WO2011090166A1 - 食道癌マーカー - Google Patents

食道癌マーカー Download PDF

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WO2011090166A1
WO2011090166A1 PCT/JP2011/051104 JP2011051104W WO2011090166A1 WO 2011090166 A1 WO2011090166 A1 WO 2011090166A1 JP 2011051104 W JP2011051104 W JP 2011051104W WO 2011090166 A1 WO2011090166 A1 WO 2011090166A1
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protein
slc38a4
esophageal cancer
antibody
cancer
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PCT/JP2011/051104
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English (en)
French (fr)
Japanese (ja)
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ウリケシ ワシュル
義朗 岸
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株式会社医学生物学研究所
ベイジンボアルマイセンウジシュユシャンゴンス
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Priority to CN201180006719.0A priority Critical patent/CN102803968B/zh
Publication of WO2011090166A1 publication Critical patent/WO2011090166A1/ja

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the present invention relates to a method for examining esophageal cancer using SLC38A4 (also known as SNAT4) protein expression as an index, a composition for detecting esophageal cancer containing an anti-SLC38A4 protein antibody, a method for producing the same, and an esophagus containing an anti-SLC38A4 protein antibody.
  • the present invention relates to a cancer detection kit and a screening method for a therapeutic agent for esophageal cancer using SLC38A4 protein.
  • Esophageal cancer is a tumor of epithelial origin that occurs in the esophagus. Histologically, it is mainly classified into squamous cell carcinoma in which the esophageal mucosal epithelium becomes cancerous and adenocarcinoma in which the esophageal gland becomes cancerous. In Japan, squamous cell carcinoma accounts for over 90% of the total, and the remaining 5% is adenocarcinoma. Therefore, most esophageal cancer in Japanese is squamous cell carcinoma. It is common among men in their 60s, and the gender ratio is 3-5: 1.
  • the proliferated cancer cells tend to invade the surroundings and metastasize easily to the lymph nodes, and thus progress rapidly. Moreover, since there are few subjective symptoms, early detection tends to be delayed.
  • the prognosis of esophageal cancer is extremely poor among gastrointestinal cancers including gastric cancer and colorectal cancer, and the overall 5-year survival rate of esophageal cancer is about 14%.
  • carcinogenic factors are unknown, smoking cessation reduces the incidence of squamous cell carcinoma of the esophagus, suggesting an association between smoking and squamous cell carcinoma of the esophagus.
  • Physiological findings, diagnostic imaging, and tumor markers are used to diagnose esophageal cancer. There are few physical findings in early cancer. In advanced cancer, sometimes right or left upper clavicular lymphadenopathy is observed. Among those diagnosed with esophageal cancer, 74% have difficulty swallowing, including esophageal discomfort, and 14% have swallowing pain.
  • Lugol's solution stains glycogen abundant in normal mucosal squamous epithelium. When the amount of glycogen is significantly reduced due to mucosal canceration or atypical epithelialization, the lesion site is not stained with Lugol's solution and becomes a white unstained zone.
  • the Lugol's solution unstained zone is not specific to cancer and becomes positive also at the site of esophagitis and atrophy.
  • Toluidine blue staining is also used, but in this case the normal mucosa becomes unstained and the cancerous lesion is stained blue. In this staining, it is essential that the cancerous lesion is exposed on the surface, and it is not positive in intradermal cancer.
  • pathological diagnosis by biopsy performed in conjunction with endoscopy is a definitive diagnosis of esophageal cancer. When esophageal cancer is confirmed, ultrasonic endoscopy and CT (computer tomography) are performed in order to determine the depth (advanced stage).
  • the PET examination is useful for the evaluation of metastases that are difficult to judge by CT.
  • SCC squamous cell carcinoma-related
  • Antigens are relatively popular.
  • the preoperative diagnosis rate with SCC alone is said to be around 30%.
  • CEA, CYFRA 21-1, etc. are used as tumor markers other than SCC.
  • the positive rate increases as the cancer progresses, but the diagnostic rate decreases in the case of early cancer.
  • the p53 antibody is characterized by a high positive rate in relatively early cases, but the positive rate is still around 20-30%.
  • Non-Patent Document 1 Cancerous cells are characterized by significant proliferation and metastasis, but since achieving these requires a large amount of nutrients, cancer cells may increase the expression level of glucose and amino acid transporters.
