WO2011105544A1 - Tumor marker, antibody to same, detection kit for same, and method for detecting same - Google Patents

Abstract

Disclosed is a novel tumor marker focusing on a sugar chain structure unique to cancer cells, an antibody to same, a detection kit for same, and a method for detecting same. Specifically, the tumor marker has the sugar chain represented in the following formula (3): NeuAc α2-6(Fuc α1-2)Gal β1-3GlcNAc β1-R (3) (In the formula, R represents sugar residue).

Description

Tumor marker, antibody thereto, detection kit and detection method thereof

The present invention relates to a tumor marker, an antibody thereto, a detection kit thereof, and a detection method thereof.

Conventionally, it has been pointed out that sugar chains are deeply involved in canceration and malignancy of cancer. For example, the sugar chain structure represented by the following formula (1) (referred to as Sialyl-Le ^a or CA19-9) is the observed specifically in cancer cells (Non-Patent Documents 1 and 2).

(In the formula, R represents a sugar residue)
Method for detecting a tumor with an antibody that recognizes the sialyl-Le ^a is the widespread (Non-Patent Document 3).

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.

(In the formula, R represents a sugar residue)
However, since the activity of fucosyltransferase 3 is not observed in individuals whose Lewis blood type is Lewis (-), Sialyl-Le ^a is not produced. Therefore, even Lewis even a cancer patient (-) Sialyl-Le ^a is from individuals is not detected. Thus, Lewis (-) in the individual, the antibody recognizing the Sialyl-Le ^a there is a problem that can not be detected tumor.

Thus, tumor detection methods that target sugar chain structures that are specifically found in cancer cells have been conventionally performed. However, the types of target sugar chain structures have been limited. Moreover, 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. In addition, 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 (-).

The present invention has been completed as a result of further studies based on such knowledge, and is described below.
Item 1.
A tumor marker having a sugar chain represented by the following formula (3).

(Where NeuAc is the N-acetylneuraminic acid residue, Fuc is the fucose residue, Gal is the galactose residue, GlcNAc is the N-acetylglucosamine residue, α2-6 is the α2-6 glycosidic bond. Α1-2 represents an α1-2 glycoside bond, β1-3 represents a β1-3 glycoside bond, and R represents a sugar residue.)
Item 2.
An antibody against the tumor marker according to Item 1.
Item 3.
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.
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. The tumor detection kit according to Item 5, wherein the material necessary for detecting the tumor marker is a lectin that recognizes the tumor marker according to Item 1.
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 8-2.
Item 8. The method according to Item 8-1, wherein the test subject and the healthy subject are both Lewis negative (Lewis (−)).
Item 9-1.
A method for determining whether or not a specimen is derived from a cancer patient, wherein the tumor marker according to item 1 is detected in the specimen, and the detected value is a detected value of the tumor marker in the specimen collected from a healthy subject. A method for determining that a sample exceeding a reference value set based on cancer patients originates.
Item 9-2.
The method according to Item 9-1, wherein the specimen is derived from Lewis negative (Lewis (-)) and the healthy subject is 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-1.
An isolated sugar chain represented by the following formula (3).

(Where NeuAc is the N-acetylneuraminic acid residue, Fuc is the fucose residue, Gal is the galactose residue, GlcNAc is the N-acetylglucosamine residue, α2-6 is the α2-6 glycosidic bond. Α1-2 represents an α1-2 glycoside bond, β1-3 represents a β1-3 glycoside bond, and R represents a sugar residue.)
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.
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.
Item 8. The method according to Item A-8-1, wherein the subject and the healthy subject are both Lewis negative (Lewis (−)).
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-9-2.
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.

(Where NeuAc is the N-acetylneuraminic acid residue, Fuc is the fucose residue, Gal is the galactose residue, GlcNAc is the N-acetylglucosamine residue, α2-6 is the α2-6 glycosidic bond. Α1-2 represents an α1-2 glycoside bond, β1-3 represents a β1-3 glycoside bond, and R represents a sugar residue.)
Term B-2.
The use according to Item B-1, wherein the sugar chain is used as a Lewis negative (Lewis (−)) tumor marker.

