WO2015068851A1 - Method for distinguishing between prostate cancer and prostatic hyperplasia - Google Patents
Method for distinguishing between prostate cancer and prostatic hyperplasia Download PDFInfo
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- WO2015068851A1 WO2015068851A1 PCT/JP2014/079863 JP2014079863W WO2015068851A1 WO 2015068851 A1 WO2015068851 A1 WO 2015068851A1 JP 2014079863 W JP2014079863 W JP 2014079863W WO 2015068851 A1 WO2015068851 A1 WO 2015068851A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- 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
- C07K16/3076—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 against structure-related tumour-associated moieties
- C07K16/3092—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 against structure-related tumour-associated moieties against tumour-associated mucins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4725—Mucins, e.g. human intestinal mucin
Definitions
- the present invention relates to prostate cancer and prostatic hypertrophy characterized by measuring mucin-1 (Mucin-1, hereinafter also referred to as “MUC1”) having a modified sugar chain among glycoprotein mucins. It relates to an identification method.
- MUC1 mucin-1
- MUC1 a type of mucin, is a high molecular weight glycoprotein that is a tumor-related antigen and is expressed in many adenocarcinomas.
- This glycoprotein is a membrane glycoprotein, and the extracellular domain has a region composed of 30-90 tandem repeats called repeat sequences. The number of repeat repeats varies from individual to individual and is genetically determined, resulting in a size polymorphism.
- CA15-3 which is a tumor marker for breast cancer, is an example of a diagnostic marker that measures MUC1 in the bloodstream.
- mucin is low organ specificity. Therefore, CA15-3 is regarded as a marker that is not suitable for the definitive diagnosis of breast cancer, and is mainly used for the purpose of determining the therapeutic effect.
- Patent Document 1 provides a relatively specific antibody 1B2 that binds when a specific sugar chain is added to the ninth threonine in the repeat sequence of MUC1 represented by SEQ ID NO: 1.
- a -1B2 sandwich immunoassay has been performed.
- Patent Document 2 provides an antibody 12D10 having a relatively high specificity when a specific sugar chain is bound to the repeat sequence of MUC1 represented by SEQ ID NO: 1, and a 12D10-12D10 sandwich assay is performed. ing.
- cancer diagnosis using these antibodies also has a problem that organ specificity is low like the antibody recognizing the core structure of MUC1, and the MUC1 recognition antibody having a sugar chain as a part of the antigen recognition site was used. No good diagnostic methods for specific cancers are provided.
- Prostate cancer is generally considered to be able to be specifically diagnosed by measuring the amount of total PSA in the blood.
- the total PSA value rises, and in particular when the total PSA value is low (3.0 to 10.0 ng / ml), there is a problem that prostate cancer and prostatic hypertrophy cannot be distinguished, There is a need for a diagnostic method that can distinguish between prostate cancer and benign prostatic hyperplasia.
- Non-Patent Document 1 it is reported that MUC1 modified with a sialyl core 1O-type sugar chain structure is expressed on the cell surface of prostate cancer tissue or prostate cancer-derived cells.
- no diagnostic technique using the amount of MUC1 that has undergone a specific sugar chain modification in body fluid as an index, or a method for distinguishing prostate cancer from hypertrophy has been reported.
- Non-Patent Document 2 it is reported that MUC1 having a sugar chain is expressed in both prostate cancer tissue and prostatic hypertrophy tissue.
- the antibody used here is an antibody that recognizes the peptide portion of MUC1, and does not recognize a sugar chain.
- cancer-related sugar chains of MUC1 there is no mention of cancer-related sugar chains of MUC1, and a method for discriminating cancer and hypertrophy using the amount of MUC1 having a sugar chain as an index has not been reported.
- An object of the present invention is to provide a method for discriminating between a prostate cancer patient and a prostatic hypertrophy patient using MUC1.
- the present invention is as follows.
- a method for discriminating between prostate cancer and benign prostatic hyperplasia which comprises measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
- (A) Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3GalNAc ⁇ , Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, and GalNAc ⁇ - One or more selected from the group consisting of: (B) Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ , Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ , and Neu5Ac ⁇ 2 ⁇ 6GalNAc ⁇
- Neu5Ac ⁇ 2 ⁇ 6GalNAc ⁇ One or more selected from the group consisting of: (However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine) (4) The method according to any one of (1) to (3) above, wherein the sugar chain of the cancer-related structure is bound to the repeat sequence of mucin-1 (SEQ ID NO: 1).
- An antibody that recognizes mucin-1 having a sugar chain (A) having the following cancer-related structure, a fragment thereof, and / or a chemically modified derivative thereof, and a mucin having a sugar chain (B) having the following cancer-related structure A kit for distinguishing prostate cancer from benign prostatic hyperplasia, comprising an antibody that recognizes -1, a fragment thereof, and / or a chemically modified derivative thereof.
- A Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3GalNAc ⁇ , Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, and GalNAc ⁇ -
- B Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ , Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ , and Neu5Ac ⁇ 2 ⁇ 6GalNAc ⁇
- a method for diagnosing prostate cancer or benign prostatic hyperplasia comprising measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
- a method for measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject for distinguishing between prostate cancer and benign prostatic hyperplasia (10) A method for measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject for distinguishing between prostate cancer and benign prostatic hyperplasia.
- a method of discriminating between prostate cancer and benign prostatic hyperplasia including a step of determining that there is.
- the method of the present invention it is possible to discriminate between prostate cancer and benign prostatic hyperplasia that were difficult with the conventional method.
- FIG. It is a figure which shows distribution of the amount of MUC1 measured by sandwich ELISA measurement in Example 1 and 2.
- FIG. It is a figure which shows the distribution of the amount of MUC1 measured by 1B2-12D10 sandwich ELISA measurement and 1B2-1B2 sandwich ELISA measurement in the serum obtained from a prostatic hypertrophy patient and a prostate cancer patient, and the distribution of a total PSA value. It is the result of ROC analysis from the amount of MUC1 measured by 1B2-12D10 sandwich ELISA measurement and 1B2-1B2 sandwich ELISA measurement, and total PSA value in serum obtained from patients with benign prostatic hyperplasia and prostate cancer.
- the identification method of the present invention is characterized in that MUC1 having a sugar chain having a cancer-related structure is measured by the above-described method, and prostate cancer and prostatic hypertrophy are identified from the obtained values.
- Mucin-1 is a tumor-associated antigen and is a high molecular weight glycoprotein that is expressed in many adenocarcinomas.
- This glycoprotein is a membrane glycoprotein, and its extracellular domain has a 30-90 tandem repeat called a repeat sequence consisting of 20 amino acid core sequences rich in serine, threonine and proline (HGVTSAPDTTRPAPGSTAPPA (SEQ ID NO: 1)).
- HGVTSAPDTTRPAPGSTAPPA SEQ ID NO: 1
- cancer-related structure refers to a structure (eg, sugar chain structure) that is highly expressed in cancer tissue or cancer cells. This structure is known to have a low expression level in normal tissues and normal cells.
- sugar chain of such a cancer-related structure include Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, GalNAc ⁇ -, Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ 3Nal6
- Examples include GalNAc ⁇ - and Neu5Ac ⁇ 2 ⁇ 6GalNAc ⁇ -.
- Neu5Ac is N-acetylneuraminic acid
- Gal is galactose
- GalNAc is N-acetylgalactosamine
- Neu5Ac ⁇ 2 ⁇ 3Gal indicates that the position 2 of Neu5Ac and the position 3 of Gal are ⁇ -glycosidically bonded
- Gal ⁇ 1 ⁇ 3GalNAc indicates that position 1 of Gal and GalNAc
- Neu5Ac ⁇ 2 ⁇ 6GalNAc indicates that the 5th position of Neu5Ac and 6th position of GalNAc are ⁇ -glycosidically bonded
- Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc is It shows that the position 3 of GalNAc in which position 2 of Neu5Ac and position 6 of GalNAc are ⁇ -glycoside-bonded and position 1 of Gal are ⁇ -glycoside-bonded.
- MUC1 having a sugar chain having a cancer-related structure refers to a MUC1 molecule having a sugar chain having the cancer-related structure. More preferably, one or more sugar chains selected from (A) Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, Gal ⁇ 1 ⁇ 3GalNAc ⁇ -, and GalNAc ⁇ - as a sugar chain of a cancer-related structure, and (B) Neu5Ac ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 3 ( MUC1 having one or more sugar chains selected from Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ -, Gal ⁇ 1 ⁇ 3 (Neu5Ac ⁇ 2 ⁇ 6) GalNAc ⁇ -, and Neu5Ac ⁇ 2 ⁇ 6GalNAc ⁇ - in the same molecule.
- the sugar chain (A) and the sugar chain (B) are bonded to the amino acid residue of MUC1 via GalNAc on the reducing end side.
- the sugar chain (A) is preferably bound to the repeat sequence (SEQ ID NO: 1) of MUC1, and specifically, preferably bound to the serine residue or threonine residue of the repeat sequence. Among them, it is more preferable that the 9th threonine of the repeat sequence (SEQ ID NO: 1) is bound.
- the sugar chain (B) is also preferably bound to a repeat sequence (SEQ ID NO: 1) of MUC1.
- the method for measuring MUC1 having a sugar chain having a cancer-related structure is not particularly limited, but an immunological method using an antibody is preferred.
- an antibody that recognizes the sugar chain (A) and an antibody that recognizes the sugar chain (B) can be used in combination.
