JP2022070974A - Method of detecting colorectal cancer - Google Patents
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Abstract
Description
特許法第３０条第２項適用申請有り 集会名 第１９回韓国がん予防学会国際会議 開催日 平成２６年１２月１２日～平成２６年１２月１３日（公開日は平成２６年１２月１２日）Application for application of Article 30, Paragraph 2 of the Patent Law Meeting name 19th Korea Cancer Prevention Society International Conference Date December 12, 2014-December 13, 2014 (Publication date is December 12, 2014) Day)
本発明は、ペプチドマーカーを用いた大腸がんの検出方法に関し、より詳細には、ペプチドマーカーを用いた大腸がんの判定、予防効果の判定、治療効果の判定、早期診断のための検査方法、及び早期治療のための検査方法に関する。 The present invention relates to a method for detecting colorectal cancer using a peptide marker, and more specifically, a test method for determining colorectal cancer using a peptide marker, determining a preventive effect, determining a therapeutic effect, and early diagnosis. , And test methods for early treatment.
大腸がんは大腸（盲腸、結腸、直腸）に発生するがん腫であり、米国では3番目に多い
がんであり、がん死の原因としては2番目に多く、生涯における罹患率は約7%にのぼって
いる。我が国でも、胃がんを抜き、肺がんに次いで多いがんとなっている。大腸がんは、早期に検出と治療が行われれば治癒が十分に望める病気である。したがって、早期発見を心がけることが重要である。
Colorectal cancer is a cancer that begins in the large intestine (cecum, colon, rectum), is the third most common cancer in the United States, the second most common cause of cancer death, and has a lifetime prevalence of about 7. It is up to%. In Japan as well, it is the second most common cancer after lung cancer, overtaking gastric cancer. Colorectal cancer is a disease that can be fully cured if detected and treated early. Therefore, it is important to keep in mind early detection.
生体内のタンパク質発現を網羅的に解析するプロテオミクス研究の進展に伴い、プロテオミクスを利用した新規バイオマーカーの探索が精力的に行われている。例えば、特許文献１又は２には特定のアミノ酸配列を有するポリペプチドを用いた大腸がんの検出方法が開示されている。 With the progress of proteomics research that comprehensively analyzes protein expression in the living body, the search for new biomarkers using proteomics is being vigorously carried out. For example, Patent Document 1 or 2 discloses a method for detecting colorectal cancer using a polypeptide having a specific amino acid sequence.
しかしながら、上記の従来技術では、がんの段階が相当進行した患者でしか検出が有効でないという問題があった。大腸がんと相関する複数のマーカーペプチドから検出精度の高いペプチド又はその組み合わせを特定できれば、早期大腸がんの検出に資する。 However, the above-mentioned conventional technique has a problem that detection is effective only in patients whose cancer stage has progressed considerably. If a peptide with high detection accuracy or a combination thereof can be identified from a plurality of marker peptides that correlate with colorectal cancer, it will contribute to the detection of early-stage colorectal cancer.
本発明の目的は、大腸がんと相関する１種又はロジスティック回帰法を含む機械学習法等の統計学的手法で選択された２種以上のペプチドを用いた、高精度な大腸がんの検出方法、該ペプチドに対する抗体を含む大腸がん検出キット、該ペプチドに対する抗体を検出試薬として含む大腸がん検出剤、及び被験者における大腸がんの罹患可能性を判定するための、その他統計学的方法を提供することにある。 An object of the present invention is highly accurate detection of colorectal cancer using one type that correlates with colorectal cancer or two or more types of peptides selected by a statistical method such as a machine learning method including a logistic regression method. Methods, colorectal cancer detection kits containing antibodies to the peptide, colorectal cancer detectors containing antibodies to the peptide as detection reagents, and other statistical methods for determining the likelihood of colorectal cancer in a subject. Is to provide.
本発明は、大腸がん患者の生体試料にて健常者と発現の差異が見出される複数のペプチドをマーカーとして用いたところ、ＲＯＣ曲線下面積（ＡＵＣ）が高い（特に５つのマーカーペプチドを用いると０．９を超える）極めて信頼性の高い大腸がんの検出又は判定が可能であることを見出した。すなわち、本発明は、以下の大腸がん以下の［１］～［１３］に記載の発明を提供するものである。
［１］被験者の生物試料中の、配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する
１種又は２種以上のペプチドを測定することからなる、該被験者における大腸がんの検出方法。
［２］被験者の生物試料中の、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、及び配列番号９で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドを測定することからなる、該被験者における大腸がんの検出方法。
［３］配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、及び配列番号９で表されるアミノ酸配列からなるペプチドを測定することからなる、［１］又は［２］に記載の方法。
［４］前記生物試料が血液、血漿、血清、唾液、尿、髄液、骨髄液、胸水、腹水、関節液、涙液、眼房水、硝子体液およびリンパ液からなる群より選択される体液からなる、［１］～［３］のいずれか一項に記載の方法。
［５］生体試料を質量分析にかけることを含む、［１］～［４］のいずれか一項に記載の方法。
［６］質量分析に内部標準物質を用いる［５］に記載の方法。
［７］内部標準物質が［１］に記載の１種又は２種以上のペプチドの安定同位体からなる［６］に記載の方法。
［８］配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有するペプチドを特異的に認識する抗体を用いることを特徴とする、［１］～［４］のいずれか一項に記載の方法。
［９］配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドに対する抗体を含む大腸がん検出キット。
［１０］配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドに対する抗体を検出試薬として含む大腸がん検出剤。
［１１］被験者における大腸がんの罹患可能性を判定するための、コンピュータにより実行される方法であって、被験者の生物試料中の、配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表される
アミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する２種以上のペプチドについての定量的データを取得する工程と、前記取得したデータを、前記２種以上のペプチドの関数である多変量ロジスティック回帰モデルに適用し、被験者における大腸がんの罹患可能性の予測確率を求める工程とを含む方法。［１２］配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、及び配列番号９で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する２種以上のペプチドの定量的データを取得し、前記取得したデータを、前記２種以上のペプチドの関数である多変量ロジスティック回帰モデルに適用する［１１］に記載の方法。
［１３］配列番号１～１４のいずれかで表されるアミノ酸配列からなるペプチド。
In the present invention, when a plurality of peptides whose expression is found to be different from that of a healthy subject in a biological sample of a colorectal cancer patient are used as markers, the area under the ROC curve (AUC) is high (especially when five marker peptides are used). We have found that it is possible to detect or determine colorectal cancer with extremely high reliability (more than 0.9). That is, the present invention provides the inventions according to the following [1] to [13] for colorectal cancer.
[1] The amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, and the amino acid sequence represented by SEQ ID NO: 4 in the biological sample of the subject. Amino acid sequence represented by SEQ ID NO: 5, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, The amino acid sequence represented by SEQ ID NO: 10, the amino acid sequence represented by SEQ ID NO: 11, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 14. A method for detecting colon cancer in a subject, which comprises measuring one or more kinds of peptides having an amino acid sequence selected from the group consisting of.
[2] The amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 6, and the amino acid sequence represented by SEQ ID NO: 7 in the biological sample of the subject. A method for detecting colon cancer in the subject, which comprises measuring one or more kinds of peptides having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 9.
[3] A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and an amino acid represented by SEQ ID NO: 7. The method according to [1] or [2], which comprises measuring a peptide consisting of a sequence and a peptide consisting of an amino acid sequence represented by SEQ ID NO: 9.
[4] From the body fluid selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, abdominal fluid, joint fluid, tear fluid, aqueous humor, vitreous fluid and lymph fluid. The method according to any one of [1] to [3].
[5] The method according to any one of [1] to [4], which comprises subjecting a biological sample to mass spectrometry.
[6] The method according to [5], which uses an internal standard substance for mass spectrometry.
[7] The method according to [6], wherein the internal standard substance is a stable isotope of one or more peptides according to [1].
[8] The amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 5. Amino acid sequence, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, and represented by SEQ ID NO: 10. It is selected from the group consisting of an amino acid sequence, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 13, and an amino acid sequence represented by SEQ ID NO: 14. The method according to any one of [1] to [4], which comprises using an antibody that specifically recognizes a peptide having an amino acid sequence.
[9] The amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 5. Amino acid sequence, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, and represented by SEQ ID NO: 10. It is selected from the group consisting of an amino acid sequence, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 13, and an amino acid sequence represented by SEQ ID NO: 14. A colon cancer detection kit containing an antibody against one or more peptides having an amino acid sequence.
[10] The amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 5. Amino acid sequence, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, and represented by SEQ ID NO: 10. It is selected from the group consisting of an amino acid sequence, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 13, and an amino acid sequence represented by SEQ ID NO: 14. A colon cancer detecting agent containing an antibody against one or more kinds of peptides having an amino acid sequence as a detection reagent.
[11] A method performed by a computer for determining the susceptibility to colorectal cancer in a subject, which is represented by the amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the subject's biological sample. Amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 5, amino acid sequence represented by SEQ ID NO: 6, and represented by SEQ ID NO: 7. Amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 11, and represented by SEQ ID NO: 12. Step to acquire quantitative data for two or more kinds of peptides having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 13, the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence represented by SEQ ID NO: 14. A method including a step of applying the acquired data to a multivariate logistic regression model which is a function of the two or more kinds of peptides to obtain a prediction probability of the possibility of developing colorectal cancer in a subject. [12] The amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, and the amino acid sequence represented by SEQ ID NO: 9. Quantitative data of two or more peptides having an amino acid sequence selected from the group consisting of amino acid sequences are obtained, and the obtained data are applied to a multivariate logistic regression model which is a function of the two or more peptides. [11].
[13] A peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 14.
本発明によれば、大腸がんを迅速かつ極めて高い信頼性で判定できるため、該疾患の判定、予防効果の判定、治療効果の判定、早期診断、及び早期治療が可能となる。 According to the present invention, since colorectal cancer can be determined rapidly and with extremely high reliability, it is possible to determine the disease, determine the preventive effect, determine the therapeutic effect, perform early diagnosis, and perform early treatment.
