA, and monoclonal antibody mAb5 was prepared as described in previous literature published by the inventors (EG Cho, MG Kim, C. Kim, SR Kim). , IS Seong, C. Chung, RH Schwartz, D. Park, N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease, J Biol Chem 276 (2001) 44581-44589). The pcDNA3 vector was purchased from Invitrogen Corporation, and the pcDNA3 / eGFP vector and the pcDNA3.1 / methtitase vector were prepared by recombining the eGFP gene and the methithetase gene in the pcDNA3 vector, respectively. In addition, BalB / c mice used to confirm the cancer metastasis to the lung was purchased from Korea Biolink (Korea, Chungcheongbuk-do) was used.

Cell Lines Used and Cell Culture

Cancer cell lines (427.1.86, 4T1, etc.) were maintained in culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum). In addition, MS1, a mouse endothelial cell, was cultured and maintained in DMEM medium supplemented with 5% FBS. In addition, to construct a 427 Epi-KD cell line knocked down (meaning inhibiting or blocking expression) in 427.1.86 cancer cells overexpressing epitin, the following two synthetic oligonucleotides were included: The pSUPER-epi construct was recombined into a pcDNA3 vector encoding a neomycin resistance gene and then transfected into 427.1.86 cancer cells. After growing for 10 days in the presence of 800 µg / ml G418, a single cell whose protein and RNA expression levels were stably knocked down was selected to construct a 427 Epi-KD cell line.

Forward strand (SEQ ID NO: 5):

5'-GATCCCC GGTGCGCTTCAAACTCTTC TTCAAGAGA GAAGAGTTTGAAGCGCACC TTTTTA-3 '

Reverse strand (SEQ ID NO: 6):

5'-TCGATAAAAA GGTGCGCTTCAAACTCTTC TCTCTTGAA GAAGAGTTTGAAGCGCACC GGG-3 '

In SEQ ID NO: 5 and SEQ ID NO: 6, the sequence portion of the bases shown in bold and underline indicates a sequence targeting epitin.

GPF-427 and GFP-427 Epi-KD cell lines were constructed in a similar manner to 427 Epi-KD cell line construction, except that the pcDNA3 / eGFP vector was used instead of the pcDNA3 vector. 4T1 cells were trackfected with control shRNA lentiviral or epitin shRNA lentiviral (which is a lentivirus encoding shRNA that silences epitin expression) and the presence of 2 μg / ml puromycin 4T1 derived clones (A7, D5, B3, C5, G3) were obtained by single cell selection. A7 and D5 clones effectively express epitin as clones of 4T1 cells obtained by transfection with control shRNA lentivirus, while B3, C5 and G3 clones were transfected with epitin shRNA lentivirus. Epilone expression is knocked down as clones. In addition, the AB-B3 clone is a clone obtained by transfecting the B3 clone with pcDNA3.1 / metlipase, and effectively expresses a human-derived metlipase. The vectors were transfected into cells using lipofectamine (purchased from Invitrogen).

Western Blotting  Analysis method

Westing blotting assays used in the examples of the present invention were performed according to methods well known in the art. Protein samples were separated on a 10% acrylamide gel under reducing conditions. A secondary antibody conjugated with horseradish peroxidase (HRP) was used for visualization of the separated proteins and treated with enhanced chemiluminescence reagents (Amersham).

Example

Example  1: endothelial cell permeation migration Assay ( Transendothelial migration assay )

In order to study the process of metastasis of epithelial-expressing cancer cells, MS1 cells were tested for the difference in the degree of transendothelial cell migration (TEM) according to the level of epitin expression. In addition, a cancer cell line expressing a fluorescent protein was prepared and tested in a mouse cancer model using an imaging device to confirm the degree of cancer cell metastasis in the animal. In addition, epitope expression knocked down cell lines (B3, C5 and G3 clones of FIGS. 6 to 8) were prepared and used in cell culture and cancer models of mice (see FIGS. 9 and 10). In this example, the relationship between the degree of epitin expression and vascular endothelial permeation migration using various cancer cell lines was examined and confirmed by the metastasis of cancer cells stained with fluorescence in the cancer model of mice. Endothelial cell permeation migration assay (Transendothelial cell migration assay) method was established (see Figure 2). This will be described in detail below.

Example  1-1: Microscopy for migration between endothelial cells

MS1 cells were cultured in gelatin-coated coverslips in 12-well plates to form a confluent monolayer. GFP-427 cells and GFP-427-Epi-KD cells stably expressing GFP fluorescent protein were isolated using Cellstripper ^™ (Cellgro) and resuspended in culture medium at a concentration of 10 ⁵ cells / ml. 1 ml of cell resuspension was added to each MS1 monolayer. After 8 hours, cells were washed and fixed, stained with Rhodamine conjugated Paloidine (actin stained red), and fluorescence with 10X Planapochromat objective lens. Observation was made under a microscope (Axiovert 200M, Zeiss). Images were analyzed using AxioVision RE software (Zeiss). Cells that migrated the endothelial cell layer were analyzed according to morphology based on GFP fluorescent protein and F-actin pattern. Optical sections were acquired with the LSM510 with a 40X / 1.2W Korr lens under 2X digital zoom settings, and the images taken using the LSM Image Browser (Carl Zeiss) were collected and organized (see Figure 3). ).

