JP2009168669A - Composition and method for diagnosing or detecting stomach cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition useful for diagnosing stomach cancer, and a detection method of stomach cancer. <P>SOLUTION: The method for detecting stomach cancer includes measurement of one or a plurality of polypeptides shown by the sequence number 1-17 in a biosample originated in a subject, variants therefrom or fragments thereof. The composition for diagnosing or detecting stomach cancer is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a composition useful for diagnosis or detection of gastric cancer.

  The present invention also relates to a method for detecting gastric cancer using the composition.

  The stomach is an important digestive organ that stores food and drink for several hours, acidifies the food and drink with the gastric acid secreted during that time to prevent spoilage, and digests food and drink with digestive enzymes.

  In Japan, the incidence of gastric cancer is about 50-60 per 100,000 population, and the male-female ratio is 1-2: 1, which tends to be more common among men. The number of deaths was about 50,000 per year, about 17% of all cancers, and the number of cancer deaths by region was the highest for a long time after World War II. Currently, the number of patients is declining year by year and is second only to lung cancer, but it remains a disease with a large number of patients. Stomach cancer is a disease with a large number of patients in Asia, such as Japan, Korea, and China, and South America. The risk factors for gastric cancer generally include smoking, high salt diet, and Helicobacter pylori infection.

  Treatment for gastric cancer includes endoscopic treatment, surgery, chemotherapy, and radiation therapy, taking into account the stage, size and depth of tumor, and degree of metastasis. In determining the treatment policy, a decision is made based on the “Gastrocancer Treatment Guidelines” created by the Japanese Gastric Cancer Society in 2004. Early gastric cancer can be completely removed by endoscopic resection or surgery, and the recurrence rate is very low. In the case of advanced gastric cancer, even if the lesion is completely removed, there are small metastases that were not known at the time of surgery, and there are many cases of recurrence.

  Thus, when gastric cancer is detected at a relatively early stage, the prognosis is relatively good, and 90% or more is cured by treatment with early stage cancer. However, the results of treatment for large tumors and tumors with metastases are inferior (the overall 5-year survival rate for gastric cancer is about 70%). Is recognized.

  However, it is difficult to detect gastric cancer early due to subjective symptoms. Most gastric cancers are asymptomatic at an early stage, and only after the cancer has progressed, often no obvious subjective symptoms appear. As subjective symptoms, soft stool, black stool, nausea, stomach discomfort, etc. are observed as gastric cancer progresses, and systemic symptoms include easy fatigue, fever, weight loss, anemia and the like. As the tumor progresses, the abdomen becomes lumped as the tumor grows. However, even if a subjective symptom exists, the patient tends to leave it alone, and is often found in a state that has already progressed to some extent by X-ray imaging at the time of medical examination.

  Examples of examination methods for gastric cancer include ultrasonic examination, CT examination, angiography examination, and X-ray imaging. Although diagnostic imaging is a useful method for detecting early small gastric cancer, it is not efficient when targeting a large number of subjects such as health examinations, and the cost required for diagnosis is relatively low. high.

  For these reasons, the discovery of blood markers that are specific and highly sensitive to gastric cancer is strongly desired. By using a blood marker, it is considered that a relatively inexpensive and high-throughput examination / diagnosis becomes possible. Known tumor markers currently used in clinical practice include CEA, BFP, NCC-ST-439, CA72-4, CA19-9 and the like. However, with these markers, cancer cannot be determined with high specificity and sensitivity, and it is used only for limited purposes such as follow-up after treatment.

  With recent advances in genome analysis or proteome analysis, various new tumor marker candidates are being discovered as a result of research in the cancer area. (For example, Patent Document 1 and Patent Document 2). In addition, in the organization, marker candidates such as pepsinogen C (Non-patent document 1), hnRNP A2 / B1 (Non-patent document 2), NSP3, transgelin, prohibitin, HSP27, protein disilicide isomerase A3, GRP58 (Non-patent document 3) have been discovered. Has been.

International Publication WO2005 / 001126 International Publication WO2003 / 060121 Melle, C.I. Journal of protein research, 2005, Vol. 5, p. 1799-1804 Lee, C.I. Proteomics, 2005, Vol. 5, p. 1160-1166 Ryu, J .; W. Et al., Journal Korean Medical Science, 2003, Vol. 18, p. 505-509

  However, since the above existing markers and marker candidates have poor specificity and / or sensitivity and their efficient detection methods from biological samples have not been established, they are generally not clinically used. Therefore, a marker for gastric cancer with higher specificity and sensitivity is desired.

  An object of the present invention is to provide a composition useful for diagnosis of gastric cancer and a method for detecting gastric cancer using the composition.

  Marker search methods include methods that compare gene expression and protein expression in gastric cancer cells and non-cancer cells, or metabolites of cells by some means, and genes contained in body fluids of gastric cancer patients and non-cancer patients. , Methods for measuring the amount of proteins, metabolites and the like.

  In order to solve the above-mentioned problems, the present inventors, for the plasma of gastric cancer patients and healthy persons, are specifically detected only in gastric cancer patients or proteins that are specifically detected only in healthy persons A marker group was found.