本発明は、新規かつ有用な大腸がんの検出マーカーペプチド（以下、包括して「本発明のペプチド」という場合もある）を提供する。
なお、本明細書において、大腸がんの「検出」には、大腸がんの判定、予防効果の判定、治療効果の判定、診断（特には早期診断）のための検査方法、及び治療（特には早期治療）のための検査方法が含まれる。大腸がんの「判定」には、大腸癌の有無を判定することのみならず、予防的に大腸がんの罹患可能性を判定することや、治療後の大腸がんの予後を予測すること、及び大腸癌の治療剤の治療効果を判定することが含まれる。
The present invention provides a novel and useful marker peptide for detecting colorectal cancer (hereinafter, may be collectively referred to as “the peptide of the present invention”).
In the present specification, "detection" of colorectal cancer includes determination of colorectal cancer, determination of preventive effect, determination of therapeutic effect, examination method for diagnosis (particularly early diagnosis), and treatment (particularly). Includes testing methods for early treatment). The "judgment" of colorectal cancer is not only to determine the presence or absence of colorectal cancer, but also to prophylactically determine the possibility of developing colorectal cancer and to predict the prognosis of colorectal cancer after treatment. , And determining the therapeutic effect of a therapeutic agent for colorectal cancer.
本発明の被験者における大腸がんの検出方法に用いられるペプチドは、ヒト血清中に見出される、配列番号１で表されるアミノ酸配列からなるペプチド、配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号４で表されるアミノ酸配列からなるペプチド、配列番号５で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、配列番号８で表されるアミノ酸配列からなるペプチド、配列番号９で表されるアミノ酸配列からなるペプチド、配列番号１０で表されるアミノ酸配列からなるペプチド、配列番号１１で表されるアミノ酸配列からなるペプチド、配列番号１２で表されるアミノ酸配列からなるペプチド、配列番号１３で表されるアミノ酸配列からなるペプチド、又は配列番号１４で表されるアミノ酸配列からなるペプチドである。これらのペプチドの測定値は大腸がんへの罹患と相関する。 The peptide used in the method for detecting colon cancer in the subject of the present invention is a peptide having an amino acid sequence represented by SEQ ID NO: 1 and a peptide consisting of an amino acid sequence represented by SEQ ID NO: 2, which are found in human serum. It consists of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6. Peptide, peptide consisting of amino acid sequence represented by SEQ ID NO: 7, peptide consisting of amino acid sequence represented by SEQ ID NO: 8, peptide consisting of amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10. Represented by a peptide consisting of, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 11, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 12, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 13, or a peptide consisting of SEQ ID NO: 14. It is a peptide consisting of an amino acid sequence. Measurements of these peptides correlate with disease of colorectal cancer.
配列番号１で表されるアミノ酸配列からなるペプチドはフィブリノーゲンα鎖の部分配列であり、配列番号２で表されるアミノ酸配列からなるペプチドはフィブリノーゲンα鎖の部分配列であり、配列番号３で表されるアミノ酸配列からなるペプチドはα－１－アンチトリプシンの部分配列であり、配列番号４で表されるアミノ酸配列からなるペプチドはα－２－ＨＳ－糖タンパク質の部分配列であり、配列番号５で表されるアミノ酸配列からなるペプチドはフィブリノーゲンα鎖の部分配列であり、配列番号６で表されるアミノ酸配列からなるペプチドはα－２－ＨＳ－糖タンパク質の部分配列であり、配列番号７で表されるアミノ酸配列からなるペプチドは血管拡張因子刺激リン酸化タンパク質の部分配列であり、配列番号８で表されるアミノ酸配列からなるペプチドは血小板因子４の全配列で
あり、配列番号９で表されるアミノ酸配列からなるペプチドは血液凝固第XIII因子の部分配列であり、配列番号１０で表されるアミノ酸配列からなるペプチドはビンキュリンの部分配列であり、配列番号１１で表されるアミノ酸配列からなるペプチドはプロトロンビンの部分配列であり、配列番号１２で表されるアミノ酸配列からなるペプチドはビンキュリンの部分配列であり、配列番号１３で表されるアミノ酸配列からなるペプチドはα－１－アンチキモトリプシンの部分配列であり、又は配列番号１４で表されるアミノ酸配列からなるペプチドはフィブリノーゲンα鎖の部分配列である。
配列番号１～１４で表されるアミノ酸配列からなるペプチドの質量分析による見かけの分子量［Ｍ＋Ｈ］+は、それぞれ約1465.66、約1616.66、約2390.26、約2739.53、約2768.23、約2858.42、約3622.78、約7759.18、約3949.98、約4038.05、約4089.02、約4152.99
、約4352.34、約5078.35である。
本発明においては、上記のインタクトなペプチドをマーカーとして用いるが、配列番号１～１４に表されるアミノ酸配列において、１個または数個（好ましくは１ないし３個,
より好ましくは１または２個）のアミノ酸が欠失、置換、付加したアミノ酸配列からなるペプチドも含み、これらの修飾ペプチドも本発明の方法においてバイオマーカーとして用いることができる。さらに、本発明のペプチドは、特定のアミノ酸に酸素原子が結合して酸化されたり、リン酸化されたり、N-アセチル化されたり、S-システイン化されたりしている場合があるが、このような場合も、配列番号１～１４で表されるアミノ酸配列を有するペプチドである限り、本発明の範囲に包含される。
The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a partial sequence of the fibrinogen α chain, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a partial sequence of the fibrinogen α chain and is represented by SEQ ID NO: 3. The peptide consisting of the amino acid sequence is a partial sequence of α-1-antitrypsin, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is a partial sequence of α-2-HS-sugar protein. The peptide consisting of the amino acid sequence represented is a partial sequence of the fibrinogen α chain, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is a partial sequence of α-2-HS-sugar protein, which is represented by SEQ ID NO: 7. The peptide consisting of the amino acid sequence to be used is a partial sequence of the vasodilator-stimulated phosphorylated protein, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 8 is the entire sequence of platelet factor 4 and is represented by SEQ ID NO: 9. The peptide consisting of the amino acid sequence is a partial sequence of blood coagulation factor XIII, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 10 is a partial sequence of vincurin, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a partial sequence. The peptide consisting of the partial sequence of prothrombin and the amino acid sequence represented by SEQ ID NO: 12 is the partial sequence of vincurin, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 13 is the partial sequence of α-1-antichimotrypsin. The peptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is a partial sequence of the fibrinogen α chain.
The apparent molecular weights [M + H] + of the peptides consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 14 are about 1465.66, about 1616.66, about 2390.26, about 2739.53, about 2768.23, about 2858.42, about 3622.78, respectively. 7759.18, about 3949.98, about 4038.05, about 4089.02, about 4152.99
, About 4352.34, about 5078.35.
In the present invention, the above-mentioned intact peptide is used as a marker, but one or several (preferably 1 to 3) are used in the amino acid sequences represented by SEQ ID NOs: 1 to 14.
More preferably, peptides consisting of amino acid sequences in which one or two) amino acids have been deleted, substituted or added are also included, and these modified peptides can also be used as biomarkers in the method of the present invention. Further, the peptide of the present invention may be oxidized, phosphorylated, N-acetylated, or S-cysteineized by binding an oxygen atom to a specific amino acid. However, as long as the peptide has the amino acid sequence represented by SEQ ID NOs: 1 to 14, it is included in the scope of the present invention.
本発明はまた、被験者の生物試料中の、上記の１４個の本発明のペプチドのうちの１種、又は２種以上を測定することからなる、該被験者における大腸がんの検出又は判定方法を包含する。
さらに好ましい一つの実施形態では、被験者における大腸がんの検出又は判定方法は、配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、及び配列番号９で表されるアミノ酸配列からなるペプチドを測定することからなる。
The present invention also relates to a method for detecting or determining colorectal cancer in a subject, which comprises measuring one or more of the above 14 peptides of the present invention in a biological sample of the subject. Include.
In one more preferred embodiment, the method for detecting or determining colorectal cancer in a subject is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: 6. It comprises measuring a peptide consisting of the amino acid sequence represented, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
被験者は、大腸がんに罹患していると疑われる患者を含み、「大腸がんに罹患していると疑われる被検者」は、被検者本人が主観的に疑いを抱く者（何らかの自覚症状がある者に限らず、単に予防検診の受診を希望する者を含む）であっても、何らかの客観的な根拠に基づく者（例えば、従来公知の臨床検査（例、便潜血検査）および／または診察の結果、大腸がんへの合理的な罹患可能性があると医師が判断した者）であってもよい。「ペプチドを測定する」とはペプチドの濃度、量、又はシグナル強度を測定することを指す。 Subjects include patients suspected of having colorectal cancer, and "subjects suspected of having colorectal cancer" are those who are subjectively suspicious of themselves (something). Not only those who have subjective symptoms, but also those who simply wish to undergo preventive screening (including those who simply wish to undergo preventive screening), but those who are based on some objective basis (for example, conventionally known clinical tests (eg, fecal occult blood test) and / Or a person who has been judged by the doctor to have a reasonable possibility of developing colorectal cancer as a result of a medical examination). "Measuring a peptide" refers to measuring the concentration, amount, or signal intensity of a peptide.
被験試料となる被検者由来の生体試料は特に限定されないが、被検者への侵襲が少ないものであることが好ましく、例えば、血液、血漿、血清、唾液、尿、涙液など生体から容易に採取できるものや、髄液、骨髄液、胸水、腹水、関節液、眼房水、硝子体液、リンパ液など比較的容易に採取されるものが挙げられる。一実施形態では、生物試料が血液、血漿、血清、唾液、尿、髄液、骨髄液、胸水、腹水、関節液、涙液、眼房水、硝子体液およびリンパ液からなる群より選択される体液からなる。血清や血漿を用いる場合、常法に従って被検者から採血し、前処理を施さず直接、又は液性成分を分離することにより分析にかける被験試料を調製することができる。検出対象である本発明のペプチドは必要に応じて、抗体カラムやスピンカラムなどを用いて、予め高分子量の蛋白質画分などを分離除去しておくこともできる。 The biological sample derived from the subject as the test sample is not particularly limited, but it is preferable that the biological sample does not invade the subject, and is easily introduced from the living body such as blood, plasma, serum, saliva, urine, and tears. The ones that can be collected in the blood plasma, the bone marrow fluid, the pleural fluid, the ascites fluid, the joint fluid, the atrioventricular fluid, the vitreous fluid, the lymph fluid, etc. can be collected relatively easily. In one embodiment, a bodily fluid selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, abdominal fluid, joint fluid, tear fluid, aqueous humor, vitreous fluid and lymph fluid. Consists of. When serum or plasma is used, a test sample to be analyzed can be prepared by collecting blood from a subject according to a conventional method and directly or by separating the liquid component without pretreatment. If necessary, the peptide of the present invention to be detected can be separated and removed in advance from a high molecular weight protein fraction or the like by using an antibody column, a spin column or the like.
