JP4748815B2 - Cancer and adenoma testing methods - Google Patents

Description

  The present invention relates to a method for examining human cancers and adenomas (excluding colorectal cancers and colorectal adenomas), and therefore a diagnostic agent.

  Cancer patients are increasing year by year, and examination methods for early detection are required. Currently, stool immune occult blood reaction and various tumor markers are used for cancer and adenoma testing, but none of them are satisfactory. That is, the positive rate of the fecal immunooccult blood test used for the examination of cancer and adenoma is 50 to 60%. For example, as a tumor marker for colon cancer, carcino-embryonic antigen (carcino-embryonic antigen, CEA), CA19-9, STX, etc. are used to determine therapeutic effects and monitor recurrence, but are not sufficient tumor markers for early colorectal cancer detection.

In normal colon, sulfation of sugar chain is plentiful, but in colorectal cancer, it is known that sulfation of sugar chain is remarkably reduced, and 3'-sulfation of galactose in the large intestine is also 6-sulfation of N-acetylglucosamine (hereinafter referred to as “GlcNAc”) is also reduced (Non-patent Document 1). Numerous isozymes of GlcNAc-6-sulfotransferase are known in patients with colorectal cancer tissue and non-cancerous colorectal tissue. Among them, I-GlcNAc6ST decreases markedly with canceration. A significant decrease in sulfation of sugar chains is explained (Non-Patent Document 2). On the other hand, GlcNAc6ST-1 which is one of the isozymes of non-cancerous large intestine does not show a significant change quantitatively with canceration. Furthermore, other isozymes, HEC-GlcNAc6ST, are markedly increased in cancer (Non-patent Document 4 ).

HEC-GlcNAc6ST, which increases in cancer, synthesizes 6-sulfated GlcNAc, but since this enzyme sulfates GlcNAc in various sugar chains, the structure and antigenicity of the sugar chains actually synthesized in cells are Very diverse. On the other hand, GlcNAc6ST-1 and I-GlcNAc6ST also synthesize 6-sulfated GlcNAc. Therefore, simply synthesizing 6-sulfated GlcNAc with HEC-GlcNAc6ST cannot be used for specific diagnosis of cancer or adenoma.
However, it has been found that the substrate specificity of GlcNAc6ST-1 and I-GlcNAc6ST is narrower than that of HEC-GlcNAc6ST (Non-patent Documents 3 and 4). Further, the present inventors have found that there is a 6-sulfated sugar chain that can be synthesized only by HEC-GlcNAc6ST and not synthesized by GlcNAc6ST-1 or I-GlcNAc6ST (Non-patent Document 5). Until now, there has been no concrete system that can diagnose cancer and adenoma.

On the other hand, it is known that a commercially available monoclonal antibody (MECA-79 antibody) (non-patent document 6) as an antibody against an immunological homing receptor for lymphocytes reacts with a chemically synthesized GlcNAc 6-sulfated sugar chain. (Non-patent Document 7). Furthermore, it has been reported that when a mouse HEC-GlcNAc6ST enzyme is introduced into CHO cells (hamster ovary cells), an antigen recognized by this antibody (MECA-79) appears on the cell surface (non-patent document). Reference 8).
The present inventors have already found that an antigen recognized by the MECA-79 antibody already exists in human colon cancer cells, and that the antibody can be used for examination of colon cancer and colon lineage (Japanese Patent Application 2003). -296216, PCT / JP2004 / 009805).

Izawa, M. et al., Cancer Res., 60: 1410-1416, 2000 Proceedings of the 22nd Japan Molecular Tumor Marker Conference, pp. 42-43, 2002 Seko, A. et al., Glycobiology, 10: 919-929, 2000 Seko, A. et al., Glycobiology, 12: 379-388, 2002 The Journal of Biological Chemistry, vol.277, No.6, 3979-3984 (2002) Streeter, P.R. et al., J. Cell Biol. 107: 1853-1862, 1988 Bruehl, R.E. et al., J. Biol. Chem. 275: 32642-32648, 2000 Yeh, J.C. et al., Cell 105: 957-969, 2001

  The present invention provides a test method and a test drug useful for diagnosing cancer and adenoma that can detect cancer patients and patients with high risk of cancer (excluding colorectal cancer patients and colorectal cancer) at a high rate. To do.