When the tumor marker of the present invention is used, the presence or absence of a tumor in a subject can be determined.

In particular, when the tumor marker of the present invention is used, the presence or absence of a tumor in a subject who is Lewis (-) can also be determined.

If the 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.

It is the separation pattern by HPLC of glycolipids of cancer cells in 2 cases (1 colorectal cancer, 1 pancreatic cancer). A and B show separation patterns of colon cancer patients, and C and D show separation patterns of pancreatic cancer patients, respectively. A and C show acidic glycolipids, and B and D show neutral glycolipid separation patterns, respectively. It is a two-dimensional sugar chain map of peak A8-2. Circles, rhombuses, triangles and squares represent monosialylated monofucosylated hexasaccharide, monosialylated pentasaccharide, monofucosylated pentasaccharide and tetrasaccharide, respectively. 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 α-sialidase derived from Arthrobacter ureafaciens, “F” is α-fucosidase derived from bovine kidney and “2,6-S” is α2-3 binding and α2-6 It shows that α2,3-sialidase (α2,3-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 α1-4 fucosylation of GlcNAc. “3F” indicates α1-3 fucosylation of GlcNAc. “2F” indicates α1-2 fucosylation of galactose. “3S” indicates α2-3 sialylation of galactose. “6S” indicates α2-6 sialylation of galactose. White circles with dots are glucose, white circles are galactose (β1-4 bond, type 2), black circles are galactose (β1-3 bond, type 1), white squares are GlcNAc, white stars are sialic acid (α2-6 bonds), black stars are Sialic acid (α2-3 bond) and white triangle indicate fucose, respectively.

1. Tumor marker The tumor marker of the present invention is a tumor marker having a sugar chain represented by the following formula (3).

Each symbol in the formula represents the following residue or bond, respectively.
NeuAc: N-acetylneuraminic acid residue
Fuc: Fucose residue
Gal: Galactose residue
GlcNAc: N-acetylglucosamine residue α2-6: α2-6 glycoside bond α1-2: α1-2 glycoside bond β1-3: β1-3 glycoside bond
R: sugar residue R (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. Sugar is exemplified. As for the bond between sugars, known bond modes can be exemplified, for example, α glycoside bond and β glycoside bond. In other words, it can be said that 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.

In the present specification, the sugar chain represented by the formula (3) may be expressed as “ST1H” (abbreviation of α2-6 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. Examples of 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.

For example, 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. In addition, when it is added to the N-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.

In addition, 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.

For example, 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. Etc.

A tumor can be detected by detecting the tumor marker of the present invention. In particular, 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. Examples of 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.

2. Tumor detection method The tumor detection method of the present invention is a method including a step of detecting a "tumor marker of the present invention".

As described above, 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.

When directly detecting the “tumor marker of the present invention” in a subject, it is possible to determine that 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.

In the case of detecting the “tumor marker of the present invention” in such a sample after collecting the sample once from the subject, 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. For example, 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.

In addition, 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. In the case of pancreatic cancer or gallbladder cancer, examples include those suspected of being abnormal by abdominal ultrasonography or CT. In the case of lung cancer, examples include those suspected of having abnormalities in chest X-ray examinations. Furthermore, in the case of pancreatic cancer, examples include those who have abnormally high levels of amylase or lipase in the blood.

As described in detail later, 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. Examples of blood-derived specimens include serum or plasma.

If 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.

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. For example, a step of performing an antigen-antibody reaction using an antibody that detects the “tumor marker of the present invention”, a step of detecting with a lectin that recognizes the “tumor marker of the present invention”, and a liquid chromatography method (LC method) And a step of detecting using a mass spectrometry method (MS method), a step of detecting using a nuclear magnetic resonance method, and the like.