生体試料中の、本発明のペプチドの検出は、例えば、生体試料を各種の分子量測定法、例えば、ゲル電気泳動や、各種の分離精製法（例：イオン交換クロマトグラフィー、疎水性クロマトグラフィー、アフィニティークロマトグラフィー、逆相クロマトグラフィーな
ど）、表面プラズモン共鳴法、イオン化法（例：電子衝撃イオン化法、フィールドディソープション法、二次イオン化法、高速原子衝突法、マトリックス支援レーザー脱離イオン化（MALDI）法、エレクトロスプレーイオン化法など）、および質量分析計（例：二重収
束質量分析計、四重極型分析計、飛行時間型質量分析計、フーリエ変換質量分析計、イオンサイクロトロン質量分析計、免疫質量分析計、安定同位体ペプチドを内部標準にした質量分析計、免疫顕微鏡計など）を組み合わせる方法等に供し、該ペプチドの分子量と一致するバンドもしくはスポット、あるいはピークを検出することにより行うことができるが、これらに限定されない。
For the detection of the peptide of the present invention in a biological sample, for example, various molecular mass spectrometry methods such as gel electrophoresis and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity) are performed on the biological sample. Chromatography, reverse phase chromatography, etc.), surface plasmon resonance method, ionization method (eg, electron impact ionization method, field dispersion method, secondary ionization method, fast atomic collision method, matrix-assisted laser desorption ionization (MALDI)) Methods, electrospray ionization methods, etc.), and mass spectrometers (eg, double-convergent mass spectrometer, quadrupole mass spectrometer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, immunity It can be used by combining a mass spectrometer, a mass spectrometer with a stable isotope peptide as an internal standard, an immunomicroscope, etc., and detecting a band, spot, or peak that matches the molecular weight of the peptide. Yes, but not limited to these.
本発明のペプチドを精製してそれらを認識する抗体を作製し、ELISA, RIA,イムノクロ
マト法、表面プラズモン共鳴法、ウェスタンブロッティング、免疫質量分析法や各種イムノアッセイ、免疫顕微鏡法により該ペプチドを検出する方法もまた、好ましく用いられ得る。さらに上記方法のハイブリッド型検出法も有効である。
A method for purifying the peptide of the present invention to prepare an antibody that recognizes them, and detecting the peptide by ELISA, RIA, immunochromatography, surface plasmon resonance, Western blotting, immunomass spectrometry, various immunoassays, and immunomicroscopy. Can also be preferably used. Further, the hybrid detection method of the above method is also effective.
本発明の検出又は判定方法における特に好ましい測定法の１つは、飛行時間型質量分析に使用するプレートの表面に被験試料を接触させ、該プレート表面に捕捉された成分の質量を飛行時間型質量分析計で測定する方法が挙げられる。飛行時間型質量分析計に適合可能なプレートは、検出対象である本発明のペプチドを効率よく吸着し得る表面構造（例：官能基付加ガラス、Si、Ge、GaAs、GaP、SiO2、SiN4、改質シリコン、種々のゲルまたは
ポリマーのコーティング）を有している限り、いかなるものであってもよい。
One of the particularly preferable measurement methods in the detection or determination method of the present invention is to bring the test sample into contact with the surface of the plate used for time-of-flight mass spectrometry, and the mass of the component captured on the plate surface is the time-of-flight mass. A method of measuring with an analyzer can be mentioned. A plate compatible with a time-of-flight mass spectrometer has a surface structure capable of efficiently adsorbing the peptide of the present invention to be detected (eg, functional group-added glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 ). , Modified silicon, various gel or polymer coatings).
好ましい実施態様においては、質量分析用プレートとして用いられる支持体は、ポリビニリデンジフロリド（PVDF）、ニトロセルロースまたはシリカゲル、特に好ましくはPVDFで薄層コーティングされた基材である（WO 2004/031759を参照）。かかる基材は、質量分析用プレートにおいて使用されているものであれば、特に限定されず、例えば、絶縁体、金属、導電性ポリマー、それらの複合体などが挙げられる。かかるPVDFで薄層コーティングされた質量分析用プレートとして、好ましくは株式会社プロトセラ社のブロットチップ（BLOTCHIP，登録商標）などが挙げられる。代わりに、質量分析用プレートは、支持体表面を塗布、噴霧、蒸着、浸漬、印刷、スパッタリング等の公知の手段でコーティングすることにより、公知の方法により調製することもできる。また、質量分析用プレート上の分子を質量分析する方法自体は公知である（例えばWO 2004/031759）。WO 2004/031759に記載の方法を、必要に応じて適宜改変して使用することができる。 In a preferred embodiment, the support used as the mass spectrometric plate is a polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably a thin layer coated substrate with PVDF (WO 2004/031759). See). Such a base material is not particularly limited as long as it is used in a mass spectrometric plate, and examples thereof include insulators, metals, conductive polymers, and composites thereof. As the mass spectrometric plate coated with a thin layer of PVDF, a blot chip (BLOTCHIP, registered trademark) manufactured by Protocera Co., Ltd. is preferable. Alternatively, the mass spectrometric plate can be prepared by a known method by coating the surface of the support by a known means such as coating, spraying, vapor deposition, dipping, printing and sputtering. Moreover, the method itself for mass spectrometric analysis of molecules on a mass spectrometric plate is known (for example, WO 2004/031759). The method described in WO 2004/031759 can be appropriately modified and used as necessary.
被験試料の質量分析用プレート（支持体）への移行は、被験試料となる被検者由来の生体試料を未処理のままで、あるいは抗体カラムその他の方法で高分子タンパク質を除去、濃縮した後に、SDS-ポリアクリルアミドゲル電気泳動もしくは等電点電気泳動に付し、泳動後ゲルをプレートと接触させて転写（ブロッティング）することにより行われる。転写の方法自体は公知であり、好ましくは電気転写が用いられる。電気転写時に使用する緩衝液としては、pH 7～9、低塩濃度の公知の緩衝液を用いることが好ましい（例えばトリス
緩衝液、リン酸緩衝液、ホウ酸緩衝液、酢酸緩衝液など）。
The transfer of the test sample to the mass analysis plate (support) is performed by leaving the biological sample derived from the subject as the test sample untreated, or after removing and concentrating the high molecular weight protein by an antibody column or other method. , SDS-polyacrylamide gel electrophoresis or isoelectric point electrophoresis, and after electrophoresis, the gel is brought into contact with a plate and transferred (brotting). The transfer method itself is known, and electric transfer is preferably used. As the buffer solution used during electrotransfer, it is preferable to use a known buffer solution having a pH of 7 to 9 and a low salt concentration (for example, Tris buffer solution, phosphate buffer solution, borate buffer solution, acetate buffer solution, etc.).
上記の方法により支持体表面上に捕捉された被験試料中の分子を質量分析することにより、分子量に関する情報から、標的分子である本発明のペプチドの存在および量を同定することができる。質量分析装置からの情報を、任意のプログラムを用いて、非大腸がん患者もしくは健常人由来の生体試料における質量分析データと比較して、示差的な（differential）情報として出力させることも可能である。そのようなプログラムは周知であり、また、当業者は、公知の情報処理技術を用いて、容易にそのようなプログラムを構築もしくは改変することができることが理解されよう。 By mass spectrometry of the molecules in the test sample captured on the surface of the support by the above method, the presence and amount of the peptide of the present invention as a target molecule can be identified from the information on the molecular weight. It is also possible to compare the information from the mass spectrometer with the mass spectrometric data in a biological sample derived from a non-colorectal cancer patient or a healthy person and output it as differential information using an arbitrary program. be. It will be appreciated that such programs are well known and that one of ordinary skill in the art can easily construct or modify such programs using known information processing techniques.
高精度な質量分析結果を得るためには、高速液体クロマトグラフィーに接続した三連四
重極型等の質量分析装置を用いて分析する。標的分子の安定同位体標識ペプチドを合成して、それを既知量の内部標準品として被験試料に混合し、逆相固相担体等でペプチド画分の粗精製を実施する。高速液体クロマトグラフィーに導入後、分離された各ペプチドは質量分析装置内でイオン化され、その後コリジョンセル内で断片化、得られたペプチドフラグメントをmultiple reaction monitoring法により定量する。この際、安定同位体標識ペプチドを内部標準として用いることでCV値が5%以下の実測データを取得できる。安定同位体標識ペプチドは、Cambridge Isotope Laboratory（MA, USA）より購入した安定同位体
標識アミノ酸（アミノ酸a(13C6,15N2)は、安定同位体炭素（原子量13）6個と、安定同位
体窒素（原子量15）2個で置換された質量数が元のアミノ酸より８原子量増加したアミノ
酸aを例示）を元のアミノ酸の配列位置に置換して既存の合成法（たとえばF-mocによる固相反応）により得られる。質量分析は株式会社プロトセラ社のBLOTCHIP（登録商標）システムでも実施可能であり、これらの方法は抗体を使用しない検出装置として臨床使用できる。
In order to obtain highly accurate mass spectrometry results, analysis is performed using a mass spectrometer such as a triple quadrupole type connected to high performance liquid chromatography. A stable isotope-labeled peptide of the target molecule is synthesized, mixed with a test sample as a known amount of an internal standard, and the peptide fraction is roughly purified using a reverse phase solid phase carrier or the like. After introduction into high performance liquid chromatography, each separated peptide is ionized in a mass spectrometer, then fragmented in a collision cell, and the obtained peptide fragment is quantified by a multiple reaction monitoring method. At this time, by using a stable isotope-labeled peptide as an internal standard, it is possible to obtain actual measurement data having a CV value of 5% or less. The stable isotope-labeled peptide is a stable isotope-labeled amino acid purchased from the Cambridge Isotope Laboratory (MA, USA) (amino acid a (13C6,15N2) has 6 stable isotope carbons (atomic weight 13) and stable isotope nitrogen (stable isotope nitrogen) Atomic weight 15) An existing synthetic method (for example, solid phase reaction by F-moc) is performed by substituting the sequence position of the original amino acid with the amino acid a whose mass number replaced by two is increased by 8 atoms from the original amino acid. Obtained by Mass spectrometry can also be performed with the BLOTCHIP® system of Protocera Co., Ltd., and these methods can be clinically used as an antibody-free detector.
