JP5114637B2 - Determination method of small cell lung cancer - Google Patents
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Description
本発明は、早期肺癌に対応できる腫瘍マーカー、詳しくは小細胞肺癌に特異的な腫瘍マーカーに関する。 The present invention relates to a tumor marker that can cope with early stage lung cancer, and more particularly to a tumor marker that is specific to small cell lung cancer.
癌治療における診断・判定として血中腫瘍マーカーの計測が挙げられる。腫瘍マーカーは腫瘍または腫瘍と反応した正常細胞が作る物質の中で、血中や尿中などに放出され腫瘍の体外診断を可能としているものである。 Diagnosis and determination in cancer treatment includes measurement of blood tumor markers. A tumor marker is a substance produced by a tumor or normal cells that have reacted with the tumor, and is released into blood, urine, etc. to enable in vitro diagnosis of the tumor.
上記腫瘍マーカーに関しては、乳癌患者と正常人の胸部組織に差異的に発現する遺伝子を使用した悪性腫瘍、特に乳癌を予想、診断 、予後判定、予防および処置するための新
規な組成物、方法および使用（例えば、特許文献１参照）や、肝炎ウイルスの感染に起因し慢性肝炎または肝硬変を発症した患者に対し、肝炎ウイルスに感染した患者のミトコンドリアＤＮＡの塩基配列を評価することによって肝臓癌の発癌リスクを評価する方法（例えば、特許文献２参照）や、その遺伝子産物がＣＬＬ細胞および前立腺癌細胞の新生物性または腫瘍形成性増殖を抑制するｍｉＲ１５およびｍｉＲ１６のコピー数、変異状態または遺伝子発現を検出することによる慢性リンパ性白血病または前立腺癌の診断方法（例えば、特許文献３参照）や、無症候性期または初期段階の患者における発癌過程の存在を検出するのに適当な迅速な癌の診断方法や、癌患者の血清中に存在する炭酸脱水酵素ＩＩの活性化化合物、すなわち腫瘍マーカー、該癌の診断方法によって、無症候性期または初期段階の患者の癌を検出することを特徴とする癌の処置方法；ならびに該癌の診断方法を行うためのキット（例えば、特許文献４参照）等が知られている。
Regarding the tumor marker, a novel composition, method and method for predicting, diagnosing, prognosing, preventing and treating malignant tumors, particularly breast cancer, using genes differentially expressed in breast tissue of breast cancer patients and normal humans Carcinogenesis of liver cancer by evaluating the base sequence of mitochondrial DNA of patients infected with hepatitis virus against patients who have developed chronic hepatitis or cirrhosis due to use (for example, see Patent Document 1) or infection with hepatitis virus A method for assessing risk (see, for example, Patent Document 2), and the copy number, mutation state, or gene expression of miR15 and miR16 whose gene product suppresses neoplastic or tumorigenic growth of CLL cells and prostate cancer cells. Diagnosis of chronic lymphocytic leukemia or prostate cancer by detection (for example, see Patent Document 3), or asymptomatic A rapid cancer diagnostic method suitable for detecting the presence of a carcinogenic process in a patient in sexual phase or early stage, or an activated compound of carbonic anhydrase II present in the serum of a cancer patient, ie a tumor marker, the cancer A method for treating cancer characterized by detecting a cancer in an asymptomatic or early stage patient according to the diagnostic method; and a kit for performing the method for diagnosing the cancer (for example, see Patent Document 4) Are known.
また、肺癌は小細胞癌と非小細胞癌の２つの型に大きく分類され、非小細胞癌はさらに腺癌、扁平上皮癌、大細胞癌の３タイプに分類され、それぞれのタイプによって発生する部位がある程度決まっており、小細胞癌と扁平上皮癌は肺門部に、腺癌と大細胞癌は肺野部にできやすい肺癌といわれている。肺癌に関して、小細胞肺癌 (ＳＣＬＣ）を患う患者を治療し、またはＳＣＬＣ腫瘍の存在を検出するための、比較的短いペプチド、詳細には、α−コノトキシンペプチドＭＩＩおよびＵ００２を使用して小細胞肺癌の存在または位置を決定することにより検出する方法（例えば、特許文献５参照）や、血清中の肺癌マーカーとして優れる血管透過性因子を測定することにより、特に化学発光検出系酵素免疫測定法を用いて高感度で測定することにより、肺癌の早期診断、さらには治療効果の判定、経過観察を行う方法（例えば、特許文献６参照）等が知られている。 In addition, lung cancer is broadly classified into two types, small cell cancer and non-small cell cancer, and non-small cell cancer is further classified into three types, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and occurs depending on each type. The site is determined to some extent, and small cell carcinoma and squamous cell carcinoma are said to be easily formed in the hilar region, and adenocarcinoma and large cell carcinoma are easily found in the lung region. With respect to lung cancer, small cells using relatively short peptides, specifically α-conotoxin peptides MII and U002, for treating patients suffering from small cell lung cancer (SCLC) or detecting the presence of SCLC tumors A method for detecting lung cancer by determining the presence or location of lung cancer (see, for example, Patent Document 5), or measuring a blood vessel permeability factor that is excellent as a lung cancer marker in serum, particularly a chemiluminescence detection system enzyme immunoassay method. A method of performing early diagnosis of lung cancer, determination of therapeutic effect, follow-up observation, and the like (for example, see Patent Document 6) by using and measuring with high sensitivity is known.