The present invention has the following features.
(1) A method for detecting gastric cancer in vitro, comprising measuring any one or more of the polypeptides represented by SEQ ID NOs: 1 to 17, variants thereof or fragments thereof in a biological sample derived from a subject. .
(2) The fragment is a polypeptide fragment comprising the amino acid sequence represented by any one of SEQ ID NOs: 18 to 39 in the amino acid sequence of the polypeptide represented by any one of SEQ ID NOs: 1 to 17, The method according to 1).
(3) The method according to (1) or (2), wherein the amount of the polypeptide, a variant or a fragment thereof or the presence thereof is measured.
(4) The amount of the polypeptide, its variant or fragment thereof is significantly increased or significantly decreased as compared with that of the control sample, (1) to (3) ) Any one of the methods.
(5) The method according to any one of (1) to (4), wherein the measurement of the polypeptide, a variant thereof or a fragment thereof is performed by an immunological method.
(6) The method according to any one of (1) to (5), wherein the measurement is performed using a substance that can bind to the polypeptide, a variant thereof, or a fragment thereof.
(7) The method according to (6), wherein the substance capable of binding is an antibody or a fragment thereof.
(8) The method according to (7), wherein the antibody is labeled.
(9) The amount of one or more of the polypeptide, variant or fragment thereof in the sample using an antibody or fragment thereof that specifically binds to the polypeptide, variant or fragment thereof Alternatively, the presence of the polypeptide, a variant thereof or a fragment thereof is measured by immunological determination, and the amount of the polypeptide, a variant or a fragment thereof is increased or decreased compared to that of a control sample, or the polypeptide, a variant thereof The method according to any one of (1) to (8), comprising detecting gastric cancer using as an index that the body or a fragment thereof is present only in one of the sample and the control sample.
(10) The method according to any one of (1) to (9), wherein the sample is blood, plasma, serum or urine.
(11) The method according to any one of (1) to (9), wherein the sample is a stomach-derived tissue or cell.
(12) The method according to any one of (7) to (9), wherein the antibody is a monoclonal antibody or a polyclonal antibody.
(13) including one or more of an antibody or a fragment thereof, or a chemically modified derivative thereof, which specifically binds to at least one of the polypeptide represented by SEQ ID NOs: 1 to 17, a variant thereof or a fragment thereof A composition for diagnosis or detection of gastric cancer.
(14) The polypeptide fragment is a polypeptide fragment comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39 in the amino acid sequence of the polypeptide represented by any of SEQ ID NOs: 1 to 17, respectively. The composition according to (13).
(15) The composition according to (13) or (14), wherein the polypeptide fragment comprises an epitope consisting of at least 7 amino acids.
(16) The composition according to any one of (13) to (15), which is in the form of a kit.
(17) A method of using the composition according to any one of (13) to (16) for in vitro detection of gastric cancer in a subject.

  The terms used herein have the following definitions:

  As used herein, “chemically modified derivative” means a derivative labeled with an enzyme, a fluorophore, a radioisotope, or the like, or a derivative containing a chemical modification such as acetylation, glycosylation, phosphorylation, or sulfation.

  As used herein, “a composition for diagnosis or detection” refers to the presence or absence of gastric cancer, the degree of morbidity, the presence or absence of improvement, and the degree of improvement, or the prevention, improvement or treatment of gastric cancer. It can be used directly or indirectly to screen candidate substances useful for.

  In this specification, a “biological sample” to be detected / diagnosed is a sample collected from a living body containing or suspected of containing a target polypeptide whose expression level increases or decreases as gastric cancer develops. Say.

  In the present specification, “specifically binds” means that an antibody or a fragment thereof forms an antigen-antibody complex only with a target polypeptide, a variant or a fragment thereof, which is a gastric cancer marker in the present invention. Peptidic or polypeptide substance means that the complex is not substantially formed. Here, “substantial” means that non-specific complex formation can occur to a lesser extent.

  As used herein, “epitope” refers to a partial amino acid region (antigenic determinant) having antigenicity or immunogenicity in the target polypeptide of the present invention, a variant thereof or a fragment thereof. An epitope usually consists of at least 5 amino acids, preferably at least 7 amino acids or at least 8 amino acids, more preferably at least 10 amino acids.

  The gastric cancer marker in the present invention is found in a biological sample such as blood of a stomach cancer patient, but is hardly or not found in that of a healthy person, or conversely, it is found only in a biological sample of a healthy person. Since it is hardly or not found in patients, it has a special effect that gastric cancer can be easily detected, for example, using blood, simply by using the presence or amount of the marker as an index.

The present invention will be described more specifically below.
<Gastric cancer marker>
A gastric cancer marker for detecting gastric cancer in vitro using the composition for diagnosis or detection of gastric cancer of the present invention is a polypeptide represented by SEQ ID NOs: 1 to 17, a variant thereof or a fragment thereof.

  Polypeptides of SEQ ID NOs: 1 to 17 of the present invention are shown in Table 1 below together with their gene names (GenBank registered names) and protein numbers (Swissprot registered numbers), and their characteristics. Among these polypeptides, the polypeptides represented by SEQ ID NOs: 1 to 4 are specifically detected in the plasma of gastric cancer patients and are not detected in the plasma of healthy subjects or detected in comparison with the plasma of gastric cancer patients. The amount was significantly decreased. In addition, the polypeptides represented by SEQ ID NOs: 5 to 17 are specifically detected in healthy human plasma and are not detected in the plasma of gastric cancer patients, or the amount detected is significantly higher than that of healthy human plasma. It was decreasing. The amino acid sequence or nucleotide (or base) sequence of these polypeptides and / or genes (cDNA) can be obtained by accessing a data bank such as NCBI, GenBank, Swissprot or the like.