  • L-type amino acid transporter LAT1 has been shown to be highly expressed in various human cancer tissues (prostate cancer, colon cancer, bladder cancer, Barrett's esophageal adenocarcinoma, oral squamous cell carcinoma, liver cancer).
  • Non-patent document 2 all of the transporters reported so far, including LAT1, are expressed in a considerable amount in normal cells, and are not considered to be cancer-specific transporters (Non-patent Documents 3 and 4).
  • Esophageal cancer is characterized by not only poor subjective symptoms but also metastasis after onset and poor prognosis. For this reason, identification of a tumor marker that is highly specific for esophageal cancer and enables early detection of esophageal cancer is desired.
  • the present invention has been made in view of such circumstances, and an object of the present invention is to provide a tumor marker that is highly specific to esophageal cancer and enables early detection of esophageal cancer. Further objects of the present invention are a method for examining esophageal cancer (particularly preferably early esophageal cancer) targeting the tumor marker, a composition for detecting esophageal cancer comprising a molecule for detecting the tumor marker, and the composition And a screening method for a therapeutic agent for esophageal cancer using the tumor marker.
  • the SLC38A4 protein (also known as SNAT4) belongs to the sodium ion-coupled amino acid transporter protein (Non-patent Document 4), and its gene is first derived from rat muscle in 2000, and in humans from liver in 2001. (Non-Patent Documents 5 to 7).
  • the SLC38A4 protein is structurally classified into the glutamine transporter family, but actually has a low affinity for glutamine, and its transport ability is much lower than other glutamine transporters. On the other hand, L alanine and asparagine are favored and transported (nonpatent literature 4).
  • Non-Patent Documents 6 to 8 the mRNA level is detected in rat muscle and liver by Northern blotting, and the liver is the highest in human tissues.
  • Non-Patent Documents 9 and 10 genes are amplified from skeletal muscle, brain, lung, heart, small intestine, kidney, pancreas, placenta, and uterus in addition to the liver (Non-patent Documents 9 and 10).
  • the protein level localization information of SLC38A4 is detected in the liver and placenta by Western blotting, and in the placenta by immunohistochemical staining (Non-patent Documents 9 and 11).
  • Non-patent Document 12 the relationship between the expression of SLC38A4 and esophagus or esophageal cancer has not been known so far.
  • the present inventors conducted an immunohistochemical staining on human cancer pathological sections and normal tissue sections for various marker candidates for the purpose of searching for cancer-specific markers.
  • antigen-specific antibodies to the transporter it was found that antibodies against the SLC38A4 protein react specifically and strongly with esophageal cancer tissues (especially early esophageal cancer tissues).
  • the present inventors can examine esophageal cancer using the expression of SLC38A4 protein as an index based on the knowledge that SLC38A4 protein is highly expressed in esophageal cancer and hardly detected in normal tissues. It was found that an immunological technique using an anti-SLC38A4 protein antibody is useful for examination of esophageal cancer.
  • screening for therapeutic agents for esophageal cancer can be performed using SLC38A4 protein.
  • the present invention relates to a method for examining esophageal cancer (particularly preferably early esophageal cancer) using the expression of SLC38A4 protein as an index, a composition for detecting esophageal cancer containing an anti-SLC38A4 protein antibody, a method for producing the same, More specifically, the present invention relates to a kit for detecting esophageal cancer containing an SLC38A4 protein antibody and a screening method for a therapeutic agent for esophageal cancer using the SLC38A4 protein.
  • a method for examining esophageal cancer comprising a step of detecting the expression of SLC38A4 protein in a cell or tissue separated from a subject, (2) The method according to (1), wherein the expression of SLC38A4 protein is detected using an antibody, (3) The method according to (2), wherein the antibody recognizes a region consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the SLC38A4 protein, (4) The method according to any one of (1) to (3), wherein the esophageal cancer is early esophageal cancer, (5) an esophageal cancer detection composition comprising an anti-SLC38A4 protein antibody, (6) The composition according to (5), wherein the antibody recognizes a region consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the SLC38A4 protein, (7) The composition according to (5) or (6), wherein the esophageal cancer is early esophageal cancer, (8) The composition according to (5), comprising: (a
  • the expression of SLC38A4 protein is specifically detected in esophageal cancer, and in particular, is detected at a high positive rate in esophageal cancer in an early highly differentiated stage. Therefore, according to the esophageal cancer test method of the present invention using the expression of SLC38A4 protein as an index, it is possible to detect esophageal cancer early with high accuracy. This makes it possible to treat esophageal cancer at an early stage of progression of esophageal cancer, and can greatly contribute to the treatment of patients and the improvement of the prognosis of patients.