上述の通り、これまでに様々な腫瘍マーカーが開発されているが、擬陽性が多く初期の癌の検出が困難であるなど改善の余地が多く残されており、特に肺癌・膵臓癌・大腸癌など難治性の癌のマーカーの開発が求められている。更に、現在日本において肺癌は男性の癌死亡率の第１位であり、女性では胃癌に次いで第２位であることから、新たな肺癌に対する腫瘍マーカーの開発による早期診断・治療成績の向上が期待されていた。本発明の課題は、早期診断が可能な小細胞肺癌に対する特異的な新規腫瘍マーカーを提供することにある。 As described above, various tumor markers have been developed so far, but there is still a lot of room for improvement such as many false positives and difficult to detect early cancers, especially lung cancer, pancreatic cancer, colon cancer, etc. There is a need to develop markers for intractable cancer. Furthermore, lung cancer is currently ranked first in Japan for cancer mortality in men and second in women after gastric cancer, so the development of new tumor markers for lung cancer is expected to improve early diagnosis and treatment results. It had been. An object of the present invention is to provide a specific novel tumor marker for small cell lung cancer capable of early diagnosis.
本発明者らは、特に早期肺癌の診断のために有用な腫瘍マーカー候補物質の探索を目的として、ヒト肺癌由来培養細胞株の培養上清に放出されるたんぱく質・ペプチドのプロテオーム解析を行った結果、小細胞肺癌培養細胞株であるＳＢＣ３の培養上清にproNeurotensin/Neuromedin N（ｐｒｏＮＴ／ＮＭＮ）が放出されているが、小細胞肺癌以外の肺癌細胞株（腺癌・大細胞癌・扁平上皮癌）の培養上清にはｐｒｏＮＴ／ＮＭＮが検出されないことを見い出し、本発明を完成するに至った。 The results of proteomic analysis of proteins and peptides released into the culture supernatant of human lung cancer-derived cell lines with the aim of searching for tumor marker candidate substances that are particularly useful for diagnosis of early lung cancer , ProNeurotensin / Neuromedin N (proNT / NMN) is released in the culture supernatant of SBC3, which is a small cell lung cancer cell line. It was found that proNT / NMN was not detected in the culture supernatant of (2), and the present invention was completed.
すなわち本発明は、（１）ヒトから採取した生体試料中の、配列番号１に示されたアミノ酸配列からなるペプチドの発現量を測定することを特徴とする小細胞肺癌の判定方法に関する。
That is, the present invention provides (1) in the biological sample taken from a human, relates to the determination how small cell lung cancer, characterized by measuring the expression level of a peptide consisting of the amino-acid sequence set forth in SEQ ID NO: 1.
また本発明は、（２）肺癌患者から採取した生体試料中の、配列番号１に示されたアミノ酸配列からなるペプチドの発現量を測定することを特徴とする小細胞肺癌再発のリスク評価方法や、（３）抗癌剤を服用している小細胞肺癌患者から採取した生体試料中の、配列番号１に示されたアミノ酸配列からなるペプチドの発現量を測定することを特徴とする抗癌剤の有効性の判定方法に関する。
The present invention also relates to ( 2 ) a risk evaluation method for recurrence of small cell lung cancer, characterized by measuring the expression level of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a biological sample collected from a lung cancer patient, (3 ) The effectiveness of the anticancer agent characterized by measuring the expression level of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in a biological sample collected from a small cell lung cancer patient taking the anticancer agent. It relates to a determination method.
さらに本発明は、（４）ヒト小細胞肺癌培養細胞株を被検物質の存在下に培養し、細胞溶解物中の、配列番号１に示されたアミノ酸配列からなるペプチドの発現量を測定し、被検物質の非存在下における場合と比較・評価することを特徴とする小細胞肺癌の抑制剤・治療剤のスクリーニング方法に関する。
The present invention further comprises (4 ) culturing a human small cell lung cancer cell line in the presence of a test substance, and measuring the expression level of a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the cell lysate. The present invention also relates to a screening method for a suppressor / therapeutic agent for small cell lung cancer, characterized in that it is compared and evaluated in the absence of a test substance.
本発明によると、小細胞肺癌の判定、小細胞肺癌再発のリスク評価、抗癌剤の小細胞肺癌に対する有効性の判定、小細胞肺癌の抑制剤・治療剤のスクリーニングを行うことができる。 According to the present invention, it is possible to determine small cell lung cancer, assess the risk of recurrence of small cell lung cancer, determine the effectiveness of an anticancer agent against small cell lung cancer, and screen for inhibitors / therapeutic agents for small cell lung cancer.