  In the present invention, the polypeptide fragment comprises at least 7, at least 8, at least 10, at least 15, preferably at least 20, at least 25, more preferably at least 30 of the amino acid sequence of the polypeptide. Consisting of at least 40, at least 50, at least 100, at least 200 consecutive amino acid residues, and retains one or more epitopes. Such a fragment is capable of immunospecifically binding to the antibody of the present invention or a fragment thereof. For example, when the polypeptide is present in blood, it is assumed that the polypeptide is present by being cleaved and fragmented by an enzyme such as protease or peptidase.

  Moreover, as shown in Example 3 below, in the present invention, the polypeptides represented by SEQ ID NOs: 18 to 20 were detected at a particularly high level in the biological samples of gastric cancer patients as compared with healthy individuals. In contrast, the polypeptides represented by SEQ ID NOs: 21 to 39 were detected at a particularly high level in the biological samples of healthy individuals as compared with gastric cancer patients. These polypeptides are fragments of the polypeptides represented by SEQ ID NOs: 1, 2, 5-9, and 11-16. Therefore, these polypeptides represented by SEQ ID NOs: 18-39 and The fragment possessed by is more preferably used as a gastric cancer marker. In Table 2 below, the polypeptides of SEQ ID NOs: 18 to 39 are shown together with the corresponding portions of the polypeptides represented by SEQ ID NOs: 1, 2, 5-9, and 11-16.

  The amino acid sequences of SEQ ID NOs: 23 to 25 and the amino acid sequences of SEQ ID NOs: 33 and 34 appear almost or completely continuously in the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 11, respectively. is doing. Therefore, the sequence in which the amino acid sequences of SEQ ID NOs: 23 to 25 are linked, that is, the sequence from the 328th threonine to the 363rd arginine in SEQ ID NO: 7 and the amino acid sequence represented by SEQ ID NOs: 33 and 34 are linked. The polypeptides represented by each of the above sequences, that is, the sequence from the 5th alanine to the 30th lysine of SEQ ID NO: 11, and fragments having them therein are more preferably used as gastric cancer markers.

  In the present invention, any of the above target polypeptides for detection of gastric cancer is significantly or exceptionally significant in gastric cancer patients, in which the level of the polypeptide in a biological sample such as blood is significantly or significantly higher in subjects suffering from gastric cancer than in healthy individuals. Characterized by high or low.

  The polypeptide in the present invention can be produced, for example, by a chemical synthesis method such as solid phase synthesis or a DNA recombination technique, which is a technique commonly used in the art. The use of DNA recombination technology is preferred from the viewpoint of procedure and ease of purification.

  First, a polynucleotide sequence encoding a partial sequence of a polypeptide in the present invention is chemically synthesized using an automatic DNA synthesizer. In general, the phosphoramidite method is used for this synthesis, and single-stranded DNA of up to about 100 bases can be automatically synthesized by this method. Automatic DNA synthesizers are commercially available from, for example, Polygen and ABI.

  Using the obtained polynucleotide as a probe or primer, total RNA extracted from living tissue such as stomach tissue in which the target gene is expressed by a known cDNA cloning, specifically, using an oligo dT cellulose column. A cDNA library is prepared from the poly A (+) RNA obtained by the treatment by RT-PCR, and the target cDNA clone is obtained from this library by screening such as hybridization screening, expression screening, and antibody screening. If necessary, the cDNA clone can be further amplified by PCR. As a result, cDNA corresponding to the target gene can be obtained.

  The probe or primer can be selected from contiguous sequences of 15 to 100 bases based on the polypeptide sequences shown in SEQ ID NOs: 1 to 17 and synthesized as described above. The cDNA cloning technique is described in, for example, Sambrook, J. et al. And Russel, D .; Written by Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, published on January 15, 2001, Volumes 7.42-7.45, Volumes 8.9-8.17. .

  Next, the cDNA clone obtained as described above is incorporated into an expression vector, and a prokaryotic or eukaryotic host cell transformed or transfected with the vector is cultured to obtain the target poly- gen from the cell or culture supernatant. Peptides can be obtained. At this time, the mature polypeptide can be secreted out of the cell by flanking the nucleotide sequence encoding the secretory signal sequence at the 5 'end of the DNA encoding the target mature polypeptide.

Vectors and expression systems are available from Novagen, Takara Shuzo, Daiichi Chemicals, Qiagen, Stratagene, Promega, Roche Diagnostics, Invitrogen, Genetics Institute, Amersham Bioscience, and the like. Examples of host cells include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis), yeast (eg, Saccharomyces cerevisiae), insect cells (eg, Sf cells), mammalian cells (eg, COS, CHO, BHK, NIH3T3, etc.) Can be used. Vectors, in addition to the DNA encoding the polypeptide, regulatory elements such as promoters (e.g. lac promoter, trp promoter, _{P L} promoter, _{P R} promoter, SV40 viral promoter, 3-phosphoglycerate kinase promoter, glycolytic System enzyme promoters), enhancers, polyadenylation signals and ribosome binding sites, replication origins, terminators, selectable markers (for example, drug resistance genes such as ampicillin resistance gene and tetracycline resistance gene; auxotrophic markers such as LEU2 and URA3) Etc. can be included.

  In order to facilitate the purification of the polypeptide, an expression product can be produced in the form of a fusion polypeptide in which a labeled peptide is bound to the C-terminus or N-terminus of the polypeptide. Representative labeled peptides include 6-10 residue histidine repeats (His tag), FLAG, myc peptide, GST polypeptide, GFP polypeptide, etc., but the labeled peptide is not limited to these.