  • composition for detecting esophageal cancer or the kit for detecting esophageal cancer of the present invention comprising an anti-SLC38A4 protein antibody is extremely useful for the examination of esophageal cancer or the screening of therapeutic agents for esophageal cancer in the present invention.
  • the X axis indicates the amount of fluorescence of the green fluorescent protein thistle green, which is an indicator that the target gene is expressed.
  • the Y axis shows the reactivity of the secondary antibody labeled with PE. It is a microscope picture which shows the result of having evaluated the performance of the anti- SLC38A4 protein antibody by immune cell staining using the cell which forcedly expressed SLC38A4. The left shows reactivity to 293T cells not expressing SLC38A4 protein, and the right shows reactivity to 293T cells transiently expressing SLC38A4 protein.
  • FIG. 5 is a photomicrograph showing the results of immunohistochemical staining with anti-SLC38A4 protein antibody using WHO pathological classification Grade II and Grade II-III esophageal cancer specimens.
  • the figure shows four typical grade II examples. It is a microscope picture which shows the result of having performed the immunohistochemical staining by the anti- SLC38A4 protein antibody using the grade III esophageal cancer specimen of the WHO pathological classification.
  • the figure shows four typical grade III examples. It is a graph which shows the score of the tissue staining according to grade of an esophageal cancer specimen by an anti-SLC38A4 protein antibody.
  • results of immunohistochemical staining for 6 gastrointestinal cancer specimens gastric adenocarcinoma, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, hepatocellular carcinoma, kidney cancer
  • results of immunohistochemical staining for 6 gastrointestinal cancer specimens gastric adenocarcinoma, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, hepatocellular carcinoma, kidney cancer
  • the present invention provides a method for examining esophageal cancer, comprising the step of detecting the expression of SLC38A4 protein in cells or tissues isolated from a subject.
  • cell or tissue refers to a cell or tissue that serves as a sample (target) when detecting the expression of SLC38A4 protein in the test method of the present invention.
  • the present invention is applied to cells or tissues separated from a living body.
  • “Separated from the subject” refers to a state in which the cells or tissues are completely isolated from the living body from which the cells or tissues are extracted by removing the cells or tissues from the living body.
  • the “subject” from which cells or tissues are removed is not limited to cancer patients, but can also be healthy subjects (including those who may have cancer). A part of an organ or tissue of a subject collected in a biopsy (biopsy) can be used for the examination method of the present invention.
  • the pathological tissue is usually prepared in a state existing in the living body, that is, in a state of being bound to surrounding cells (as a tissue piece) and used in the method of the present invention, but the pathological tissue is separated from the surrounding cells. Then, it may be used in the method of the present invention.
  • tissue judged to be cancer by other diagnostic methods a tissue judged to have a high probability of being cancer, Or the tissue which has a possibility of being cancer is used suitably.
  • the tissue used is preferably a tissue that is determined to be cancerous by other diagnostic methods, or a tissue that is highly likely to be cancerous.
  • examples of other diagnostic methods include X-ray contrast examination, endoscopy, ultrasonic examination, CT examination, MRI examination, PET examination, and a diagnostic method using a tumor marker.
  • a tissue suspected of having cancer due to one or more of these is used.
  • the “SLC38A4 protein” for detecting expression is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.
  • the amino acid sequence of a protein can be mutated in nature (ie, non-artificially) due to mutations in the gene that it encodes. Therefore, in the present invention, such a natural mutant of the SLC38A4 protein can also be a detection target.
  • detecting the expression of SLC38A4 protein means both detection of the presence or absence of expression of SLC38A4 protein and detection of the degree of expression.
  • the expression level of SLC38A4 protein can be grasped as an absolute amount or a relative amount. In the case of grasping the relative amount, for example, it can be determined by comparing with the expression level of the SLC38A4 protein of the prepared standard sample.
  • the “standard sample” is a sample in which whether or not the SLC38A4 protein is expressed is specified in advance. For example, a pathological tissue in which a site where esophageal cancer already exists can be used as the standard sample of the present invention. Moreover, the tissue (normal tissue) which does not suffer from cancer can also be used as the standard sample of the present invention.