上記の質量分析による検出において、タンデム質量分析（MS/MS）法を用いてペプチド
を同定することができ、かかる同定法としては、MS/MSスペクトルを解析してアミノ酸配
列を決定するde novo sequencing法と、MS/MSスペクトル中に含まれる部分的な配列情報
（質量タグ）を用いてデータベース検索を行い、ペプチドを同定する方法等が挙げられる。また、MS/MS法を用いることにより、直接本発明のペプチドのアミノ酸配列を同定し、
該配列情報に基づいて該ペプチドの全部もしくは一部を合成し、これを以下の抗体に対する抗原として利用することもできる。
In the above mass spectrometric detection, peptides can be identified using tandem mass spectrometry (MS / MS), such as de novo sequencing, which analyzes MS / MS spectra to determine amino acid sequences. Examples thereof include a method and a method of identifying a peptide by performing a database search using partial sequence information (mass tag) contained in the MS / MS spectrum. In addition, by using the MS / MS method, the amino acid sequence of the peptide of the present invention was directly identified.
It is also possible to synthesize all or part of the peptide based on the sequence information and use it as an antigen against the following antibodies.
本発明のペプチドの測定は、それに対する抗体を用いて行うこともできる。よって、本発明は、ペプチドを特異的に認識する抗体を用いた大腸がんの検出又は判定方法、かかる抗体を含む大腸がん検出又は判定剤、ならびにかかる抗体を含む大腸がん検出又は判定キットを含む。かかる方法は、最適化されたイムノアッセイ系を構築してこれをキット化すれば、上記質量分析装置のような特殊な装置を使用することなく、高感度かつ高精度に該ペプチドを検出することができる点で、特に有用である。 The peptide of the present invention can also be measured using an antibody against it. Therefore, the present invention relates to a method for detecting or determining colorectal cancer using an antibody that specifically recognizes a peptide, a colorectal cancer detection or determination agent containing such an antibody, and a colorectal cancer detection or determination kit containing such an antibody. including. In such a method, if an optimized immunoassay system is constructed and this is made into a kit, the peptide can be detected with high sensitivity and high accuracy without using a special device such as the above-mentioned mass spectrometer. It is especially useful in that it can be done.
本発明のペプチドに対する抗体は、例えば、本発明のペプチドを、これを発現する患者由来の生体試料から単離・精製し、該ペプチドを抗原として動物を免疫することにより調製することができる。あるいは、得られるペプチドの量が少量である場合は、RT-PCRによる該ペプチドをコードするcDNA断片の増幅等の周知の遺伝子工学的手法によりペプチドを大量に調製することができ、あるいはかかるcDNAを鋳型として、無細胞転写・翻訳系を用いて本発明のペプチドを取得することもできる。さらに有機合成法により大量に調製することも可能である。 An antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a biological sample derived from a patient expressing the peptide and immunizing an animal with the peptide as an antigen. Alternatively, if the amount of peptide obtained is small, the peptide can be prepared in large quantities by well-known genetic engineering techniques such as amplification of the cDNA fragment encoding the peptide by RT-PCR, or such cDNA can be obtained. The peptide of the present invention can also be obtained using a cell-free transcription / translation system as a template. Furthermore, it can be prepared in large quantities by an organic synthesis method.
本発明のペプチドに対する抗体（以下、「本発明の抗体」と称する場合がある）は、ポリクローナル抗体またはモノクローナル抗体のいずれであってもよく、周知の免疫学的手法により作製することができる。また、該抗体は完全抗体分子だけでなくそのフラグメントをも包含し、例えば、Fab、F(ab')2、ScFv、およびminibody等が挙げられる。 The antibody against the peptide of the present invention (hereinafter, may be referred to as "antibody of the present invention") may be either a polyclonal antibody or a monoclonal antibody, and can be produced by a well-known immunological method. Further, the antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab') 2, ScFv, and minibody.
例えば、ポリクローナル抗体は、本発明のペプチドを抗原として、市販のアジュバント（例えば、完全または不完全フロイントアジュバント）とともに、動物の皮下あるいは腹腔内に2～3週間おきに2～4回程度投与し、最終免疫後に全血を採取して抗血清を精製することにより取得できる。抗原を投与する動物としては、ラット、マウス、ウサギ、ヤギ、ヒツジ、ウマ、モルモット、ハムスターなど、目的の抗体を得ることができる哺乳動物が挙げられる。 For example, the polyclonal antibody using the peptide of the present invention as an antigen is administered subcutaneously or intraperitoneally in an animal 2 to 4 times every 2 to 3 weeks together with a commercially available adjuvant (for example, a complete or incomplete Freund's adjuvant). It can be obtained by collecting whole blood after final immunization and purifying the antiserum. Examples of the animal to which the antigen is administered include mammals capable of obtaining the antibody of interest, such as rats, mice, rabbits, goats, sheep, horses, guinea pigs, and hamsters.
また、モノクローナル抗体は、細胞融合法により作成することができる。モノクローナ
ル抗体を調製するための技法の説明は、Stites et al, Basic and Clinical Immunology.
(Lang Medical Publications Los Altos. CA. 4th Edition） and references therein,
、in particular Koehler, G. & Milstein, C. Nature 256, 495-497 (1975).に見出さ
れ得る。例えば、本発明のペプチドを市販のアジュバントと共にマウスに2～4回皮下あるいは腹腔内に投与し、最終投与後に脾臓あるいはリンパ節を採取し、白血球を採取する。この白血球と骨髄腫細胞（例えば、NS-1, P3X63Ag8など）を細胞融合して該ペプチドに対するモノクローナル抗体を産生するハイブリドーマを得る。所望のモノクローナル抗体を産生するハイブリドーマは、周知のEIAまたはRIA法等を用いて抗原と特異的に結合する抗体を、培養上清中から検出することにより選択できる。モノクローナル抗体を産生するハイブリドーマの培養は、インビトロ、またはマウスもしくはラット、このましくはマウス腹水中等のインビボで行うことができ、抗体はそれぞれハイブリドーマの培養上清および動物の腹水から取得することができる。
In addition, the monoclonal antibody can be produced by a cell fusion method. A description of techniques for preparing monoclonal antibodies can be found in Stites et al, Basic and Clinical Immunology.
(Lang Medical Publications Los Altos. CA. 4th Edition) and references herein,
, In particular Koehler, G. & Milstein, C. Nature 256, 495-497 (1975). For example, the peptide of the present invention is administered subcutaneously or intraperitoneally to mice 2 to 4 times together with a commercially available adjuvant, and after the final administration, the spleen or lymph node is collected and leukocytes are collected. These leukocytes and myeloma cells (eg, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. A hybridoma that produces a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. Culture of hybridomas producing monoclonal antibodies can be performed in vitro or in vivo, such as in mouse or rat, or mouse ascites, and the antibodies can be obtained from the hybridoma culture supernatant and animal ascites, respectively. ..
抗体を用いる本発明の検出又は判定方法は、特に制限されるべきものではなく、被験試料中の抗原量に対応した抗体、抗原もしくは抗体－抗原複合体の量を化学的または物理的手段により検出し、これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、いずれの測定法を用いてもよい。例えば、ネフロメトリー、競合法、イムノメトリック法およびサンドイッチ法等が好適に用いられる。測定に際し、抗体または抗原は、放射性同位元素、酵素、蛍光物質、または発光物質等の標識剤と結合され得る。さらに、抗体あるいは抗原と標識剤との結合にビオチン-アビジン系を用いることも
できる。これら個々の免疫学的測定法は、当業者の通常の技能により、本発明の定量方法に適用可能である。
The detection or determination method of the present invention using an antibody is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is detected by chemical or physical means. However, any measurement method may be used as long as it is a measurement method for calculating this from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method, sandwich method and the like are preferably used. Upon measurement, the antibody or antigen may be bound to a labeling agent such as a radioisotope, enzyme, fluorescent substance, or luminescent substance. Furthermore, a biotin-avidin system can also be used for binding an antibody or antigen to a labeling agent. These individual immunoassays can be applied to the quantification method of the present invention by the ordinary skill of those skilled in the art.
本発明のペプチドはタンパク質分解産物からなるため、未分解のタンパク質や、切断部位が共通の類似ペプチド等様々な分子が測定値に影響を与える可能性がある。そこで、第１工程において、生体試料を抗体により免疫アフィニティ精製し、抗体に結合したフラクションを、第２工程において質量分析に付し、精緻な分子量を基準に同定、定量する、いわゆる免疫質量分析法を利用することができる（例えば、Rapid Commun. Mass Spectrom.
2007, 21: 352-358を参照）。免疫質量分析法によれば、未分解のタンパク質も類似ペプチドも、質量分析計で完全に分離され、バイオマーカーの正確な分子量を基準に高い特異性と感度で定量が可能となる。
Since the peptide of the present invention consists of proteolytic products, various molecules such as undegraded proteins and similar peptides having a common cleavage site may affect the measured values. Therefore, in the first step, a biological sample is immunoaffinished with an antibody, and the fraction bound to the antibody is subjected to mass spectrometry in the second step to identify and quantify based on a precise molecular weight, a so-called immunomass spectrometry method. Can be used (eg Rapid Commun. Mass Spectrom.
2007, 21: 352-358). According to immunomass spectrometry, undegraded proteins and similar peptides are completely separated by mass spectrometry, and can be quantified with high specificity and sensitivity based on the accurate molecular weight of the biomarker.