本発明の肺癌の判定方法としては、ヒトから採取した血清、痰、肺組織等の生体試料中の、配列番号１に示されたアミノ酸配列からなるペプチド（ｐｒｏＮＴ／ＮＭＮ）の発現量を測定する方法であれば特に制限されるものではなく、また本発明の肺癌の判定用キットとしては、ｐｒｏＮＴ／ＮＭＮに特異的に結合する抗体、又はそれらの標識物を備えたキットや、ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプライマーセット若しくはプローブ、又はそれらの標識物を備えたキットであれば特に制限されるものではなく、また、ｐｒｏＮＴ／ＮＭＮの発現量の測定方法としては、血清、痰、肺組織溶解物等を対象サンプルとして、上記本発明の肺癌の判定用キットを用いる、ＥＬＩＳＡ法等の免疫反応を利用する方法や、ドットブロット法、ノーザンブロット法、ＲＮアーゼプロテクションアッセイ法、ＲＴ（Reverse Transcription）−ＰＣＲ法等の発現している遺伝子を転写レベルで検出する方法の他、二次元電気泳動に供試して、所定箇所に現れるスポットの大きさを測定する方法を挙げることができる。そして、ｐｒｏＮＴ／ＮＭＮの発現を検出した場合、対象者は小細胞肺癌患者と判定されることになる。 As a method for determining lung cancer of the present invention, the expression level of a peptide (proNT / NMN) comprising the amino acid sequence shown in SEQ ID NO: 1 in a biological sample such as serum, sputum, lung tissue or the like collected from a human is measured. The method is not particularly limited, and the lung cancer determination kit of the present invention includes an antibody that specifically binds to proNT / NMN, or a kit comprising a label thereof, or proNT / NMN. The kit is not particularly limited as long as it is a kit provided with a primer set or probe for detecting the presence of mRNA, or a label thereof, and the method for measuring the expression level of proNT / NMN includes serum, sputum , Using a lung cancer lysate or the like as a target sample, using the above-described lung cancer determination kit of the present invention, a method utilizing an immune reaction such as an ELISA method, or a dot In addition to methods for detecting the expressed gene at the transcription level, such as lot method, Northern blot method, RNase protection assay method, RT (Reverse Transcription) -PCR method, etc. A method of measuring the size of the spot that appears can be mentioned. When the expression of proNT / NMN is detected, the subject is determined to be a small cell lung cancer patient.
本発明の肺癌再発のリスク評価方法としては、肺癌患者から採取した血清、痰、肺癌組織等の生体試料中のｐｒｏＮＴ／ＮＭＮの発現量を測定する方法であれば特に制限されるものではなく、また本発明の肺癌再発のリスク評価用キットとしては、ｐｒｏＮＴ／ＮＭＮに特異的に結合する抗体、又はそれらの標識物を備えたキットや、ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプライマーセット若しくはプローブ、又はそれらの標識物を備えたキットであれば特に制限されるものではなく、また、ｐｒｏＮＴ／ＮＭＮの発現量の測定方法としては、血清、痰、肺癌細胞溶解物等を対象サンプルとして、上記本発明の小細胞肺癌再発のリスク評価用キットを用いる、ＥＬＩＳＡ法等の免疫反応を利用する方法や、ドットブロット法、ノーザンブロット法、ＲＮアーゼプロテクションアッセイ法、ＲＴ−ＰＣＲ法等の発現している遺伝子を転写レベルで検出する方法の他、二次元電気泳動に供試して、所定箇所に現れるスポットの大きさを測定する方法を挙げることができる。そして、ｐｒｏＮＴ／ＮＭＮの発現を検出した場合、対象者は小細胞肺癌が再発していると判定されることになる。 The risk assessment method for lung cancer recurrence of the present invention is not particularly limited as long as it is a method for measuring the expression level of proNT / NMN in a biological sample such as serum, sputum, lung cancer tissue collected from a lung cancer patient, The kit for assessing the risk of recurrence of lung cancer of the present invention includes a kit comprising antibodies that specifically bind to proNT / NMN, or a label thereof, and a primer set for detecting the presence of proNT / NMN mRNA. Alternatively, the kit is not particularly limited as long as it is a kit equipped with a probe or a labeled product thereof. In addition, as a method for measuring the expression level of proNT / NMN, serum, sputum, lung cancer cell lysate or the like is used as a target sample. A method using an immune reaction such as ELISA using the kit for evaluating the risk of recurrence of small cell lung cancer of the present invention, or a dot blot method In addition to methods for detecting expressed genes at the transcription level, such as Northern blotting, RNase protection assay, RT-PCR, etc., the size of spots appearing at predetermined locations is measured by two-dimensional electrophoresis The method of doing can be mentioned. When proNT / NMN expression is detected, the subject is determined to have relapsed small cell lung cancer.
本発明の抗癌剤の有効性の判定方法としては、抗癌剤を服用している小細胞肺癌患者から採取した血清、痰、肺癌組織等の生体試料中の、ｐｒｏＮＴ／ＮＭＮの発現量を測定する方法であれば特に制限されるものではなく、また本発明の小細胞肺癌に対する抗癌剤の有効性判定用キットとしては、ｐｒｏＮＴ／ＮＭＮに特異的に結合する抗体、又はそれらの標識物を備えたキットや、ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプライマーセット若しくはプローブ、又はそれらの標識物を備えたキットであれば特に制限されるものではなく、抗癌剤の有効性を判定することにより、各患者個人に適した治療、いわゆるテーラーメイド治療が可能となる。また、上記ペプチドｐｒｏＮＴ／ＮＭＮの発現量の測定方法としては、血清、痰、肺癌細胞溶解物等を対象サンプルとして、上記本発明の抗癌剤の小細胞肺癌における有効性判定用キットを用いる、ＥＬＩＳＡ法等の免疫反応を利用する方法や、ドットブロット法、ノーザンブロット法、ＲＮアーゼプロテクションアッセイ法、ＲＴ−ＰＣＲ法等の発現している遺伝子を転写レベルで検出する方法の他、二次元電気泳動に供試して、所定箇所に現れるスポットの大きさを測定する方法を挙げることができる。そして、抗癌剤の服用の結果、ｐｒｏＮＴ／ＮＭＮの発現量が減少した場合、当該抗癌剤は小細胞肺癌の治療に有効であると判定されることになる。 The method for determining the effectiveness of the anticancer agent of the present invention is a method of measuring the expression level of proNT / NMN in a biological sample such as serum, sputum, lung cancer tissue collected from a small cell lung cancer patient taking an anticancer agent. There is no particular limitation as long as it is a kit for determining the effectiveness of an anticancer agent for small cell lung cancer of the present invention, a kit comprising an antibody that specifically binds to proNT / NMN, or a label thereof, It is not particularly limited as long as it is a kit provided with a primer set or probe for detecting the presence of proNT / NMN mRNA, or a label thereof, and by determining the effectiveness of an anticancer agent, The so-called tailor-made treatment can be performed. In addition, as a method for measuring the expression level of the peptide proNT / NMN, an ELISA method using serum, sputum, lung cancer cell lysate or the like as a target sample, and a kit for determining the effectiveness of the anticancer agent of the present invention in small cell lung cancer In addition to methods using immune reactions such as dot blot method, northern blot method, RNase protection assay method, RT-PCR method, etc. A method of measuring the size of a spot appearing at a predetermined place by a test can be mentioned. When the expression level of proNT / NMN decreases as a result of taking the anticancer agent, it is determined that the anticancer agent is effective for the treatment of small cell lung cancer.