  When a polypeptide according to the present invention is produced without a labeled peptide, the polypeptide can be purified and isolated by means well known to those skilled in the art using its physical or chemical properties. . For example, ion exchange chromatography, gel filtration chromatography, normal phase chromatography, reverse phase chromatography, hydrophobic interaction chromatography, isoelectric point chromatography, high performance liquid chromatography (HPLC), electrophoresis, ammonium sulfate. Examples thereof include a method of combining one or a plurality of methods such as fractionation, salting out, salt solution, ultrafiltration, and dialysis. Furthermore, when a labeled peptide such as histidine repeat, FLAG, myc, GST, or GFP is attached to the polypeptide, a method by affinity chromatography suitable for each generally used labeled peptide can be mentioned. In this case, an expression vector that facilitates isolation and purification may be constructed. In particular, if an expression vector is constructed so that it is expressed in the form of a fusion polypeptide of a polypeptide and a labeled peptide, and the polypeptide is genetically engineered, isolation and purification are easy.

  The polypeptide in the present invention includes a variant thereof. “Mutant” means a variant comprising a deletion, substitution, addition or insertion of one or more, preferably 1 or several amino acids in the amino acid sequence represented by SEQ ID NOs: 1 to 17 or a partial sequence thereof, or About 80% or more, about 85% or more, preferably about 90% or more, more preferably about 95% or more, about 97% or more, about 98% or more, about 99% or more of the amino acid sequence or a partial sequence thereof It means a mutant exhibiting sex. Such mutants include, for example, natural mutants such as homologues of mammal species different from humans, and variants based on polymorphic mutations between the same species of mammals (eg, race).

  Here, “several” refers to an integer of about 10, 9, 8, 7, 6, 5, 4, 3 or 2.

  Further, “% identity” can be determined using the above-mentioned BLAST or FASTA protein or nucleic acid homology search program with or without introducing a gap (Karlin, S. et al., 1993, Proceedings of the National Academic Sciences USA, Vol. 90, p. 5873-5877; Altschul, SF et al., 1990, Journal of Molecular Biology, vol. 410; Pearson, WR et al., 1988, Proceedings of the National Academic Sciences USA, Vol. 85, pp. 2444-2448; Nomunetto database usage of the (second edition), 1998, Kyoritsu Shuppan Co., Ltd.).

<Composition for diagnosis or detection of gastric cancer>
The present invention includes one or more of an antibody or a fragment thereof, or a chemically modified derivative thereof, which specifically binds to the polypeptide represented by SEQ ID NOs: 1 to 17, a variant thereof or a fragment thereof. A composition for diagnosis or detection of gastric cancer is provided.

  More preferably, the composition for diagnosis or detection of gastric cancer in the present invention is a fragment of the polypeptide represented by SEQ ID NOs: 1 to 17, and is represented by any one of SEQ ID NOs: 18 to 39, respectively. A composition comprising one or more of an antibody or fragment thereof, or a chemically modified derivative thereof capable of specifically binding to a polypeptide fragment comprising an amino acid sequence.

  An antibody that recognizes a polypeptide that is a gastric cancer marker is capable of specifically binding to the polypeptide via the antigen-binding site of the antibody. The antibody that can be used in the present invention is prepared by a conventional technique using a polypeptide having the amino acid sequence of SEQ ID NOs: 1 to 17, a variant or fragment thereof, or a fusion polypeptide thereof as one or a plurality of immunogens. can do. These polypeptides, fragments, variants or fusion polypeptides used as immunogens are at least 7, at least 8, at least 10, at least in the amino acid sequence of each polypeptide represented by SEQ ID NOs: 1 to 17. A polypeptide fragment consisting of 15, preferably at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200 consecutive amino acid residues, or SEQ ID NOs: 1-17 It consists of each polypeptide full length represented by these. More preferably, it is a polypeptide fragment comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39, respectively.

  These polypeptides, fragments, variants or fusion polypeptides contain epitopes that elicit antibody formation, but these epitopes may be linear or higher order (intermittent). The epitope can be identified by any epitope analysis method known in the art, for example, phage display method, reverse immunogenetics method and the like.

  Examples of the antibody in the present invention include, but are not limited to, an antiserum, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, a human-type antibody, and a human antibody.

  The antibodies that can be used in the present invention include any type, class, and subclass. Such antibodies include, for example, IgG, IgE, IgM, IgD, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2.

  Furthermore, antibodies of all aspects are induced by the polypeptide according to the present invention. If all or part of the polypeptide or epitope is isolated, both polyclonal and monoclonal antibodies can be prepared using conventional techniques. Methods include, for example, those mentioned in Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, New York, 1980.

  Polyclonal antibodies can be produced by immunizing animals such as birds (for example, chickens) and mammals (for example, rabbits, goats, horses, sheep, mice, etc.) with the polypeptide of the present invention. The target antibody can be purified from the blood of an immunized animal by appropriately combining techniques such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and the like.

  Monoclonal antibodies can be obtained by techniques including producing hybridoma cell lines that produce monoclonal antibodies specific for each polypeptide in mice by conventional techniques. One method for producing such hybridoma cell lines is to immunize an animal with a polypeptide according to the invention, collect spleen cells from the immunized animal, fuse the spleen cells to a myeloma cell line, Generating hybridoma cells and identifying a hybridoma cell line that produces a monoclonal antibody that binds to the polypeptide. Monoclonal antibodies can be recovered by conventional techniques.

