WO2011105544A1 - Marqueur tumoral, anticorps dirigé contre ce marqueur, kit de détection de celui-ci, et procédé de détection de celui-ci - Google Patents

Marqueur tumoral, anticorps dirigé contre ce marqueur, kit de détection de celui-ci, et procédé de détection de celui-ci Download PDF

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WO2011105544A1
WO2011105544A1 PCT/JP2011/054261 JP2011054261W WO2011105544A1 WO 2011105544 A1 WO2011105544 A1 WO 2011105544A1 JP 2011054261 W JP2011054261 W JP 2011054261W WO 2011105544 A1 WO2011105544 A1 WO 2011105544A1
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tumor marker
tumor
present
antibody
cancer
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PCT/JP2011/054261
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English (en)
Japanese (ja)
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泰豪 宮本
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地方独立行政法人大阪府立病院機構
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • C07K16/303—Liver or Pancreas
    • 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
    • C07K16/3046—Stomach, Intestines
    • 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
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the present invention relates to a tumor marker, an antibody thereto, a detection kit thereof, and a detection method thereof.
  • Non-Patent Documents 1 and 2 are the observed specifically in cancer cells.
  • Sialyl-Le a is a sugar chain structure represented by the following formula (2) by the action of fucosyltransferase 3 (FUT3) encoded by the Lewis gene in vivo (referred to as Sialyl-Le c or Du-PAN-2) It is generated by adding fucose to.
  • FUT3 fucosyltransferase 3
  • tumor detection methods that target sugar chain structures that are specifically found in cancer cells have been conventionally performed.
  • the types of target sugar chain structures have been limited.
  • the tumor detection method using them has a problem that subjects to be targeted are limited.
  • An object of the present invention is to provide a new tumor marker having a sugar chain structure specifically found in cancer cells, an antibody against the same, a detection kit thereof, and a detection method thereof.
  • the present inventor has come up with a novel human-derived sugar chain structure that has not been known so far after extensive trial and error.
  • the present inventor has further intensively studied and found that this novel sugar chain structure is a sugar chain structure specifically observed in cancer cells.
  • the present inventor has also found that this novel sugar chain structure is a sugar chain structure also found in cancer cells derived from individuals of Lewis (-).
  • Item 1 A tumor marker having a sugar chain represented by the following formula (3).
  • Item 2 An antibody against the tumor marker according to Item 1.
  • Item 3. The antibody according to Item 2, which is a monoclonal antibody.
  • Item 4. A lectin that recognizes the tumor marker according to Item 1.
  • Item 5. A tumor detection kit comprising a material necessary for detecting the tumor marker according to Item 1.
  • Item 6 The tumor detection kit according to Item 5, wherein the material necessary for detecting the tumor marker is an antibody against the tumor marker according to Item 1.
  • Item 7. Item 6.
  • Item 8-1 The subject in which the tumor marker according to item 1 is detected in a sample collected from a subject and the detected value exceeds a reference value set based on the detected value of the tumor marker in a sample collected from a healthy subject To determine that the patient is a cancer patient.
  • Item 8. The method according to Item 8-1, wherein the test subject and the healthy subject are both Lewis negative (Lewis ( ⁇ )).
  • Item 10. (I) a step of applying a candidate compound to a tumor cell in which the tumor marker according to item 1 is expressed; and (ii) a candidate compound used when the expression of the tumor marker in the tumor cell is reduced or eliminated. Selecting an antitumor compound as an antitumor compound.
  • Term A-2 An antibody against the sugar chain according to Item A-1.
  • Term A-3 The antibody according to Item A-2, which is a monoclonal antibody.
  • Term A-4 A lectin that recognizes the sugar chain according to Item A-1.
  • Term A-5 A lectin that recognizes the sugar chain according to Item A-1.
  • the sugar chain detection kit comprising a material necessary for detecting the sugar chain according to Item A-1.
  • Term A-6 The sugar chain detection kit according to Item A-5, wherein the material necessary for detecting the sugar chain is an antibody against the sugar chain according to Item A-1 (that is, the antibody according to Item A-2).
  • Term A-7 The tumor detection kit according to Item A-5, wherein the material necessary for detecting the sugar chain is a lectin that recognizes the sugar chain according to Item A-1 (ie, the lectin according to Item A-4).