As a result of the study, the present inventors have found that there is a marked difference in the distribution of the glycosylsulfatase GlcNAc-6-sulfotransferase isozyme between non-cancerous tissues and cancer and adenoma tissues, and GlcNAc6ST- Cancer and adenomas (excluding colorectal cancer and colorectal adenoma) by detecting 6-sulfated glycans from patients and stool specimens that are not synthesized by 1 or I-GlcNAc6ST and can be synthesized only by HEC-GlcNAc6ST. It was clarified that can be detected specifically.
Conventional antibodies that react with GlcNAc-6-sulfated sugar chains include AG223 (Biochem. (Tokyo), 124: 670-678, 1998), G152, G72, AG97, AG107, AG273, G2706, G27011, G27039 (or more, J. Biol. Chem., 273: 11225-11233, 1998). On the other hand, the MECA-79 antibody (catalog number 09961D manufactured by Pharmingen, sold by Becton Dickinson) marketed as an antibody against the immunological homing receptor of lymphocytes also reacts with some GlcNAc-6-sulfated sugar chain. It is known to do (Non Patent Literature 6). Of these antibodies, GlcNAc-6-sulfated glycans that are commonly expressed in normal cells and weakly or not react with cells that express GlcNAc-6-sulfated glycans well. Screening for antibodies that are highly reactive with the cells to be expressed, and searching the obtained antibodies using patient-derived specimens, found that they are highly positive for various cancers. It came to complete.

That is, the present invention relates to the reaction of MECA-79 antibody (Farmingen catalog number 09961D) or an antibody having the same antigen specificity as the MECA-79 antibody against the tissue, body fluid or stool of a subject or an extract thereof. This is a method for examining cancer and adenoma (excluding colorectal cancer and colorectal adenoma).
Further, the present invention is the above test method, further comprising reacting a probe with a label to this antibody and detecting the label qualitatively or quantitatively.
A preferred test method is to immobilize an antigen present in a patient's tissue, body fluid or stool or an extract thereof on a support, react an antibody with the antigen, react a labeled probe with the antigen, Consisting of detecting. It is preferable to insert a washing step as appropriate between each step. Examples of the probe include anti-human IgG antibody, protein G, protein A, and protein L. This probe is usually labeled. Examples of the label include a radioisotope (125I) and an enzyme (peroxidase, alkaline phosphatase). When an enzyme antibody is used, the change (coloring or the like) may be observed by reacting the substrate.
The present invention also relates to cancers and adenomas (not including colorectal cancers and colorectal adenomas) mainly composed of MECA-79 antibody (Farmingen catalog number 09961D) or antibodies having the same antigen specificity as MECA-79 antibody. )).

In addition, cancers and adenomas (excluding colorectal cancer and colorectal adenomas) are examined by examining the presence or absence of reactivity or the reaction intensity of antibodies against the tissues, body fluids or feces of these subjects or their extracts. This antibody is present in cells that express the GlcNAc-6-sulfotransferase HEC-GlcNAc6ST gene and is absent or only in trace amounts in cells that express the GlcNAc6ST-1 or I-GlcNAc6ST gene Reacts specifically with antigen.
This antigen is present in cells into which the GlcNAc-6-sulfotransferase HEC-GlcNAc6ST gene has been introduced, and is not present in cells into which the GlcNAc6ST-1 or I-GlcNAc6ST gene has been introduced, or is present only in trace amounts. May be.

This antigen has the general formula
R1-Galβ1-3 / 4 (SO ₃ -6) GlcNAcβ1-R2
(In the formula, R1 is a sugar residue added by another enzyme group, and the structure is not particularly limited. Galβ represents β galactose, GlcNAcβ represents βN-acetylglucosamine, and Galβ1-3 / 4 represents Galβ. 1 indicates that GlcNAcβ is bonded to 3rd and / or 4th position, (SO ₃ -6) indicates that a sulfate group is added to 6th position of GlcNAcβ, and R2 represents −3GalNAcα, −3Galβ Or -2Manα, which binds to position 1 of GlcNAcβ.

In addition, an antigen having a sugar chain that is present in a cell that expresses the GlcNAc-6-sulfate transferase HEC-GlcNAc6ST gene and that is not present in a cell that expresses the GlcNAc6ST-1 or I-GlcNAc6ST gene, or is present only in a trace amount It can be used as a diagnostic agent for cancer and adenoma (excluding colorectal cancer and colorectal adenoma).
In addition, an antigen having a sugar chain that is present in a cell into which the GlcNAc-6-sulfotransferase HEC-GlcNAc6ST gene has been introduced and is not present in a cell into which the GlcNAc6ST-1 or I-GlcNAc6ST gene has been introduced, or is present only in a trace amount It can be used as a diagnostic agent for cancer and adenoma (excluding colorectal cancer and colorectal adenoma).