When using an antibody that detects the “tumor marker of the present invention”, the specific technique is not particularly limited as long as the “tumor marker of the present invention” is detected. For example, in addition to the RIA (Radioimmunoassay) method or the EIA (Enzyme immunoassay) method, it can be performed using a known method. The ELISA method can be used as the EIA method. As the ELISA method, for example, a sandwich ELISA method can be used. As the sandwich ELISA method, for example, a two-step sandwich method can be used.

The 2-step sandwich method can be performed, for example, by the following procedure. As a monoclonal antibody (hereinafter sometimes simply referred to as “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. As a first reaction, 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. As a second reaction, 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. Examples of antibody-bound particles include, but are not limited to, ferrite particles to which antibodies are bound. Examples of enzyme-labeled antibodies include, but are not limited to, antibodies labeled with alkaline phosphatase (ALP). When an ALP-labeled antibody is used, 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).

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.

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. For example, 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. Alternatively, for example, “average value of healthy persons” + (α × standard deviation) can be set. Here, α can be set as appropriate.

3. Tumor detection kit The 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”.

For example, when 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. Further, for example, when 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. In this case, a substrate solution (a solution containing the enzyme substrate in the enzyme-labeled antibody) 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.

4). Antibody The antibody of the present invention 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”.

By utilizing an antigen-antibody reaction using the antibody of the present invention, 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. For example, the following method can be mentioned.

Examples of immunogens include the following. For example, 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, immunized with ST1H-added glycolipid adsorbed on cells such as Salmonella minnesota. In addition, there is also a method using an immunogen that is not a glycolipid but a glycan portion bound to BSA (bovineserumalbumin) using a carrier.

Examples of 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.

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. For example, the following method can be mentioned.

Examples of 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.

In the ELISA method, 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 method for producing a monoclonal antibody that recognizes a sugar chain can also be carried out with reference to the following literature.
(1) Kannagi, R. 2000. Monoclonal anti-glycoshingolipid antibodies. Methods Enzymol. 312: 160-179.
(2) Yasuo Suzuki, Susumu Ando, Biochemical Experimental Method 35, Ganglioside Research Method I, Academic Publishing Center, p209-p237.

“Antibody-binding particles” or “enzyme-labeled antibodies” can be produced according to known methods, respectively.

5. Lectin 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. For the purification, 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. Create 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. In addition to affinity chromatography, 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. For the measurement of lectin activity, 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. Next, 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.

6). Screening Method for Antitumor Compound 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.

Specifically, 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.

More specifically, for example, 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. After the measurement, 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.

7). Method for Monitoring Cancer State (Disease State) 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.

Note that the above-described tumor detection kit can also be used to detect the tumor marker of the present invention in the monitoring method.

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the following examples.

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.

(1) Extraction of cancer cells from cancer tissue (i) Slice fresh cancer tissue (colon cancer, pancreatic cancer) and normal tissue (colon normal mucosa, normal pancreas tissue) as thin as possible with a feather blade under a stereomicroscope did.
(Ii) About 10 ml of DMEM / F12 medium containing 2 mg / ml Collagenase was added to the sliced piece and incubated at 37 ° C. for 30 minutes.
(Iii) The reaction solution was rubbed with a 500 μm stainless mesh to remove undigested matter, and the filtered solution was washed with PBS, centrifuged at 1500 rpm for 5 minutes to completely remove sup, and a cell pellet was obtained.
(Iv) Suspension the cell pellet with 1300 μl of PBE (PBS, 0.5% BSA, 2 mM EDTA), add 100 μl of FcR Blocking Reagent and 100 μl of CD326 MicroBeads (Milteny Biotec), and incubate at 4 ° C. for 30 minutes (Milteny Biotec protocol I followed.)
(V) The reaction solution was washed with PBE, and CD326 positive cells were separated with a MACS separation column (CD326 positive corresponds to cancer cells or normal mucosal epithelial cells).