  • Term A-8-1 The sugar chain described in item A-1 in the sample collected from the subject is detected, and the detected value exceeds the reference value set based on the detected value of the sugar chain in the sample collected from the healthy subject.
  • a method of determining an examiner as a cancer patient Term A-8-2.
  • Term A-9-1. A method for determining whether or not a specimen is derived from a cancer patient, wherein the sugar chain according to item A-1 in the specimen is detected, and the detected value is a detection of the sugar chain in the specimen collected from a healthy subject A method for determining that a specimen exceeding a reference value set based on a value is derived from a cancer patient.
  • Term A-10 The method according to Item A-9-1, wherein the specimen is derived from Lewis negative (Lewis (-)) and the healthy subject is Lewis negative (Lewis (-)).
  • Term A-10 (I) a step of applying a candidate compound to a tumor cell in which the sugar chain described in item A-1 is expressed; and (ii) when the sugar chain expression of the tumor cell is reduced or eliminated. Selecting a candidate compound as an anti-tumor compound.
  • Term B-1 Use of a sugar chain represented by the following formula (3) as a tumor marker.
  • Term B-2 The use according to Item B-1, wherein the sugar chain is used as a Lewis negative (Lewis ( ⁇ )) tumor marker.
  • the tumor marker of the present invention When used, the presence or absence of a tumor in a subject can be determined.
  • the tumor marker of the present invention when used, the presence or absence of a tumor in a subject who is Lewis (-) can also be determined.
  • tumor marker of the present invention is used, variations in tumor determination means can be increased. By using the tumor marker of the present invention in combination with a conventional determination means, a more reliable determination can be performed.
  • Black circles indicate A8-2, and black triangles, black diamonds, and black squares indicate glycosidase degradation products of A8-2, respectively.
  • the starting point of the arrow indicates the starting material, and the end point indicates the decomposition product.
  • Each glycosidase used for the degradation is shown next to the arrow.
  • “N” is ⁇ -sialidase derived from Arthrobacter ureafaciens
  • “F” is ⁇ -fucosidase derived from bovine kidney
  • 2,6-S is ⁇ 2-3 binding and ⁇ 2-6 It shows that ⁇ 2,3-sialidase ( ⁇ 2,3-sialidase) was used under conditions that decompose sialic acid in the bond.
  • Figure 2 shows the expected biosynthetic pathway of major glycolipids in cancer cells and normal epithelial cells. Solid arrows indicate the dominant pathway in normal epithelial cells. The wavy arrow indicates the pathway that increases in cancer cells. “4F” indicates ⁇ 1-4 fucosylation of GlcNAc. “3F” indicates ⁇ 1-3 fucosylation of GlcNAc. “2F” indicates ⁇ 1-2 fucosylation of galactose. “3S” indicates ⁇ 2-3 sialylation of galactose. “6S” indicates ⁇ 2-6 sialylation of galactose.
  • White circles with dots are glucose, white circles are galactose ( ⁇ 1-4 bond, type 2), black circles are galactose ( ⁇ 1-3 bond, type 1), white squares are GlcNAc, white stars are sialic acid ( ⁇ 2-6 bonds), black stars are Sialic acid ( ⁇ 2-3 bond) and white triangle indicate fucose, respectively.
  • Tumor marker of the present invention is a tumor marker having a sugar chain represented by the following formula (3).
  • sugar residue is a group consisting of 0, 1 or multiple (preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars. is there.
  • the sugar here may be a known sugar that constitutes a sugar chain present in the living body, and examples thereof include pentose, hexose, amino sugar, uronic acids, deoxy sugar, and the like.
  • the bond between sugars known bond modes can be exemplified, for example, ⁇ glycoside bond and ⁇ glycoside bond.
  • the tumor markers of the present invention include those in which R does not exist, those in which R is a monosaccharide, and those in which R is a sugar chain.
  • the end of the sugar residue may be PA (pyridylamino), for example, or may be bound to a protein or peptide.
  • the sugar chain represented by the formula (3) may be expressed as “ST1H” (abbreviation of ⁇ 2-6 sialylated type 1H).
  • ST1H is a sugar chain structure that is present more in cancer cells than in normal cells. ST1H is also characterized in that it is also present in cancer cells derived from individuals that are Lewis (-).
  • the tumor marker of the present invention is not particularly limited as long as it has ST1H.