Also present in the tissues, body fluids or stool of cancer or adenoma patients,
R1-Galβ1-3 / 4 (SO ₃ -6) GlcNAcβ1-R2
(In the formula, R1 is a sugar residue added by another enzyme group, and the structure is not particularly limited. Galβ represents β galactose, GlcNAcβ represents βN-acetylglucosamine, and Galβ1-3 / 4 represents Galβ. 1 indicates that GlcNAcβ is bonded to 3rd and / or 4th position, (SO ₃ -6) indicates that a sulfate group is added to 6th position of GlcNAcβ, and R2 represents −3GalNAcα, −3Galβ Alternatively, it represents -2Manα and binds to position 1 of GlcNAcβ.) Cancers and adenomas (except for colorectal cancers and colorectal adenomas) whose main component is an antibody that specifically reacts with an antigen having a sugar chain represented by GlcNAcβ. It can be used as a test drug.

The structure of 6-sulfated sugar chain that can be synthesized only by HEC-GlcNAc6ST and not synthesized by GlcNAc6ST-1 or I-GlcNAc6ST is
_{R1-Galβ1-3 / 4 (SO 3} - -6) GlcNAcβ1-R2
It is represented by

In the body, GlcNAcβ, a partner with which GlcNAc-6-sulfate acts, is carried by various sugar chain carriers. R2 represents the carrier.
From studies by us and other researchers, it has been found that HEC-GlcNAc6ST has the ability to transfer sulfate groups to all GlcNAcβ1-R2 tested so far (Non-Patent Documents 4, 7, etc.). In contrast, GlcNAc6ST-1 and I-GlcNAc6ST have the ability to transfer a sulfate group only when having a specific R2 moiety.
While HEC-GlcNAc6ST to transfer sulfate group, the GlcNAc6ST-1 and I-GlcNAc6ST can not less transfer sulfate group refers to the structure after the case (sulfation R2 is -3GalNAcα is SO ₃ ^- -6GlcNAcβ1-3GalNAcα and become), if it is -3Galbeta (structure after sulfation SO ₃ ^- and the -6GlcNAcbeta1-3Galbeta), if it is -2Manarufa (structure after sulfation SO ₃ ^- a -6GlcNAcbeta1-2Manarufa) (J. Biol. Chem., 277: 3979-3984, 2002 and Glycobiology, 12: 379-388, 2002). In the test method of the present invention, an antibody specific for any of these three may be used, or an antibody that cross-reacts with all of the sugar chains of the three.

GlcNAc-6-sulfate enzyme in cells, the GlcNAc sugar chain terminus by adding a sulfate group as above 6-sulfated GlcNAc (i.e., SO ₃ ^- -6GlcNAc) synthesize. However, even after 6-sulfated GlcNAc is generated at the sugar chain end, a sugar residue (R1) is further added to this by other enzymes in the cell, so it is finally synthesized and produced from the cell. The structure and antigenicity of sugar chains vary widely. Usually, the structures first added to 6-sulfated GlcNAc are Galβ1-4 and Galβ1-3 (denoted as Galβ1-3 / 4). In addition to ^{_{this, NeuAcα2-3 / 6, SO 3 -}} -3 / 6, that Fucα1-2 / 3/4 is added are generally known. This R1 portion is added later after the synthesis of 6-sulfated GlcNAc by GlcNAc-6-sulfating enzyme is completed. For this reason, the R1 portion is not related to the substrate specificity of GlcNAc-6-sulfating enzymes such as HEC-GlcNAc6ST, GlcNAc6ST-1 and I-GlcNAc6ST.

  Such an antigen having a sugar chain is present in a cancer tissue obtained from a colorectal cancer patient by biopsy or surgery, or a sample such as serum, ascites, or feces containing a substance derived therefrom. From these, the antigen can be easily extracted with phosphate buffered saline. An antibody against this sugar chain antigen can be obtained using a known antibody production technique (for example, Methods in Enzymology, 312: 160-179, 2000; Methods in Molecular Biology, 199: 203-218, 2002, etc.). An example of this antibody is MECA-79 antibody (Pharmingen catalog number 09961D, shown in FIGS. 4 and 5).