Example  1-2: Boyden Of the Chamber Chamber (Falcon)  How to use

The cells were cultured in an 8 μm pore chamber (Insert, Falcon) until 5 × 10 ⁴ MS1 cells reached a dense state. 4T1 cells were stained with Molecular Probes ^™ (CFSE) and resuspended in DMEM medium without serum. 10 ⁵ stained with CFSE 4T1 cells were added to the MS1 cell layer and incubated for 4 hours. The cells were fixed with 3.7% formaldehyde (Sigma) and then unmigrated 4T1 cells were removed and washed. Images of CFSE-positive cells were obtained using an IX-51 microscope (Olympus) equipped with AxioCam MRm (Zeiss), and the cell number was analyzed by ImageJ (National Institute of Health) (FIG. 2, 7 and 8). Reference).

Example  1-3: Epitin  Correlation between expression level and vascular endothelial cell penetration

As shown in FIG. 3, rounded cells (FIG. 3C) based on the form visualized by GFP fluorescence protein and F-actin were GFP-427 cells or GFP-427 − attached to the MS1 endothelial layer. Epi-KD cells were counted, and spindle or polygonal cells (FIGS. 3 f and i) were counted as GFP-427 cells or GFP-427-Epi-KD cells that permeate the MS1 endothelial layer. In the image taken with the vertical section, round-shaped cells are observed as hemispheres (c 'in FIG. 3) attached on the MS1 cell layer, while spindle or polygonal cells are dispersed in the MS1 cell layer or located between MS1 cells. Was observed (f 'and i' in Figure 3). "*" Mark in f of FIG. 3 represents actin stress fiber. In three experiments, the average value of the number of GFP-427 cells attached to the MS1 endothelial cell layer and the number of GFP-427 cells permeated through the MS1 endothelial cell layer in the cells of GFP fluorescence protein positive cells was obtained. The average value of the number of GFP-427-Epi-KD cells attached to the MS1 endothelial cell layer per cell and the number of GFP-427-Epi-KD cells permeated through the MS1 endothelial cell layer was calculated. The 427 cell number and the GFP-427-Epi-KD cell number were compared (see FIG. 4).

Fig. 4 compares the number of cells permeated and migrated relative to the total number of cells in epidermal expressing cells GFP-427 and epitin expressing knocked down cells GFP-427-Epi-KD. As a result, 63% of epitin-expressing cells (GFP-427) penetrated the MS1 cell layer, whereas only 42% of epitin-expressing cells (GFP-427 Epi-KD) penetrated the MS1 cell layer. It can be seen that shifting reduced expression of epitin reduced TEM ability. As can be seen again from Figure 5, TEM assay significantly reduced TEM ability in 427 Epi-KD cells knocked down epitope expression by transfecting thyroid carcinoma cell line 427 expressing a large amount of epitin with shRNA. From this, it can be seen that epitin plays an important role in the endothelial cell migration of cancer cells, it can be seen that epitin is also involved in cancer metastasis. In addition, this shows that the TEM assay can be effectively used to identify cancer metastasis-related target substances and cancer metastasis inhibitors. On the other hand, in Figures 4 and 5, the error bar (error bar) represents the standard deviation, the asterisk (*) has a statistically significant level of P It shows that it is <0.01.

Example  1-4: Epitin  4, not overexpressing cancer cell lines T1  Through experiments with breast cancer cell lines TEM Assy  Reliability Verification as Experimental Model for Cancer Transition

Experiments were performed using clones derived from 4T1 cell line (A7, D5, B3, C5, G3) and mouse breast cancer cell line 4T1 as described above. First, Western blotting using the anti-epitin antibody mAb5 against 4T1 and A7, D5, B3, C5, G3 clones was performed to check the expression level of epitin and whether the same amount was loaded with anti-tubulin band (See FIG. 6). For each clone derived from the 4T1 cell line, a TEM assay using the Vödene chamber method described in Example 1-2 was performed to count the number of cells permeated through the MS1 endothelial cell layer, and then numerically graphed for each clone. As shown in FIG. 7, cells of the A7 and D5 clones that effectively express epitin migrate effectively through the endothelial cell layer, while cells of the B3, C5 and G3 clones that have knocked down epithelial expression express the endothelial cell layer. It can be seen that the transmission does not move easily. In addition, cells of AB-B3 clone obtained by transfecting B3 clone knocked down with epitin expression with pcDNA3.1 / metlipase effectively restore the TEM ability by penetrating the endothelial cell layer by the expression of metlipase. One could confirm (see FIG. 8). On the other hand, each point shown in Figures 7 and 8 represents the number of cells permeated in the microscopic field, the average value and the standard deviation for them are shown in the graph, the asterisk (*) has a statistically significant level p <0.001 and an asterisk (**) indicate that the statistically significant level is p <0.01.