  • Detecting the expression of SLC38A4 protein in the present invention is preferably performed by an immunological technique.
  • the immunological technique include immunohistochemical staining method, ELISA method, radioimmunoassay, FCM, immunoprecipitation method, immunoblotting and the like.
  • an anti-SLC38A4 protein antibody is used, the antibody is brought into contact with the SLC38A4 protein, and the SLC38A4 protein is detected using the binding property (binding amount) of the antibody to the SLC38A4 protein as an index.
  • “contacting” means placing the antibody and the SLC38A4 protein under physiological conditions where the anti-SLC38A4 protein antibody can recognize the SLC38A4 protein.
  • the cell or tissue separated from the subject is immersed in a solution containing the antibody, or the antibody is applied to the cell or tissue. It means that the contained solution is sufficiently dripped or sprayed and the physiological condition is set such that the antibody can recognize SLC38A4 present in cells or tissues.
  • the immunological detection method rapid and sensitive detection is possible, and the operation is simple.
  • the inspection method of the present invention is also advantageous in that the burden on the patient's body is small.
  • the anti-SLC38A4 protein antibody used in the test method of the present invention is not particularly limited in its type and origin as long as it has a specific binding property to the SLC38A4 protein. It is preferably an antibody that recognizes the amino acid sequence “GIGNSEKAAMSSQFANEDT” (SEQ ID NO: 2) at positions 29 to 47 of the SLC38A4 protein (SEQ ID NO: 1).
  • an antibody to which a labeling substance is bound is used as the anti-SLC38A4 protein antibody, the amount of antibody bound to the SLC38A4 protein can be directly measured by detecting the label, which is simple.
  • this method has a problem of preparing an antibody to which a labeling substance is bound, and has a problem that detection sensitivity is generally lowered. Therefore, in the present invention, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound.
  • the “secondary antibody” is an antibody exhibiting specific binding to the anti-SLC38A4 protein antibody.
  • an anti-SLC38A4 protein antibody when an anti-SLC38A4 protein antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody.
  • Labeled secondary antibodies that can be used are commercially available for antibodies derived from various biological species such as rabbits, goats, and mice, and appropriate secondary antibodies are available depending on the biological species from which the anti-SLC38A4 protein antibody is derived. Can be selected and used in the present invention.
  • protein G or protein A to which a labeling substance is bound can be used.
  • the labeling substance examples include peroxidase, ⁇ -D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substance.
  • HRP horseradish peroxidase
  • FITC fluorescein isothiocyanate
  • RVC rhodamine isothiocyanate
  • alkaline phosphatase biotin
  • biotin by using biotin as a labeling substance and reacting this with avidin peroxidase, it is possible to detect an antibody bound to the SLC38A4 protein with high sensitivity.
  • immunohistochemical staining of a living tissue is performed by the following procedures (1) to (10).
  • Various documents and books can be referred to for immunohistochemical staining of living tissues (for example, “Enzyme Antibody Method, Revised 3rd Edition”, edited by Keiichi Watanabe, Kazuho Nakane, interdisciplinary planning).
  • (1) Fixation / embedding in paraffin Fix tissue surgically collected from a living body with formalin, paraformaldehyde, anhydrous ethyl alcohol, or the like. Then embedded in paraffin. Generally dehydrated with alcohol, treated with xylene, and finally embedded in paraffin.
  • a specimen embedded in paraffin is sliced into a desired thickness (for example, 3 to 5 ⁇ m thick) and spread on a glass slide.
  • a desired thickness for example, 3 to 5 ⁇ m thick
  • an alcohol-fixed specimen, a dry-sealed specimen, a frozen specimen, or the like may be used.
  • Pretreatment antigen activation
  • enzyme treatment heat treatment and / or pressure treatment are performed for antigen activation.
  • removal of endogenous peroxidase When peroxidase is used as a labeling substance at the time of staining, it is treated with hydrogen peroxide to remove endogenous peroxidase activity.
  • Nonspecific reaction inhibition The section is treated with a bovine serum albumin solution (for example, 1% solution) for several minutes to several tens of minutes to inhibit nonspecific reaction. Note that this step may be omitted by carrying out the next primary antibody reaction using an antibody solution containing bovine serum albumin.