The test method and test drug using the antibody of the present invention include not only colorectal cancer and colorectal adenoma, but also cancer in general and adenoma that is the origin of the cancer, that is, malignant tumor in general, that is, epithelial cancer and non-epithelial Applicable to cancer, preferably epithelial cancer.
Malignant tumors are classified into epithelial and non-epithelial cancers. Epithelial cancer is classified into adenocarcinoma, squamous cell carcinoma, and other epithelial cancers. Adenocarcinoma includes colon cancer, breast cancer, gallbladder cancer, stomach cancer, kidney cancer, ovarian cancer, prostate cancer, pancreatic cancer, Some lung cancer, thyroid cancer, bronchial cancer, bile duct cancer, fallopian tube cancer, salivary gland cancer, sputum, round cancer, etc. include squamous cell carcinoma, esophageal cancer, part of lung cancer, uterine cancer, oral cancer Tongue cancer, laryngeal cancer, pharyngeal cancer, skin cancer, vaginal cancer, penile cancer and the like, and other epithelial cancers include liver cancer, bladder cancer and the like. Non-epithelial cancers include leukemia, malignant lymphoma, brain tumor, as well as osteosarcoma, malignant melanoma, and fibrosarcoma.

The following examples illustrate the invention, but are not intended to limit the invention.
Reference example 1
First, Colo 201 cells were used as a human colon cancer cell line, and RT- was used as a normal colon epithelial cell line using TSA-SW480 cells treated with trichostatin A in SW480 cells (obtained from Tohoku University Medical Cell Resource Center). The gene expression of GlcNAc-6-sulfatase isozyme was examined by PCR.
In RT-PCR analysis, as a PCR primer for detection of HEC-GlcNAc6ST gene (Genebank AF131235) expression, a synthetic oligonucleotide of SEQ ID NO: 1 is used on the upper strand side and SEQ ID NO: 2 is used on the lower strand side (Tm = 59 ° C.) As a primer for detecting the expression of GlcNAc6ST-1 gene (Genebank AB011451), the synthetic strand of SEQ ID NO: 3 is used on the upper strand side and SEQ ID NO: 4 is used on the lower strand side (Tm = 62 ° C), and the I-GlcNAc6ST gene (Genebank AF176838) As a PCR primer for detecting expression, a synthetic oligonucleotide of SEQ ID NO: 5 was used on the upper strand side and SEQ ID NO: 6 was used on the lower strand side (Tm = 60 ° C.).
The result is shown in FIG. The colon cancer cell line (Colo 201 cell) was found to be a cell exhibiting a typical colon cancer pattern that strongly expresses the HEC-GlcNAc6ST gene and hardly expresses the GlcNAc6ST-1 and I-GlcNAc6ST genes. In addition, it was found that TSA-SW480 cells show typical normal epithelial patterns that hardly express HEC-GlcNAc6ST gene and significantly express GlcNAc6ST-1 and I-GlcNAc6ST gene.

Reference example 2
HEC-GlcNAc6ST (Genebank NM_005769), GlcNAc6ST-1 (Genebank NM_004267), I-GlcNAc6ST (Genebank NM_012126) gene cDNA along with drug-resistant neo gene in human colon cancer cell line SW480 (obtained from Tohoku Univ. Introduced. After drug selection and cloning, gene expression was confirmed by RT-PCR. In these cultures, stable expression was confirmed by monitoring gene expression by periodically detecting the 6-sulfotransferase gene product during maintenance culture.
These cancer cells are used to immunize mice according to conventional methods and react with cancer cells transfected with GlcNAc-6-sulfotransferase. However, GlcNAc-6-sulfotransferase GlcNAc6ST-1 or I-GlcNAc6ST Monoclonal antibodies that did not react with the transfected cancer cells were prepared. As a result, KN173, KN101, KN439, 7A4 and other antibodies satisfying the above conditions were obtained.

Indirect fluorescent antibody method for screening the reactivity between cells and antibodies (primary antibody 1.0 μg / ml, 4 ° C, 30 min. Secondary antibody is Zymed Laboratories rabbit anti-rat IgM antibody, 4 ° C, 30 min.) Then, flow cytometry analysis was performed with FACScan (Becton Dickinson).
After the monoclonal antibody obtained above was reacted at 4 ° C. for 30 min. As a primary antibody, FITC-labeled rabbit anti-mouse immunoglobulin antibody (Zymed Laboratories, 4 ° C., 30 min.) Was used as a secondary antibody. The cells were stained as in the method and analyzed with a Becton Dickinson instrument FACScan. The result is shown in FIG.
None of the antibodies reacted with cancer cells transfected with HEC-GclNAc6ST, and did not react with cancer cells transfected with GlcNAc6ST-1 or I-GlcNAc6ST.
In FIG. 2, the positive control antibody KN412 is a pan-6-sulfated antibody that reacts with any 6-sulfotransferase gene product, and is a control showing 6-sulfotransferase gene expression in the transfected cells. It is an antibody.