The production of monoclonal and polyclonal antibodies is described in detail below.
A. Preparation of monoclonal antibody (1) Immunization and collection of antibody-producing cells The immunogen obtained as described above is administered to mammals such as rats, mice (for example, Balb / c of inbred mice), rabbits, and the like. . The single dose of the immunogen is appropriately determined depending on the type of immunized animal, the route of administration, etc., and is about 50 to 200 μg per animal. Immunization is performed mainly by injecting an immunogen subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and after the first immunization, booster immunization is performed 2 to 10 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 4 weeks. After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA (Enzyme-Linked Immuno Sorbent Assay) method. When the antibody titer reaches a plateau, the immunogen is intravenously or intraperitoneally. Inject and give final immunization. Then, antibody-producing cells are collected 2 to 5 days, preferably 3 days after the last immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

(2) Cell fusion Hybridoma cell lines that produce monoclonal antibodies specific for each protein can be produced and identified by conventional techniques. One method for producing such a hybridoma cell line is to immunize an animal with a polypeptide of the invention, collect spleen cells from the immunized animal, and fuse the spleen cells to a myeloma cell line, thereby Generating hybridoma cells and identifying a hybridoma cell line producing a monoclonal antibody that binds to the enzyme. As a myeloma cell line to be fused with an antibody-producing cell, a generally available cell line of an animal such as a mouse can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, thymine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. The cell line is preferably derived from an animal of the same species as the immunized animal. Specific examples of myeloma cell lines include P3X63-Ag., Which is a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient cell line derived from BALB / c mice. 8 strains (ATCC TIB9).

  Next, the myeloma cell line and antibody-producing cells are fused. For cell fusion, antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture medium such as serum-free DMEM, RPMI-1640 medium, etc. The fusion reaction is performed in the presence of an accelerator. As a cell fusion promoter, polyethylene glycol having an average molecular weight of 1500 to 4000 daltons can be used at a concentration of about 10 to 80%. In some cases, an auxiliary agent such as dimethyl sulfoxide may be used in combination in order to increase the fusion efficiency. Furthermore, antibody-producing cells and myeloma cell lines can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(3) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As a method for this, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 2 million cells / well, a selective medium is added to each well, and thereafter Cultivate by changing the selective medium. The culture temperature is 20 to 40 ° C, preferably about 37 ° C. When the myeloma cells are of HGPRT-deficient strain or thymidine kinase-deficient strain, cells having the ability to produce antibodies and myeloma cell lines can be obtained by using a selective medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine. Only those hybridomas can be selectively cultured and propagated. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, it is screened whether the target antibody exists in the culture supernatant of the hybridoma which has proliferated. Hybridoma screening is not particularly limited, and may be carried out according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and performed by enzyme immunoassay (EIA: Enzyme Immuno Assay and ELISA), radioimmunoassay (RIA: Radio Immuno Assay) or the like. Can do. Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established. The hybridoma is stable in culture in a basic medium such as RPMI-1640 or DMEM, and produces and secretes a monoclonal antibody that specifically reacts with the polypeptide cancer marker of the present invention.

(4) Antibody recovery Monoclonal antibodies can be recovered by conventional techniques. That is, as a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration). Cultivate for 2 to 10 days and obtain antibody from the culture supernatant. In the case of the ascites formation method, about 10 million hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. And ascites or serum is collected after 1-2 weeks.

  In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate salting out method, ion exchange chromatography, affinity chromatography, gel filtration chromatography or the like is appropriately selected, or these In combination, a purified monoclonal antibody of the present invention can be obtained.

B. Preparation of polyclonal antibody In the case of preparing a polyclonal antibody, an animal is immunized in the same manner as described above, and 6 to 60 days after the final immunization, enzyme immunoassay (EIA and ELISA), radioimmunoassay (RIA), etc. The antibody titer is measured and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.

In the present invention, an antigen-binding fragment of the above antibody can also be used. Examples of antigen-binding fragments that can be produced by conventional techniques include, but are not limited to, Fab and Fab ′, F (ab ′) ₂ , Fv, scFv, dsFv, and the like. Also included are antibody fragments and derivatives that can be produced by genetic engineering techniques. Such antibodies include, for example, synthetic antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), single chain antibodies, and the like.

  The antibody of the present invention can be used in an assay for detecting the presence of a polypeptide or a (poly) peptide fragment thereof in the present invention both in vitro and in vivo. The use of monoclonal antibodies is preferred to allow specific detection in the assay, but even polyclonal antibodies can be identified by the so-called absorption method, which involves binding the antibody to an affinity column to which the purified polypeptide is bound. Antibodies can be obtained.

  Accordingly, the composition of the present invention comprises at least one antibody or fragment thereof that can specifically bind to the polypeptide of SEQ ID NOs: 1 to 17, a variant thereof, or a fragment thereof, preferably a plurality, more preferably All types can be included. More preferably, the composition of the present invention is a fragment of the polypeptide represented by SEQ ID NOs: 1 to 17, each comprising a polypeptide fragment comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39, and It can include at least one antibody or a fragment thereof that can specifically bind, preferably multiple types, more preferably all types.

  Preferably, the composition of the present invention is in the form of a kit. In such a kit, the antibodies or fragments thereof that can specifically bind to each of the above polypeptides are individually or appropriately mixed and mixed as a mixture into individual containers (for example, vials) and packaged. be able to. The antibody or fragment thereof may be bound to a solid support or may be in a free form. When bound to a solid support, for example, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon , Multi-well plates made of materials such as polymethacrylate, polymethyl methacrylate, polytetrafluoroethylene, polyvinylidene fluoride, latex, agarose, cellulose, sepharose, glass, metal, ceramics, magnetic materials, beads, test tubes, sticks, The antibody or fragment thereof may be attached or bound on a solid phase carrier in the form of a test piece or the like.