  • Antibody reaction An antibody diluted to an appropriate concentration is dropped onto a section on a slide glass, and then allowed to react for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer. (7) Addition of labeling reagent Peroxidase is frequently used as a labeling substance. In the above antibody reaction, an anti-SLC38A4 protein antibody conjugated with peroxidase can also be used.
  • a secondary antibody conjugated with peroxidase a protein G or protein conjugated with peroxidase.
  • a or the like can also be used.
  • a secondary antibody to which peroxidase is bound is dropped onto a section on a slide glass, and then reacted for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer.
  • Color reaction DAB (3,3'-diaminobenzidine) is dissolved in Tris buffer. Subsequently, hydrogen peroxide is added.
  • the coloring solution thus prepared is allowed to permeate the section for several minutes (for example, 5 minutes) to develop a color. After color development, the section is thoroughly washed with tap water to remove DAB.
  • Nuclear staining Nuclear staining is performed by reacting Meyer's hematoxylin for several seconds to several tens of seconds. Wash with running water and color (usually for a few minutes).
  • Dehydration, clearing, sealing After dehydrating with alcohol, clearing with xylene, and finally sealing with synthetic resin, glycerin, rubber syrup or the like.
  • the probability that a cell or tissue is cancer or a cancerous site in the tissue has a strong correlation with the staining intensity by immunostaining and the proportion of cells stained by immunostaining. For this reason, in the method of the present invention, by detecting the expression of the SLC38A4 protein, it is possible to evaluate the probability that cells or tissues separated from the subject are cancerous and to identify cancerous sites in the tissues. . Thereby, the site
  • the stage of pathological tissue can be determined. It can.
  • Well differentiated cancer cells have many similarities to normal squamous epithelial cells, and it is difficult to distinguish between cancer and normal compared to advanced cancers that have advanced grades.
  • this book uses anti-SLC38A4 protein antibody.
  • the tissue staining of the invention is extremely useful for detection of early cancer that is difficult to distinguish from normal by visual observation.
  • “early esophageal cancer” specifically refers to esophageal cancer at a stage that does not reach grade III, preferably grade I or II esophageal cancer.
  • Information obtained by implementing the above method for cancer patients can be used for evaluation or grasping of the patient's disease state, evaluation of therapeutic effects, and the like.
  • the method of the present invention is performed in parallel with cancer treatment, the therapeutic effect can be evaluated based on the resulting information.
  • changes in the staining properties in the pathological tissue can be examined, and the therapeutic effect can be determined from the change in increase or decrease in the stained site.
  • cancer can be diagnosed based on an index that is excellent in objectivity such as dyeability.
  • Diagnosis of cancer in a subject is usually performed by a doctor (including those who have received instructions from the doctor; the same applies hereinafter), but data relating to the expression level of SLC38A4 protein in a pathological tissue obtained by the method of the present invention. Is useful for diagnosis by doctors. Therefore, the method of the present invention can also be expressed as a method of collecting and presenting data useful for diagnosis by a doctor.
  • the present invention provides a composition for detecting esophageal cancer comprising an anti-SLC38A4 protein antibody.
  • the anti-SLC38A4 protein antibody is not particularly limited in its type and origin as long as it has specific binding properties for the SLC38A4 protein.
  • the antibody used in the composition for detecting esophageal cancer of the present invention may be a monoclonal antibody or a polyclonal antibody.
  • the antibody used in the composition for detecting esophageal cancer of the present invention can be a functional fragment of the antibody or a multimeric form of the functional fragment (eg, dimer, trimer, tetramer, polymer).
  • Examples of such functional fragments and multimers thereof include Fab, Fab ′, F (ab ′) 2, Fv, scFv, sc (Fv) 2, dsFv, and diabody.
  • Fab means a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and part of a heavy chain.
  • Fab ′ differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody.
  • F (ab ′) 2 means a bivalent antigen-binding fragment of an immunoglobulin that consists of both light chains and parts of both heavy chains.
  • Fv is the minimum antibody fragment which has a complete antigen recognition and binding site. Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a noncovalent bond.
  • “ScFv” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain.
  • “Sc (Fv) 2” is a chain formed by joining two heavy chain variable regions and two light chain variable regions with a linker or the like.
  • “DsFv” is a disulfide stabilized Fv.
  • a “diabody” is a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, each region comprising a separate It forms a pair with the complementary region of the strand.