- an antibody that also recognizes the amino acid of MUC1 can be used as at least one of the two types of antibodies.
- both antibodies may also recognize the amino acid of MUC1.
- MUC1 having the sugar chain (A) and the sugar chain (B) in the same molecule can be reliably measured.
- Examples of such a measurement method using two types of antibodies include a sandwich method using two types of antibodies, an immunoprecipitation / immunostaining method in which immunoprecipitation is performed with one antibody and then immunostaining with the other antibody. Can give.
- the “sandwich method” is a method for tracking an antigen by reacting two antibodies that specifically bind to the antigen with the antigen. For example, in a method in which one of the antibodies used is bound to a carrier and reacted with an antigen in a sample to supplement the antigen, and the other antibody is also reacted to the antigen to form an antigen-antibody complex. is there.
- the formed antigen-antibody complex is detected by labeling at least one antibody or reacting a secondary antibody.
- detecting a molecule having a multivalent antigenic epitope such as MUC1 it is also possible to sandwich using the same antibody.
- the “immunoprecipitation” method of the “immunoprecipitation / immunostaining method” refers to a method of specifically precipitating a target antigen from a solution with an antibody.
- an antigen can be easily purified and detected by using an antibody bound to a particulate carrier made of a polymer such as Sepharose.
- the “immunoprecipitation method” in the “immunoprecipitation / immunostaining method” means that proteins are separated by electrophoresis according to the properties of the protein such as molecular weight and isoelectric point, and then transferred to a highly hydrophobic membrane. This refers to an immunological technique for detecting a protein with an antibody. In the present invention, these immunoprecipitation methods and immunostaining methods can be used in combination.
- the antibody used in the present invention is not particularly limited, but can be prepared by a known technique (for example, the methods described in Patent Documents 1 and 2).
- an antibody that recognizes the structure in which the sugar chain (A) is bound to the 9th threonine of the MUC1 repeat sequence (SEQ ID NO: 1) is preferable.
- Patent Document 1 describes antibody 1B2.
- Patent Document 2 describes antibody 12D10 as an example of such an antibody.
- the sample derived from the subject used in the present invention is not particularly limited, and examples thereof include tissue, blood, plasma, and urine, and serum and plasma are particularly preferable. Further, a body fluid (serum, plasma, etc.) of a patient suspected of having prostate cancer is preferable, and a body fluid of a patient whose blood total PSA value is 3.0 to 10.0 ng / ml is particularly preferable. In the PSA test in Japan, the normal value is 3.0 ng / ml or less in the youngest age group of 50 to 64 years in the test group.
- the total PSA value is 3.0 to 10.0 ng / ml
- prostate cancer and benign prostatic hyperplasia cannot be distinguished by the conventional method using only the blood total PSA value as an index.
- PSA refers to a prostate specific antigen (Prostatic Specific Antigen).
- total PSA mainly refers to bound PSA and free PSA bound to antichymotrypsin, that is, the sum of bound PSA and free PSA.
- Free PSA refers to free PSA that is not complexed.
- the present invention also provides an antibody that recognizes MUC1 having the sugar chain (A), an antibody fragment that can bind to the antigen, and / or a chemically modified derivative thereof, and an antibody that recognizes MUC1 having the sugar chain (B). Also included are kits comprising antibody fragments capable of binding to the antigen and / or chemically modified derivatives thereof. By using this kit, it is possible to easily distinguish between prostate cancer and prostatic hypertrophy.
- a method for discriminating between prostate cancer and benign prostatic hyperplasia distinguishes (or determines) prostate cancer from benign prostatic hyperplasia in vitro based on the amount of MUC1 having a sugar chain of a cancer-related structure in a sample. It is a method to do. For example, when the value of the amount of MUC1 having a sugar chain of a cancer-related structure in a sample derived from a subject is larger than a cutoff value calculated in advance, the subject suffers from prostate cancer rather than prostatic hypertrophy Alternatively, it is possible to identify (or determine) that there is a possibility.
- the method for determining the cut-off value is not particularly limited.
- the value ⁇ 3SD, the average value of patients with benign prostatic hyperplasia may be used as the cutoff value.
- the mean value of patients with benign prostatic hyperplasia is ⁇ 2 SD.
- the cutoff value may be determined using a Roc curve.
- An example of a method using an ROC curve is to measure a target marker in biological samples of patients with benign prostatic hyperplasia and prostate cancer, and measure sensitivity (positive rate), specificity, and false positive rate (1 -Specificity) is determined, and the ROC curve is created by plotting the false positive rate (1-specificity) on the X axis and the sensitivity (positive rate) on the Y axis.
- the cutoff value may be determined by a method using Youden index.
- the cut-off value varies depending on the definition of each patient and the method of measuring a marker. Therefore, it is preferable to confirm the measurement value of the prostatic hypertrophy patient and the prostate cancer patient in advance, determine the cutoff value, and detect the target marker accordingly.
- Example 1 Measurement of MUC1 in human serum samples by sandwich ELISA method Measurement of MUC1 in serum samples of prostate cancer patients and patients with benign prostatic hyperplasia
- Measurement of MUC1 in human serum specimens was performed by the sandwich ELISA method described below. 50 ⁇ l of a phosphate buffer solution (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) containing 25 ng of anti-MUC1 antibody 1B2 (see Patent Document 1) was added to each well of a 96-well microtiter plate (made by Greiner). The temperature was fixed for 16 hours.
- a phosphate buffer solution 50 mM sodium phosphate, 150 mM NaCl, pH 7.4
- anti-MUC1 antibody 1B2 see Patent Document 1
- Each well was washed 3 times with a washing solution (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) containing 300 ⁇ l of a surfactant, and then 25 ng of biotin-labeled anti-MUC1 antibody 12D10 (Patent Document) 2) was added, and the mixture was allowed to stand at room temperature for 1 hour.
- a washing solution (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4
- biotin-labeled anti-MUC1 antibody 12D10 Patent Document 2
- Example 2 As in Example 1, except that the serum as the specimen was diluted 10-fold, and the reaction was performed using HRP-labeled anti-MUC1 antibody 1B2 instead of biotin-labeled anti-MUC1 antibody 12D10, and each well was treated with a 300 ⁇ l interface. After washing with a washing solution containing an activator three times, 50 ⁇ l of TMB solution was added to develop color for 10 minutes, and then the reaction was stopped by adding a 1M phosphoric acid solution, and the absorbance at 450 nm was measured. The result is shown in FIG.
- Comparative Example 1 The total PSA of the sample similar to that in Example 1 was measured.
- the total PSA value of the specimen is a value measured using an immunoassay device Roche E170 and a PSA measurement reagent at the time of blood collection. The results are shown in FIG.
- the 1B2-12D10 sandwich ELISA measurement of Example 1 showed a larger value in the serum of prostate disease (prostate cancer and benign prostatic hyperplasia) patients than in the healthy subject. Furthermore, when prostate cancer and benign prostatic hypertrophy patients are compared, it is clearly high in prostate cancer patients, and it becomes clear that 1B2-12D10 sandwich measurement can distinguish prostate cancer from prostatic hypertrophy. It was. This indicates that there is a significant difference in the p-value of less than 0.05 in the results of the significant difference test between the two groups when divided into the prostate cancer and prostatic hypertrophy patient groups (FIG. 1). It was.
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Abstract
Provided is a method for distinguishing between prostate cancer and prostatic hyperplasia which has hereto been difficult to achieve. By measuring the quantity of MUC1, which contains sugar chains of cancer-associated structures, it is possible to distinguishing between prostate cancer and prostatic hyperplasia. The present invention provides a method for distinguishing between prostate cancer and prostatic hyperplasia from a value obtained by measuring MUC1 having, inside the same molecule, sugar chains of cancer-associated structures such as Neu5Acα2→3Galβ1→3GalNAcα-, and sugar chains of cancer-associated structures such as Neu5Acα2→3Galβ1→3(Neu5Acα2→6)GalNAcα-, by sandwich assay etc. using two antibodies.
Description
本発明は、糖タンパク質ムチンの中でも、修飾されている糖鎖の異なるムチン－１（Ｍｕｃｉｎ－１、以下、「ＭＵＣ１」とも称する）を測定することを特徴とする前立腺癌と前立腺肥大症との識別方法に関する。
The present invention relates to prostate cancer and prostatic hypertrophy characterized by measuring mucin-1 (Mucin-1, hereinafter also referred to as “MUC1”) having a modified sugar chain among glycoprotein mucins. It relates to an identification method.