あるいは、本発明の抗体を用いる別の本発明の検出又は判定方法として、該抗体を上記したような質量分析計に適合し得るチップの表面上に固定化し、該チップ上の該抗体に被検試料を接触させ、該抗体に捕捉された生体試料成分を質量分析にかけ、該抗体が認識するマーカーペプチドの分子量に相当するピークを検出する方法が挙げられる。 Alternatively, as another detection or determination method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a chip that can be compatible with a mass spectrometer as described above, and the antibody on the chip is tested. Examples thereof include a method in which a sample is brought into contact with each other, the biological sample component captured by the antibody is subjected to mass spectrometry, and a peak corresponding to the molecular weight of the marker peptide recognized by the antibody is detected.
上記のいずれかの方法により測定された被検者由来試料中の本発明のペプチドのレベルが、非大腸がん患者もしくは健常人由来の対照試料中の該ペプチドレベルに比べて有意に変動している場合、該被検者は大腸がんに罹患している可能性が高いと判定することができる。 The level of the peptide of the present invention in the subject-derived sample measured by any of the above methods was significantly different from the peptide level in the control sample derived from a non-colorectal cancer patient or a healthy person. If so, it can be determined that the subject is likely to have colorectal cancer.
本発明のペプチドは、それぞれ単独でも大腸がんの検出マーカーとして利用することができるが、2種以上を組み合わせることにより、感度（有病正診率）および特異度（無病
正診率）をより高めることができる。
Each of the peptides of the present invention can be used alone as a detection marker for colorectal cancer, but by combining two or more types, the sensitivity (presence accuracy rate) and specificity (disease-free accuracy rate) can be further increased. Can be enhanced.
２種以上のペプチドをマーカーとして用いる場合の検出手法としては、例えば、(1) 測定対象であるすべてのペプチドについてレベルが有意に変動する場合に大腸がんであると判定し、いずれかのペプチドについてレベルが有意に変動しない場合に大腸がんでないと判定する方法、(2) 測定対象であるすべてのペプチドについてレベルが有意に変動しない
場合に大腸がんでないと判定し、いずれかのペプチドについてレベルが有意に変動した場合に大腸がんであると判定する方法、(3) 測定対象であるn個のペプチドのうち、例えば
、2～(n-1)個以上のペプチドについて、レベルが有意に変動する場合に大腸がんであると判定する方法、さらに各ペプチド間で重みを持たせる方法、ならびに(4) バギング法、ブースティング法、ランダムフォレスト法などの機械学習法、などが考えられるが、特には複数のマーカーペプチドを１つのマーカーセットとして取り扱うことが出来る解析手法である多変量ロジスティック回帰分析を用いることが好ましい。この場合、マーカーとして用いるペプチドの数は特に限定されないが、好ましくは２～14種、より好ましくは3～10
種、さらにより好ましくは5種である。
As a detection method when two or more kinds of peptides are used as markers, for example, (1) it is determined that the peptide is colon cancer when the level of all the peptides to be measured fluctuates significantly, and one of the peptides is used. A method for determining that there is no colon cancer when the level does not change significantly, (2) It is determined that there is no colon cancer when the level does not change significantly for all the peptides to be measured, and the level for any of the peptides Method for determining colon cancer when there is a significant change in, (3) Of the n peptides to be measured, for example, the level of 2 to (n-1) or more peptides changes significantly In this case, a method for determining colon cancer, a method for giving weight between each peptide, and (4) machine learning methods such as bagging method, boosting method, and random forest method can be considered, but in particular. It is preferable to use multivariate logistic regression analysis, which is an analysis method capable of treating a plurality of marker peptides as one marker set. In this case, the number of peptides used as a marker is not particularly limited, but is preferably 2 to 14, more preferably 3 to 10.
Species, and even more preferably five.
一つの実施形態において、検出又は測定されるペプチドは、配列番号１～配列番号１４で表されるアミノ酸配列からなるペプチドの１４種のペプチドのうちの少なくとも１種であり、好ましくは２種以上である。 In one embodiment, the peptide to be detected or measured is at least one of 14 peptides of the peptide consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 14, preferably two or more. be.
好ましい実施形態において、検出又は測定されるペプチドは、配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、及び配列番号９で表されるアミノ酸配列からなるペプチドのうちの少なくとも１種であり、好ましくは２種以上、より好ましくは３種以上、さらに好ましくは４種以上である。 In a preferred embodiment, the peptide to be detected or measured comprises a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and an amino acid sequence represented by SEQ ID NO: 6. It is at least one of a peptide, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9, preferably two or more, more preferably three or more. More preferably, it is 4 or more.
さらに好ましい実施形態において、検出又は測定されるペプチドは、配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、及び配列番号９で表されるアミノ酸配列からなるペプチドの５種である。 In a further preferred embodiment, the peptide to be detected or measured is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6. , A peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
本願では、質量分析により特定された候補ペプチドの多変量ロジスティック回帰モデルを最尤法により構築したところ、ＲＯＣの曲線下面積（ＡＵＣ）が高い（５つのマーカーペプチドで０．９を超える）極めて信頼性の高い大腸がんの検出又は判定が可能であることを見出した。検出又は測定されるペプチドの数が多いほど、検査の精度は向上する。例えば、配列番号２で表されるアミノ酸配列からなるペプチド（分子量1616.66）、配列番
号３で表されるアミノ酸配列からなるペプチド（分子量2390.26）、配列番号６で表され
るアミノ酸配列からなるペプチド（分子量2858.42）、配列番号７で表されるアミノ酸配
列からなるペプチド（分子量3622.78）、及び配列番号９で表されるアミノ酸配列からな
るペプチド（分子量3949.98）の５つのペプチドの血中濃度の関数であるロジスティック
回帰モデル（式１）：
予測罹患確率=1/{1+exp(-(3.836581e-05×[m/z 1616.66]-5.079913e-04×[m/z 2390.26]-3.288524e-05×[m/z 2858.42]+5.243605e-04×[m/z 3622.78]-5.477792e-05×[m/z 3949.98]+4.379456)} ・・・ （式１）
によれば、特異度に対して感度をプロットしたＲＯＣの曲線下面積（ＡＵＣ）が０．９を超え、高い精度で大腸がんを検出することができる。なお、[mz1616.66] は配列番号２で表されるアミノ酸配列からなるペプチドの血中濃度、[mz2390.26]は配列番号３で表され
るアミノ酸配列からなるペプチドの血中濃度、[mz2858.42] は配列番号６で表されるアミノ酸配列からなるペプチドの血中濃度、[mz3622.78] は配列番号７で表されるアミノ酸配列からなるペプチドの血中濃度、[mz3949.98] は配列番号９で表されるアミノ酸配列からなるペプチドの血中濃度を表す。
In the present application, when a multivariate logistic regression model of candidate peptides identified by mass spectrometry was constructed by the most likely method, the area under the curve (AUC) of ROC is high (more than 0.9 for 5 marker peptides) and extremely reliable. It has been found that it is possible to detect or determine highly sexual colorectal cancer. The greater the number of peptides detected or measured, the better the accuracy of the test. For example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (molecular weight 1616.66), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (molecular weight 2390.26), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 (molecular weight). 2858.42), a logistic that is a function of the blood concentration of five peptides: the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (molecular weight 3622.78) and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 (molecular weight 3949.98). Regression model (Equation 1):
Predicted morbidity probability = 1 / {1 + exp (-(3.836581e-05 × [m / z 1616.66] -5.079913e-04 × [m / z 2390.26]-3.288524e-05 × [m / z 2858.42] +5.243605 e-04 × [m / z 3622.78]-5.477792 e-05 × [m / z 3949.98] +4.379456)} ・ ・ ・ (Equation 1)
According to the report, the area under the curve (AUC) of the ROC plotting the sensitivity with respect to the specificity exceeds 0.9, and colorectal cancer can be detected with high accuracy. [Mz1616.66] is the blood concentration of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and [mz2390.26] is the blood concentration of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, [mz2858]. .42] is the blood concentration of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, [mz3622.78] is the blood concentration of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and [mz3949.98] is It represents the blood concentration of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
検出又は測定されるペプチドの数は、本発明の検査方法におけるＡＵＣが或る閾値を超える値となる数であることが好ましい。通常、閾値は０．９であり、好ましくは０．９２である。ペプチドの数を増やすほど約１に近づけることが可能である。 The number of peptides detected or measured is preferably such that the AUC in the test method of the present invention exceeds a certain threshold value. The threshold is usually 0.9, preferably 0.92. It is possible to approach about 1 as the number of peptides increases.
本発明の検出方法は、患者から時系列で生体試料を採取し、各試料における本発明のペプチドの発現の経時変化を調べることにより行うこともできる。生体試料の採取間隔は特に限定されないが、患者のQOLを損なわない範囲でできるだけ頻繁にサンプリングするこ
とが望ましく、例えば、血漿もしくは血清を試料として用いる場合、約1日～約1月間の間隔で採血を行うことが好ましい。本発明のペプチドは、大腸がんが進行するに従って血清・血漿レベルが各ペプチドで低下又は上昇する傾向にある。従って、これらのマーカーのレベルが経時的に上昇又は低下した場合には、該患者における大腸がんが改善または悪化している可能性が高いと判定することができる。
The detection method of the present invention can also be carried out by collecting biological samples from patients in chronological order and examining the time course of expression of the peptide of the present invention in each sample. The collection interval of the biological sample is not particularly limited, but it is desirable to sample as often as possible within the range that does not impair the QOL of the patient. For example, when plasma or serum is used as a sample, blood is collected at intervals of about 1 day to about 1 month. It is preferable to do. In the peptide of the present invention, serum / plasma levels tend to decrease or increase in each peptide as colorectal cancer progresses. Therefore, if the levels of these markers increase or decrease over time, it can be determined that there is a high possibility that colorectal cancer in the patient has improved or worsened.