  • the anti-SLC38A4 protein antibody is preferably an antibody that recognizes the amino acid sequence “GIGNSEKAAMSSQFANEDT” (SEQ ID NO: 2) at positions 29 to 47 of the SLC38A4 protein (SEQ ID NO: 1).
  • the method for producing a composition for detecting esophageal cancer of the present invention includes a step of immunizing SLC38A4 protein or an immunogenic part thereof, and a step of separating and purifying an antibody that binds to SLC38A4 protein. Furthermore, the process of mixing the other component accept
  • Polyclonal antibodies can be prepared by the following procedure.
  • An antigen for example, the full length of SLC38A4 protein described in SEQ ID NO: 1 or a part thereof
  • the SLC38A4 protein as an antigen can be prepared by separating and purifying from a biological sample.
  • SLC38A4 protein prepared as a recombinant protein can be used as an antigen.
  • Recombinant protein for example, inserts a gene encoding SLC38A4 protein or a part thereof into a vector in an expressible state, introduces the vector into an appropriate host, and isolates and purifies the protein expressed in the host To be prepared.
  • SLC38A4 protein preferably a peptide region consisting of amino acid sequence “GIGNSEKAAMSSQFANEDT” (SEQ ID NO: 2) at positions 29 to 47 is used as a fusion protein with GST, ⁇ -galactosidase, maltose-binding protein, histidine (His) tag or the like What was prepared can also be used as an antigen.
  • a fusion protein can be easily separated and purified by a general-purpose method.
  • Immunization is repeated as necessary, and blood is collected when the antibody titer has sufficiently increased, and serum is obtained by centrifugation or the like.
  • the obtained antiserum can be made into an IgG fraction by affinity chromatography using protein G, protein A or the like.
  • affinity purification using the SLC38A4 protein or a part thereof, an antibody that binds to the SLC38A4 protein or a part thereof used as an immunogen can be further separated and purified from the antiserum or IgG fraction.
  • the polyclonal antibody used in the composition for detecting esophageal cancer of the present invention particularly preferably immunizes a peptide consisting of the amino acid sequence “GIGNSEKAAMSSQFANEDT” (SEQ ID NO: 2) at positions 29 to 47 of the SLC38A4 protein and binds to the peptide.
  • GIGNSEKAAMSSQFANEDT amino acid sequence “GIGNSEKAAMSSQFANEDT”
  • monoclonal antibodies can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, a clone that produces an antibody having high specificity for the target protein is selected. After the hybridoma is monocloned, the target antibody can be obtained by purifying the culture medium of the selected clone.
  • the hybridoma is proliferated to a desired number or more, it is transplanted into the abdominal cavity of an animal (for example, a mouse) and allowed to proliferate in ascites, and then the target antibody can be obtained by purifying the ascites.
  • affinity chromatography using protein G, protein A or the like is preferably used.
  • affinity chromatography in which an antigen is immobilized may be used.
  • methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.
  • the obtained anti-SLC38A4 protein antibody is directly labeled with an enzyme, fluorescence, or the like.
  • the obtained anti-SLC38A4 protein antibody (primary antibody) is not labeled and the antibody is recognized. Secondary antibodies may be labeled.
  • the composition for detecting esophageal cancer of the present invention may contain other components acceptable as a composition in addition to the anti-SLC38A4 protein antibody.
  • other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, labeled compounds, and secondary antibodies. It is done.
  • excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
  • disintegrant starch, carboxymethylcellulose, calcium carbonate and the like can be used.
  • buffering agent phosphate, citrate, acetate and the like can be used.
  • emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
  • suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • stabilizer propylene glycol, diethylin sulfite, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • the present invention also provides a kit for detecting esophageal cancer comprising the composition for detecting esophageal cancer of the present invention.
  • the kit of the present invention may be combined with a substrate necessary for detection of the label, a positive control or negative control, or a buffer used for dilution or washing of the sample. it can.
  • the kit of the present invention contains a substance labeled with a substance (for example, secondary antibody, protein G, protein A, etc.) that binds to the antibody. Can be combined. Furthermore, the kit of the present invention can include instructions for using the kit.
  • the kit of the present invention is useful, for example, in the diagnosis of esophageal cancer. ⁇ Screening method for therapeutic agents for esophageal cancer> The present invention also provides a screening method for a therapeutic agent for esophageal cancer.