(2) Structural analysis of glycolipids of cancer cells and normal mucosal epithelial cells The analysis was performed as described in the following paper.
Misonou, Y .; Shida, K .; Korekane, H .; Seki, Y .; Noura, S .; Ohue, M .; Miyamoto, Y., Comprehensive Clinico-Glycomic Study of 16 Colorectal Cancer Specimens: Elucidation of Aberrant Glycosylation and Its Mechanistic Causes in Colorectal Cancer Cells.J Proteome Res 2009, 8, (6), 2990-3005.

(3) Glycolipid extraction method (i) Chloroform: methanol (2: 1) 600 μl was added to the cell pellet and incubated at 37 ° C. for 2 hours. After centrifugation, the supernatant was collected. Again, 550 μl of chloroform: methanol: water (1: 2: 0.8) was added to the pellet, incubated at 37 ° C. for 2 hours, centrifuged, and the supernatant was recovered. The two extracts were combined to give a total lipid (total lipid).
(Ii) Neutral lipids and acidic lipids were separated by an ion exchange column (DEAE). DEAE Sephadex A25 (Amersham-Pharmacia Biotec) gel (0.1 ml) was packed in a minicolumn and equilibrated with chloroform: methanol: water (30: 60: 8).
(Iii) The extracted total lipid was applied to the column at 1 ml / min. This was washed with 0.8 ml of 100% methanol, and the eluates were combined to obtain a neutral lipid fraction. The acidic lipid fraction was eluted with 1 ml of methanol containing 200 mM ammonium acetate. Both were dried with a centrifugal concentrator.
(Iv) The dried acidic lipid fraction was dissolved in 50 μl of chloroform: methanol: water (5: 5: 1). The dried neutral lipid fraction was completely dissolved in 50 μl of chloroform: methanol (2: 1), added with 1000 μl of acetone, allowed to stand at 4 ° C. for 2 hours, and then centrifuged at 15000 rpm for 15 minutes. The supernatant was removed and the precipitate was quickly dried with a centrifugal concentrator. Add 50 μl of chloroform: methanol: water (5: 5: 1) containing 0.1N NaOH, incubate at 37 ° C for 1 hour, and then dilute the acetic acid stock solution 20 times with chloroform: methanol (5: 5: 1). 6 μl was added to neutralize.
(V) Desalting and removal of tissue-derived impurities by gel filtration chromatography.
(Vi) The support for gel filtration chromatography Toyopearl HW40C (TOSOH) was equilibrated with chloroform: methanol: water (5: 5: 1), and 1 ml was packed in the column.
(Vii) The neutral lipid fraction and acidic lipid fraction were applied to a column and eluted with chloroform: methanol: water (5: 5: 1). Glycolipid is eluted in 300μl-800μl. They were dried with a centrifugal concentrator.

(4) Glycan chain excision from glycolipid (i) 45 μl of 30 mM sodium acetate (pH 5.0), 0.1% taurodeoxycholate (1 mg / ml) was added to the dried acidic and neutral glycolipid and dissolved.
(Ii) 5 μl of recombinant Endoglycoceramidase II (TAKARA, 2 mU / μl) was added and reacted at 37 ° C. overnight.

(5) Fluorescent labeling of sugar chains with 2-aminopyridine (PA)
(I) The reaction solution was transferred to Reacti Vial (Plerce) and freeze-dried.
(Ii) 20 μl of a pyridylamination reagent (prepared by dissolving 552 mg of 2-aminopyridine in 200 μl of acetic acid) was added to the lyophilized sample and reacted at 90 ° C. for 1 hour.
(Iii) Thereafter, 70 μl of a reducing reagent (prepared by dissolving 100 mg of borane-dimethylamine in 40 μl of acetic acid and 25 μl of water) was added and reacted at 80 ° C. for 35 minutes.
(Iv) After completion of the reaction, excess 2-aminopyridine was removed using phenol-chloroform extraction, cation exchange resin Dowex 50W-X8 (NH4 + type) and the like.