本発明の小細胞肺癌の抑制剤・治療剤のスクリーニング方法としては、ヒト肺小細胞癌培養細胞株（例えば、ＳＢＣ３、ＳＢＣ５）を、抗癌剤、抗癌候補物質、低分子化合物、天然有機高分子物質、天然の動植物の抽出物、ペプチドなどの被検物質の存在下に培養し、細胞溶解物中のｐｒｏＮＴ／ＮＭＮの発現量を測定し、被検物質の非存在下における場合と比較・評価する方法であれば特に制限されず、上記ペプチドの発現量を測定する方法としては、ヒト肺癌培養細胞株（ＳＢＣ３、ＳＢＣ５）の細胞溶解物を二次元電気泳動に供試して所定箇所に現れるスポットの大きさを測定する方法の他、ｐｒｏＮＴ／ＮＭＮに特異的に結合する抗体、又はそれらの標識物を用いるＥＬＩＳＡ法等の免疫反応を利用する方法や、ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプライマーセット若しくはプローブ、又はそれらの標識物を用いるドットブロット法、ノーザンブロット法、ＲＮアーゼプロテクションアッセイ法、ＲＴ−ＰＣＲ法等の発現している遺伝子を転写レベルで検出する方法を挙げることができる。そして、被検物質の存在下に培養した細胞溶解物中のｐｒｏＮＴ／ＮＭＮの発現量が、被検物質の非存在下における場合と比較して減少している場合、当該被検物質は小細胞肺癌の治療に有効な小細胞肺癌の抑制剤・治療剤である可能性が大きい。 As a screening method for the inhibitor / therapeutic agent for small cell lung cancer of the present invention, human lung small cell carcinoma cell lines (for example, SBC3, SBC5) are used as anticancer agents, anticancer candidate substances, low molecular compounds, natural organic polymers. Culture in the presence of test substances such as substances, natural animal and plant extracts, peptides, etc., measure the expression level of proNT / NMN in cell lysates, and compare and evaluate in the absence of test substances The method for measuring the expression level of the peptide is a spot that appears in a predetermined place after two-dimensional electrophoresis of a cell lysate of a human lung cancer cell line (SBC3, SBC5) is used. In addition to a method for measuring the size of the antibody, a method using an immune reaction such as an ELISA method using an antibody that specifically binds to proNT / NMN or a label thereof, or proNT / NMN Detection of expressed genes at the transcription level such as dot blot method, Northern blot method, RNase protection assay method, RT-PCR method using primer sets or probes for detecting the presence of mRNA, or labels thereof The method of doing can be mentioned. When the expression level of proNT / NMN in the cell lysate cultured in the presence of the test substance is decreased as compared to the case in the absence of the test substance, the test substance is a small cell. There is a high possibility that it is an inhibitor / therapeutic agent for small cell lung cancer that is effective in the treatment of lung cancer.
上記ｐｒｏＮＴ／ＮＭＮに特異的に結合する抗体としては、モノクローナル抗体、ポリクローナル抗体、一本鎖抗体、ヒト化抗体、キメラ抗体、２つのエピトープを同時に認識することができる二機能性抗体等を例示することができる。これら抗体は、慣用のプロトコールを用いて、動物（好ましくはヒト以外）に、配列番号１に示されたアミノ酸配列からなるペプチド又はエピトープを含む断片を投与することにより産生され、例えばモノクローナル抗体の調製には、連続細胞系の培養物により産生される抗体をもたらす、ハイブリドーマ法（Nature 256, 495-497, 1975）、トリオーマ法、ヒトＢ細胞ハイブリドーマ法（Immunology Today 4, 72, 1983）及びＥＢＶ−ハイブリドーマ法（MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R.Liss, Inc.,1985）など任意の方法を用いることができる。 Examples of antibodies that specifically bind to proNT / NMN include monoclonal antibodies, polyclonal antibodies, single chain antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies that can simultaneously recognize two epitopes, and the like. be able to. These antibodies are produced by administering a fragment comprising the peptide or epitope consisting of the amino acid sequence shown in SEQ ID NO: 1 to an animal (preferably non-human) using a conventional protocol. For example, preparation of a monoclonal antibody Include the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV-, which give rise to antibodies produced by continuous cell line cultures. Any method such as a hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used.