  • One aspect thereof is a method for screening SLC38A4 protein as a target, the step of providing SLC38A4 protein or part thereof, the step of contacting a candidate compound with SLC38A4 protein or part thereof, and SLC38A4 protein or part thereof Selecting a compound that binds to.
  • a compound that binds to the SLC38A4 protein can be obtained, and a therapeutic drug for esophageal cancer can be screened.
  • Candidate compounds include chemically synthesized or natural low molecular weight compounds, natural or synthetic proteins, peptides, antibodies (including the antibody of the present invention), cell extracts, culture supernatants, and the like.
  • the binding activity of the candidate compound can be measured by EIA, ELISA, or the like. Screening artificially synthesized compounds that bind to proteins using high-throughput combinatorial chemistry techniques is also well known to those skilled in the art (Verdine GL. Nature (ENGLAND) 1996 Nov 7; 384: 11-13, Hogan JC Jr. Nature (ENGLAND) 1996 Nov 7; 384: 17-19). The compound thus obtained can also be used for targeting esophageal cancer.
  • Another embodiment is a method for screening using the expression of SLC38A4 protein as an indicator, and administering a candidate compound or a control to an esophageal cancer model animal (excluding humans), and separating the esophageal tissue of the model animal And detecting the expression of SLC38A4 protein in the separated tissue and selecting a compound that reduces the expression of SLC38A4 protein compared to a control.
  • This method can be carried out independently or in addition to the above-described method of screening using the SLC38A4 protein as a target.
  • an esophageal cancer model animal is prepared.
  • ethanol or acetaldehyde can be injected subcutaneously into ALDH2 knockout mice to cause squamous cell carcinoma, and a model mouse for esophageal cancer can be obtained (see, for example, JP-A-2005-110601).
  • An esophageal cancer model animal can also be produced by transplanting human esophageal cancer cells into immunodeficient mice.
  • Candidate compounds include chemically synthesized or natural low molecular weight compounds, natural or synthetic proteins, peptides, antibodies (including the antibody of the present invention), cell extracts, culture supernatants, and the like.
  • a negative control or a positive control can be used as a control.
  • physiological saline or the like is usually used as a negative control. It may be a substance that has already been found to have no therapeutic effect.
  • a positive control a substance or the like that has already been found to have a therapeutic effect is used.
  • the method of administration is well known to those skilled in the art, and can be appropriately selected from oral, intravenous injection, intradermal, or subcutaneous.
  • the tissue of the esophagus part of the model animal is separated.
  • the separated tissue is subjected to immunostaining, it can be subjected to treatment such as fixation and paraffin embedding as described above.
  • the expression of SLC38A4 protein in the separated tissue is detected, and a compound that reduces the expression of SLC38A4 protein is selected compared to the control.
  • a compound that reduces the expression of SLC38A4 protein is selected compared to the control.
  • the stained image of the positive control administration group and the staining image of the candidate compound administration group are compared, and the staining of the candidate compound administration group is made more negative (for example, staining Select a compound that reduces the intensity or reduces the proportion of cancer cells that are strongly stained.
  • a stained image of the control instead of the stained image of the negative control administration group, a stained image of the positive control administration group (stained image of the treatment group of the substance having a therapeutic effect; standard staining image) is prepared, and is equal or more negative A compound that produces a stained image may be selected.
  • the peptide consisting of the amino acid sequence at positions 29 to 47 was artificially synthesized and bound to KLH, then mixed with complete floyd adjuvant, and immunized to two or more rabbits six times every other week.
  • Antigen-specific IgG was purified from the antiserum obtained after immunization using an agarose bead column on which an antigen peptide was immobilized.
  • an ELISA using a 96-well plate with an antigen peptide immobilized thereon was performed, and when the antigen concentration was 0.4 ⁇ g / ml, the titer (absorbance OD450) was 1.0 or more. This was used for the subsequent experiments.
  • the titer of the antigen-specific IgG fraction obtained in this example was 1.2 (FIG. 2).
  • FCM flow cytometry
  • the obtained antibody desirably exhibits reactivity in cell staining and flow cytometry (FCM) on 293T cultured cells in which the full length of the SLC38A4 protein is forcibly expressed.
  • PcDNA3.1 (Invitrogen) was used as an expression vector. Downstream of the SLC38A4 gene, an IRES sequence, which is an entry site for ribosomes, and a gene for green fluorescent protein thistle green (Amalgam) were mounted immediately below.