  The antibody or fragment thereof used in the present invention may be bound with a label, for example, a fluorophore, an enzyme, a radioisotope, or the like, if necessary, or such a label may be bound to a secondary antibody. Also good.

  Examples of fluorophores include fluorescein and its derivatives, rhodamine and its derivatives, dansyl chloride and its derivatives, umbelliferone, and the like.

  Examples of the enzyme include horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, and amylase.

Radioisotopes include, for example, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), phosphorus ( ³² P), sulfur ( ³⁵ S), metals (eg, ⁶⁸ Ga, ⁶⁷ Ga, ⁶⁸ Ge, ⁵⁴ Mn, ⁹⁹ Mo, ⁹⁹ Tc, ¹³³ Xe, etc.), tritium and the like.

Other labels include, for example, luminescent substances such as NADH ⁻ , FMNH ²⁻ , acridinium ester, luminol, and bioluminescent substances such as luciferase and luciferin.

  If necessary, an avidin-biotin system or a streptavidin-biotin system can be used. In this case, for example, biotin can be bound to the antibody of the present invention or a fragment thereof.

  The kit of the present invention may further comprise a labeled secondary antibody, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, a marker polypeptide as a purified standard substance, instructions for use, and the like.

<Detection of gastric cancer>
According to the present invention, using the substance capable of binding to the gastric cancer marker, the polypeptide represented by SEQ ID NOs: 1 to 17, a variant thereof or a fragment thereof, preferably one of them in a biological sample derived from a subject Alternatively, gastric cancer can be detected by a method comprising examining the quantity or presence of a plurality. By the method of the present invention, the polypeptide represented by SEQ ID NO: 1 to 4, the variant or fragment thereof is detected in the biological sample of the subject, or the polypeptide, variant or When it is determined that the expression level of the fragment is significantly high, or the polypeptide represented by SEQ ID NOs: 5 to 17, the variant or the fragment thereof is not detected, or the polypeptide compared to the control, the When it is determined that the expression level of the variant or fragment thereof is significantly low, the subject can be diagnosed as having gastric cancer.

  More preferably, any one or more of the polypeptide fragments represented by SEQ ID NOs: 1 to 17, each comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39 Can be detected by a method comprising examining its amount or presence. By the method of the present invention, a polypeptide fragment comprising the amino acid sequence represented by SEQ ID NO: 18-20 is detected in a biological sample of a subject, or those polypeptides, variants thereof or fragments thereof compared to a control When the expression level of the polypeptide is determined to be significantly high, or a fragment containing the polypeptide represented by SEQ ID NO: 21 to 39 is not detected, or the polypeptide, its variant or its fragment is compared with the control When it is determined that the expression level is significantly low, the subject can be diagnosed as having gastric cancer.

  In the method of the present invention, the detection of the gastric cancer marker may be a single marker, but is preferably performed for a plurality of markers, for example, 2 or more, 3 or more, 4 or more, 5 or more to 37, 38 or 39 or less. Good. This is to avoid unexpected detection of non-specific complexes, in other words, misdiagnosis. The gastric cancer marker of the present invention may be used in combination with other cancer diagnostic markers known to those skilled in the art.

  The composition of the present invention is useful for diagnosis, determination or detection of gastric cancer, that is, diagnosis of the presence or absence of disease and the degree of disease. In the diagnosis of gastric cancer, a comparison is made with controls such as normal tissue (or cells), non-cancerous tissue (or cells), normal body fluids, etc., and the presence or amount of the above-mentioned gastric cancer marker in a biological sample of a subject is detected. If the difference in presence or amount is significant, the subject is suspected of having gastric cancer.

  The specimen sample used in the method of the present invention is, for example, stomach tissue, surrounding tissues, stomach cells, or body fluid such as blood, serum, plasma, stomach fluid, urine. The biological tissue of the subject can be collected by biopsy or obtained by surgery.

  In the present invention, the subject is a mammal including a human, preferably a human.

  The substance capable of binding to the gastric cancer marker is, for example, the antibody or fragment thereof, aptamer, Affibody (trademark of Affibody), each gastric cancer marker receptor, each gastric cancer marker specific action inhibitor, each gastric cancer marker It contains a specific action activator, and is preferably an antibody or a fragment thereof, or a chemically modified derivative thereof.

  In an embodiment of the present invention, the measurement is carried out by contacting an antibody or fragment optionally labeled with a conventional enzyme or fluorophore with a tissue section or homogenized tissue or body fluid, qualitatively or with an antigen-antibody complex. A step of quantitatively measuring can be included. Detection is, for example, a method for measuring the presence and level of a target polypeptide by immunoelectron microscopy, an enzyme antibody method (for example, ELISA), a fluorescent antibody method, a radioimmunoassay method, a homogeneous method, a heterogeneous method, a solid phase method, a sandwich method Or the like by a method of measuring the presence or level of the target polypeptide by a conventional method. Furthermore, in the present invention, in order to easily detect the reaction between the antibody of the present invention and the target polypeptide in the sample, the reaction of the present invention is directly detected by labeling the antibody of the present invention, or the label is labeled. Detection is indirectly by using a secondary antibody. In the detection method of the present invention, it is preferable to use the latter indirect detection in terms of sensitivity.

  Depending on the type of gastric cancer marker described above, if the target polypeptide is present or disappears in bodily fluids or gastric cancer tissues or cells, preferably blood, obtained from a subject, or compared to a control, If the level is significantly increased or decreased, it is determined to be gastric cancer. Here, “significantly” means that there is a statistically significant difference.