ムチンの一種であるＭＵＣ１は、腫瘍関連抗原であり、多くの腺癌で発現する高分子量糖タンパク質である。この糖タンパク質は、膜糖タンパク質であり、細胞外ドメインには、リピート配列と呼ばれる３０～９０タンデム型反復から構成される領域が存在する。リピート配列の反復回数は、個体によって異なり、遺伝的に決定され、サイズ多型を生じる。リピート配列内にはＯ型糖鎖修飾可能なアミノ酸であるセリン、トレオニンが計５個あり、それぞれのアミノ酸にＯ型糖鎖が付加することは可能であるが、付加しているＯ型糖鎖の種類や、付加しているアミノ酸の部位は、癌の種類によって様々な組み合わせがあり、不均一である。一般的に、癌では糖鎖付加量が減少するためペプチド骨格のコア配列が露出することが知られ、露出したペプチド骨格に対する抗体を用いた免疫診断が確立されている。特に、乳癌の腫瘍マーカーであるＣＡ１５－３は血流中のＭＵＣ１を測定する診断マーカーの一例である。一般に、ムチンを診断マーカーとする欠点として臓器特異性の低さが挙げられる。そのため、ＣＡ１５－３は乳癌の確定診断には適さないマーカーと見なされ、主に治療効果を判定する目的で利用されている。
MUC1, a type of mucin, is a high molecular weight glycoprotein that is a tumor-related antigen and is expressed in many adenocarcinomas. This glycoprotein is a membrane glycoprotein, and the extracellular domain has a region composed of 30-90 tandem repeats called repeat sequences. The number of repeat repeats varies from individual to individual and is genetically determined, resulting in a size polymorphism. There are a total of five serine and threonine amino acids that can modify O-type sugar chains in the repeat sequence, and it is possible to add O-type sugar chains to each amino acid. There are various combinations of the types of amino acids and added amino acid sites depending on the type of cancer, and they are uneven. In general, it is known that the core sequence of the peptide backbone is exposed because the amount of glycosylation decreases in cancer, and immunodiagnosis using an antibody against the exposed peptide backbone has been established. In particular, CA15-3, which is a tumor marker for breast cancer, is an example of a diagnostic marker that measures MUC1 in the bloodstream. In general, a drawback of using mucin as a diagnostic marker is low organ specificity. Therefore, CA15-3 is regarded as a marker that is not suitable for the definitive diagnosis of breast cancer, and is mainly used for the purpose of determining the therapeutic effect.
糖鎖を抗原認識部位の一部とするＭＵＣ１認識抗体としては、ＭＹ．１Ｅ１２やＫＬ６が知られている。さらに、特許文献１では、配列番号１で示されたＭＵＣ１のリピート配列中の９番目のトレオニンに特定の糖鎖が付加した場合に結合する、比較的特異性の高い抗体１Ｂ２が提供され、１Ｂ２－１Ｂ２サンドイッチイムノアッセイが行われている。一方、特許文献２では、配列番号１で示されたＭＵＣ１のリピート配列に特定の糖鎖が結合した場合に結合する比較的特異性の高い抗体１２Ｄ１０が提供され、１２Ｄ１０－１２Ｄ１０サンドイッチアッセイが行われている。しかしながらこれらの抗体を使用した癌の診断も、ＭＵＣ１のコア構造を認識する抗体と同様に臓器特異性が低いという問題があり、糖鎖を抗原認識部位の一部とするＭＵＣ１認識抗体を使用した特定の癌に対する性能の良い診断方法は提供されていない。
As the MUC1-recognizing antibody having a sugar chain as a part of the antigen recognition site, MY. 1E12 and KL6 are known. Furthermore, Patent Document 1 provides a relatively specific antibody 1B2 that binds when a specific sugar chain is added to the ninth threonine in the repeat sequence of MUC1 represented by SEQ ID NO: 1. A -1B2 sandwich immunoassay has been performed. On the other hand, Patent Document 2 provides an antibody 12D10 having a relatively high specificity when a specific sugar chain is bound to the repeat sequence of MUC1 represented by SEQ ID NO: 1, and a 12D10-12D10 sandwich assay is performed. ing. However, cancer diagnosis using these antibodies also has a problem that organ specificity is low like the antibody recognizing the core structure of MUC1, and the MUC1 recognition antibody having a sugar chain as a part of the antigen recognition site was used. No good diagnostic methods for specific cancers are provided.
前立腺癌は、一般に血中トータルＰＳＡ量を測定することによって特異的に診断することが可能であると考えられている。しかし前立腺肥大症患者においてもトータルＰＳＡ値は上昇し、特にトータルＰＳＡ値が低値（３．０～１０．０ｎｇ／ｍｌ）の場合において、前立腺癌と前立腺肥大症を識別できないという問題があり、前立腺癌と前立腺肥大症を識別可能な診断方法が必要とされている。
Prostate cancer is generally considered to be able to be specifically diagnosed by measuring the amount of total PSA in the blood. However, even in patients with benign prostatic hyperplasia, the total PSA value rises, and in particular when the total PSA value is low (3.0 to 10.0 ng / ml), there is a problem that prostate cancer and prostatic hypertrophy cannot be distinguished, There is a need for a diagnostic method that can distinguish between prostate cancer and benign prostatic hyperplasia.
非特許文献１によれば、前立腺癌組織または前立腺癌由来細胞の細胞表面にはシアリルコア１Ｏ型糖鎖構造によって修飾されたＭＵＣ１が発現することが報告されている。しかし、体液中の特定の糖鎖修飾を受けたＭＵＣ１量を指標とした診断技術もしくは、前立腺癌と肥大症を識別する方法は報告されていない。
According to Non-Patent Document 1, it is reported that MUC1 modified with a sialyl core 1O-type sugar chain structure is expressed on the cell surface of prostate cancer tissue or prostate cancer-derived cells. However, no diagnostic technique using the amount of MUC1 that has undergone a specific sugar chain modification in body fluid as an index, or a method for distinguishing prostate cancer from hypertrophy has been reported.
非特許文献２によれば、前立腺癌組織及び前立腺肥大症組織のいずれにおいても、糖鎖を有するＭＵＣ１が発現していると報告されている。しかし、ここで用いられている抗体は、ＭＵＣ１のペプチド部分を認識する抗体であって、糖鎖を認識するものではない。また、ＭＵＣ１の癌関連糖鎖についての言及はなく、糖鎖を有するＭＵＣ１の量を指標とした癌と肥大症を識別する方法は報告されていない。
According to Non-Patent Document 2, it is reported that MUC1 having a sugar chain is expressed in both prostate cancer tissue and prostatic hypertrophy tissue. However, the antibody used here is an antibody that recognizes the peptide portion of MUC1, and does not recognize a sugar chain. Moreover, there is no mention of cancer-related sugar chains of MUC1, and a method for discriminating cancer and hypertrophy using the amount of MUC1 having a sugar chain as an index has not been reported.
本発明は、ＭＵＣ１を用いた前立腺癌患者と前立腺肥大症患者を識別する方法を提供することを目的とする。
An object of the present invention is to provide a method for discriminating between a prostate cancer patient and a prostatic hypertrophy patient using MUC1.
本発明者は上記課題について鋭意検討した結果、本発明に到達した。即ち本発明は、以下の通りである。
（１）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定することを特徴とする、前立腺癌と前立腺肥大症を識別する方法。
（２）前記ムチン－１が、下記の群より選択される１種以上の癌関連構造の糖鎖を有するものである、前記（１）に記載の方法。
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、
ＧａｌＮＡｃα－、
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（３）前記ムチン－１が、下記の癌関連構造の糖鎖（Ａ）及び（Ｂ）を有するものである、前記（１）又は（２）に記載の方法。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（４）癌関連構造の糖鎖がムチン－１のリピート配列（配列番号１）に結合している、前記（１）～（３）いずれかに記載の方法。
（５）癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、及び癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体を組み合わせて用いる、前記（３）に記載の方法。
（６）サンドイッチ法又は免疫沈降／免疫染色法により測定することを特徴とする、前記（１）～（５）のいずれかに記載の方法。
（７）前記被検体は血中トータルＰＳＡ値が３.０～１０.０ｎｇ/ｍｌである、前記（１）～（６）のいずれかに記載の方法。
（８）下記の癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体、並びに下記の癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体を含有することを特徴とする、前立腺癌と前立腺肥大症の識別用キット。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（９）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定することを特徴とする、前立腺癌又は前立腺肥大症の診断方法。
（１０）前立腺癌と前立腺肥大症との識別のための被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定する方法。
（１１）前立腺癌又は前立腺肥大症の診断用医薬の製造のための、被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体の使用。
（１２）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定する工程およびその測定値がカットオフ値よりも大きい場合に前記試料が前立腺癌患者由来であると判断する工程を含む前立腺癌と前立腺肥大症を識別する方法。 As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.
(1) A method for discriminating between prostate cancer and benign prostatic hyperplasia, which comprises measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
(2) The method according to (1) above, wherein the mucin-1 has one or more sugar chains having a cancer-related structure selected from the following group.
Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-,
GalNAcα-,
Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−;
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(3) The method according to (1) or (2) above, wherein the mucin-1 has sugar chains (A) and (B) having the following cancer-related structures.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(4) The method according to any one of (1) to (3) above, wherein the sugar chain of the cancer-related structure is bound to the repeat sequence of mucin-1 (SEQ ID NO: 1).
(5) The above (3), wherein an antibody that recognizes mucin-1 having a sugar chain (A) having a cancer-related structure and an antibody that recognizes mucin-1 having a sugar chain (B) having a cancer-related structure are used in combination. The method described in 1.
(6) The method according to any one of (1) to (5) above, wherein measurement is performed by a sandwich method or an immunoprecipitation / immunostaining method.
(7) The method according to any one of (1) to (6), wherein the subject has a blood total PSA value of 3.0 to 10.0 ng / ml.
(8) An antibody that recognizes mucin-1 having a sugar chain (A) having the following cancer-related structure, a fragment thereof, and / or a chemically modified derivative thereof, and a mucin having a sugar chain (B) having the following cancer-related structure A kit for distinguishing prostate cancer from benign prostatic hyperplasia, comprising an antibody that recognizes -1, a fragment thereof, and / or a chemically modified derivative thereof.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(9) A method for diagnosing prostate cancer or benign prostatic hyperplasia, comprising measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
(10) A method for measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject for distinguishing between prostate cancer and benign prostatic hyperplasia.