また、一本鎖抗体をつくるために、一本鎖抗体の調製法（米国特許第4,946,778 号、米国特許第5,260,203号、米国特許第5,091,513号、米国特許第5,455,030号）を用いることができ、ヒト化抗体をつくるために、ヒト化抗体の調製法（米国特許第5,585,089号、Nature, 321, 522-525, 1986、Protein Engineering, 4, 773-783, 1991）を用いることができ、キメラ抗体をつくるために、キメラ抗体の調製法（米国特許第4,816,567号、Science, 229, 1202-1207, 1985、BioTechniques, 4, 214, 1986、Nature, 312, 643-646, 1984、Nature, 314, 268.270, 1985）を用いることができる。二機能性抗体は、２つの関連した抗体を産生する２つのモノクローナル細胞系同士のハイブリッド、または２つの抗体の断片の化学結合によって産生することができる。 In addition, a single chain antibody preparation method (US Pat. No. 4,946,778, US Pat. No. 5,260,203, US Pat. No. 5,091,513, US Pat. No. 5,455,030) can be used to produce a single chain antibody, and human In order to make a humanized antibody, a method for preparing a humanized antibody (US Pat. No. 5,585,089, Nature, 321, 522-525, 1986, Protein Engineering, 4, 773-783, 1991) can be used. To produce a chimeric antibody (US Pat. No. 4,816,567, Science, 229, 1202-1207, 1985, BioTechniques, 4, 214, 1986, Nature, 312, 643-646, 1984, Nature, 314, 268.270, 1985) can be used. Bifunctional antibodies can be produced by chemical coupling of hybrids between two monoclonal cell lines that produce two related antibodies, or fragments of two antibodies.
また、上記抗体のＦａｂ断片やＦ（ａｂ’）２断片等も、上記抗体と同様に用いることができる。例えば、Ｆａｂ断片は抗体をパパイン等で処理することにより、またＦ（ａｂ’）２断片はペプシン等で処理することにより調製することができる。 In addition, Fab fragments and F (ab ′) 2 fragments of the above antibodies can be used in the same manner as the above antibodies. For example, the Fab fragment can be prepared by treating an antibody with papain or the like, and the F (ab ′) 2 fragment can be prepared with pepsin or the like.
これら抗体に、例えば、ＦＩＴＣ（フルオレセインイソシアネート）又はテトラメチルローダミンイソシアネート等の蛍光物質や、１２５Ｉ、３２Ｐ、１４Ｃ、３５Ｓ又は３Ｈ等のラジオアイソトープや、アルカリホスファターゼ、ペルオキシダーゼ、β−ガラクトシダーゼ又はフィコエリトリン等の酵素で標識したものや、グリーン蛍光タンパク質（ＧＦＰ）等の蛍光発光タンパク質などを融合させた融合タンパク質を用いることによって、上記配列番号１に示されるアミノ酸からなるペプチドを免疫学的測定方法により測定することができる。かかる免疫学的測定方法としては、ＲＩＡ法、ＥＬＩＳＡ法、蛍光抗体法、プラーク法、スポット法、血球凝集反応法、オクタロニー法等の方法を挙げることができる。 These antibodies include, for example, fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioisotopes such as 125 I, 32 P, 14 C, 35 S or 3 H, alkaline phosphatase, peroxidase, β-galactosidase Alternatively, an immunological measurement of the peptide consisting of the amino acid shown in SEQ ID NO: 1 by using a fusion protein labeled with an enzyme such as phycoerythrin or a fluorescent protein such as green fluorescent protein (GFP). It can be measured by the method. Examples of such immunological measurement methods include RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, octalony method and the like.
上記ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプライマーセットとしては、これらペプチドのｍＲＮＡ又はｃＤＮＡ（配列番号３）のうち、上流及び下流の配列の一部とハイブリダイズしうる相補的なプライマーセットであれば、プライマー配列の長さ及びｍＲＮＡ又はｃＤＮＡのどの部位と相補的であるかなどは特に制限されず、例えば、これらのプライマーは、５’側又は３’側、又はその両方に、上記ペプチドのｍＲＮＡ又はｃＤＮＡ配列と一部に相補的ではない配列を含んでも、これらとハイブリダイズできる限り、プライマーとして用いることができる。また、非特異的な増幅を防ぐためや、適当な制限酵素認識部位を導入するために、これらのｍＲＮＡ又はｃＤＮＡと相補的でないミスマッチ配列を持つプライマーを使用することができる。 As a primer set for detecting the presence of the proNT / NMN mRNA, a complementary primer set capable of hybridizing with a part of the upstream and downstream sequences of the mRNA or cDNA (SEQ ID NO: 3) of these peptides. If so, the length of the primer sequence and which region of the mRNA or cDNA is complementary is not particularly limited. For example, these primers may be placed on the 5 ′ side, 3 ′ side, or both, as described above. Even if it contains a sequence that is not partially complementary to the mRNA or cDNA sequence of the peptide, it can be used as a primer as long as it can hybridize with it. Further, in order to prevent non-specific amplification or to introduce an appropriate restriction enzyme recognition site, a primer having a mismatch sequence that is not complementary to these mRNA or cDNA can be used.