さらに、上記時系列的なサンプリングによる大腸がんの検出方法は、前回サンプリングと当回サンプリングとの間に、被検者である患者に対して該疾患の治療措置が講じられた場合に、当該措置による治療効果を評価するのに用いることができる。即ち、治療の前後にサンプリングした試料について、治療後の状態が治療前の状態と比較して病態の改善が認められると判定された場合に、当該治療の効果があったと評価することができる。一方、治療後の状態が治療前の状態と比較して病態の改善が認められない、あるいはさらに悪化していると判定された場合には、当該治療の効果がなかったと評価することができる。 Furthermore, the above-mentioned method for detecting colorectal cancer by time-series sampling is applicable when a therapeutic measure for the disease is taken for the patient who is the subject between the previous sampling and the current sampling. It can be used to evaluate the therapeutic effect of the measure. That is, when it is determined that the condition after the treatment is improved as compared with the condition before the treatment for the samples sampled before and after the treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after the treatment does not improve or is worsened as compared with the condition before the treatment, it can be evaluated that the treatment was not effective.
さらに、上記時系列的なサンプリングによる大腸がんの検出のための検査方法は、健康食品等の摂取、禁煙、有機溶媒（ジクロロメタン等）等の有害環境からの隔離等大腸がん罹患リスク低減措置後の大腸がんリスクの予防効果を評価するのに用いることができる。即ち、罹患リスクの防止方法施行の前後にサンプリングした試料について、施行後の状態が施行前の状態と比較して病態の発症もしくは進行が認められないと判定された場合に、当該防止方法施行の効果があったと評価することができる。一方、治療後の状態が治療前の状態と比較して病態の改善が認められない、あるいはさらに悪化していると判定された場合には、当該防止方法施行の効果がなかったと評価することができる。 Furthermore, the test methods for detecting colorectal cancer by the above time-series sampling include measures to reduce the risk of developing colorectal cancer, such as ingestion of health foods, quitting smoking, and isolation from harmful environments such as organic solvents (dichloromethane, etc.). It can be used to evaluate the preventive effect of later colorectal cancer risk. That is, when it is determined that the condition after the treatment does not show the onset or progression of the pathological condition in the sample sampled before and after the treatment of the risk of morbidity compared with the condition before the treatment, the prevention method is performed. It can be evaluated that it was effective. On the other hand, if it is determined that the condition after treatment does not improve or worsens compared to the condition before treatment, it can be evaluated that the prevention method was not effective. can.
従って、本発明のペプチドならびに方法は、大腸がんを診断または検出するマーカーのみならず、大腸がんの予後を予測するマーカー、ならびに治療効果判定のマーカーともなり得る。すなわち、本発明のペプチドならびに方法は、大腸がん治療の創薬標的分子のスクリーニングに、および／または患者（リスポンダー）の選別もしくは薬の投与量（用量）の調節のためのコンパニオン診断薬として使用することができる。さらに、本発明のペプチドならびに方法は、健康食品等の摂取、禁煙、有機溶媒（ジクロロメタン等）等の有害環境からの隔離等大腸がん罹患リスク低減措置後の大腸がんリスクの予防効果を評価するのに用いることができる。 Therefore, the peptide and method of the present invention can be not only a marker for diagnosing or detecting colorectal cancer, but also a marker for predicting the prognosis of colorectal cancer and a marker for determining the therapeutic effect. That is, the peptides and methods of the present invention are used for screening drug discovery target molecules for the treatment of colorectal cancer and / or as companion diagnostic agents for selecting patients (responders) or adjusting drug doses. can do. Furthermore, the peptide and method of the present invention evaluate the preventive effect of colorectal cancer risk after measures to reduce the risk of colorectal cancer such as ingestion of health foods, quitting smoking, isolation from harmful environments such as organic solvents (dioxide, etc.), etc. Can be used to do.
一実施形態において、本発明は、配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドに対する抗体を含む大腸がん検出キットを包含する。
別の実施形態において、本発明は、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、及び配列番号９で表されるアミノ酸配列からなる群から選択されるアミノ酸配
列を有する１種又は２種以上のペプチドに対する抗体を含む大腸がん検出キットを包含する。
In one embodiment, the present invention has an amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 4, and a sequence. Amino acid sequence represented by No. 5, amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, and sequence. From the amino acid sequence represented by No. 10, the amino acid sequence represented by SEQ ID NO: 11, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 14. Includes a colon cancer detection kit comprising an antibody against one or more peptides having an amino acid sequence selected from the group.
In another embodiment, the present invention comprises an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 6, and an amino acid sequence represented by SEQ ID NO: 7. And a colon cancer detection kit comprising an antibody against one or more peptides having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 9.
別の実施形態において、本発明は、 配列番号１で表されるアミノ酸配列、配列番号２
で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドに対する抗体を検出試薬として含む大腸がん検出剤を包含する。
別の実施形態において、本発明は、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、及び配列番号９で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドに対する抗体を検出試薬として含む大腸がん検出剤を包含する。
In another embodiment, the present invention is the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2.
, Amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 5, amino acid sequence represented by SEQ ID NO: 6, and amino acid sequence represented by SEQ ID NO: 7. , Amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 11, and amino acid sequence represented by SEQ ID NO: 12. Detects antibodies against one or more peptides having an amino acid sequence selected from the group consisting of the amino acid sequence represented by, the amino acid sequence represented by SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 14. Includes colorectal cancer detectors included as reagents.
In another embodiment, the present invention comprises an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 6, and an amino acid sequence represented by SEQ ID NO: 7. And a colon cancer detecting agent containing an antibody against one or more kinds of peptides having an amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 9 as a detection reagent.
また別の実施形態において、本発明は、被験者における大腸がんの罹患可能性を判定するための、コンピュータにより実行される方法であって、被験者の生物試料中の、配列番号１で表されるアミノ酸配列、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号４で表されるアミノ酸配列、配列番号５で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、配列番号８で表されるアミノ酸配列、配列番号９で表されるアミノ酸配列、配列番号１０で表されるアミノ酸配列、配列番号１１で表されるアミノ酸配列、配列番号１２で表されるアミノ酸配列、配列番号１３で表されるアミノ酸配列、及び配列番号１４で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する２種以上のペプチドについての定量的データを取得する工程と、前記取得したデータを、前記２種以上のペプチドの関数である多変量ロジスティック回帰モデルに適用し、被験者における大腸がんの罹患可能性の予測確率を求める工程とを含む方法を包含する。ここで、ペプチドの定量的データとは、例えば質量分析やペプチドに対する抗体を用いて測定されたペプチドの発現量、血中濃度等の定量的な測定値を指す。
別の実施形態において、本発明は、被験者における大腸がんの罹患可能性を判定するための、コンピュータにより実行される方法であって、被験者の生物試料中の、配列番号２で表されるアミノ酸配列、配列番号３で表されるアミノ酸配列、配列番号６で表されるアミノ酸配列、配列番号７で表されるアミノ酸配列、及び配列番号９で表されるアミノ酸配列からなる群から選択されるアミノ酸配列を有する１種又は２種以上のペプチドについての定量的データを取得する工程と、前記取得したデータを、前記１種又は２種以上のペプチドの関数である多変量ロジスティック回帰モデルに適用し、被験者における大腸がんの罹患可能性の予測確率（予測罹患確率）を求める工程とを含む方法を包含する。
好ましい実施形態において、定量的データを取得するペプチドは、配列番号２で表されるアミノ酸配列からなるペプチド、配列番号３で表されるアミノ酸配列からなるペプチド、配列番号６で表されるアミノ酸配列からなるペプチド、配列番号７で表されるアミノ酸配列からなるペプチド、及び配列番号９で表されるアミノ酸配列からなるペプチドの５つのペプチドである。
In yet another embodiment, the invention is a computer-executed method for determining the susceptibility to colorectal cancer in a subject, represented by SEQ ID NO: 1 in the subject's biological sample. Amino acid sequence, amino acid sequence represented by SEQ ID NO: 2, amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 5, and represented by SEQ ID NO: 6. Amino acid sequence, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10, represented by SEQ ID NO: 11. For two or more peptides having an amino acid sequence selected from the group consisting of an amino acid sequence, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 13, and an amino acid sequence represented by SEQ ID NO: 14. And the step of applying the obtained data to a multivariate logistic regression model which is a function of the two or more kinds of peptides to obtain the prediction probability of the possibility of developing colorectal cancer in a subject. Includes methods including. Here, the quantitative data of the peptide refers to quantitatively measured values such as the expression level of the peptide and the blood concentration measured by mass analysis or using an antibody against the peptide.
In another embodiment, the invention is a computerized method for determining the likelihood of developing colorectal cancer in a subject, the amino acid represented by SEQ ID NO: 2 in the subject's biological sample. An amino acid selected from the group consisting of a sequence, an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 7, and an amino acid sequence represented by SEQ ID NO: 9. The step of acquiring quantitative data for one or more peptides having a sequence and the acquired data are applied to a multivariate logistic regression model which is a function of the one or more peptides. It includes a method including a step of obtaining a predicted probability of morbidity of colorectal cancer (predicted morbidity probability) in a subject.
In a preferred embodiment, the peptide for which quantitative data is obtained is from a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and the amino acid sequence represented by SEQ ID NO: 6. , A peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
上記コンピュータにより実行される方法は、予測確率を求めた後で、該予測確率に基づいて被験者における大腸がんの罹患可能性を判定する工程をさらに含んでもよい。例えば求めた予測確率が或る閾値を超えた場合に、その被験者を大腸がんに罹患していると判定する。通常、閾値は０．５であり、好ましくは０．５６８である。 The method performed by the computer may further include determining the likelihood of colorectal cancer in the subject based on the predicted probabilities after determining the predicted probabilities. For example, when the obtained prediction probability exceeds a certain threshold value, the subject is determined to have colorectal cancer. The threshold is usually 0.5, preferably 0.568.
別の実施形態において、本発明のペプチドは、検出以外に積極的な大腸がんの創薬ターゲットを提供することもできる。即ち、該ペプチドそれ自体が該疾患の治療（寛解）方向に生理機能を持つ（「治療ペプチド」という）場合、該ペプチドの量もしくは活性を増大させる物質を患者に投与することにより、また、該ペプチドそれ自体が該疾患の増悪方向に生理機能を持つ場合（「増悪ペプチド」という）、該ペプチドの量もしくは活性を低減させる物質を投与することにより、それぞれ該疾患を治療することができる。 In another embodiment, the peptides of the invention can also provide an aggressive drug discovery target for colorectal cancer in addition to detection. That is, when the peptide itself has a physiological function in the direction of treatment (remission) of the disease (referred to as "therapeutic peptide"), by administering to the patient a substance that increases the amount or activity of the peptide, and also When the peptide itself has a physiological function in the direction of exacerbation of the disease (referred to as "exacerbation peptide"), the disease can be treated by administering a substance that reduces the amount or activity of the peptide.