(11) An antibody recognizing mucin-1 having a sugar chain of a cancer-related structure contained in a sample derived from a subject for the manufacture of a diagnostic cancer for prostate cancer or benign prostatic hyperplasia, a fragment thereof, and / or Use of its chemically modified derivatives.
(12) a step of measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject, and when the measured value is greater than a cutoff value, the sample is derived from a prostate cancer patient A method of discriminating between prostate cancer and benign prostatic hyperplasia including a step of determining that there is.
（１）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定することを特徴とする、前立腺癌と前立腺肥大症を識別する方法。
（２）前記ムチン－１が、下記の群より選択される１種以上の癌関連構造の糖鎖を有するものである、前記（１）に記載の方法。
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、
ＧａｌＮＡｃα－、
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（３）前記ムチン－１が、下記の癌関連構造の糖鎖（Ａ）及び（Ｂ）を有するものである、前記（１）又は（２）に記載の方法。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（４）癌関連構造の糖鎖がムチン－１のリピート配列（配列番号１）に結合している、前記（１）～（３）いずれかに記載の方法。
（５）癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、及び癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体を組み合わせて用いる、前記（３）に記載の方法。
（６）サンドイッチ法又は免疫沈降／免疫染色法により測定することを特徴とする、前記（１）～（５）のいずれかに記載の方法。
（７）前記被検体は血中トータルＰＳＡ値が３.０～１０.０ｎｇ/ｍｌである、前記（１）～（６）のいずれかに記載の方法。
（８）下記の癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体、並びに下記の癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体を含有することを特徴とする、前立腺癌と前立腺肥大症の識別用キット。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す）
（９）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定することを特徴とする、前立腺癌又は前立腺肥大症の診断方法。
（１０）前立腺癌と前立腺肥大症との識別のための被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定する方法。
（１１）前立腺癌又は前立腺肥大症の診断用医薬の製造のための、被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体の使用。
（１２）被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定する工程およびその測定値がカットオフ値よりも大きい場合に前記試料が前立腺癌患者由来であると判断する工程を含む前立腺癌と前立腺肥大症を識別する方法。 As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.
(1) A method for discriminating between prostate cancer and benign prostatic hyperplasia, which comprises measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
(2) The method according to (1) above, wherein the mucin-1 has one or more sugar chains having a cancer-related structure selected from the following group.
Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-,
GalNAcα-,
Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−;
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(3) The method according to (1) or (2) above, wherein the mucin-1 has sugar chains (A) and (B) having the following cancer-related structures.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(4) The method according to any one of (1) to (3) above, wherein the sugar chain of the cancer-related structure is bound to the repeat sequence of mucin-1 (SEQ ID NO: 1).
(5) The above (3), wherein an antibody that recognizes mucin-1 having a sugar chain (A) having a cancer-related structure and an antibody that recognizes mucin-1 having a sugar chain (B) having a cancer-related structure are used in combination. The method described in 1.
(6) The method according to any one of (1) to (5) above, wherein measurement is performed by a sandwich method or an immunoprecipitation / immunostaining method.
(7) The method according to any one of (1) to (6), wherein the subject has a blood total PSA value of 3.0 to 10.0 ng / ml.
(8) An antibody that recognizes mucin-1 having a sugar chain (A) having the following cancer-related structure, a fragment thereof, and / or a chemically modified derivative thereof, and a mucin having a sugar chain (B) having the following cancer-related structure A kit for distinguishing prostate cancer from benign prostatic hyperplasia, comprising an antibody that recognizes -1, a fragment thereof, and / or a chemically modified derivative thereof.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
(9) A method for diagnosing prostate cancer or benign prostatic hyperplasia, comprising measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
(10) A method for measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject for distinguishing between prostate cancer and benign prostatic hyperplasia.
(11) An antibody recognizing mucin-1 having a sugar chain of a cancer-related structure contained in a sample derived from a subject for the manufacture of a diagnostic cancer for prostate cancer or benign prostatic hyperplasia, a fragment thereof, and / or Use of its chemically modified derivatives.
(12) a step of measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject, and when the measured value is greater than a cutoff value, the sample is derived from a prostate cancer patient A method of discriminating between prostate cancer and benign prostatic hyperplasia including a step of determining that there is.
本発明の方法によれば、従来法では難しかった前立腺癌と前立腺肥大症とを識別することができる。
According to the method of the present invention, it is possible to discriminate between prostate cancer and benign prostatic hyperplasia that were difficult with the conventional method.
以下に本発明を更に詳細に説明する。本発明の識別方法は、上述の方法により癌関連構造の糖鎖を有するＭＵＣ１を測定し、得られた値から前立腺癌と前立腺肥大症とを識別することを特徴とするものである。
Hereinafter, the present invention will be described in more detail. The identification method of the present invention is characterized in that MUC1 having a sugar chain having a cancer-related structure is measured by the above-described method, and prostate cancer and prostatic hypertrophy are identified from the obtained values.
ムチン－１（ＭＵＣ１）は、腫瘍関連抗原であり、多くの腺癌で発現する高分子量糖タンパク質である。この糖タンパク質は、膜糖タンパク質であり、細胞外ドメインには、セリン、トレオニンおよびプロリンに富む２０個のアミノ酸コア配列からなるリピート配列（ＨＧＶＴＳＡＰＤＴＲＰＡＰＧＳＴＡＰＰＡ（配列番号１））と呼ばれる３０～９０タンデム型反復から構成される領域が存在する。
Mucin-1 (MUC1) is a tumor-associated antigen and is a high molecular weight glycoprotein that is expressed in many adenocarcinomas. This glycoprotein is a membrane glycoprotein, and its extracellular domain has a 30-90 tandem repeat called a repeat sequence consisting of 20 amino acid core sequences rich in serine, threonine and proline (HGVTSAPDTTRPAPGSTAPPA (SEQ ID NO: 1)). There is an area composed of
ここで「癌関連構造」とは、癌組織または癌細胞において高発現している構造（例えば、糖鎖構造）をいう。この構造は、正常組織や正常細胞において発現量が低いことが知られている。このような癌関連構造の糖鎖としては、例えば、Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、Ｇａｌβ１→３ＧａｌＮＡｃα－、ＧａｌＮＡｃα－、Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及びＮｅｕ５Ａｃα２→６ＧａｌＮＡｃα－などを挙げることができる。
Here, “cancer-related structure” refers to a structure (eg, sugar chain structure) that is highly expressed in cancer tissue or cancer cells. This structure is known to have a low expression level in normal tissues and normal cells. Examples of the sugar chain of such a cancer-related structure include Neu5Acα2 → 3Galβ1 → 3GalNAcα-, Galβ1 → 3GalNAcα-, GalNAcα-, Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−3Nal6 Examples include GalNAcα- and Neu5Acα2 → 6GalNAcα-.
このとき、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸であり、Ｇａｌはガラクトースであり、ＧａｌＮＡｃはＮ－アセチルガラクトサミンである。
At this time, Neu5Ac is N-acetylneuraminic acid, Gal is galactose, and GalNAc is N-acetylgalactosamine.
さらに、本明細書において、糖鎖の結合様式について、Ｎｅｕ５Ａｃα２→３Ｇａｌは、Ｎｅｕ５Ａｃの２位とＧａｌの３位とがα－グリコシド結合することを示し、Ｇａｌβ１→３ＧａｌＮＡｃは、Ｇａｌの１位とＧａｌＮＡｃの３位とがβ－グリコシド結合することを示し、Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃは、Ｎｅｕ５Ａｃの２位とＧａｌＮＡｃの６位とがα－グリコシド結合することを示し、Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃは、Ｎｅｕ５Ａｃの２位とＧａｌＮＡｃの６位がα－グリコシド結合したＧａｌＮＡｃの３位とＧａｌの１位とがβ－グリコシド結合することを示す。
Furthermore, in the present specification, regarding the binding mode of the sugar chain, Neu5Acα2 → 3Gal indicates that the position 2 of Neu5Ac and the position 3 of Gal are α-glycosidically bonded, and Galβ1 → 3GalNAc indicates that position 1 of Gal and GalNAc And Neu5Acα2 → 6GalNAc indicates that the 5th position of Neu5Ac and 6th position of GalNAc are α-glycosidically bonded, and Galβ1 → 3 (Neu5Acα2 → 6) GalNAc is It shows that the position 3 of GalNAc in which position 2 of Neu5Ac and position 6 of GalNAc are α-glycoside-bonded and position 1 of Gal are β-glycoside-bonded.
本発明において「癌関連構造の糖鎖を有するＭＵＣ１」とは、前記の癌関連構造の糖鎖を有するＭＵＣ１分子をいう。より好ましくは、癌関連構造の糖鎖として（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、Ｇａｌβ１→３ＧａｌＮＡｃα－、及びＧａｌＮＡｃα－から選択される１種以上の糖鎖及び、（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及びＮｅｕ５Ａｃα２→６ＧａｌＮＡｃα－から選択される１種以上の糖鎖のそれぞれを同一分子内に有するＭＵＣ１である。
In the present invention, “MUC1 having a sugar chain having a cancer-related structure” refers to a MUC1 molecule having a sugar chain having the cancer-related structure. More preferably, one or more sugar chains selected from (A) Neu5Acα2 → 3Galβ1 → 3GalNAcα-, Galβ1 → 3GalNAcα-, and GalNAcα- as a sugar chain of a cancer-related structure, and (B) Neu5Acα2 → 3Galβ1 → 3 ( MUC1 having one or more sugar chains selected from Neu5Acα2 → 6) GalNAcα-, Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα-, and Neu5Acα2 → 6GalNAcα- in the same molecule.