Given that vascular endothelial permeation migration is an essential step in cancer metastasis, TEM ability is maintained in cells of A7 and D5 clones (epitin expression) induced by transfection of breast cancer cells 4T1 with control shRNAs. However, the lack of TEM ability in cells of B3, C5, and G3 clones (epitin knockdown) induced by transfection of breast cancer cells 4T1 with epitin shRNA has shown that epitin plays an important role in endothelial cell migration. Suggests deep involvement in cancer metastasis. In particular, the transfection of epitope knocked down B3 clones with Matriptase vectors further reinforces the fact that the TEM ability is restored, which indicates that the TEM assay can be used as a cancer metastasis model with confidence. To prove that it can be.

Example  2: In vivo ( in vivo Metastasis Assay  through Epitin  Correlation between expression level and vascular endothelial cell penetration

Clones derived from 5 x 10 ⁴ 4T1 cell lines in PBS solution (A7, D5, B3, C5, G3) were each routed through the tail vein of six Balb / c mice (purchased from Korea Biolink). Injected. Mice injected with clones derived from the 4T1 cell line were sacrificed after 14 days and lungs were stained by injecting Indian ink dissolved in PBS (available regardless of manufacturer as a general ink) through trachea. Stained lungs were dissected, washed appropriately with PBS, and images were taken for each clone to count tumor nodules (see Figure 9). The counted results were numerically graphed for each clone (see FIG. 10).

As shown in FIG. 9, in the lungs of mice injected with cells of A7 and D5 clones that effectively express epitin, the entire surface is covered with tumor nodules, while epitope expression is knocked down B3, C5 and G3 It can be seen that the number of tumor nodules covering the lungs of mice injected with the cells of the clones is significantly reduced. FIG. 10 shows that the results confirmed by the naked eye are quantified, and epitin effectively induces cancer metastasis. In FIG. 10, the average value and standard deviation of the number of tumor nodules are shown in the graph for each clone, with an asterisk (*) indicating a statistical significance level p <0.001 and an asterisk (**) indicating a statistical significance level p. It shows that it is <0.01.

These in vivo experimental results were consistent with the endothelial cell permeation migration assay results of Example 1. Therefore, the present Example confirms again that epitin is an important substance related to cancer progression and metastasis, and shows that the endothelial cells of Example 1 have a close correlation with epithelial expression level and vascular endothelial cell permeation migration. Permeation transfer assays have proven to be an effective way to screen cancer metastasis-related targets and cancer metastasis inhibitors.

Example  3: Cancer metastasis  Inhibitory antigen Of peptide  Confirmation of antibody induction and transfer of vascular endothelial cells using this antibody Assay

In order to control cancer metastasis using immunity, we tested whether the antigenicity of epitin can produce immune antibodies.Epitin is immune because epitine proteins are expressed in the thymus microenvironment, which plays an important role in causing immune tolerance. Responses are the most important identification process for cancer metastasis vaccine development and the possibility of monoclonal antibody development. Oligopeptides having high antigenic potential and capable of being immunogens in both humans and mice were selected and tested for antibody formation by immunization with mice.

For this purpose, an oligopeptide having an amino acid sequence of KQARVVGGTNADEGEWPWQ, an oligopeptide having an amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), or an oligopeptide having an amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) is selected from KLH Three mice were immunized three times after binding to the mice, and after 3 days, serum was collected and subjected to ELISA analysis. Three mice with high antibody-forming amounts were selected. And to determine whether these antibodies (AG22-1, AG22-3, AG22-5) inhibits cancer metastasis by epitin, the endothelial cell permeation transfer assay (TEM) described in Example 1-2 was carried out It was.