別の実施形態において、本発明はまた、本発明のペプチドが治療ペプチドとして作用する場合に、該ペプチドの量もしくは活性を増大させる、および／または、本発明のペプチドが増悪ペプチドとして作用する場合に、該ペプチドの量もしくは活性を低減させることによる、大腸がんの治療方法を提供する。該治療方法は、具体的には、治療ペプチドとしての本発明のペプチドの量もしくは活性を増大させる物質および／または増悪ペプチドとしての本発明のペプチドの量もしくは活性を低減させる物質の有効量を、大腸がん患者に投与することを含む。従って、本発明はまた、治療ペプチドとしての本発明のペプチドの量もしくは活性を増大させる物質および／または増悪ペプチドとしての本発明のペプチドの量もしくは活性を低減させる物質を含有してなる、大腸がん治療剤を提供する。 In another embodiment, the invention also increases the amount or activity of the peptide when the peptide of the invention acts as a therapeutic peptide, and / or when the peptide of the invention acts as an exacerbating peptide. , Provide a method for treating colon cancer by reducing the amount or activity of the peptide. The therapeutic method specifically comprises an effective amount of a substance that increases the amount or activity of the peptide of the invention as a therapeutic peptide and / or a substance that reduces the amount or activity of the peptide of the invention as an exacerbating peptide. Includes administration to patients with colorectal cancer. Accordingly, the present invention also comprises a substance that increases the amount or activity of the peptide of the invention as a therapeutic peptide and / or a substance that reduces the amount or activity of the peptide of the invention as an exacerbating peptide. Provide a therapeutic agent.
具体的には、治療ペプチドとしての本発明のペプチドの活性を増大させる物質としては、該ペプチド自体あるいはそれと同様のアゴニスト作用を有する分子が挙げられる。あるいは、治療ペプチドとしての本発明のペプチドの活性を増大させる物質として、該ペプチドの非中和抗体、好ましくはアゴニスト抗体なども挙げることができる。一方、増悪ペプチドとしての本発明のペプチドの活性を低減させる物質としては、該ペプチドのアンタゴニスト作用を有する分子、あるいは該ペプチドに対する中和抗体などが挙げられる。 Specifically, examples of the substance that increases the activity of the peptide of the present invention as a therapeutic peptide include the peptide itself or a molecule having an agonistic action similar thereto. Alternatively, as a substance that increases the activity of the peptide of the present invention as a therapeutic peptide, a non-neutralizing antibody of the peptide, preferably an agonist antibody and the like can also be mentioned. On the other hand, examples of the substance that reduces the activity of the peptide of the present invention as an exacerbating peptide include a molecule having an antagonistic action on the peptide, a neutralizing antibody against the peptide, and the like.
治療ペプチドとしての本発明のペプチドの量もしくは活性を増大させる物質および増悪ペプチドとしての本発明のペプチドの量もしくは活性を低減させる物質は、常套手段に従って製剤化することができる。 A substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and a substance that reduces the amount or activity of the peptide of the present invention as an exacerbating peptide can be formulated according to conventional means.
例えば、経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤などがあげられる。かかる組成物は自体公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有するものである。例えば、錠剤用の担体、賦形剤としては、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムなどが用いられる。 For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules). , Syrups, emulsions, suspensions and the like. Such compositions are produced by methods known per se and contain carriers, diluents or excipients commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
非経口投与のための組成物としては、例えば、注射剤、坐剤などが用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤、関節内注射剤などの剤形を包含する。注射剤、坐剤などでは、有効成分（該ペプチド）の血中濃度の延長や吸収効率の増加を目的に、既存の方法による化学修飾（糖鎖、ＰＥＧその他）体が使用される。かかる注射剤は、自体公知の方法に従って、例えば、上記化合物またはその塩を通常注射剤に用いられる無菌の水性もしくは油性液に溶解、懸濁または乳化することによって調製する。調製された注射液は、通常、適当なアンプルに充填される。直腸投与に用いられる坐剤は、上記化合物またはその塩を通常の坐薬用基剤に混合することによって調製される。 As the composition for parenteral administration, for example, injections, suppositories and the like are used, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, intra-articular injection. Includes dosage forms such as agents. In injections, suppositories and the like, chemically modified (sugar chains, PEG, etc.) bodies by existing methods are used for the purpose of prolonging the blood concentration of the active ingredient (the peptide) and increasing the absorption efficiency. Such injections are prepared according to methods known per se, for example, by dissolving, suspending or emulsifying the above compounds or salts thereof in a sterile aqueous or oily liquid usually used for injections. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration are prepared by mixing the above compounds or salts thereof with a conventional suppository base.
上記の経口用または非経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。かかる投薬単位の剤形としては、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤などが例示され、それぞれの投薬単位剤形当たり非生物学的製剤では通常5～500ｍｇ、とりわけ注射剤では5～100ｍｇ、その他の剤形で
は10～250ｍｇの上記化合物が含有され、生物学的製剤の注射剤では10～50000mgの上記化合物が含有されていることが好ましい。
It is convenient that the oral or parenteral pharmaceutical compositions described above are prepared in dosage forms of dosage units that are compatible with the dosage of the active ingredient. Dosage forms of such dosage units include tablets, pills, capsules, injections (ampoules), suppositories, etc., and the non-biological product for each dosage unit dosage form is usually 5 to 500 mg, especially injection. It is preferable that the agent contains 5 to 100 mg of the above compound, the other dosage form contains 10 to 250 mg of the above compound, and the injection of the biologic contains 10 to 50000 mg of the above compound.
なお前記した各組成物は、上記治療ペプチドとしての本発明のペプチドの量もしくは活性を増大させる物質または増悪ペプチドとしての本発明のペプチドの量もしくは活性を低減させる物質との配合により、好ましくない相互作用を生じない限り、他の活性成分を含有してもよい。 It should be noted that each of the above-mentioned compositions is not preferable due to the combination with a substance that increases the amount or activity of the peptide of the present invention as the therapeutic peptide or a substance that reduces the amount or activity of the peptide of the present invention as an exacerbating peptide. Other active ingredients may be contained as long as they do not cause an action.
このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトに対して経口的にまたは非経口的に投与することができる。 Since the pharmaceutical product thus obtained is safe and has low toxicity, it can be administered orally or parenterally to humans, for example.
治療ペプチドとしての本発明のペプチドの量もしくは活性を増大させる物質および増悪ペプチドとしての本発明のペプチドの量もしくは活性を低減させる物質の投与量は、その作用、投与ルート、患者の重篤度、年齢、体重、薬物受容性などにより差異はあるが、例えば、成人１日あたり活性成分量として非生物学的製剤では約0.0008～約25ｍｇ／ｋｇ、好ましくは約0.008～約2ｍｇ／ｋｇの範囲であり、これを1回もしくは数回に分けて投与
することができる。生物学的製剤の注射剤では10～5000mg/kg, 好ましくは約10～約2000
ｍｇ／ｋｇの範囲であり、これを1回もしくは数回に分けて投与することができる。
The dose of the substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and the substance that decreases the amount or activity of the peptide of the present invention as an exacerbating peptide is the action, administration route, severity of the patient, and so on. Although there are differences depending on age, body weight, drug acceptability, etc., for example, the amount of active ingredient per day for adults is in the range of about 0.0008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg for non-biological preparations. There is, and this can be administered once or in several divided doses. Biopharmacy injections 10-5000 mg / kg, preferably about 10-2000
It is in the range of mg / kg and can be administered once or in several divided doses.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明がこれらに限定されないことは言うまでもない。 Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited thereto.
本明細書中に引用されているすべての特許出願および文献の開示は、それらの全体が参照により本明細書に組み込まれるものとする。 All patent applications and literature disclosures cited herein are incorporated herein by reference in their entirety.
実施例１ BLOTCHIP（登録商標）を用いたプロファイリング解析
（１）検体
京都府立医科大学でサンプリングした大腸がん（UICC7分類、ステージIIからIV）患者
の血清72例および健常者の血清63例1.5μLをドデシル硫酸ナトリウム（SDS）ポリアクリ
ルアミドゲル電気泳動（PAGE）に供し、ペプチドをタンパク質と分離した。
Example 1 Profiling analysis using BLOTCHIP (registered trademark) (1) Specimen Serum of 72 patients with colorectal cancer (UICC7 classification, stage II to IV) sampled at Kyoto Prefectural University of Medicine and 63 cases of healthy subjects 1.5 μL Was subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to separate the peptide from the protein.
（２）BLOTCHIP（登録商標）による質量分析およびディファレンシャルプロファイリング解析
電気泳動終了後、ゲルを切り出し、BLOTCHIP（登録商標）（Protosera, Inc.）に電気
転写した。転写終了後、チップの表面を超純水でリンスし、チップ全体にマトリックス（α-Cyano-4-hydroxy cinnamic acid, Sigma-Aldrich Co., アメリカ合衆国ミズーリ州）
を塗布後、matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectrometer (Bruker Daltonics社製 UltraFlexII)で、Proteomics 11:2727-2737.に記載された通りに質量分析を行った。積算スペクトルをClinProTools2.2(Bruker Daltonik GmbH) を用いて、大腸がん患者血清と非大腸がん対照血清の間でディファレンシャルプロファイリング解析を行った。解析手法は以下の通りである。
(2) Mass spectrometry and differential profiling analysis by BLOTCHIP (registered trademark) After the completion of electrophoresis, the gel was cut out and electrotransferred to BLOTCHIP (registered trademark) (Protosera, Inc.). After the transfer is completed, the surface of the chip is rinsed with ultrapure water, and the entire chip is matrixed (α-Cyano-4-hydroxy cinnamic acid, Sigma-Aldrich Co., Missouri, USA).