このとき前記糖鎖（Ａ）及び前記糖鎖（Ｂ）は、還元末端側のＧａｌＮＡｃを介してＭＵＣ１のアミノ酸残基に結合している。特に前記糖鎖（Ａ）は、ＭＵＣ１のリピート配列（配列番号１）に結合していることが好ましく、具体的にはリピート配列のセリン残基又はトレオニン残基に結合していることが好ましい。中でもリピート配列（配列番号１）の９番目のトレオニンに結合していることがさらに好ましい。また前記糖鎖（Ｂ）もＭＵＣ１のリピート配列（配列番号１）に結合していることが好ましい。
At this time, the sugar chain (A) and the sugar chain (B) are bonded to the amino acid residue of MUC1 via GalNAc on the reducing end side. In particular, the sugar chain (A) is preferably bound to the repeat sequence (SEQ ID NO: 1) of MUC1, and specifically, preferably bound to the serine residue or threonine residue of the repeat sequence. Among them, it is more preferable that the 9th threonine of the repeat sequence (SEQ ID NO: 1) is bound. The sugar chain (B) is also preferably bound to a repeat sequence (SEQ ID NO: 1) of MUC1.
癌関連構造の糖鎖を有するＭＵＣ１の測定方法としては特に限定されるものではないが、抗体を用いた免疫学的方法が好ましい。例えば、前記糖鎖（Ａ）を認識する抗体及び前記糖鎖（Ｂ）を認識する抗体を組み合わせて用いることができる。但し、その２種類の抗体の少なくとも一方として、ＭＵＣ１のアミノ酸をも認識する抗体を用いることができる。別の態様では、両方の抗体が、ＭＵＣ１のアミノ酸をも認識してもよい。これにより前記糖鎖（Ａ）及び前記糖鎖（Ｂ）を同一分子内に有するＭＵＣ１を確実に測定することができる。このような２種類の抗体を用いた測定方法としては、例えば２種類の抗体を用いたサンドイッチ法や、一方の抗体で免疫沈降させ、次いで他方の抗体で免疫染色する免疫沈降／免疫染色法などをあげることができる。
The method for measuring MUC1 having a sugar chain having a cancer-related structure is not particularly limited, but an immunological method using an antibody is preferred. For example, an antibody that recognizes the sugar chain (A) and an antibody that recognizes the sugar chain (B) can be used in combination. However, an antibody that also recognizes the amino acid of MUC1 can be used as at least one of the two types of antibodies. In another aspect, both antibodies may also recognize the amino acid of MUC1. Thereby, MUC1 having the sugar chain (A) and the sugar chain (B) in the same molecule can be reliably measured. Examples of such a measurement method using two types of antibodies include a sandwich method using two types of antibodies, an immunoprecipitation / immunostaining method in which immunoprecipitation is performed with one antibody and then immunostaining with the other antibody. Can give.
ここで前記「サンドイッチ法」とは、抗原に対して特異的に結合する２つの抗体を抗原に反応させて抗原を追跡する方法である。例えば、使用される抗体の一方を担体に結合した状態にし、試料中の抗原と反応させて抗原を補足し、またもう一方の抗体も抗原に反応させて、抗原抗体複合体を形成する方法である。形成された抗原抗体複合体は、少なくとも１つの抗体を標識するか、または二次抗体を反応させることによって検出される。ＭＵＣ１のように抗原エピトープが多価の分子を検出する場合は、同一の抗体を用いてサンドイッチすることも可能である。抗原に対して２つの抗体を同時に反応させる方法や、順次反応させる方法等があるが、順次反応させる方法の方が好ましい。
Here, the “sandwich method” is a method for tracking an antigen by reacting two antibodies that specifically bind to the antigen with the antigen. For example, in a method in which one of the antibodies used is bound to a carrier and reacted with an antigen in a sample to supplement the antigen, and the other antibody is also reacted to the antigen to form an antigen-antibody complex. is there. The formed antigen-antibody complex is detected by labeling at least one antibody or reacting a secondary antibody. When detecting a molecule having a multivalent antigenic epitope such as MUC1, it is also possible to sandwich using the same antibody. There are a method of reacting two antibodies to an antigen simultaneously, a method of reacting sequentially, and the like, but a method of reacting sequentially is preferable.
また前記「免疫沈降／免疫染色法」の「免疫沈降」法とは、抗体によって目的の抗原を、溶液中から特異的に沈降せしめる方法のことをいう。例えば、抗体をセファロースなどの高分子からなる粒子状の担体と結合して使用することで、容易に抗原を精製及び検出することができる方法である。また前記「免疫沈降／免疫染色法」の「免疫染色法」とは、タンパク質を電気泳動等によって分子量や等電点などのタンパク質の性質に応じて分離した後に、疎水性の高い膜に転写されたタンパク質を抗体によって検出する免疫学的手法のことをいう。本発明ではこれらの免疫沈降法と免疫染色法とを組み合わせて用いることができる。
In addition, the “immunoprecipitation” method of the “immunoprecipitation / immunostaining method” refers to a method of specifically precipitating a target antigen from a solution with an antibody. For example, an antigen can be easily purified and detected by using an antibody bound to a particulate carrier made of a polymer such as Sepharose. The “immunoprecipitation method” in the “immunoprecipitation / immunostaining method” means that proteins are separated by electrophoresis according to the properties of the protein such as molecular weight and isoelectric point, and then transferred to a highly hydrophobic membrane. This refers to an immunological technique for detecting a protein with an antibody. In the present invention, these immunoprecipitation methods and immunostaining methods can be used in combination.
本発明に用いられる抗体は、特に限定されるものではないが公知の手法（例えば特許文献１、２に記載の方法）で作製することができる。中でも、ＭＵＣ１のリピート配列（配列番号１）の９番目のトレオニンに前記糖鎖（Ａ）が結合した構造を認識する抗体が好ましく、このような抗体の一例として、特許文献１に抗体１Ｂ２が記載されている。またＭＵＣ１のリピート配列（配列番号１）に前記糖鎖（Ｂ）が結合した構造を認識する抗体が好ましく、このような抗体の一例として特許文献２に抗体１２Ｄ１０が記載されている。
The antibody used in the present invention is not particularly limited, but can be prepared by a known technique (for example, the methods described in Patent Documents 1 and 2). Among them, an antibody that recognizes the structure in which the sugar chain (A) is bound to the 9th threonine of the MUC1 repeat sequence (SEQ ID NO: 1) is preferable. As an example of such an antibody, Patent Document 1 describes antibody 1B2. Has been. An antibody that recognizes the structure in which the sugar chain (B) is bound to the repeat sequence (SEQ ID NO: 1) of MUC1 is preferable, and Patent Document 2 describes antibody 12D10 as an example of such an antibody.
本発明に用いられる被検体由来の試料は特に限定されるものではないが、例えば、組織、血液、血漿、尿が挙げられるが、特に血清や血漿が好ましい。また、前立腺癌が疑われる患者の体液（血清、血漿等）が好ましく、特に血中トータルＰＳＡ値が３．０～１０．０ｎｇ／ｍｌである患者の体液であることが好ましい。日本国内のＰＳＡ検査では、検査対象群の最も若い年齢層である５０～６４歳において、正常値は３．０ｎｇ／ｍｌ以下とされている。特にトータルＰＳＡ値が３．０～１０．０ｎｇ／ｍｌの場合は、血中トータルＰＳＡ値だけを指標とした従来法では前立腺癌と前立腺肥大症とを識別できないという問題があったが、本発明によればこれらを識別することができ、さらには、前立腺癌の存在が疑われる患者の前立腺癌の有無を鑑別する方法としても期待される。
The sample derived from the subject used in the present invention is not particularly limited, and examples thereof include tissue, blood, plasma, and urine, and serum and plasma are particularly preferable. Further, a body fluid (serum, plasma, etc.) of a patient suspected of having prostate cancer is preferable, and a body fluid of a patient whose blood total PSA value is 3.0 to 10.0 ng / ml is particularly preferable. In the PSA test in Japan, the normal value is 3.0 ng / ml or less in the youngest age group of 50 to 64 years in the test group. In particular, when the total PSA value is 3.0 to 10.0 ng / ml, there is a problem that prostate cancer and benign prostatic hyperplasia cannot be distinguished by the conventional method using only the blood total PSA value as an index. Can be identified, and is also expected as a method for differentiating the presence or absence of prostate cancer in patients suspected of having prostate cancer.
本明細書において「ＰＳＡ」とは、前立腺特異抗原（Ｐｒｏｓｔａｔｉｃ Ｓｐｅｃｉｆｉｃ Ａｎｔｉｇｅｎ）をさす。本明細書中「トータルＰＳＡ」とは、主にアンチキモトリプシンと結合した結合型ＰＳＡ及びフリーＰＳＡをさし、即ち、結合型ＰＳＡ及びフリーＰＳＡの合計をさす。「フリーＰＳＡ」とは、複合体を形成していない遊離型ＰＳＡをさす。
In this specification, “PSA” refers to a prostate specific antigen (Prostatic Specific Antigen). In the present specification, “total PSA” mainly refers to bound PSA and free PSA bound to antichymotrypsin, that is, the sum of bound PSA and free PSA. “Free PSA” refers to free PSA that is not complexed.