In other words, vascular endothelial cells (MS1) constituting the vascular endothelial cells were cultured in a Boyden chamber to form a dense layer, and 1x10 ⁵ cells of a D5 clone effectively expressing epitin derived from a 4T1 cancer cell line. In order to distinguish it from vascular endothelial cells, it was labeled with a fluorescent substance called CFSE and administered on an endothelial cell layer. At this time, the concentration of serum was given to promote the movement of cancer cells, and endothelial cell permeation migration assay was performed by controlling protein expression of cells or administering antibodies to determine factors involved in the movement of cancer cells. And, counting the number of D5 cancer cells that migrate to the other side of the vascular endothelial cells, when the antibody is not added, when the antibodies (AG22-1, AG22-3, AG22-5) induced in the previous experiment, respectively, and It was measured by dividing the case with the positive control H270 antibody. The amounts of antibodies AG22-1, AG22-3, AG22-5 and H270 were used in this experiment based on the amount of serum generally used. Based on the amount (0.5 ml) of the cell culture medium used in this experiment, 5 (1: 100 dilution ratio). The measured results are shown in FIG. 11, and it was confirmed that the migration of cancer cells was inhibited by the antibodies AG22-1, AG22-3, and AG22-5. In FIG. 12, the number of cancer cell migrations at the time of antibody administration was expressed as a percentage when the antibody was not administered. As a result, mouse antibodies AG22-1, AG22-3, and AG22-5 immunized with the antigen oligopeptide of the present invention inhibit cancer cell migration by approximately 63%, 48%, and 34%, respectively, against cancer cells derived from the 4T1 cell line. It was observed that. Thus, the antigen oligopeptides of the present invention induce an immune response to produce antibodies and the resulting antibodies inhibit cancer cell migration in endothelial cell permeation migration assays, thereby making the antigen oligopeptides of cancer cells a cancer metastasis vaccine. It can be said to have an effect of inhibiting the metastasis process.

Example  4: Cancer metastasis  Inhibitory antigen Peptides  Animal experiment

Based on the results of Example 3, an oligopeptide having an amino acid sequence of KQARVVGGTNADEGEWPWQ, an oligopeptide having an amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG) or an oligopeptide having an amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) Balb / c mice were used to determine whether cancer metastasis by induction of immune responses functions as a vaccine.

Example  4-1: Vaccine efficacy test through primary animal experiment

Oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ) (hereinafter abbreviated as "Epi-SP"), oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG) (hereinafter referred to as "Epi-2LDL") ) Or oligopeptides having the amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN) (hereinafter abbreviated as "Epi-4LDL") consisted of 10 mouse Balb / c per group to investigate the effect on 4T1 cancer cell metastasis A total of four groups (Adj, Adj + Epi-2LDL, Adj + Epi-4LDL, Adj + EpiSP) were assigned to the immune enhancers (Adjuvant), "Immune Enhancers + Epi-2LDL", "Immune Enhancers + Epi-4LDL" and " Immunity Enhancer + Epi-SP ″ was injected three times, once every three weeks. At the first injection, 50 μg oligo peptide and CFA were mixed (50 μg oligo peptide + 100 μl CFA in 100 μl PBS solution) to each mouse. Intraperitoneal injection and from the second injection 20 μg oligo peptide was mixed with IFA (20 μg oligo peptide + 100 μl IFA in 100 μl PBS solution) and injected intraperitoneally into each mouse (volume ratio of immune enhancer and oligo peptide is 1: Adjusted to 1). And 3 days after the third injection, 4T1 cancer cells in each mouse 5 × 10 ⁵ cells / 200 μl PBS were injected intravenously. On the 19th day, the weight of the mouse, the lungs were removed separately, and the weight of the lungs and the number of nodules formed in the lungs were counted (see FIG. 14). To determine the weight of the lungs and the number of nodules, the mouse was killed, the lungs were removed separately, and the lungs were taken on a 35 mm dish. Counted. Marked "dead" in Figure 14 is the lung of a mouse that has already died before confirmation. Regardless of the size of the humps, they were counted, and when the humps were divided, they were counted as different humps. In addition, blood was drawn from the eyes to obtain serum.

Specifically, using the Graphpad prism 5 program, a survival curve for the aforementioned experimental results was drawn for each group (see FIG. 13). On day 17 of injection of 4T1 cancer cells, 4 of 9 mice in the Adj group died and 1 of 10 mice in the Adj + Epi-2LDL group died. On day 18, one more died in the Adj + Epi-2LDL group and one of eight mice in the Adj + Epi-4LDL group died. All nine of the nine mice in the Adj + EpiSP group survived by the 19th day of confirming metastasis. This is summarized in Table 1 below.

On the 19th day after injection of 4T1 cancer cells, the survival rate for each group was 55.6% in Adj, 80% in Adj + Epi-2LDL, 87.5% in Adj + Epi4LDL, and 100% in Adj + EpiSP. The survival rate was the highest. In addition, using the Graphpad prism 5 program, the lung weight and nodule number of the Adj, Adj + Epi-2LDL, Adj + Epi-4LDL, and Adj + EpiSP groups were graphed (FIG. 15). 15 is shown using the mean value and the standard error. Lung weight was the heaviest in the Adj group, followed by Adj + Epi-2LDL, Adj + Epi-4LDL, and Adj + EpiSP. The number of nodules was highest in Adj group, followed by Adj + Epi-2LDL group, Adj + Epi-4LDL group, and Adj + EpiSP group. Percentages were obtained by dividing the entire individual by the average value based on the average value of the number of nodules in the Adj group, and again, a graph was drawn for each group by using the Prism 5 program (FIG. 16). 16 is shown using the mean value and the standard error. The number of nodules decreased by 14% in the Adj + 2LDL group, 20% in the Adj + 4LDL group and 42% in the Adj + SP group compared to the Adj group. Therefore, when immunized with the oligopeptide having the amino acid sequence of SEQ ID NO: 1 it can be seen that the most excellent cancer metastasis inhibitory effect.