  • the tumor marker of the present invention include a glycoprotein (ST1H-added glycoprotein) obtained by adding ST1H to an O-linked sugar chain or N-linked sugar chain on a glycoprotein, and ST1H added to a lipid.
  • Glycolipid (ST1H addition glycolipid) and the like As an example of the tumor marker of the present invention, ST1H itself separated from them may be used. The excision of the tumor marker of the present invention from ST1H-added glycoprotein or ST1H-added glycolipid can be performed by a known method.
  • excision of the tumor marker of the present invention from ST1H-added glycoprotein can be performed by a hydrazine decomposition method, an alkaline decomposition method, or the like when added to an O-linked sugar chain on the glycoprotein.
  • N-glycan is degraded by the hydrazine degradation method or the action of an enzyme having an activity of cleaving GlcNAc-Asn bond, such as N-glycanase and glycopeptidase. This can be done by cleaving the bond between the conjugated sugar chain and the protein.
  • glycolipid when added to a glycolipid, it can be carried out by the action of an enzyme having an activity of hydrolyzing a glycosidic bond, such as endoglycoceramidase.
  • excision of the tumor marker of the present invention from glycolipid cleaves the glycosidic bond between the glycolipid sugar chain and ceramide by the action of an enzyme such as endoglycoceramidase, which has an activity of hydrolyzing glycosidic bonds.
  • an enzyme such as endoglycoceramidase, which has an activity of hydrolyzing glycosidic bonds.
  • a tumor can be detected by detecting the tumor marker of the present invention.
  • a tumor derived from an individual that is Lewis (-) can also be detected. Details of this tumor detection method will be described later.
  • the “tumor” in the present invention is preferably a malignant tumor. Although it does not specifically limit as a malignant tumor, for example, an adenocarcinoma is preferable.
  • adenocarcinoma is preferable.
  • preferable adenocarcinoma in the present invention include colon cancer, pancreatic cancer, gastric cancer, gallbladder cancer, bile duct cancer, prostate cancer, uterine cancer, ovarian cancer, esophageal cancer, kidney cancer, bladder cancer, breast cancer, laryngeal cancer, pharyngeal cancer, Examples include liver cancer and lung cancer. Among these, colon cancer and pancreatic cancer are preferable.
  • the tumor detection method of the present invention is a method including a step of detecting a "tumor marker of the present invention”.
  • the “tumor marker of the present invention” is present more in cancer cells than in normal cells. For this reason, there is a causal relationship between the presence of the “tumor marker of the present invention” and the tumor.
  • the “tumor marker of the present invention” in the subject may be directly detected, or the “tumor marker of the present invention” in such a sample may be detected after the sample is once collected from the subject.
  • the area where the detected value of the “tumor marker of the present invention” exceeds the reference value is the area where the tumor is present it can.
  • the test subject to which the detected value of the “tumor marker of the present invention” exceeds the reference value It can be determined that the person is a cancer patient. In contrast, it is possible to determine that the subject who is the provider of the specimen whose detected value of the “tumor marker of the present invention” is below the reference value is not a cancer patient. Preferably, it is possible to determine whether or not the subject who is the specimen provider is a cancer patient based on the presence or absence of the tumor marker of the present invention.
  • the subject is not particularly limited.
  • the subject may be a health checkup patient who is regularly performed in the same manner as current tumor markers (CA19-9, CEA, PSA, etc.).
  • the fact that such health check-up recipients can be subjects means that all people can be subjects.
  • examples of the subject include a person who needs to determine whether or not a tumor exists, or a person who has undergone cancer surgery and needs a diagnosis of recurrence.
  • the person who needs to determine whether or not there is a tumor is not particularly limited.For example, symptoms such as loss of appetite, rapid weight loss, cough, or melena, or abnormalities in various images, blood tests, etc. You can list people who have left. For example, in the case of colorectal cancer, fecal occult blood is positive, or a person suspected of having an abnormality in image diagnosis or the like. In the case of gastric cancer, a person who is suspected of being abnormal in a stomach X-ray examination or the like can be mentioned.
  • pancreatic cancer or gallbladder cancer examples include those suspected of being abnormal by abdominal ultrasonography or CT.
  • lung cancer examples include those suspected of having abnormalities in chest X-ray examinations.
  • pancreatic cancer examples include those who have abnormally high levels of amylase or lipase in the blood.