After application, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
Mass spectrometry was performed on a mass spectrometer (UltraFlex II from Bruker Daltonics) as described in Proteomics 11: 2727-2737. A differential profiling analysis was performed between colorectal cancer patient sera and non-colorectal cancer control sera using ClinProTools 2.2 (Bruker Daltonik GmbH) for the integrated spectrum. The analysis method is as follows.
（ａ）ClinProTools 2.2(Bruker)による解析
解析ソフトClinProTools 2.2を使用し、2群間で比較を行い、有意差のあったピークを
抽出した。ClinProTools 2.2にてノーマライゼーション（標準化）後、各ピークについてウィルコクソン検定を実施し、p値が0.05以下の場合に有意差ありと判定した。
(A) Analysis by ClinProTools 2.2 (Bruker) Using the analysis software ClinProTools 2.2, comparisons were made between the two groups, and peaks with significant differences were extracted. After normalization (standardization) with ClinProTools 2.2, Wilcoxon test was performed for each peak, and it was judged that there was a significant difference when the p-value was 0.05 or less.
（ｂ）BlotMate2.0（Protosera）によるMSスペクトルの作成と目視によるノイズピークの除去
解析ソフトFlexAnalysis2.4を用いて一検体あたり4回繰り返し測定により得られた4つ
の積算スペクトルをさらに積算し、全平均積算スペクトルを得た。一つの全平均積算スペクトルは一つの血清検体に対応している。解析ソフトBlotMate2.0を用いて、全平均積算
スペクトルのノーマライゼーションを行った後、前項目にて実施したClinProTools2.2の
有意差検定により有意差ありとなったピークについて、MSスペクトルを描画した。目視によりピーク形状、ピーク強度について精査を実施し、ノイズを含むピークを除外した。
(B) Creation of MS spectrum by BlotMate2.0 (Protosera) and removal of noise peak by visual inspection Using the analysis software FlexAnalysis 2.4, the four integrated spectra obtained by repeated measurement four times per sample are further integrated and totaled. An average integrated spectrum was obtained. One total average integrated spectrum corresponds to one serum sample. After normalizing the total average integrated spectrum using the analysis software BlotMate 2.0, the MS spectrum was drawn for the peaks that had a significant difference by the significance test of ClinProTools 2.2 performed in the previous item. The peak shape and peak intensity were visually inspected, and peaks containing noise were excluded.
以上のディファレンシャルプロファイリング解析の結果、ClinProTools2.2の解析と、
その後の目視による精査により14個のピークをピックアップし、下記の分子量Ｍを有する14種のペプチドをバイオマーカー候補（ターゲットペプチド）群とした（表１は分子量（[M+H]+）で示す）。
約1465.66、約1616.66、約2390.26、約2739.53、約2768.23、約2858.42、約3622.78、約7759.18、約3949.98、約4038.05、約4089.02、約4152.99、約4352.34、約5078.35
As a result of the above differential profiling analysis, ClinProTools 2.2 analysis and
After that, 14 peaks were picked up by visual inspection, and 14 kinds of peptides having the following molecular weight M were used as a biomarker candidate (target peptide) group (Table 1 shows the molecular weight ([M + H] +). ).
Approx. 1465.66, Approx. 1616.66, Approx. 2390.26, Approx. 2739.53, Approx. 2768.23, Approx.
（３）ペプチドシーケンシング
上記の番号１～１４のペプチドのアミノ酸配列を周知のペプチド配列決定法により同定した（表２）。なお、配列番号１２と配列番号１４のペプチドは質量分析中にメチオニンが酸化された可能性がある。
（４）多変量ロジスティック回帰モデル
ここで、表３に示される１４個のペプチドでは、最もＡＵＣが高い９番目の血液凝固第XIII因子 Ａ鎖由来のペプチドでも、この一つのペプチドではＡＵＣが０．８０３以下で
あったため、１４個のペプチドからランダムに選択した２～５個のペプチドを用いてモデルを構築した。モデルの構築には統計解析ソフトR（R Core Team (2013). R: A language
and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.）を使用した。ロジスティック
回帰モデル構築はRのパッケージである”ResourceSelection パッケージ“（http://cran.r-project.org/package=ResourceSelection）を使用した。15,302のペプチドの組の組み合わせを調べた後、以下の配列番号２で表されるアミノ酸配列からなるペプチド（分子量1616.66）、配列番号３で表されるアミノ酸配列からなるペプチド（分子量2390.26）、配列番号６で表されるアミノ酸配列からなるペプチド（分子量2858.42）、配列番号７で表
されるアミノ酸配列からなるペプチド（分子量3622.78）、及び配列番号９で表されるア
ミノ酸配列からなるペプチド（分子量3949.98）の５つのペプチドの血中濃度の関数であ
るロジスティック回帰モデルを得た：
予測罹患確率=1/{1+exp(-(3.836581e-05×[mz1616.66]-5.079913e-04×[mz2390.26]-3.288524e-05×[mz2858.42]+5.243605e-04×[mz3622.78]-5.477792e-05×[mz3949.98]+4.3794
56)}・・・ （１）
(4) Multivariate logistic regression model Here, among the 14 peptides shown in Table 3, even the peptide derived from the 9th blood coagulation factor XIII A chain having the highest AUC has an AUC of 0. Since it was 803 or less, a model was constructed using 2 to 5 peptides randomly selected from 14 peptides. Statistical analysis software R (R Core Team (2013). R: A language for model construction
and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.) Was used. The logistic regression model was constructed using the R package "ResourceSelection Package" (http://cran.r-project.org/package=ResourceSelection). After investigating the combination of the peptide pairs of 15,302, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 below (molecular weight 1616.66), the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (molecular weight 2390.26), and SEQ ID NO: A peptide consisting of the amino acid sequence represented by 6 (molecular weight 2588.42), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (molecular weight 3622.78), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 (molecular weight 3949.98). A logistic regression model, which is a function of blood concentrations of 5 peptides, was obtained:
Predicted morbidity probability = 1 / {1 + exp (-(3.836581e-05 × [mz1616.66]-5.079913e-04 × [mz2390.26]-3.288524e-05 × [mz2858.42] +5.243605e-04 × [mz3622.78] -5.477792e-05 × [mz3949.98] +4.3794
56)} ・ ・ ・ (1)
構築したモデルの診断能の評価のためにROC分析を実施した。Rのパッケージである”Epi パッケージ“（A package for statistical analysis in epidemiology、Version 1.149、http://cran.r-project.org/web/packages/Epi/index.html）を用いた。構築したモデルの大腸がんステージ（II期+IIIa期、IIIb期+IV期、およびII～IV期）のAUC、SN、SP、cutoff値について95%信頼区間とともに表３に示した。95%信頼区間は、ブートストラップ
抽出を用いて推定し、サンプリング回数は10,000回、復元抽出によりサンプリングを行った。
ROC analysis was performed to evaluate the diagnostic ability of the constructed model. The R package "Epi package" (A package for statistical analysis in epidemiology, Version 1.149, http://cran.r-project.org/web/packages/Epi/index.html) was used. Table 3 shows the AUC, SN, SP, and cutoff values of the colorectal cancer stages (stage II + IIIa, stage IIIb + stage IV, and stages II to IV) of the constructed model together with 95% confidence intervals. The 95% confidence interval was estimated using bootstrap extraction, the number of samplings was 10,000, and sampling was performed by restoration extraction.
実施例４ 検出性能の評価
実施例３において得られたロジスティック回帰モデルを用いて、健常者群(n=63)、大腸がん患者群（n=72）について、検出性能の評価を実施した。5個のマーカーの多変量ロジ
スティック予測モデルを用いた場合、大腸がん患者群全体におけるROCの曲線下面積（AUC）は0.924(95%信頼区間)であり、ステージIIとIIIaの患者ではAUCは0.917、ステージIIIbとIVの患者ではAUCは0.945であった（図１，表３）。
Example 4 Evaluation of detection performance Using the logistic regression model obtained in Example 3, the detection performance was evaluated for the healthy subject group (n = 63) and the colorectal cancer patient group (n = 72). Using a 5-marker multivariate logistic prediction model, the ROC subcurve area (AUC) for the entire colon cancer patient population was 0.924 (95% confidence interval), and the AUC was for stage II and IIIa patients. Patients with 0.917, stage IIIb and IV had an AUC of 0.945 (Figures 1 and 3).
本発明の新規な大腸がん検出マーカーを利用した臨床検査方法は、大腸がんを迅速且つ的確に判断できるので、該疾患の早期発見、早期治療が可能となる点で有用である。 The clinical examination method using the novel colorectal cancer detection marker of the present invention is useful in that it can quickly and accurately determine colorectal cancer, and thus enable early detection and early treatment of the disease.
Claims (13)
に対する抗体を含む大腸がん検出キット。 Amino acid sequence represented by SEQ ID NO: 1, amino acid sequence represented by SEQ ID NO: 2, amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 5, Amino acid sequence represented by SEQ ID NO: 6, amino acid sequence represented by SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10. An amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 11, the amino acid sequence represented by SEQ ID NO: 12, the amino acid sequence represented by SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 14. A colon cancer detection kit containing an antibody against one or more kinds of peptides having.
を含む方法。 A method performed by a computer for determining the susceptibility to colorectal cancer in a subject, which is the amino acid sequence represented by SEQ ID NO: 1 and the amino acid represented by SEQ ID NO: 2 in the subject's biological sample. Sequence, amino acid sequence represented by SEQ ID NO: 3, amino acid sequence represented by SEQ ID NO: 4, amino acid sequence represented by SEQ ID NO: 5, amino acid sequence represented by SEQ ID NO: 6, amino acid represented by SEQ ID NO: 7. Sequence, amino acid sequence represented by SEQ ID NO: 8, amino acid sequence represented by SEQ ID NO: 9, amino acid sequence represented by SEQ ID NO: 10, amino acid sequence represented by SEQ ID NO: 11, amino acid represented by SEQ ID NO: 12. The step of acquiring quantitative data for two or more peptides having an amino acid sequence selected from the group consisting of the sequence, the amino acid sequence represented by SEQ ID NO: 13, and the amino acid sequence represented by SEQ ID NO: 14, and the above-mentioned step. A method including a step of applying the acquired data to a multivariate logistic regression model which is a function of the two or more kinds of peptides to obtain a prediction probability of the possibility of developing colorectal cancer in a subject.
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