上記ｐｒｏＮＴ／ＮＭＮのｍＲＮＡの存在を検出するためのプローブとしては、これらペプチドをコードするＤＮＡ（ｃＤＮＡ）又はＲＮＡ（ｃＲＮＡ）とハイブリダイズしうるアンチセンス鎖の全部又は一部であり、プローブとして成立する程度の長さ（少なくとも２０ベース以上）を有するものが好ましく、例えば、これらのプローブは、５’側又は３’側、又はその両方に、上記ペプチドのｍＲＮＡ又はｃＤＮＡ配列と一部に相補的ではない配列を含んでも、これらとハイブリダイズできる限り、プローブとして用いることができ、また、検出しやすいように、任意の配列を付加したものを用いることができる他、検出しやすいように５’末端を標識したものを用いることもでき、そのような標識としては、たとえば、ビオチン、蛍光、又は３２Ｐなどを例示することができる。 The probe for detecting the presence of the proNT / NMN mRNA is all or part of the antisense strand that can hybridize with DNA (cDNA) or RNA (cRNA) encoding these peptides, and is established as a probe. For example, these probes are partially complementary to the mRNA or cDNA sequence of the peptide on the 5 'side, 3' side, or both. As long as it can be hybridized with these, it can be used as a probe as long as it can be hybridized with them. In addition, a sequence to which an arbitrary sequence is added so that it can be easily detected can be used. End-labeled ones can also be used, and examples of such labels include biotin, fluorescence, and Such as 32 P can be exemplified.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
（培養細胞株）
実施例では９種類の培養細胞株を用いた。いずれの培養細胞株もヒト肺癌由来のものであり、小細胞癌としてＳＢＣ−３、ＳＢＣ−５、腺癌としてＮＣＩ−Ｈ６５０、ＮＣＩ−Ｈ１６５０、大細胞癌としてＮＣＩ−Ｈ６６１、ＮＣＩ−Ｈ１５８１、ＮＣＩ−Ｈ４６０、扁平上皮癌としてＮＣＩ−Ｈ２１７０、ＮＣＩ−Ｈ１８６９を用いた。ＳＢＣ−３の培養上清を分析し、他の培養上清と比較することで腫瘍マーカーの候補を検索した。これらの細胞株は、American Type Culture Collection (ATCC社）又は財団法人ヒューマン振興財団から入手することができる。
(Cultured cell line)
In the examples, nine types of cultured cell lines were used. All the cultured cell lines are derived from human lung cancer, SBC-3 and SBC-5 as small cell carcinoma, NCI-H650 and NCI-H1650 as adenocarcinoma, NCI-H661, NCI-H1581 and NCI as large cell carcinoma -H460, NCI-H2170 and NCI-H1869 were used as squamous cell carcinoma. SBC-3 culture supernatants were analyzed and compared with other culture supernatants to search for tumor marker candidates. These cell lines can be obtained from the American Type Culture Collection (ATCC) or the Human Promotion Foundation.
（培養上清の調整）
培養上清は以下のように回収した。各々の細胞を１×１０６となるよう１０ｃｍシャーレに播種し、一晩インキュベートして細胞をシャーレに接着させた。培地は１０％のウシ胎児血清（fetal bovine serum：ＦＢＳ）を含むＲＰＭＩ１６４０（Sigma社製）を用いた。次にリン酸緩衝生理食塩水（phosphate buffered saline：ＰＢＳ）で２回洗浄した後、ＦＢＳを含まないＲＰＭＩ１６４０培地に交換した。系内にＦＢＳが存在すると測定誤差が大きくなるため、実施例では無血清培地を使用した。ＲＰＭＩ１６４０培地で１時間インキュベートした後、培地を捨て、ＰＢＳでさらに２回洗浄し、ＲＰＭＩ１６４０培地を７ｍｌ加えた。４８時間インキュベートした後この培地を回収し、実験に用いた。
(Adjustment of culture supernatant)
The culture supernatant was collected as follows. Each cell was seeded in a 10 cm petri dish at 1 × 10 6 and incubated overnight to adhere the cells to the petri dish. RPMI1640 (manufactured by Sigma) containing 10% fetal bovine serum (FBS) was used as the medium. Next, after washing twice with phosphate buffered saline (PBS), the medium was replaced with RPMI1640 medium containing no FBS. When FBS is present in the system, the measurement error increases, so a serum-free medium was used in the examples. After incubation with RPMI 1640 medium for 1 hour, the medium was discarded, washed twice more with PBS, and 7 ml of RPMI 1640 medium was added. After incubation for 48 hours, the medium was collected and used for experiments.
（培養上清のＭＳスペクトル測定）
ＳＢＣ３培養細胞株の培養上清のＭＳスペクトルを測定した。培養上清１ｍｌに対し、ＺｉｐＴｉｐ（Millipore社製）を用いて脱塩・濃縮を行い、ＭＳスペクトルを測定した。ＭＳスペクトルはApplied Biosystems社製４７００ＭＡＬＤＩ ＴＯＦ／ＴＯＦ質量分析計を用いて測定した。対照として細胞と接触していない培地を上述と同様の処理を行い、ＭＳスペクトルを測定した。結果を図１に示す。図のように培養上清では分子量１０００−１００００付近に培地には存在しない分子が多く観測された。これらの分子はＳＢＣ３細胞が生成し、放出したものであると考えられる。また分子量から類推すると直接ＭＳ／ＭＳスペクトルを測定し、タンパク質の配列を決定するためには分子量が大きすぎることが分かった。
(Measurement of MS spectrum of culture supernatant)
The MS spectrum of the culture supernatant of the SBC3 cultured cell line was measured. 1 ml of the culture supernatant was desalted and concentrated using ZipTip (Millipore), and the MS spectrum was measured. MS spectra were measured using an Applied Biosystems 4700 MALDI TOF / TOF mass spectrometer. As a control, a medium not in contact with cells was treated in the same manner as described above, and an MS spectrum was measured. The results are shown in FIG. As shown in the figure, in the culture supernatant, many molecules not present in the culture medium were observed in the vicinity of a molecular weight of 1000-10000. These molecules are thought to have been generated and released by SBC3 cells. Further, by analogy from the molecular weight, the MS / MS spectrum was directly measured, and it was found that the molecular weight was too large to determine the protein sequence.