  • the tumor marker of the present invention is highly expressed particularly in the tumor of Lewis (-) individuals. Therefore, by detecting the tumor marker of the present invention, it is possible to preferably detect a tumor derived from an individual who is especially Lewis ( ⁇ ). Therefore, a person who is Lewis (-) can be preferably a subject.
  • the specimen is not particularly limited, and examples thereof include blood-derived specimens, pancreatic juice-derived specimens, bile-derived specimens, and urine-derived specimens.
  • blood-derived specimens include serum or plasma.
  • the blood-derived specimen is derived from a cancer patient, the blood-derived specimen contains ST1H-added glycoprotein and ST1H-added glycolipid secreted from cancer cells. Therefore, when a blood-derived specimen is used as a specimen, a tumor can be detected by directly detecting ST1H-added glycoprotein or ST1H-added glycolipid.
  • an anticoagulant When using a blood-derived sample as a sample, an anticoagulant may be added in advance if necessary.
  • the anticoagulant is not particularly limited, and examples thereof include EDTA-dipotassium, sodium citrate, and heparin sodium.
  • the step of detecting the “tumor marker of the present invention” is not limited as long as the “tumor marker of the present invention” can be detected.
  • a step of detecting using a mass spectrometry method (MS method) a step of detecting using a nuclear magnetic resonance method, and the like.
  • the specific technique is not particularly limited as long as the “tumor marker of the present invention” is detected.
  • the RIA Radioimmunoassay
  • EIA Enzyme immunoassay
  • the ELISA method can be used as the EIA method.
  • a sandwich ELISA method can be used.
  • a two-step sandwich method can be used.
  • the 2-step sandwich method can be performed, for example, by the following procedure.
  • a monoclonal antibody for detecting the “tumor marker of the present invention” (hereinafter sometimes simply referred to as “tumor marker”)
  • it can be recovered by a method such as a magnet.
  • Two types are prepared: “antibody-bound particles” bound to particles and “enzyme-labeled antibodies” bound to enzymes.
  • a primary immune complex is formed by a monoclonal antibody bound to antibody-bound particles and a tumor marker contained in a specimen. Thereafter, the primary immune complex is recovered with a magnet or the like, the reaction solution is removed, and the primary immune complex is washed.
  • a secondary immune complex is formed by an enzyme-labeled antibody and a tumor marker bound to antibody-bound particles via a monoclonal antibody. Thereafter, the secondary immune complex is recovered again with a magnet or the like, the reaction solution is removed, and the secondary immune complex is washed. Finally, a “substrate solution” (solution containing the enzyme substrate in the enzyme-labeled antibody) is added to the secondary immune complex, and the amount of tumor marker present in the sample is measured by detecting the enzyme reaction. To do.
  • antibody-bound particles include, but are not limited to, ferrite particles to which antibodies are bound.
  • enzyme-labeled antibodies include, but are not limited to, antibodies labeled with alkaline phosphatase (ALP).
  • an ALP-labeled antibody as a substrate solution, for example, 3- (2′- spiroadamantane) -4- methoxy-4- (3 ′′-phosphoryloxy) phenyl-1,2- dioxetane ⁇ 2 sodium salt ( 3- (2′-spiroadamantane) -4-methoxy-4- (3 ′′ -phosphoryloxy) phenyl-1,2-dioxetane disodium salt; AMPPD).
  • the LC method When using the LC method, for example, it may be detected by performing analysis by a two-dimensional sugar chain mapping method after separation by HPLC of a normal phase column and a reverse phase column. In detail, it can carry out by the method as described in an Example. Moreover, you may carry out combining several processes as needed. For example, a method (LC-MS method) combining a step of detecting using liquid chromatography and a step of detecting using mass spectrometry may be used. In detail, it can carry out by the method as described in an Example.
  • LC-MS method combining a step of detecting using liquid chromatography and a step of detecting using mass spectrometry may be used. In detail, it can carry out by the method as described in an Example.
  • a lectin When a lectin is used, it can be detected by, for example, lectin column chromatography or lectin blot.
  • the detection value differs depending on the detection method, but may be a measurement value directly detected by a detection device, or may be a calculation value calculated according to a specific calculation formula based on the measurement value. .
  • Examples of the calculated value include the amount and concentration of the “tumor marker of the present invention”.
  • the amount or concentration of the “tumor marker of the present invention” is, for example, a standard curve (standard curve; based on a detection value obtained using a standard specimen in which the amount or concentration of the “tumor marker of the present invention” is known in advance. It can be calculated by creating a Standard curve.