(6) Separation and identification of pyridylaminated (PA) sugar chain Pyridylaminated (PA) sugar chain was separated by normal phase column and reverse phase column HPLC, two-dimensional sugar chain mapping method, mass spectrometry The structure is identified using the method and enzymatic digestion method.
When separating by normal phase and reverse phase HPLC, PA-oligoglucose oligomers (1 to 15) were simultaneously analyzed, and the elution position (minute) of each oligosaccharide was measured to calculate glucose.Unit.
A two-dimensional sugar chain map was created by plotting glucose.Unit on the reverse phase column on the horizontal axis and glucose.Unit on the normal phase column on the vertical axis. 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.

First, PA sugar chains were separated by normal phase HPLC.
TSK amide-80 2.0 X 250mm (TOSOH) was used.
Separation liquid
A: acetonitrile: 0.5M triethylamine acetate (pH 7.3), 10% acetonitrile 75: 15B: acetonitrile: 0.5M triethylamine acetate (pH 7.3), 10% acetonitrile 40:50 Flow rate is 0.2ml / min, separation B is 0 %, And 100 minutes after injection of the sample, the separation liquid B was linearly raised to 100%.

The fluorescence detector was set to an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm.

Each peak (PA sugar chain) separated by normal-phase HPLC was collected, dried with a centrifugal concentrator, dissolved in an appropriate amount of water, and further separated by reverse-phase HPLC.
ODS-80TS 2.0 X 150mm (TOSOH) was used.
Separation liquid
A: 50 mM triethylamine acetate (pH 6.0)
B: 50 mM triethylamine acetate (pH 6.0), 20% acetonitrile
The flow rate was 0.2 ml / min, the separation liquid B was 0%, and after the sample was injected, the separation liquid B was linearly increased to 18% in 54 minutes.

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.

(7) Discovery of a novel tumor marker and its detection In two cases (one colorectal cancer and one pancreatic cancer), a novel sugar chain structure was discovered.
The separation pattern of glycolipids of cancer cells of two patients is shown in FIG.

All PA sugar chains other than A8-2 in the peak match the PA standard sugar chain that is a known standard in the 2D sugar chain map and also in mass spectrometry, so the structure can be estimated. (Tables 1 to 4).

However, 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 (α-sialidase from Arthrobacter ureafaciens) (Nacalai), it matched with the standard Type1H on a two-dimensional map. In addition, when A8-2 was cleaved with α-fucosidase (from bovine kidney) (Sigma), it was changed to an unknown sialic acid-added sugar chain, which was further converted into α2-3 sialydase from Salmonella typhimurium (Takara Bio Inc.). When cut, it matches the standard Lc4 on the 2D map. However, although it was cleaved with α2-3 sialydase, sialic acid was not cleaved under conditions that specifically cleave α2-3 bonds, but under conditions that cleave both α2-3 and α2-6 bonds. Cleavage showed that sialic acid was α2-6 linked to terminal galactose. From the above results, this novel sugar chain structure could be identified as NeuAcα2-6 (Fucα1-2) Galβ1-3GlcNAcβ1-3Galβ1-4Glc.

This new sugar chain structure

(Wherein R represents a sugar residue (same as above))
Was named ST1H (abbreviation of α2-6 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 α2,3-sialidase from Salmonella typhimurium (Takara) or 2 U / ml α-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, α2,3-sialidase specifically cleaved α2,3-linked sialic acid to terminal galactose, but did not cleave α2,6-linked sialic acid. However, Arthrobacter α-sialidase cleaved both α2,3 and α2,6 -linked sialic acids regardless of the position of sialic acid addition. When α2,3-sialidase from Salmonella phi typhimurium (Takara) was reacted at 10 U / ml for 16 terminal h (condition 2), both sialic acids α2,3 and α2,6-linked to terminal galactose were cleaved.

10 / ml of α-fucosidase from bovine kidney (Sigma) under conditions of 100 mM sodium acetate buffer, pH 5.5, for 16 hr at 37 ° C, fucose, N-acetylglucosamine bound to galactose, α1,2 It is possible to cut fucose bonded to α1,3 and α1,4.