また、本発明には前記糖鎖（Ａ）を有するＭＵＣ１を認識する抗体、その抗原に結合できる抗体断片、及び／又はその化学修飾誘導体、並びに前記糖鎖（Ｂ）を有するＭＵＣ１を認識する抗体、その抗原に結合できる抗体断片、及び／又はその化学修飾誘導体を含むキットも含まれる。本キットを用いることで前立腺癌と前立腺肥大症の識別を容易に行うことが可能となる。
The present invention also provides an antibody that recognizes MUC1 having the sugar chain (A), an antibody fragment that can bind to the antigen, and / or a chemically modified derivative thereof, and an antibody that recognizes MUC1 having the sugar chain (B). Also included are kits comprising antibody fragments capable of binding to the antigen and / or chemically modified derivatives thereof. By using this kit, it is possible to easily distinguish between prostate cancer and prostatic hypertrophy.
本明細書において「前立腺癌と前立腺肥大症を識別する方法」とは、試料中の癌関連構造の糖鎖を有するＭＵＣ１の量に基づいてインビトロで前立腺癌と前立腺肥大症を識別（又は決定）する方法である。例えば、予め算出されたカットオフ値よりも被検体由来の試料中における癌関連構造の糖鎖を有するＭＵＣ１の量の値が大きいとき、被検体が前立腺肥大症ではなく前立腺癌に罹患しているもしくはその可能性がある、と識別（又は決定）することができる。
As used herein, “a method for discriminating between prostate cancer and benign prostatic hyperplasia” distinguishes (or determines) prostate cancer from benign prostatic hyperplasia in vitro based on the amount of MUC1 having a sugar chain of a cancer-related structure in a sample. It is a method to do. For example, when the value of the amount of MUC1 having a sugar chain of a cancer-related structure in a sample derived from a subject is larger than a cutoff value calculated in advance, the subject suffers from prostate cancer rather than prostatic hypertrophy Alternatively, it is possible to identify (or determine) that there is a possibility.
本発明において、カットオフ値の決定方法は、特に限定されないが、例えば、前立腺肥大症患者の平均値±２ＳＤ（標準偏差）、あるいは前立腺肥大症患者の平均値±１ＳＤ、前立腺肥大症患者の平均値±３ＳＤ、前立腺肥大症患者の平均値をカットオフ値として用いてもよい。好ましくは前立腺肥大症患者の平均値±２ＳＤである。
また、例えば、Ｒｏｃ曲線を用いてカットオフ値を決定してもよい。ＲＯＣ曲線を用いる方法の一例を示すと、前立腺肥大症患者と前立腺癌患者の生体試料における目的とするマーカーを測定して、各測定値における感度（陽性率）、特異度、偽陽性率（１－特異度）を求めて、Ｘ軸に偽陽性率（１－特異度）、Ｙ軸に感度（陽性率）をプロットしてＲＯＣ曲線を作成する。このとき、感度と特異度の優れた理想的なマーカーのＲＯＣ曲線は、左上隅に近づくため、左上隅との距離が最小となる点をカットオフ値とすることができる。また、Ｙｏｕｄｅｎ ｉｎｄｅｘを用いた方法によりカットオフ値を決定してもよい。
カットオフ値を用いて前立腺肥大症患者と前立腺癌患者を区別する場合、それぞれの患者の定義やマーカーの測定方法によって、カットオフ値が変わってくる。従って、目的とするマーカーについて、前立腺肥大症患者、前立腺癌患者の測定値を事前に確認して、カットオフ値を決めて、それに従って検出することが好ましい。 In the present invention, the method for determining the cut-off value is not particularly limited. For example, the mean value ± 2SD (standard deviation) of prostatic hypertrophy patients, or the mean value ± 1SD of prostatic hypertrophy patients, the mean of prostatic hypertrophy patients The value ± 3SD, the average value of patients with benign prostatic hyperplasia may be used as the cutoff value. The mean value of patients with benign prostatic hyperplasia is ± 2 SD.
Further, for example, the cutoff value may be determined using a Roc curve. An example of a method using an ROC curve is to measure a target marker in biological samples of patients with benign prostatic hyperplasia and prostate cancer, and measure sensitivity (positive rate), specificity, and false positive rate (1 -Specificity) is determined, and the ROC curve is created by plotting the false positive rate (1-specificity) on the X axis and the sensitivity (positive rate) on the Y axis. At this time, since the ROC curve of an ideal marker having excellent sensitivity and specificity approaches the upper left corner, the point at which the distance from the upper left corner is minimum can be set as the cutoff value. Further, the cut-off value may be determined by a method using Youden index.
When a prostatic hypertrophy patient and a prostate cancer patient are distinguished using a cut-off value, the cut-off value varies depending on the definition of each patient and the method of measuring a marker. Therefore, it is preferable to confirm the measurement value of the prostatic hypertrophy patient and the prostate cancer patient in advance, determine the cutoff value, and detect the target marker accordingly.
また、例えば、Ｒｏｃ曲線を用いてカットオフ値を決定してもよい。ＲＯＣ曲線を用いる方法の一例を示すと、前立腺肥大症患者と前立腺癌患者の生体試料における目的とするマーカーを測定して、各測定値における感度（陽性率）、特異度、偽陽性率（１－特異度）を求めて、Ｘ軸に偽陽性率（１－特異度）、Ｙ軸に感度（陽性率）をプロットしてＲＯＣ曲線を作成する。このとき、感度と特異度の優れた理想的なマーカーのＲＯＣ曲線は、左上隅に近づくため、左上隅との距離が最小となる点をカットオフ値とすることができる。また、Ｙｏｕｄｅｎ ｉｎｄｅｘを用いた方法によりカットオフ値を決定してもよい。
カットオフ値を用いて前立腺肥大症患者と前立腺癌患者を区別する場合、それぞれの患者の定義やマーカーの測定方法によって、カットオフ値が変わってくる。従って、目的とするマーカーについて、前立腺肥大症患者、前立腺癌患者の測定値を事前に確認して、カットオフ値を決めて、それに従って検出することが好ましい。 In the present invention, the method for determining the cut-off value is not particularly limited. For example, the mean value ± 2SD (standard deviation) of prostatic hypertrophy patients, or the mean value ± 1SD of prostatic hypertrophy patients, the mean of prostatic hypertrophy patients The value ± 3SD, the average value of patients with benign prostatic hyperplasia may be used as the cutoff value. The mean value of patients with benign prostatic hyperplasia is ± 2 SD.
Further, for example, the cutoff value may be determined using a Roc curve. An example of a method using an ROC curve is to measure a target marker in biological samples of patients with benign prostatic hyperplasia and prostate cancer, and measure sensitivity (positive rate), specificity, and false positive rate (1 -Specificity) is determined, and the ROC curve is created by plotting the false positive rate (1-specificity) on the X axis and the sensitivity (positive rate) on the Y axis. At this time, since the ROC curve of an ideal marker having excellent sensitivity and specificity approaches the upper left corner, the point at which the distance from the upper left corner is minimum can be set as the cutoff value. Further, the cut-off value may be determined by a method using Youden index.
When a prostatic hypertrophy patient and a prostate cancer patient are distinguished using a cut-off value, the cut-off value varies depending on the definition of each patient and the method of measuring a marker. Therefore, it is preferable to confirm the measurement value of the prostatic hypertrophy patient and the prostate cancer patient in advance, determine the cutoff value, and detect the target marker accordingly.
以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的のみに提供される。従って、本発明の範囲は、上記実施形態にも下記実施例にも限定されるものではなく、添付の特許請求の範囲によってのみ限定される。
Hereinafter, the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above-described embodiment and the following examples, but is limited only by the appended claims.
実施例１ サンドイッチＥＬＩＳＡ法によるヒト血清検体中のＭＵＣ１の測定
前立腺癌患者および前立腺肥大症患者の血清検体中のＭＵＣ１の測定 Example 1 Measurement of MUC1 in human serum samples by sandwich ELISA method Measurement of MUC1 in serum samples of prostate cancer patients and patients with benign prostatic hyperplasia
前立腺癌患者および前立腺肥大症患者の血清検体中のＭＵＣ１の測定 Example 1 Measurement of MUC1 in human serum samples by sandwich ELISA method Measurement of MUC1 in serum samples of prostate cancer patients and patients with benign prostatic hyperplasia
ヒト血清検体中のＭＵＣ１量の測定には、健常人男性血清（３２検体）、前立腺癌患者血清（６２検体、ＳＲＬ社）、前立腺肥大症患者血清（５２検体、ＳＲＬ社）を用意した（表１）。日本国内のＰＳＡ検査では、検査対象群の最も若い年齢層である５０～６４歳において、正常値は３．０ｎｇ／ｍｌ以下とされていることを考慮して、前立腺癌患者血清、前立腺肥大症患者血清はともに、トータルＰＳＡ量が３．０～１０．０ｎｇ／ｍｌの範囲であるものを使用した。
For the measurement of the amount of MUC1 in human serum samples, healthy human serum (32 samples), prostate cancer patient serum (62 samples, SRL), and prostatic hypertrophy patient serum (52 samples, SRL) were prepared (Table). 1). In the PSA test in Japan, considering that the normal value is 3.0 ng / ml or less in the youngest age group of the test group, 50 to 64 years old, prostate cancer patient serum, prostatic hypertrophy Both patient sera used had a total PSA amount in the range of 3.0 to 10.0 ng / ml.