  • the reference value that serves as an index for determination can be set based on the detected value of the “tumor marker of the present invention” using normal tissues of the same subject or other healthy subjects as controls.
  • a plurality of types of controls may be prepared and set based on the average value of the detected values of the “tumor marker of the present invention”.
  • the number of controls prepared for calculating the average value is not particularly limited.
  • “average value of healthy persons” + ( ⁇ ⁇ standard deviation) can be set.
  • ⁇ can be set as appropriate.
  • Tumor detection kit of the present invention is a kit containing a material necessary for detecting the aforementioned "tumor marker of the present invention”.
  • the tumor detection kit of the present invention is used for the aforementioned “tumor detection method of the present invention”.
  • the material necessary for detecting the “tumor marker of the present invention” refers to a part or all of reagents or equipment substantially necessary for the “tumor detection method of the present invention”. It may be part or all of the reagent, or part or all of the device. Further, a combination of a part or all of the reagent and a part or all of the device may be used.
  • the material necessary for detecting the “tumor marker of the present invention” varies depending on the type of the step of detecting the tumor marker in the “tumor detection method of the present invention”.
  • the step of detecting a tumor marker in the “tumor detection method of the present invention” is an antigen-antibody reaction using an antibody, as an example of the material necessary for detecting “the tumor marker of the present invention”
  • An antibody is mentioned.
  • the antigen-antibody reaction is the above-described sandwich ELISA method (two-step sandwich method)
  • the antibody is included as an example of a material necessary for detecting the “tumor marker of the present invention”.
  • Antibody-binding particles and enzyme-labeled antibodies may further be included.
  • the material necessary for detecting the “tumor marker of the present invention” may further include instructions indicating the procedure for performing the detection.
  • Antibody is an antibody against the aforementioned “tumor marker of the present invention”.
  • the antibody of the present invention is preferably a specific antibody against the “tumor marker of the present invention”.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.
  • a monoclonal antibody is preferable.
  • the antibody of the present invention may be incorporated in the aforementioned “antibody-binding particle” or “enzyme-labeled antibody”.
  • the “tumor marker of the present invention” contained in a specimen can be detected.
  • the antigen-antibody reaction using the antibody of the present invention can be performed as described above.
  • the method for producing the polyclonal antibody of the present invention is not particularly limited as long as the polyclonal antibody of the present invention can be produced.
  • the following method can be mentioned.
  • immunogens include the following.
  • ST1H-added glycolipid is immunized with a liposome comprising cholesterol and phosphatidylcholine as a carrier substance together with an adjuvant such as complete Freund® or lipid A.
  • immunized with ST1H-added glycolipid adsorbed on cells such as Salmonella minnesota.
  • an immunogen that is not a glycolipid but a glycan portion bound to BSA (bovineserumalbumin) using a carrier.
  • BSA bovineserumalbumin
  • immunization methods include the following. Rabbits and chickens are injected subcutaneously with the immunogen, boosted several times every 3-4 days, and blood is collected one week after the final immunization.
  • ELISA As a means for confirming polyclonal antibodies, for example, ELISA can be used.
  • the method for producing the monoclonal antibody of the present invention is not particularly limited as long as the monoclonal antibody of the present invention can be produced.
  • the following method can be mentioned.
  • immunogens include the following. There is a method in which ST1H-added glycolipid is immunized with a liposome comprising cholesterol and phosphatidylcholine as a carrier substance together with an adjuvant such as complete Freund or lipid A. Alternatively, an ST1H-added glycolipid adsorbed on cells such as Salmonella minnesota is used as an immunogen. Examples of immunization methods include the following. Several mice are given the immunogen intraperitoneally, boosted several times every 3-4 days, and blood is collected one week after the final immunization. Serum antibody titer is measured by ELISA, and the mouse with the highest titer is selected and used for normal cell fusion.
  • Spleen cells are extracted from mice, fused with myeloma cells by the PEG (polyethylene glycol) method, etc., cultured in a hybridoma selection medium (HAT medium), and hybridomas are selected.
  • the ELISA method is mainly used for screening positive clones, cloning, and antibody titer measurement, but there are also complement binding reaction methods.