  As a measuring method replacing the immunological method, a method using mass spectrometry is included. Specifically, this method can be performed by the technique described in the examples. That is, a biological sample such as serum or plasma is filtered to remove impurities, diluted with a buffer solution (for example, pH of about 8) and adjusted to a concentration of about 10 mg / ml to about 15 mg / ml, and then a molecular weight of 5 Molecular weight fractionation is performed through a hollow fiber filter (Reference Example (1) below) or a centrifugal flat membrane filter capable of removing more than 10,000 proteins, and the fraction is treated with protease (for example, trypsin) to be peptideized, and mass spectrometry is performed. Is applied to a patient before gastric cancer removal surgery based on the mass / charge number and intensity of a specific peak derived from the polypeptide of interest by applying a meter (a type using matrix-assisted laser desorption ionization or electrospray ionization) Differences in the amount of polypeptide present in a sample between healthy individuals can be measured.

  The present invention is more specifically described by the following examples. However, the present invention is not limited by this example.

<Reference example>
(1) Production of hollow fiber filter 100 polysulfone hollow fibers having a pore size of about 50,000 on the membrane surface are bundled, and both ends are fixed to a glass tube with an epoxy potting agent so as not to block the hollow part of the hollow fiber. And created a mini-module. The minimodule (module A) is used to remove high molecular weight proteins in serum or plasma, and has a diameter of about 7 mm and a length of about 17 cm. Similarly, a minimodule (module B) used for concentration of low molecular weight proteins was prepared using a membrane having a pore size of about 3,000 for the molecular weight cut off. The mini module has an inlet connected to the hollow fiber lumen at one end, and an outlet at the opposite end. The hollow fiber inlet and outlet are closed circulation system flow paths using silicon tubes, and the liquid is driven and circulated through the flow path by a peristaltic pump. Further, the glass tube of the hollow fiber mantle is provided with a port for discharging the liquid leaking from the hollow fiber, and one module set is configured. Modules were connected by T-shaped connectors in the middle of the flow path, and three modules A and one module B were connected in tandem to form one hollow fiber filter. The hollow fiber filter was washed with distilled water and filled with an aqueous solution of PBS (phosphate buffer containing 0.15 mM NaCl, pH 7.4). The fraction raw material serum or plasma is injected from the flow channel inlet of the hollow fiber filter, and is discharged from the flow channel outlet after fractionation and concentration. Serum or plasma injected into the hollow fiber filter is subjected to molecular sieving with a molecular weight of about 50,000 for each module A, and components having a molecular weight lower than 50,000 are concentrated and prepared in module B. Yes.

<Example 1>
(1) Protein Identification of Healthy Person and Gastric Cancer Patient Plasma EDTA plasma was obtained from 19 gastric cancer patients before resection surgery in the 50-70s and 8 healthy persons of the same age, and each plasma was measured. Plasma was filtered through a filter having a pore size of 0.22 μm to remove contaminants, and the protein concentration was adjusted to 50 mg / mL. This plasma was further diluted to 12.5 mg / mL of 25 mM ammonium bicarbonate solution (pH 8.0), and fractionated by molecular weight using the hollow fiber filter shown in Reference Example (1). The fractionated plasma sample (total volume 1.8 mL, containing up to 250 μg protein) is separated into 7 fractions by ProteomeLab® PF2D System (Beckman Coulter) reverse phase chromatography, and each fraction is lyophilized After that, the sample was redissolved in 100 μL of 25 mM ammonium bicarbonate solution (pH 8.0). This sample was digested with trypsin in an amount of 1/50 of the total protein at 37 ° C. for 2 to 3 hours for peptideization. The peptides of each fraction were further fractionated into 4 fractions by an ion exchange column (KYA Technologies). Each fraction was further fractionated on a reverse phase column (KYA Technologies), and the eluted peptide was surveyed using a mass spectrometer Q-TOF Ultimate (Micromass) linked online. Measured with

  In this field, the mass / charge ratio is usually corrected by using a standard substance at the time of measurement. In the analysis of the present invention, albumin, α -Further refinement of the data was achieved by using several proteins such as fibrinogen as internal standards and recalibrating the measured data using the theoretical mass values of those proteins. Data refined in this way was analyzed using protein analysis software MASCOT. As a standard for identifying a blood protein, (i) at least one of the peptides belonging to the protein is detected with high reliability of a Mowse score of 34 or more, (ii) a measured value of MS data of the peptide, and Using two criteria that the error between the measured value of the MS / MS data and the theoretical value of the peptide is 0.03 dalton or less, analysis was performed under conditions that could eliminate erroneous protein identification as much as possible.

(2) Proteins with detected or increased number of plasma in pre-cancerous plasma of gastric cancer patients compared to healthy individuals' plasma Compare this data between healthy and cancer patients, and more than 3 of the identified proteins A protein that was detected in gastric cancer patients and not detected at all in healthy individuals was found as a protein whose expression was significantly enhanced in the plasma of patients before gastric cancer removal surgery compared to healthy individuals. These proteins are polypeptides represented by SEQ ID NOs: 1 to 4 shown in Table 3 below. In addition, among the identified proteins, the number of proteins detected in healthy individuals is 3 or more than the number of individuals detected in gastric cancer patients, and its expression is significantly reduced in the plasma of patients before gastric cancer removal surgery compared to healthy individuals. Found as a protein. These proteins are polypeptides represented by SEQ ID NOs: 5 to 17 shown in Table 3 below. Therefore, it was found that the polypeptides represented by these SEQ ID NOs: 1 to 17 are useful in the detection of gastric cancer as gastric cancer markers.