測定は以下に示す方法で実施した。
Measurement was carried out by the following method.
ヒト血清検体中のＭＵＣ１の測定は、以下に記載するサンドイッチＥＬＩＳＡ法により測定した。９６穴マイクロタイタープレート（グライナー社製）の各ウェルに２５ｎｇの抗ＭＵＣ１抗体１Ｂ２（特許文献１参照）を含むリン酸緩衝液（５０ｍＭリン酸ナトリウム、１５０ｍＭ ＮａＣｌ、ｐＨ７．４）を５０μｌ加えて４℃１６時間固定した。これらのウェルを３００μｌの洗浄液（２０ｍＭ Ｔｒｉｓ－ＨＣｌ、１５０ｍＭ ＮａＣｌ、ｐＨ７．４）で３回洗浄した後、３％ＢＳＡを含むＴＢＳ溶液を２００μｌ加えて室温で２時間放置してブロッキングを行った（抗ＭＵＣ１抗体１Ｂ２固相化プレート）。
Measurement of MUC1 in human serum specimens was performed by the sandwich ELISA method described below. 50 μl of a phosphate buffer solution (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) containing 25 ng of anti-MUC1 antibody 1B2 (see Patent Document 1) was added to each well of a 96-well microtiter plate (made by Greiner). The temperature was fixed for 16 hours. These wells were washed three times with 300 μl of a washing solution (20 mM Tris-HCl, 150 mM NaCl, pH 7.4), and then 200 μl of a TBS solution containing 3% BSA was added and left standing at room temperature for 2 hours to perform blocking ( Anti-MUC1 antibody 1B2 solid phase plate).
各ウェルを３００μｌの洗浄液で３回洗浄した後、希釈液（１％ＢＳＡ、２０ｍＭ Ｔｒｉｓ－ＨＣｌ、１５０ｍＭ ＮａＣｌ、０．０５％Ｔｗｅｅｎ－２０、ｐＨ７．４）で４倍に希釈した前立腺疾患患者（前立腺癌患者、前立腺肥大症患者）から採取された血清、又は対照被検体として健常人から採取された血清を加え、室温で１時間放置した。各ウェルを３００μｌの界面活性剤を含む洗浄液（２０ｍＭ Ｔｒｉｓ－ＨＣｌ、１５０ｍＭ ＮａＣｌ、０．０５％Ｔｗｅｅｎ－２０、ｐＨ７．４）で３回洗浄した後、２５ｎｇのビオチン標識抗ＭＵＣ１抗体１２Ｄ１０（特許文献２参照）を含む希釈液を５０μｌ加え、室温で１時間放置した。次に、各ウェルを３００μｌの界面活性剤を含む洗浄液で３回洗浄した後、希釈液で５０００倍に希釈したｐｏｌｙＨＲＰ８０－ストレプトアビジン溶液を５０μｌ加え、室温で１時間放置した。最後に、各ウェルを３００μｌの洗浄液で３回洗浄した後、５０μｌのＴＭＢ溶液を添加して１０分間発色させた後に、１Ｍのリン酸溶液を添加することで反応を停止し、４５０ｎｍにおける吸光度を測定した。図１にその結果を示す。
Each well was washed 3 times with 300 μl of washing solution and then diluted 4 times with diluent (1% BSA, 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) ( Serum collected from prostate cancer patients and patients with benign prostatic hyperplasia) or serum collected from healthy individuals as control subjects was added and allowed to stand at room temperature for 1 hour. Each well was washed 3 times with a washing solution (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) containing 300 μl of a surfactant, and then 25 ng of biotin-labeled anti-MUC1 antibody 12D10 (Patent Document) 2) was added, and the mixture was allowed to stand at room temperature for 1 hour. Next, each well was washed three times with a washing solution containing 300 μl of a surfactant, and then 50 μl of a polyHRP80-streptavidin solution diluted 5000 times with a diluted solution was added and left at room temperature for 1 hour. Finally, after each well was washed 3 times with 300 μl of washing solution, 50 μl of TMB solution was added to develop color for 10 minutes, and then the reaction was stopped by adding 1 M phosphoric acid solution, and the absorbance at 450 nm was measured. It was measured. The result is shown in FIG.
実施例２
実施例１と同様にして、但し、被検体である血清は１０倍に希釈し、ビオチン標識抗ＭＵＣ１抗体１２Ｄ１０の代わりにＨＲＰ標識抗ＭＵＣ１抗体１Ｂ２を用いて反応を行い、各ウェルを３００μｌの界面活性剤を含む洗浄液で３回洗浄した後、５０μｌのＴＭＢ溶液を添加して１０分間発色させた後に、１Ｍのリン酸溶液を添加することで反応を停止し、４５０ｎｍにおける吸光度を測定した。図１にその結果を示す。 Example 2
As in Example 1, except that the serum as the specimen was diluted 10-fold, and the reaction was performed using HRP-labeled anti-MUC1 antibody 1B2 instead of biotin-labeled anti-MUC1 antibody 12D10, and each well was treated with a 300 μl interface. After washing with a washing solution containing an activator three times, 50 μl of TMB solution was added to develop color for 10 minutes, and then the reaction was stopped by adding a 1M phosphoric acid solution, and the absorbance at 450 nm was measured. The result is shown in FIG.
実施例１と同様にして、但し、被検体である血清は１０倍に希釈し、ビオチン標識抗ＭＵＣ１抗体１２Ｄ１０の代わりにＨＲＰ標識抗ＭＵＣ１抗体１Ｂ２を用いて反応を行い、各ウェルを３００μｌの界面活性剤を含む洗浄液で３回洗浄した後、５０μｌのＴＭＢ溶液を添加して１０分間発色させた後に、１Ｍのリン酸溶液を添加することで反応を停止し、４５０ｎｍにおける吸光度を測定した。図１にその結果を示す。 Example 2
As in Example 1, except that the serum as the specimen was diluted 10-fold, and the reaction was performed using HRP-labeled anti-MUC1 antibody 1B2 instead of biotin-labeled anti-MUC1 antibody 12D10, and each well was treated with a 300 μl interface. After washing with a washing solution containing an activator three times, 50 μl of TMB solution was added to develop color for 10 minutes, and then the reaction was stopped by adding a 1M phosphoric acid solution, and the absorbance at 450 nm was measured. The result is shown in FIG.
比較例１
実施例１と同様の検体のトータルＰＳＡを測定した。検体のトータルＰＳＡ値は、採血時に免疫測定装置ＲｏｃｈｅＥ１７０とＰＳＡ測定試薬を用いて測定された値である。図２にその結果を示す。 Comparative Example 1
The total PSA of the sample similar to that in Example 1 was measured. The total PSA value of the specimen is a value measured using an immunoassay device Roche E170 and a PSA measurement reagent at the time of blood collection. The results are shown in FIG.
実施例１と同様の検体のトータルＰＳＡを測定した。検体のトータルＰＳＡ値は、採血時に免疫測定装置ＲｏｃｈｅＥ１７０とＰＳＡ測定試薬を用いて測定された値である。図２にその結果を示す。 Comparative Example 1
The total PSA of the sample similar to that in Example 1 was measured. The total PSA value of the specimen is a value measured using an immunoassay device Roche E170 and a PSA measurement reagent at the time of blood collection. The results are shown in FIG.
実施例１の１Ｂ２－１２Ｄ１０サンドイッチＥＬＩＳＡ測定では、前立腺疾患（前立腺癌及び前立腺肥大症）患者血清では健常人血清に比べて大きい値が示された。さらに、前立腺癌と前立腺肥大症患者を比較すると、前立腺癌患者において明らかに高値を示しており、１Ｂ２－１２Ｄ１０サンドイッチ測定により前立腺癌を前立腺肥大症から識別することが可能であることが明らかとなった。このことは、前立腺癌と前立腺肥大症の患者群で分けた場合の２群間有意差検定の結果も、ｐ値が０．０５未満であり（図１）、有意差があることが示された。
The 1B2-12D10 sandwich ELISA measurement of Example 1 showed a larger value in the serum of prostate disease (prostate cancer and benign prostatic hyperplasia) patients than in the healthy subject. Furthermore, when prostate cancer and benign prostatic hypertrophy patients are compared, it is clearly high in prostate cancer patients, and it becomes clear that 1B2-12D10 sandwich measurement can distinguish prostate cancer from prostatic hypertrophy. It was. This indicates that there is a significant difference in the p-value of less than 0.05 in the results of the significant difference test between the two groups when divided into the prostate cancer and prostatic hypertrophy patient groups (FIG. 1). It was.
一方、実施例２の１Ｂ２抗体のみを使用した１Ｂ２－１Ｂ２サンドイッチＥＬＩＳＡ測定では、前立腺癌と前立腺肥大症の患者群で有意に差があるものの、実施例１の１Ｂ２－１２Ｄ１０サンドイッチＥＬＩＳＡ測定と比べて明確な差ではないことが分かる。このことは、前立腺癌と前立腺肥大症を識別するには、実施例１の１Ｂ２－１２Ｄ１０サンドイッチＥＬＩＳＡ測定がより優れており、即ち、糖鎖（Ａ）及び（Ｂ）を同一分子内に有するＭＵＣ１を測定することが望ましいといえる。また図２によれば、本発明による１Ｂ２－１２Ｄ１０サンドイッチＥＬＩＳＡ測定の結果は、現行品であるトータルＰＳＡの結果よりも、前立腺癌と前立腺肥大症との識別にすぐれていることが明らかである。
On the other hand, in the 1B2-1B2 sandwich ELISA measurement using only the 1B2 antibody of Example 2, there is a significant difference between the group of patients with prostate cancer and benign prostatic hyperplasia, but compared with the 1B2-12D10 sandwich ELISA measurement of Example 1. It turns out that it is not a clear difference. This indicates that the 1B2-12D10 sandwich ELISA measurement of Example 1 is better for discriminating between prostate cancer and benign prostatic hyperplasia, that is, MUC1 having sugar chains (A) and (B) in the same molecule. It can be said that it is desirable to measure. Also, according to FIG. 2, it is clear that the result of the 1B2-12D10 sandwich ELISA measurement according to the present invention is superior to the result of the total PSA which is the current product, in distinguishing between prostate cancer and prostatic hypertrophy.