（培養上清中のタンパク質のカラムクロマトグラフィーによる分離とＭＳスペクトル測定）
ＳＢＣ３培養上清中のタンパク質を、カラムクロマトグラフィーを用いて分離・精製した。カラムクロマトグラフィーシステムとしてＰｒｏｔｅｏｍｅＬａｂ ＰＦ２Ｄ（ベックマン社製）を、カラムとしてＭｉｇｈｔｙｓｉｌ ＲＰ−１８ＧＰ２５０−４．６（５μｍ：関東化学社製）を用いた。溶出曲線を図２に示す。図のように保持時間が１８−１９分前後に培地には無いピークが検出された。これらの全画分を回収し、ＭＳスペクトルを測定した。一例として１８−１９分前後に溶出した画分のＭＳスペクトルを図３に示す。図のように分子量１７３００前後の分子が観測された。
(Separation of protein in culture supernatant by column chromatography and MS spectrum measurement)
Proteins in the SBC3 culture supernatant were separated and purified using column chromatography. ProteomeLab PF2D (manufactured by Beckman) was used as the column chromatography system, and Mightysil RP-18GP250-4.6 (5 μm: manufactured by Kanto Chemical Co., Inc.) was used as the column. The elution curve is shown in FIG. As shown in the figure, a peak not present in the culture medium was detected at a retention time of around 18-19 minutes. All these fractions were collected and MS spectra were measured. As an example, FIG. 3 shows an MS spectrum of a fraction eluted around 18-19 minutes. As shown in the figure, molecules having a molecular weight of about 17300 were observed.
（タンパク質のトリプシン消化とＭＳ／ＭＳスペクトル測定）
カラムクロマトグラフィーで得られた１８−１９分前後の画分中のタンパク質をトリプシン消化し、ＭＳ／ＭＳ測定を行うことによって、タンパク質の配列を決定した。トリプシン消化は以下のように行った。得られた画分を減圧下で遠心濃縮し、０．２％ ＲａｐｉＧｅｓｔ（Waters社製）を１５μｌ加えタンパク質を溶解した。これにトリプシン溶液（８．３ｎｇ／μｌ、５０ｍＭ 炭酸水素アンモニウム）を１５μｌ加え３７℃で１時間インキュベートした。５００ｍＭ 塩酸３μｌを加えて反応を停止した後、ＺｉｐＴｉｐを用いて脱塩を行い、ＭＳスペクトルを測定した。ＭＳスペクトルの結果を図４に示す。分子量１７３００前後のピークは観測されなかったことから、トリプシン消化が行われ、タンパク質が断片化していることが分かる。
(Protein trypsin digestion and MS / MS spectrum measurement)
Proteins in fractions around 18-19 minutes obtained by column chromatography were trypsin digested and subjected to MS / MS measurement to determine the protein sequence. Trypsin digestion was performed as follows. The obtained fraction was centrifuged and concentrated under reduced pressure, and 15 μl of 0.2% RapiGest (manufactured by Waters) was added to dissolve the protein. To this, 15 μl of a trypsin solution (8.3 ng / μl, 50 mM ammonium bicarbonate) was added and incubated at 37 ° C. for 1 hour. After stopping the reaction by adding 3 μl of 500 mM hydrochloric acid, desalting was performed using ZipTip, and the MS spectrum was measured. The results of MS spectrum are shown in FIG. Since a peak with a molecular weight of around 17300 was not observed, it was found that trypsin digestion was performed and the protein was fragmented.
さらに上記タンパク質断片のペプチドに対し、ＭＳ／ＭＳスペクトルの測定を行った。一例としてｍ／ｚが１０４７のフラグメントに対するＭＳ／ＭＳスペクトルを図５に示す。図のように１０４７の親ピークが無くなりアミノ酸単位に開裂したスペクトルが得られた。このスペクトルをデータベースと照らし合わせることによって、ペプチド配列を決定した。その結果、このペプチドの配列はＱＬＹＥＮＫＰＲであることが分かった。同定されたｙ系列およびｂ系列の分子量を図５に示す。 Furthermore, MS / MS spectra were measured for the peptides of the above protein fragments. As an example, an MS / MS spectrum for a fragment having an m / z of 1047 is shown in FIG. As shown in the figure, 1047 parent peak disappeared and a spectrum cleaved into amino acid units was obtained. The peptide sequence was determined by comparing this spectrum with a database. As a result, the sequence of this peptide was found to be QLYENKPR. The molecular weights of the identified y series and b series are shown in FIG.
さらに、図４で検出されたｍ／ｚが７８９．５、９０２．６、９４５．６、２５１３．１に対して同様にペプチド配列を決定した。結果を表１に示す。 Furthermore, peptide sequences were similarly determined for m / z detected in FIG. 4 of 789.5, 902.6, 945.6, and 2513.1. The results are shown in Table 1.