Example 1
HEC-GlcNAc6ST (Genebank NM_005769), GlcNAc6ST-1 (Genebank NM_004267), I-GlcNAc6ST (Genebank NM_012126) gene cDNA along with drug-resistant neo gene in human colon cancer cell line SW480 (obtained from Tohoku Univ. Introduced. After drug selection and cloning, gene expression was confirmed by RT-PCR. In these cultures, stable expression was confirmed by monitoring gene expression by periodically detecting the 6-sulfotransferase gene product during maintenance culture.
Indirect fluorescent antibody method for screening the reactivity between cells and antibodies (primary antibody 1.0 μg / ml, 4 ° C, 30 min. Secondary antibody is Zymed Laboratories rabbit anti-rat IgM antibody, 4 ° C, 30 min.) Then, flow cytometry analysis was performed with FACScan (Becton Dickinson).
After reacting MECA-79 antibody (Pharmingen catalog number 09961D) as the primary antibody at 4 ° C for 30 min., FITC-labeled rabbit anti-mouse immunoglobulin antibody (Zymed Laboratories, 4 ° C, 30 min.) As the secondary antibody. .) And was analyzed with a Becton Dickinson instrument FACScan. The result is shown in FIG.
MECA-79 antibody reacted slightly with cancer cells transfected with GlcNAc6ST-1 in addition to cancer cells transfected with HEC-GclNAc6ST.

Reference example 3
The 6-sulfated sugar chain in the sera of various cancer patients was measured by a sandwich ELISA system using the antibody secreted by clone 7A4 obtained in Reference Example 2. A patient serum sample is reacted on a microplate on which monoclonal antibody 7A4 secreted by clone 7A4 is immobilized, followed by reaction with the same antibody labeled with biotin, followed by reaction with streptavidin-labeled Horse Radish Peroxidase. The reaction was measured at an absorbance of 450 nm using the absorbance of the sample at 620 nm as a control. The average (normal) + 2SD was taken as the cut-off line, and positive and negative were determined.
The result is shown in FIG. Breast cancer, pancreatic cancer, gall bladder, esophageal cancer, gastric cancer, gastric cancer, hepatocellular, renal cancer (kidney), prostate cancer (prostate), lung cancer (lung), It was found that the sulfated sugar chain increased in a wide range of cancers such as ovarian cancer and uterine cancer. All healthy subjects were negative.

It is a figure which shows the flow cytometry analysis result using a MECA-79 antibody about a colon cancer cell strain (COLO201 cell) and a normal colon epithelial cell strain (SW480 cell treated with trichostatin A). It is a figure which shows the reactivity with various antibodies by a flow cytometry method. The vertical axis represents cell frequency (cell number), and the horizontal axis represents fluorescence intensity (Arbitrary Unit). Transfectant indicates a transgenic cell. It is a figure which shows the example of a measurement of the sugar_chain | carbohydrate in the sera of various cancer patients by the sandwich ELISA system using antibody 7A4. It is a figure which shows the catalog of MECA-79 antibody (Pharmingen company catalog number 09961D). It is a figure which shows the catalog of MECA-79 antibody (Pharmingen company catalog number 09961D).

Claims (4)

From examining the reactivity of MECA-79 antibody (Farmingen catalog number 09961D) or antibody having the same antigen specificity as MECA-79 antibody against the tissues, body fluids or stool of the subject or extracts thereof A test method for cancer and adenomas (excluding colorectal cancer and colorectal adenoma). The test method according to claim 1, comprising reacting a probe with a label to the antibody and detecting the label qualitatively or quantitatively. The test method according to claim 1 or 2, wherein the cancer and adenoma are epithelial cancer and adenoma which is the origin of the cancer. A test agent for cancer and adenoma (excluding colorectal cancer and colorectal adenoma) mainly composed of MECA-79 antibody (Farmingen catalog number 09961D) or an antibody having the same antigen specificity as MECA-79 antibody .