Example  4-2: Vaccine efficacy test through secondary animal experiment

It was confirmed by the test in Example 4-1 that the antigen oligopeptide of the present invention effectively suppresses cancer metastasis in mice. The test was repeated using 20 mice in each group, increasing the number of mice. A total of four groups (Adj, Adj + 2LDL, Adj + 4LDL, Adj + SP) consisting of 20 mouse Balb / c per group, respectively, were used as immune enhancers (Adjuvant), "Immune Enhancer + Epi-2LDL", "Immune Enhancers" + Epi-4LDL "and" Immune Enhancer + Epi-SP "were injected three times every three weeks. At the first injection, 50 μg oligopeptide and CFA were mixed (50 μg oligopeptide in 100 μl PBS solution plus 100 μl CFA) intraperitoneally injected into each mouse, and from the second injection 20 μg oligopeptide was mixed with IFA (second injection). 20 μg oligo peptide + 100 μl IFA in 100 μl PBS solution; 20 μg oligo peptide + 50 μl IFA in 50 μl PBS solution at each injection intraperitoneally to each mouse (volume ratio of immunopotentiator and oligo peptide 1: 1 adjustment). On the third day after the third injection, 1 × 10 ⁵ cells / 200 μl PBS of 4T1 cancer cells were injected intravenously into each mouse. On the 28th day after the injection of the cancer cells, the mice were weighed and the lungs were stained black by adding 1 ml of black ink to the trachea. The lungs were stained with black ink in the trachea, and each lung was removed and photographed in a dish. Staining made clear the distinction between lungs and nodules, and counted the white nodules as nodules. 17 is a photograph of the surface of the lung in which nodules occur in each group. On the 28th day after the cancer cell injection, the number of nodules was highest in the Adj group, followed by the Adj + 4LDL group, the Adj + 2LDL group, and the Adj + SP group.

Using the Graphpad prism 5 program, the number of nodules in the Adj group, Adj + 2LDL group, Adj + 4LDL group, and Adj + SP group was graphed. In addition, based on the average value of the number of nodules in the Adj group, the total individual was divided by the average value to obtain a percentage, and again, a graph was drawn for each group by using the Prism 5 program (FIG. 18). 18 is shown using the mean value and the standard error. The number of nodules decreased by 58% in Adj + 4LDL group, 62% in Adj + 2LDL group and 70% in Adj + SP group compared to Adj group. Therefore, the results of experiments using 20 mice per group were also almost identical to those of Example 4-1, and it was confirmed once again that the antigen oligopeptide of the present invention can be used as an effective vaccine. there was.