これらの事実は、測定の特異性、感度を統計的に解析したＲＯＣ解析結果からも明らかである（図３、表２）。すなわち、１Ｂ２－１２Ｄ１０サンドイッチ測定では、最適化されたカットオフ値（ＡＢＳ４５０ｎｍ＝０．２９４）での特異性は７８．８％、感度は９０．３％であり、診断性能の１つの指標となるＡＵＣは０．９２５と充分に高い値を示した。一方、１Ｂ２－１Ｂ２サンドイッチ測定では特異性が８８．５％とトータルＰＳＡ測定よりも高い値を示したが、感度はほぼ同じであった。診断性能の１つの指標となるＡＵＣも、トータルＰＳＡ測定よりも良い値を示したが、１Ｂ２－１２Ｄ１０サンドイッチ測定の性能には及ばなかった。
These facts are also apparent from the results of ROC analysis that statistically analyzed the specificity and sensitivity of the measurement (FIG. 3, Table 2). That is, in the 1B2-12D10 sandwich measurement, the specificity at the optimized cutoff value (ABS 450 nm = 0.294) is 78.8%, and the sensitivity is 90.3%, which is one index of diagnostic performance. AUC showed a sufficiently high value of 0.925. On the other hand, the 1B2-1B2 sandwich measurement showed a specificity of 88.5%, which was higher than the total PSA measurement, but the sensitivity was almost the same. AUC, which is one index of diagnostic performance, also showed a better value than the total PSA measurement, but did not reach the performance of 1B2-12D10 sandwich measurement.
Claims (8)
- 被検体由来の試料中に含まれる癌関連構造の糖鎖を有するムチン－１の量を測定することを特徴とする、前立腺癌と前立腺肥大症を識別する方法。 A method for discriminating between prostate cancer and benign prostatic hyperplasia characterized by measuring the amount of mucin-1 having a sugar chain having a cancer-related structure contained in a sample derived from a subject.
- 前記ムチン－１が、下記の群より選択される１種以上の癌関連構造の糖鎖を有するものである、請求項１に記載の方法。
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、
ＧａｌＮＡｃα－、
Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す） 2. The method according to claim 1, wherein the mucin-1 has one or more types of sugar chains having a cancer-related structure selected from the following group.
Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-,
GalNAcα-,
Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−;
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine) - 前記ムチン－１が、下記の癌関連構造の糖鎖（Ａ）及び（Ｂ）を有するものである、請求項１又は２に記載の方法。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す） The method according to claim 1 or 2, wherein the mucin-1 has sugar chains (A) and (B) having the following cancer-related structures.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine) - 癌関連構造の糖鎖がムチン－１のリピート配列（配列番号１）に結合している、請求項１～３いずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein a sugar chain having a cancer-related structure is bound to a repeat sequence of mucin-1 (SEQ ID NO: 1).
- 癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、及び癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体を組み合わせて用いる、請求項３に記載の方法。 The method according to claim 3, wherein an antibody that recognizes mucin-1 having a sugar chain (A) having a cancer-related structure and an antibody that recognizes mucin-1 having a sugar chain (B) having a cancer-related structure are used in combination. .
- サンドイッチ法又は免疫沈降／免疫染色法により測定することを特徴とする、請求項１～５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein the measurement is carried out by a sandwich method or immunoprecipitation / immunostaining method.
- 前記被検体は血中トータルＰＳＡ値が３.０～１０.０ｎｇ/ｍｌである、請求項１～６のいずれかに記載の方法。 The method according to any one of claims 1 to 6, wherein the subject has a blood total PSA value of 3.0 to 10.0 ng / ml.
- 下記の癌関連構造の糖鎖（Ａ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体、並びに下記の癌関連構造の糖鎖（Ｂ）を有するムチン－１を認識する抗体、その断片、及び／又はその化学修飾誘導体を含有することを特徴とする、前立腺癌と前立腺肥大症の識別用キット。
（Ａ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３ＧａｌＮＡｃα－、
Ｇａｌβ１→３ＧａｌＮＡｃα－、及び
ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（Ｂ）Ｎｅｕ５Ａｃα２→３Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、
Ｇａｌβ１→３（Ｎｅｕ５Ａｃα２→６）ＧａｌＮＡｃα－、及び
Ｎｅｕ５Ａｃα２→６ＧａｌＮＡｃα－
からなる群より選択される１種以上；
（但し、Ｎｅｕ５ＡｃはＮ－アセチルノイラミン酸、Ｇａｌはガラクトース、ＧａｌＮＡｃはＮ－アセチルガラクトサミンを示す） An antibody that recognizes mucin-1 having a sugar chain (A) having the following cancer-related structure, a fragment thereof, and / or a chemically modified derivative thereof, and mucin-1 having a sugar chain (B) having the following cancer-related structure: A kit for distinguishing prostate cancer from benign prostatic hyperplasia, comprising an antibody to be recognized, a fragment thereof, and / or a chemically modified derivative thereof.
(A) Neu5Acα2 → 3Galβ1 → 3GalNAcα−,
Galβ1 → 3GalNAcα-, and GalNAcα-
One or more selected from the group consisting of:
(B) Neu5Acα2 → 3Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−,
Galβ1 → 3 (Neu5Acα2 → 6) GalNAcα−, and Neu5Acα2 → 6GalNAcα−
One or more selected from the group consisting of:
(However, Neu5Ac represents N-acetylneuraminic acid, Gal represents galactose, and GalNAc represents N-acetylgalactosamine)
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|---|---|---|---|---|
| JP2003519096A (en) * | 1999-08-18 | 2003-06-17 | アルタレックス コーポレーション | Therapeutic antibodies to MUC-1 antigen and methods of using the same |
| US20080071148A1 (en) * | 2006-04-03 | 2008-03-20 | Massachusetts Institute Of Technology | Glycomic patterns for the detection of disease |
| JP2011038952A (en) * | 2009-08-14 | 2011-02-24 | National Institute Of Advanced Industrial Science & Technology | Method for detecting cancer by detection of glycoprotein having specific sugar chain structure |
| WO2011052244A1 (en) * | 2009-10-30 | 2011-05-05 | Tokyo Institute Of Technology | A method for analyzing PSA, and a method for distinguishing prostate cancer from prostatic hypertrophy using that method for analyzing PSA |
| JP2012255736A (en) * | 2011-06-10 | 2012-12-27 | J-Oil Mills Inc | Glycoprotein detection method |
| JP2013156245A (en) * | 2011-05-12 | 2013-08-15 | Shizuokaken Koritsu Daigaku Hojin | Lectin having target directivity to cancer |
-
2013
- 2013-11-11 JP JP2013232746A patent/JP2017009287A/en active Pending
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2014
- 2014-11-11 WO PCT/JP2014/079863 patent/WO2015068851A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003519096A (en) * | 1999-08-18 | 2003-06-17 | アルタレックス コーポレーション | Therapeutic antibodies to MUC-1 antigen and methods of using the same |
| US20080071148A1 (en) * | 2006-04-03 | 2008-03-20 | Massachusetts Institute Of Technology | Glycomic patterns for the detection of disease |
| JP2011038952A (en) * | 2009-08-14 | 2011-02-24 | National Institute Of Advanced Industrial Science & Technology | Method for detecting cancer by detection of glycoprotein having specific sugar chain structure |
| WO2011052244A1 (en) * | 2009-10-30 | 2011-05-05 | Tokyo Institute Of Technology | A method for analyzing PSA, and a method for distinguishing prostate cancer from prostatic hypertrophy using that method for analyzing PSA |
| JP2013156245A (en) * | 2011-05-12 | 2013-08-15 | Shizuokaken Koritsu Daigaku Hojin | Lectin having target directivity to cancer |
| JP2012255736A (en) * | 2011-06-10 | 2012-12-27 | J-Oil Mills Inc | Glycoprotein detection method |
Non-Patent Citations (2)
| Title |
|---|
| CHANDRASEKARAN E. V. ET AL.: "Analysis of the Specificity of Sialyltransferases toward Mucin Core 2, Globo, and Related Structures. Identification of the Sialylation Sequence and the Effects of Sulfate, Fucose, Methyl, and Fluoro Substituents of the Carbohydrate Chain in the Biosynthesis of Selectin and Siglec Ligands, and Novel", BIOCHEMISTRY, vol. 44, no. 47, 2005, pages 15619 - 15635 * |
| XIONG CHENG-YI ET AL.: "Development of tumor targeting anti-MUC-I multimer: effects of di- scFv unpaired cysteine location on PEGylation and tumor binding", PROTEIN ENG DES SEL, vol. 19, no. 8, 2006, pages 359 - 367 * |
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