  • a mixture of antigen glycolipid, cholesterol and phosphatidylcholine is immobilized on a 96-well plate, and the hybridoma supernatant is added and reacted. After washing, for example, a Goat anti-mouse immunoglobulin labeled with peroxidase or the like is reacted. Further, after washing, color is developed using a peroxidase chromogenic substrate such as TMB (3,3 ', 5,5'-tetramethylbenzidine), and the color development is read with a plate reader.
  • a peroxidase chromogenic substrate such as TMB (3,3 ', 5,5'-tetramethylbenzidine
  • a method for producing a monoclonal antibody that recognizes a sugar chain can also be carried out with reference to the following literature.
  • Antibody-binding particles or “enzyme-labeled antibodies” can be produced according to known methods, respectively.
  • the lectin of the present invention is a lectin that recognizes the aforementioned “tumor marker of the present invention”.
  • the lectin of the present invention can be obtained, for example, as follows.
  • Lectin extraction and purification methods are basically the same as general protein purification methods. For example, it can be carried out according to the method described in “Nobuyuki Yamazaki, Shiro Yagi, Tatsuya Oda, Tomomitsu Hatakeyama, Tomohisa Ogawa, Biochemical Experimental Method 52, Lectin Research Method, Society Publishing Center, p19-p77.”
  • Extraction materials include animal tissues, plant seeds, invertebrate body fluids and shells, and fungi. They are homogenized in, for example, phosphate buffer, Tris-HCl buffer or unbuffered saline (which may contain detergent such as Triton X-100, EDTA or various protease inhibitors as appropriate). Extract the lectin.
  • affinity chromatography using a sugar chain as a ligand is mainly used.
  • affinity chromatography using a sugar chain as a ligand is mainly used.
  • affinity chromatography using a sugar chain as a ligand is mainly used.
  • For the activation of the resin for immobilizing the sugar chain on the carrier as a ligand there is a method using carbonyldiimidazole or divinylsulfone in addition to epoxy activation.
  • an affinity gel immobilized with ST1H using the above method, apply a crude extract containing lectin to the gel, bind the lectin, wash the gel, and a solution containing excess ST1H, or Lectins can be obtained by elution with a strongly acidic solution or the like.
  • purification can be performed by combining gel filtration chromatography, ion exchange chromatography, and the like in the same manner as normal protein purification.
  • the measurement of lectin activity is, for example, the method described in “Nobuyuki Yamazaki, Shiro Yagi, Tatsuya Oda, Tomomitsu Sasayama, Tomohisa Ogawa, Biochemical Experimental Method 52, Lectin Research Method, Society Publishing Center, p19-p77.” It can therefore be done.
  • a method of measuring the binding between lectin and a sugar ligand ST1H is used.
  • ST1H is immobilized on a microtiter plate using divinyl sulfone or the like.
  • the lectin solution is added to the wells on the plate, allowed to react, washed, and the colloidal gold solution is added to the wells to react, and then the absorbance at 620 nm is measured to determine the amount of bound lectin.
  • the tumor marker of the present invention can be used as an index for screening an antitumor compound. Therefore, the present invention also includes a method for screening an antitumor compound using the tumor marker of the present invention as an index.
  • An antitumor compound is a compound having an antitumor effect.
  • the screening method is used when a candidate compound (preferably a plurality) is applied to tumor cells expressing the tumor marker of the present invention, and the amount of the tumor marker decreases or disappears.
  • This is a method for selecting a candidate compound as an antitumor compound. That is, the screening method includes (i) a step of applying a candidate compound to a tumor cell in which the tumor marker of the present invention is expressed, and (ii) when the expression of the tumor marker of the present invention in the tumor cell is reduced or eliminated And a step of selecting the used candidate compound as an antitumor compound.
  • a colon cancer cell derived from a person of Lewis (-) (cultured with the tumor marker of the present invention) is cultured and established, and the expression level of the tumor marker of the present invention in the cell is determined.
  • a procedure is exemplified in which the candidate compound is added to the medium and cultured for a certain period of time, and then the expression level of the tumor marker of the present invention is measured again.
  • the tumor detection kit described above can also be used for detecting the tumor marker of the present invention in the screening method.
  • the tumor marker of the present invention can be used as an index for monitoring a disease state (cancer state) of a cancer patient. Therefore, the present invention also includes a method for monitoring the pathology of cancer patients using the tumor marker of the present invention as an index.