The predicted biosynthesis pathway of ST1H is shown in FIG. 3 along with other major glycolipid pathways. 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 . In contrast, Lewis (-) key glycolipid in normal cells is Lc ₄ and Type1H. In malignant tumors, Type 2 abundance, α2-3 and / or α2-6 sialylation, and α1-2 fucosylation rates are increased. For this reason, various sugar chain structures such as Le ^x , Le ^y , LST-c, SLe ^x and ST2H as Type 2 and SLe ^a , SLe ^c and ST1H as Type 1 appear. And it should be noted that Lewis (-) cancer cells do not show the activity of fucosyltransferase 3 (FUT3: advancing the reaction of the pathway represented by "4F" in FIG. 3), so Le ^a , SLe ^a and Le ^b are not produced, but instead SLe ^c and / or ST1H are specifically increased.

SLe ^a sugar chain structure (NeuAcα2-3Galβ1-3 (Fucα1-4) GlcNAcβ1-R ) and sLe ^c carbohydrate structures (NeuAcα2-3Galβ1-3GlcNAcβ1-R), depending respectively CA19-9 antibody and DU-PAN-2 antibody Be recognized.

In all 65 patients, (example colon cancer 56, pancreatic cancer 3 cases) 59 cases, (Reason 1) very high that Le ^a structure was observed in the sugar chain, also is (Reason 2) CA19-9 All 5U Since it was more than / ml, it was judged to be Lewis positive (Lewis (+)). 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.

At present, ST1H was expressed only in 3 of the 65 cases. This frequency is by no means low, assuming that ST1H や す い 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.

For the following reasons, ST1H is likely to be expressed in Lewis (-) human cancer. As shown in FIG. 3, in the Lewis (+) person, 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 . In cancer, it becomes SLe ^a via SLe ^c . Therefore, the antibody CA19-9 that recognizes SLe ^a as a tumor marker and recognizes it is used. On the other hand, it is known that 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. In the case of Lewis (-), Lc ₄ and Type 1H are the main products because Lewis enzyme (FUT3) has no activity. In cancer, SLe ^c is created from Lc ₄ , but it is not converted to SLe ^a , so the amount of SLe ^c increases. Thus, Lewis (-) with respect to recognize SLe ^c structure DU-PAN-2 antibodies are used in place of CA19-9 antibody. This discovery suggests that 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.

That is, like DU-PAN-2, ST1H can be used as a general tumor marker, and at the same time can be a better tumor marker for Lewis (-) people.

Claims (8)

1. A tumor marker having a sugar chain represented by the following formula (3).
   

   

   (Where NeuAc is the N-acetylneuraminic acid residue, Fuc is the fucose residue, Gal is the galactose residue, GlcNAc is the N-acetylglucosamine residue, α2-6 is the α2-6 glycosidic bond. Α1-2 represents an α1-2 glycoside bond, β1-3 represents a β1-3 glycoside bond, and R represents a sugar residue.)
2. An antibody against the tumor marker according to claim 1.
3. The antibody according to claim 2, which is a monoclonal antibody.
4. A lectin that recognizes the tumor marker according to claim 1.
5. A tumor detection kit comprising a material necessary for detecting the tumor marker according to claim 1.
6. The tumor detection kit according to claim 5, wherein the material necessary for detecting the tumor marker is an antibody against the tumor marker according to claim 1 or a lectin that recognizes the tumor marker according to claim 1.
7. A method for determining whether or not a specimen is derived from a cancer patient, wherein the tumor marker according to claim 1 is detected in the specimen, and the detected value is a detected value of the tumor marker in the specimen collected from a healthy subject A method for determining that a sample that exceeds a reference value set based on the above is derived from a cancer patient.
8. (I) a step of applying a candidate compound to the tumor cell in which the tumor marker according to claim 1 is expressed; and (ii) a candidate used when the expression of the tumor marker in the tumor cell is reduced or eliminated. A method for screening an antitumor compound, comprising a step of selecting the compound as an antitumor compound.

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