Example  4-3: Vaccine efficacy test through tertiary animal experiment

Oligopeptide (Epi-SP) having an amino acid sequence of SEQ ID NO: 1 and a control thereof to confirm whether the experimental results of Example 4-1 and Example 4-2 are due to the immunity of the antigen oligopeptide of the present invention An scrambled oligo peptide Epi-SP-Sc (EQGKGARDWPEWAVQGVNT; SEQ ID NO: 4) was made and the effects were compared. In order to test whether the cancer metastasis suppression model can be applied not only to mice but also to humans, immunization was performed using the injection alum, an adjuvant that can be used in both mice and humans. In addition, for comparison with cancer cell lines in which epitin expression was knocked down, 4T1 Con-D5 cell line expressing epitin and cancer cell line 4T1 EpiKD-B3 in which epitin expression was knocked down were used. A total of 11 groups of 5 Balb / c mice per group (Adj, Adj + SP, Adj + SP + 4T1Con-D5, Adj + SP + 4T1EpiKD-B3, Adj + Sc + 4T1Con-D5, Adj + Sc + 4T1EpiKD-B3, Adj + 4T1Con-D5, Adj + 4T1EpiKD-B3, 4T1Con-D5, 4T1EpiKD-B3, None) 8 groups (Adj, Adj + SP, Adj + SP + 4T1Con-D5, Adj + SP + 4T1EpiKD -B3, Adj + Sc + 4T1Con-D5, Adj + Sc + 4T1EpiKD-B3, Adj + 4T1Con-D5, Adj + 4T1EpiKD-B3) as an immune enhancer (Adjuvant), "immune enhancer + Epi-SP" or "immune enhancer A total of three injections of + Epi-SP-sc "were administered once every three weeks. 50 μg oligopeptide plus 50 μg oligopeptide in the first injection and 20 μg oligopeptide in the injection alum with the first injection (50 μg oligopeptide + 50 in 50 μl PBS solution) Μl injection alum; second injection 20 μg oligo peptide + 50 μl injection alum in 50 μl PBS solution Each mouse was injected intraperitoneally (volume ratio of immunopotentiator and oligo peptide was adjusted to 1: 1). On the third day after the third injection, 5 × 10 ⁵ cells / 200 μl PBS of 4T1 cancer cells (4T1 Con-D5 or 4T1 EpiKD-B3) were intravenously injected into each mouse. On the 17th day after the cancer cell injection, the mice were weighed, 1 ml of black ink was added to the trachea to stain the lungs, and each lung was detached and taken in a dish. The photograph of FIG. 19 is a photograph of the surface of the lung with nodules in each group. Staining made clear the distinction between lungs and nodules, and counted the white nodules as nodules. On day 17 after cancer injection, the nodule number of mice immunized with immune enhancer + Epi-SP and injected with 4T1 con-D5 was immunized with immune enhancer + Epi-SP-Sc and injected with 4T1 con-D5. It was less than the number of nodules and less than that of mice injected with 4T1 con-D5 with or without immunization. In addition, the number of nodules in mice immunized with immune enhancer + Epi-SP and injected with 4T1 con-D5 was similar to the number of nodules in mice injected with 4T1 EpiKD-B3 knocked down.

Graphpad prism 5 program was used to graph the number of nodules for each mouse group. 20 is shown using the mean value and the standard error. Nodules of mice immunized with Epi-SP were found in the Adj + SP + 4T1 group except for those that significantly exceeded the standard error of the mean value. The lowest number was found, and much more nodules were counted in the group of mice immunized with Epi-SP-Sc, mice immunized only with an adjuvant, and mice not immunized. Similar numbers of nodules were counted in the group of mice injected with 4T1 EpiKD-B3 knocked down with epitin expression, similar to the number of nodules in the Adj + SP + 4T1 group. In addition, the average value of the number of nodules in the Adj + 4T1 group was used as a reference to calculate the percentage by dividing the entire individual by the average value, and again, a graph was drawn for each group using the Prism 5 program (FIG. 21). 21 is shown using the mean value and the standard error. The number of nodules in the Adj + SP + 4T1 group was 74% lower than that in the Adj + 4T1 group, which was similar to that of other groups of mice injected with 4T1 EpiKD-B3 knocked down.

In the case of using Alum, which is an immunoadjuvant used in humans, the results were similar to those using CFA and IFA of Examples 4-1 and 4-2, and it was confirmed that the present experiment could be applied to humans. Epi-SP antigen oligopeptide of the present invention and epi-SP-sc oligopeptide as a control group were immunized with mice and cancer cells were injected to compare the number of nodules metastasized to the lungs. Only the peptides had a reduced number of metastasized nodules. This shows that the Epi-SP antigen oligopeptides of the present invention can specifically inhibit cancer metastasis. In addition, the formation of nodules occurred less in the group of mice injected with 4T1 EpiKD-B3, a cancer cell line in which epitin was knocked down, than in the group injected with 4T1 con-D5. The number of nodules in the mouse group injected with 4T1 EpiKD-B3 was similar to that of the mouse group injected with 4T1 con-D5 after immunization with the Epi-SP antigen oligopeptide of the present invention. In inhibiting the nodules metastasized to the lung after cancer cell injection, the method of immunizing with the Epi-SP antigen oligopeptide of the present invention and inhibiting the activity of epitin is similar to the injection of 4T1 EpiKD-B3 knocked down the epitin expression. It can be seen that the effect can be obtained.

Meanwhile, cell lysates of MCF7 cells, which are human breast cancer cell lines, were electrophoresed on a 10% gel and then immunized with anti-metlipase antibody (H-270, Santacruz) as a primary antibody and Adj + SP as described above. Serum 1-4 from the treated mice and 5 serum from mice subjected to post-immune cancer metastasis experiments were used. The H-270 antibody was used as a control, indicated by the arrow is a band indicating the full size of the antigen. That is, as shown in Figure 22, when Western blotting with serum obtained from the immunized treated mice, the band at the same size as in the H-270 treatment that confirms the human metlipase upon electrophoresis Indicated. Eventually, the antibody to the Epi-SP antigen oligopeptide of the present invention can bind to metlipase (or epitin) in MCF7 by showing a band at the same size as the band of H-270 for MCF7. It is known that anti-Epi-SP antigen oligopeptide antibodies formed in mice can also bind to metlipase (or epitin) in human cells, affecting the metastasis of cancer cells and inhibiting cancer progression and metastasis. Show that it is.