  • this expression vector is transiently introduced into a 293T cell using a lipofection reagent (Invitrogen), a cell with the fluorescence of thistle green is identified as a cell into which the SLC38A4 gene has been introduced.
  • the cells were fixed with 4% paraformaldehyde / phosphate buffer (PBS) at 4 ° C. for 15 minutes, and then 0.1% Of Triton X-100 / PBS for 15 minutes at room temperature.
  • PBS paraformaldehyde / phosphate buffer
  • 1 ⁇ g / ml antibody solution diluted in PBS containing 100 ⁇ l of 1% BSA and 0.1% Triton X-100
  • the cells were reacted at room temperature for 1 hour.
  • a PE-labeled anti-rabbit IgG antibody manufactured by Beckman Coulter, diluted 200-fold.
  • the diluted solution is the same as the primary antibody diluted solution) was reacted as a secondary antibody.
  • the antibody reacts with antigen-expressing cells, cells that glow with thistle green (green) will glow with PE (red) at the same time (that is, the dots shift to the upper right in the FCM data).
  • the dot did not shift to the upper right, while the myc tag antibody (manufactured by MBL: PL14) was used as the positive control.
  • the anti-SLC38A4 protein antibody was used, the dot shifted to the upper right (FIG. 3).
  • 293T cells introduced with a mock gene as a control and 293T cells introduced with an SLC38A4 / SNAT4 gene were treated in the same manner as described above, and then UV fluorescence microscope (OLYMPUS OLYMPUS, IX71 fluorescence microscope system system). The observation was performed. As a result, almost no fluorescence (red) was confirmed in the control cells, and fluorescence (red) was confirmed in the 293T cells introduced with the SLC38A4 / SNAT4 gene (FIG. 4). From the results of FIGS. 2 to 4, it was determined that the prepared antibody specifically recognized the SLC38A4 protein.
  • Com01-D7 and Com01-D8 are both WHO pathological classification grade I esophageal squamous cell carcinoma. To deparaffinize tissue sections, 3 x 5 minutes in xylene, 2 x 5 minutes in 100% ethanol, 1 x 5 minutes in 95% ethanol, 1 x 5 minutes in 90% ethanol, 5 min in 80% ethanol The treatment was performed once, 70% ethanol once for 5 minutes, and PBS three times for 5 minutes (all at room temperature).
  • the tissue section was immersed in a 10 mM citrate buffer solution (pH 6) containing 0.05% Tween 20 and treated in an autoclave at 125 ° C. for 5 minutes.
  • a 10 mM citrate buffer solution pH 6) containing 0.05% Tween 20
  • PBS containing 3% hydrogen peroxide was treated for 10 minutes at room temperature, and then PBS containing 5% normal goat serum and 0.5% BSA (blocking) Solution) at room temperature for 30 minutes. After the excess solution was wiped with a cloth, an appropriate amount of an anti-SLC38A4 protein antibody diluted to 1 ⁇ g / ml with a blocking solution was added (the tissue section was sufficiently immersed) and allowed to react at room temperature for 2 hours.
  • the degree of staining was divided into four stages according to the staining intensity and distribution. That is, when the entire test cancer tissue is stained very strongly (++), when the entire test tissue is stained positive (+), when the entire test tissue is stained, or the entire test tissue is stained weakly Was positive (+-) and negative (-) when the test tissue was not stained at all.
  • 10 were strongly positive, 24 were positive, 18 were weak positive, 44 were negative, The total number of positive cases including all weak positives was 52 cases, corresponding to 54% of the total.
  • tissue staining for gastric adenocarcinoma, colon adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, hepatocellular carcinoma, and kidney cancer was carried out according to the method of FIG. All these specimens were purchased from Shanghai OutDo. As a result, the staining in these 6 cancer types was not confirmed (FIG. 11). Staining with an anti-SLC38A4 protein antibody was shown to be a reaction specific to esophageal squamous cell carcinoma.
  • esophageal cancer can be detected early with high accuracy using the expression of SLC38A4 protein as an index. This makes it possible to treat esophageal cancer at an early stage of progression of esophageal cancer, increase the success rate of the patient's treatment, and improve the prognosis of the patient.
  • the present invention can greatly contribute to diagnosis, treatment, and development of therapeutic agents for esophageal cancer.

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