  • the monitoring method is a method for monitoring the medical condition of a patient having a tumor in which the tumor marker of the present invention is expressed. Specifically, a tumor in which the tumor marker of the present invention is expressed from the patient (preferably periodically) is collected as a sample, and the expression of the tumor marker of the present invention in the sample is measured. It is a method of monitoring the cancer state (degree of progression) of the patient by comparing with. That is, the monitoring method comprises (i) a step of collecting the tumor as a specimen from a patient having a tumor in which the tumor marker of the present invention is expressed, and (ii) measuring the expression of the tumor marker of the present invention in the specimen, Or a step of comparing with subsequent measurement results.
  • tumor detection kit can also be used to detect the tumor marker of the present invention in the monitoring method.
  • Tissue samples derived from all 65 cases 60 cases of colon cancer and 5 cases of pancreatic cancer were used. Cancer cells were extracted from the cancer tissue, and glycolipids were further extracted from the cancer cells. A sugar chain was cut out from the glycolipid and further fluorescently labeled. Fluorescently labeled sugar chains were separated by HPLC using normal phase columns and reverse phase columns by HPLC, and analyzed by a two-dimensional sugar chain mapping method to verify the presence or absence of new sugar chains that did not match the standard. Furthermore, this novel sugar chain structure was identified using mass spectrometry and enzyme digestion. Each procedure was performed as follows.
  • the sugar chain structure was estimated by comparison with a standard two-dimensional sugar chain map of a known PA sugar chain (see FIG. 2). Furthermore, mass spectrometry was performed and it was confirmed that the mass was the same as that estimated. For sugar chains that could not be estimated by the two-dimensional sugar chain map, the PA sugar chain was decomposed with an enzyme (exoglycosidase) that cleaves a specific sugar chain structure as necessary, and the structure was estimated.
  • an enzyme exoglycosidase
  • the fluorescence detector was set to an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm.
  • the fluorescence detector was set at an excitation wavelength of 315 nm and a fluorescence wavelength of 400 nm. Recover each peak (PA sugar chain) separated by reversed-phase HPLC, dry with a centrifugal concentrator, dissolve in an appropriate amount of water, and then add nano-LC / LCQ Deca XP (ESI-ion trap) The mass spectrometric measurement was performed.
  • A8-2 did not match the standard that it had so far, and the possibility of an unknown sugar chain was great (Fig. 2). In mass spectrometry, it was an isomer with Sialyl Le x , Sialyl Le a , ST2H. To determine the structure, A8-2 was cleaved with exoglycosidase (FIG. 2; method will be described later). When A8-2 was cut with Neuraminidase ( ⁇ -sialidase from Arthrobacter ureafaciens) (Nacalai), it matched with the standard Type1H on a two-dimensional map.
  • Neuraminidase ⁇ -sialidase from Arthrobacter ureafaciens
  • ST1H (abbreviation of ⁇ 2-6 sialylated type 1H). ST1H could not be detected in any case of normal mucosal epithelial cells.
  • Cleavage with exoglycosidase was performed as follows. Using 2 U / ml ⁇ 2,3-sialidase from Salmonella typhimurium (Takara) or 2 U / ml ⁇ -sialidase from Arthrobacter ureafaciens (Nacalai) as exoglycosidase, 100 mM sodium acetate buffer, pH 5.5, for 2 hr The reaction was carried out at 37 ° C. (condition 1). Under condition 1, ⁇ 2,3-sialidase specifically cleaved ⁇ 2,3-linked sialic acid to terminal galactose, but did not cleave ⁇ 2,6-linked sialic acid.
  • FIG. 3 shows a predicted glycolipid synthesis pathway in cancer cells and normal cells. Solid arrows indicate pathways in normal cells. On the other hand, a wavy arrow indicates a route in cancer cells.
  • ST1H is considered to be produced by sialylating the galactose in its precursor Type 1H. This reaction is considered to be a cancer cell specific reaction.
  • the major glycolipids in Lewis (+) normal cells are Le a and Le b .
  • Lewis (-) key glycolipid in normal cells is Lc 4 and Type1H.
  • SLe a sugar chain structure (NeuAc ⁇ 2-3Gal ⁇ 1-3 (Fuc ⁇ 1-4) GlcNAc ⁇ 1-R ) and sLe c carbohydrate structures (NeuAc ⁇ 2-3Gal ⁇ 1-3GlcNAc ⁇ 1-R), depending respectively CA19-9 antibody and DU-PAN-2 antibody Be recognized.