(3) Proteins with increased or decreased MS peak intensity in plasma before cancer removal surgery for gastric cancer patients compared with healthy human plasma Further, MS peak intensity at the time of detection of peptides belonging to the protein identified in (2) above Was analyzed. Although this MS peak intensity is not strictly quantitative, it is a numerical value that reflects the amount of protein present to some extent, and it is understood by those skilled in the art that quantitative comparative analysis using MS peak intensity is possible. It is well known. The MS peak intensity at the time of detection of each peptide was compared between a healthy person and a cancer patient, and among the detected peptides, a peptide whose peak intensity in the gastric cancer patient group was significantly larger than that of a healthy person was determined to be healthy. It was found as a peptide whose abundance was significantly increased in the plasma of patients before gastric cancer removal surgery compared to humans. These peptides are the polypeptides represented by any of SEQ ID NOs: 18 to 20, and are any one fragment of the polypeptides represented by SEQ ID NOs: 1 and 2. Similarly, among the detected peptides, a protein whose peak intensity in the group of gastric cancer patients is significantly smaller than that of healthy persons is present in the plasma of patients before gastric cancer removal surgery compared to that of healthy persons. Found as a significantly reduced peptide. These peptides are polypeptides represented by SEQ ID NOs: 21 to 39, and these peptides are fragments of any one of the polypeptides represented by SEQ ID NOs: 5-9 and 11-16. Therefore, it was found that the polypeptides represented by these SEQ ID NOs: 18 to 39, and fragments having them therein are useful in the detection of gastric cancer as gastric cancer markers. The polypeptide fragments represented by these SEQ ID NOs: 18 to 39 are respectively the gene name, the fragment of the sequence represented by SEQ ID NOs: 1, 2, 5-9, and 11-16, the peptide length, Table 4 shows the amino acid sequences (indicated by one letter). In addition, the MS peak intensities of the polypeptide fragments represented by SEQ ID NOs: 18 to 39 in gastric cancer patients and healthy individuals are shown in FIGS. The numbers on the MS peak intensity of gastric cancer patients and healthy individuals in each sequence indicate the number of each individual.

  The present invention is particularly useful in the pharmaceutical and pharmaceutical industries because it can provide a composition for diagnosis or detection of gastric cancer having excellent specificity and sensitivity.

The MS peak intensity of polypeptide represented by sequence number 18-20 in this invention is shown. The MS peak intensity of the polypeptide represented by SEQ ID NOs: 21 and 22 in the present invention is shown. The MS peak intensity of polypeptide represented by sequence number 23-25 in this invention is shown. The MS peak intensity of polypeptide represented by sequence number 26-29 in this invention is shown. The MS peak intensity of polypeptide represented by sequence number 30-32 in this invention is shown. The MS peak intensity of polypeptide represented by sequence number 33-36 in this invention is shown. The MS peak intensity of polypeptide represented by sequence number 37-39 in this invention is shown.

Claims (17)

  A method for detecting gastric cancer in vitro, comprising measuring any one or more of the polypeptides represented by SEQ ID NOs: 1 to 17, variants thereof or fragments thereof in a biological sample derived from a subject.   The fragment is a polypeptide fragment comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39 in the amino acid sequence of the polypeptide represented by any of SEQ ID NOs: 1 to 17, respectively. The method described.   The method according to claim 1 or 2, wherein the amount of the polypeptide, a variant thereof or a fragment thereof or the presence thereof is measured.   4. The method according to claim 1, wherein the amount of the polypeptide, a variant or a fragment thereof is significantly increased or significantly decreased compared to that of a control sample. The method according to item.   The method according to any one of claims 1 to 4, wherein measurement of the polypeptide, a variant thereof or a fragment thereof is performed by an immunological method.   The method according to any one of claims 1 to 5, wherein the measurement is performed using a substance capable of binding to the polypeptide, a variant thereof or a fragment thereof.   The method according to claim 6, wherein the bindable substance is an antibody or a fragment thereof.   8. The method of claim 7, wherein the antibody is labeled.   Using an antibody or fragment thereof that specifically binds to the polypeptide, variant or fragment thereof, the amount or presence of one or more of the polypeptide, variant or fragment thereof in the sample is determined. Immunologically measured and indicated that the amount of the polypeptide, variant or fragment thereof is increased or decreased relative to that of a control sample, or the polypeptide, variant or fragment thereof The method according to any one of claims 1 to 8, comprising detecting gastric cancer using as an index that a fragment is present only in one of the sample and the control sample.   The method according to claim 1, wherein the sample is blood, plasma, serum or urine.   The method according to any one of claims 1 to 9, wherein the sample is a stomach-derived tissue or cell.   The method according to any one of claims 7 to 9, wherein the antibody is a monoclonal antibody or a polyclonal antibody.   A gastric cancer comprising one or more of an antibody or a fragment thereof or a chemically modified derivative thereof that specifically binds to at least one of the polypeptide represented by SEQ ID NOs: 1 to 17, a variant thereof or a fragment thereof. Composition for diagnosis or detection.   The polypeptide fragment is a polypeptide fragment comprising the amino acid sequence represented by any of SEQ ID NOs: 18 to 39 in the amino acid sequence of the polypeptide represented by any of SEQ ID NOs: 1 to 17, respectively. Item 14. The composition according to Item 13.   The composition according to claim 13 or 14, wherein the fragment of the polypeptide comprises an epitope consisting of at least 7 amino acids.   The composition according to any one of claims 13 to 15, which is in the form of a kit.   Use of the composition according to any one of claims 13 to 16 for in vitro detection of gastric cancer in a subject.

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