Example  5: cell migration inhibition test

In vitro to confirm the effect of the antibody on the movement of cells among the functional effects of the antibody produced through the process of Example 4-3 ( in vitro experiments were performed. The cells used 427.1.86 cells of the mouse expressing epitin. 427.1.86 Cells were densely cultured, and then the blue layer was used to cut the cell layers, and the cloned antibodies were added at a 3: 2 ratio with the culture media. Then 20 hours of incubation and the movement of the cells were observed under a microscope. In the case of a which was not inserted into the antibody, cell migration was observed, whereas in the case of the addition of four antibodies (c to f) to the antigen oligopeptide of the present invention obtained through the procedure of Example 4-3, the cells were added. It can be confirmed that the movement of is suppressed (see FIG. 23). b is the result of using a negative clone antibody which is considered to be ineffective, and g is the result of using serum from an immunized mouse to obtain hybridoma. From these results, it can be seen that the monoclonal antibody obtained through the process of Example 4-3 inhibits the migration of epithelial cancer cells itself in an in vitro experiment.

Next, the same experiment was conducted on endothelial cells, which are important for blood formation and metastasis of cancer. The cells used MS1 and proceeded in the same manner as above. In addition, CM was added by adding a conditioned medium (CM) in which 427.1.86 cells overexpressing epitin were added in consideration of the experimental purpose of confirming whether cell migration by epitin occurs and whether the antibody of the present invention inhibits it. Water-soluble epitin contained in was added and the migration of endothelial cells observed at this time was observed. As shown in FIG. 24, a shows that MS1 cells alone did not migrate when both antibody and 427.1.86 CM were not added, and b added 427.1.86 CM to cells with water-soluble epitin. Show that the movement was induced. d to g show that the migration of cells is suppressed when the four antibodies to the antigen oligopeptide of the present invention are added, c is a negative clone antibody, and h is a hybridoma (hybridoma) to obtain Results using serum from immunized mice are shown. Referring to Figure 24, the endothelial cell in the endothelial cell migration occurs by the soluble epitin, it can be confirmed that the movement of the cell is inhibited by the antibody to the antigen oligopeptide of the present invention.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Antigen peptide inhibiting cancer metastasis, cancer metastasis          vaccine and composition for inhibiting and treating cancer          metastasis comprising the same, and method for screening target          material related to cancer metastasis and cancer metastasis          inhibitor <130> OP-2010-0056 <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 1 (SP cancer metastasis vaccine; mouse          origin) <400> 1 Lys Gln Ala Arg Val Val Gly Gly Thr Asn Ala Asp Glu Gly Glu Trp   1 5 10 15 Pro Trp Gln             <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 2 (2LDL cancer metastasis vaccine; mouse          origin) <400> 2 Cys Gly Asp Gly Ser Asp Glu Glu Gly   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> Antigen oligopeptide 3 (4LDL cancer metastasis vaccine; mouse          origin) <400> 3 Cys Ser Asp Gly Ser Asp Glu Lys Asn   1 5 <210> 4 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Scrambled control <400> 4 Glu Gln Gly Lys Gly Ala Arg Asp Trp Pro Glu Trp Ala Val Gln Gly   1 5 10 15 Val Asn Thr             <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pSUPER-epi construct forward strand <400> 5 gatccccggt gcgcttcaaa ctcttcttca agagagaaga gtttgaagcg caccttttta 60                                                                           60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> pSUPER-epi construct reverse strand <400> 6 tcgataaaaa ggtgcgcttc aaactcttct ctcttgaaga agagtttgaa gcgcaccggg 60                                                                           60 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 4 (SP cancer metastasis vaccine; human          origin) <400> 7 Arg Gln Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp   1 5 10 15 Pro Trp Gln             <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 5 (2LDL cancer metastasis vaccine; human          origin) <400> 8 Cys Gly Asp Asn Ser Asp Glu Gln Gly   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 6 (4LDL cancer metastasis vaccine; human          origin) <400> 9 Cys Ser Asp Gly Ser Asp Glu Lys Asp   1 5 <210> 10 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Antigen oligopeptide 7 (SP cancer metastasis vaccine; rabbit          origin) <400> 10 Arg Gln Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp   1 5 10 15 Pro Trp Gln             <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> Antigen oligopeptide 8 (SP cancer metastasis vaccine; rat origin) <400> 11 Lys Gln Ala Arg Val Val Gly Gly Thr Asn Ala Asp Glu Gly Glu Trp   1 5 10 15 Pro Trp Gln            

Claims (14)

Oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), oligopeptide having the amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN), amino acid sequence of the SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ) Oligopeptide having an amino acid sequence of SEQ ID NO: 8, oligopeptide having an amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), oligopeptide having an amino acid sequence of SEQ ID NO: 10 (RQARVVGGTDADEGEWPWQ), and SEQ ID NO: Antigenic peptides that inhibit cancer metastasis, characterized in that at least one oligopeptide selected from the group consisting of oligopeptides having an amino acid sequence of 11 (KQARVVGGTNADEGEWPWQ). Oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), oligopeptide having the amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN), amino acid sequence of the SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ) Oligopeptide having an amino acid sequence of SEQ ID NO: 8, oligopeptide having an amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), oligopeptide having an amino acid sequence of SEQ ID NO: 10 (RQARVVGGTDADEGEWPWQ), and SEQ ID NO: Cancer metastasis vaccine comprising at least one oligopeptide selected from the group consisting of oligopeptides having an amino acid sequence of 11 (KQARVVGGTNADEGEWPWQ). The method of claim 2,
Cancer metastasis vaccine further comprising an adjuvant or an immune enhancer. The method according to claim 2 or 3,
Cancer metastasis vaccine further comprising at least one substance selected from the group consisting of aluminum sulfate, aluminum hydroxide, magnesium hydroxide, aluminum phosphate, phosphorus lecithin, Pluronic polyol, polyanion and oily emulsion . Oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), oligopeptide having the amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN), amino acid sequence of the SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ) Oligopeptide having an amino acid sequence of SEQ ID NO: 8, oligopeptide having an amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), oligopeptide having an amino acid sequence of SEQ ID NO: 10 (RQARVVGGTDADEGEWPWQ), and SEQ ID NO: A cancer metastasis vaccine comprising DNA encoding at least one oligopeptide selected from the group consisting of oligopeptides having an amino acid sequence of 11 (KQARVVGGTNADEGEWPWQ). Oligopeptide having the amino acid sequence of SEQ ID NO: 1 (KQARVVGGTNADEGEWPWQ), oligopeptide having the amino acid sequence of SEQ ID NO: 2 (CGDGSDEEG), oligopeptide having the amino acid sequence of SEQ ID NO: 3 (CSDGSDEKN), amino acid sequence of the SEQ ID NO: 7 (RQARVVGGTDADEGEWPWQ) Oligopeptide having an amino acid sequence of SEQ ID NO: 8, oligopeptide having an amino acid sequence of SEQ ID NO: 9 (CSDGSDEKD), oligopeptide having an amino acid sequence of SEQ ID NO: 10 (RQARVVGGTDADEGEWPWQ), and SEQ ID NO: A composition for inhibiting and treating cancer metastasis comprising an antibody induced by an immune response against at least one antigenic peptide selected from the group consisting of oligopeptides having an amino acid sequence of 11 (KQARVVGGTNADEGEWPWQ) or serum containing such an antibody. (a) culturing endothelial cells to prepare an endothelial cell layer, (b) culturing epithelial expressing cells and preparing (c) epithelial expressing cells on the endothelial cell layer (D) counting the number of cells moved through the endothelial cell layer to determine the cancer metastasis reference value, and (e) adding a cancer metastasis inhibiting candidate material to the above (a) to ( c) counting the number of cells moved through the endothelial cell layer after performing step c) and comparing the cancer metastasis reference value obtained in step (d). The method of claim 7, wherein
The endothelial cell layer is a screening method of cancer metastasis inhibitor, characterized in that the MS1 cell layer of mouse endothelial cells (Mouse endothelial cells). 9. The method according to claim 7 or 8,
The cells added to the endothelial cell layer is 427.1.86 cells which are thymic epithelial cell cancer cells or 4T1 cells which are breast cancer cells. 9. The method according to claim 7 or 8,
The method of screening a cancer metastasis suppressor, characterized in that the cells added to the endothelial cell layer is labeled with a label. (a) culturing endothelial cells to prepare an endothelial cell layer, (b) culturing and preparing cells expressing a cancer metastasis related candidate target material, and (c) expressing the cancer metastasis related candidate target material Adding cells to the endothelial cell layer, (d) counting the number of cells that have migrated through the endothelial cell layer, and determining cancer metastasis reference values; Preparing a cell having reduced expression or activity and adding the same to the endothelial cell layer, counting the number of cells that have migrated through the endothelial cell layer, and comparing the cancer metastasis reference value obtained in step (d). Screening method for cancer metastasis related target material. 12. The method of claim 11,
The endothelial cell layer is a method for screening cancer metastasis-related target material, characterized in that the MS1 cell layer of mouse endothelial cells (Mouse endothelial cells). 13. The method according to claim 11 or 12,
The cells added to the endothelial cell layer is 427.1.86 cells which are thymic epithelial cell cancer cells or 4T1 cells which are breast cancer cells. 13. The method according to claim 11 or 12,
The method of screening cancer metastasis-related target material, characterized in that the cells added to the endothelial cell layer is labeled with a label.

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