  • Colon cancer 60 cases in 4 cases pancreatic cancer cells five cases 2 cases (Reason 1) or not acknowledged Le a structure in sugar chain structure, or very that the expression was low, (Reason 2) CA19- 9 was OU / ml, (Reason 3) Examining the genome, all 6 were homozygous of mutant allele, and (Reason 4) Lewis enzyme activity was below detection limit Therefore, it was judged as Lewis negative (Lewis (-)). Note that the presence ratio of Lewis (-) is said to be about 10%, and six out of 65 cases are said to be Lewis (-), which is in good agreement with the ratio.
  • ST1H was expressed only in 3 of the 65 cases. This frequency is by no means low, assuming that ST1H ⁇ ⁇ ⁇ is likely to develop in cancer in Lewis ( ⁇ ) people. This is because 4 out of 60 cases of colorectal cancer and 2 out of 5 cases of pancreatic cancer cells were Lewis ( ⁇ ), and ST1H was expressed in 3 out of 6 cases of Lewis ( ⁇ ). However, by increasing the number of cases analyzed, there is a good possibility that ST1H expression is observed even in Lewis (+) human cancer.
  • ST1H is likely to be expressed in Lewis (-) human cancer.
  • the activity of Lewis enzyme is strong, so that Lc 4 and Type 1H rarely exist as they are, and change to Le a and Le b .
  • the antibody CA19-9 that recognizes SLe a as a tumor marker and recognizes it is used.
  • SLe c (DU-PAN-2 value), which is a precursor of SLe a , is increased in Lewis ( ⁇ ) human cancer, and is used as a tumor marker.
  • Lc 4 and Type 1H are the main products because Lewis enzyme (FUT3) has no activity.
  • FUT3 Lewis enzyme
  • SLe c is created from Lc 4 , but it is not converted to SLe a , so the amount of SLe c increases.
  • Lewis (-) with respect to recognize SLe c structure DU-PAN-2 antibodies are used in place of CA19-9 antibody.
  • ST1H may be synthesized from Type1H in Lewis (-) human cancer. This indicates that ST1H can be used as a better tumor marker for Lewis (-) individuals, similar to the SLe c structure.
  • ST1H can be used as a general tumor marker, and at the same time can be a better tumor marker for Lewis (-) people.

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Abstract

L'invention porte sur un nouveau marqueur tumoral basé sur une structure de chaîne de sucres unique aux cellules cancéreuses, sur un anticorps dirigé contre celui-ci, sur un kit de détection de celui-ci, et sur un procédé de détection de celui-ci. Spécifiquement, le marqueur tumoral comprend la chaîne de sucres représentée par la formule suivante (3) : NeuAc α2-6(Fuc α1-2)Gal β1-3GlcNAc β1-R (3) (dans la formule, R représente un résidu de sucre).
PCT/JP2011/054261 2010-02-26 2011-02-25 Marqueur tumoral, anticorps dirigé contre ce marqueur, kit de détection de celui-ci, et procédé de détection de celui-ci WO2011105544A1 (fr)

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WO2018143336A1 (fr) * 2017-02-03 2018-08-09 住友化学株式会社 Procédé de détection du cancer du pancréas
JP2020516892A (ja) * 2017-04-14 2020-06-11 ジュノー セラピューティクス インコーポレイテッド 細胞表面グリコシル化を評価するための方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015091953A (ja) * 2013-10-01 2015-05-14 Ｊｃｒファーマ株式会社 ２−アミノピリジンによる糖鎖の標識法
WO2018143336A1 (fr) * 2017-02-03 2018-08-09 住友化学株式会社 Procédé de détection du cancer du pancréas
JPWO2018143336A1 (ja) * 2017-02-03 2019-11-21 住友化学株式会社 膵臓癌の検査方法
JP2020516892A (ja) * 2017-04-14 2020-06-11 ジュノー セラピューティクス インコーポレイテッド 細胞表面グリコシル化を評価するための方法
JP7355650B2 (ja) 2017-04-14 2023-10-03 ジュノー セラピューティクス インコーポレイテッド 細胞表面グリコシル化を評価するための方法
US11796534B2 (en) 2017-04-14 2023-10-24 Juno Therapeutics, Inc. Methods for assessing cell surface glycosylation

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