これらのペプチドはアミノ酸１３個からなる直鎖ペプチドであるニューロテンシンの前駆体（ｐｒｏＮＴ／ＮＭＮ）の一部であることが分かった。図６にｐｒｏＮＴ／ＮＭＮの配列を示す（配列番号１）。太字で示したものが検出されたペプチドである。さらにトリプシン消化前のタンパク質の分子量は図３より１７３００前後と推定されていた。ｐｒｏＮＴ／ＮＭＮの分子量は１７２２５であり、高分子のマススペクトルの誤差を許容するとほぼ一致していることが分かる。以上のことよりＳＢＣ３培養上清にはｐｒｏＮＴ／ＮＭＮが放出されていることが明らかとなった。ニューロテンシン（ＮＴ）は「ＬＹＥＮＫＰＲＲＰＹＩＬ」（配列番号２）の構造を持つ。これまでの研究によりＮＴはペプチドホルモンとして機能することが分かっている。しかしながらヒト肺癌由来の腫瘍細胞がＮＴの前駆体であるｐｒｏＮＴ／ＮＭＮを放出しているという報告が無く、本研究によってはじめて明らかとなった。一般的にペプチドホルモンは血中で不安定である場合が多く、腫瘍マーカーとしては不適切である場合が多かったが、ｐｒｏＮＴ／ＮＭＮはペプチドホルモンの前駆体であるため血中安定性が高く腫瘍マーカー候補として適切である可能性が高い。 These peptides were found to be part of the neurotensin precursor (proNT / NMN), a linear peptide consisting of 13 amino acids. FIG. 6 shows the sequence of proNT / NMN (SEQ ID NO: 1). The peptide shown in bold is the detected peptide. Furthermore, the molecular weight of the protein before trypsin digestion was estimated to be around 17300 from FIG. It can be seen that the molecular weight of proNT / NMN is 17225, which is almost in agreement with an error in the mass spectrum of the polymer. From the above, it was revealed that proNT / NMN was released in the SBC3 culture supernatant. Neurotensin (NT) has the structure of “LYENKPRRPYIL” (SEQ ID NO: 2). Previous studies have shown that NT functions as a peptide hormone. However, there is no report that human lung cancer-derived tumor cells release proNT / NMN, which is a precursor of NT, and this study was revealed for the first time. In general, peptide hormones are often unstable in blood and often unsuitable as tumor markers. However, proNT / NMN is a precursor of peptide hormones and has high blood stability and tumors. There is a high possibility that it is suitable as a marker candidate.
（種々の肺癌由来培養細胞の比較）
他の小細胞癌としてＳＢＣ５の培養上清を上述の方法を用いて分析したところ、ＳＢＣ３と同様にｐｒｏＮＴ／ＮＭＮの放出が確認できた。さらに、３種類の大細胞癌、２種類の腺癌、２種類の扁平上皮癌ではｐｒｏＮＴ／ＮＭＮの放出は検出できなかった。以上のことよりｐｒｏＮＴ／ＮＭＮは小細胞肺癌特異的なマーカーとして期待できると言える。なお、正常者の肺由来の細胞からｐｒｏＮＴ／ＮＭＮの放出は報告されていない。
(Comparison of various lung cancer-derived cultured cells)
When the culture supernatant of SBC5 was analyzed using the above-mentioned method as another small cell carcinoma, the release of proNT / NMN could be confirmed in the same manner as SBC3. Furthermore, release of proNT / NMN could not be detected in 3 types of large cell carcinoma, 2 types of adenocarcinoma, and 2 types of squamous cell carcinoma. From the above, it can be said that proNT / NMN can be expected as a marker specific to small cell lung cancer. In addition, release of proNT / NMN from normal lung cells has not been reported.
（培養上清中のタンパク質のカラムクロマトグラフィーによる分離とｎａｎｏＬＣ−ＥＳＩ−ＬＩＴ−ＴＯＦ質量分析）
また、ＳＢＣ３培養上清を用いてカラムクロマトグラフィーを行い、得られたタンパク質画分をトリプシンのみ、あるいはトリプシンとＶ８プロテアーゼの２種類の酵素で消化した。消化されたタンパク質断片（ペプチド）に対し、ｎａｎｏＬＣ−ＥＳＩ−ＬＩＴ−ＴＯＦ質量分析計 ＮａｎｏＦｒｏｎｔｉｅｒ（（株）日立ハイテクノロジーズ社製）での測定を行った。トリプシン消化は以下のように行った。
(Separation of protein in culture supernatant by column chromatography and nanoLC-ESI-LIT-TOF mass spectrometry)
In addition, column chromatography was performed using the SBC3 culture supernatant, and the obtained protein fraction was digested with trypsin alone or with two types of enzymes, trypsin and V8 protease. The digested protein fragment (peptide) was measured with a nanoLC-ESI-LIT-TOF mass spectrometer NanoFrontier (manufactured by Hitachi High-Technologies Corporation). Trypsin digestion was performed as follows.
カラムクロマトグラフィーで得られた１８−１９分前後の画分を減圧下で遠心濃縮し、トリプシン溶液３０μｌ（トリプシンが０．５μｇ含まれた５０ｍＭ炭酸水素アンモニウム溶液）を加え、３７℃で１８時間インキュベートし、その反応液５μｌを分析した。
また、トリプシンとＶ８プロテアーゼの２種類の酵素での処理には、トリプシン溶液３０μｌのかえて、トリプシン・Ｖ８プロテアーゼ混合液３０μｌ（トリプシンが１μｇ、Ｖ８プロテアーゼが１μｇ含まれた５０ｍＭ炭酸水素アンモニウム溶液）を用いた。
The fraction obtained by column chromatography around 18-19 minutes was concentrated by centrifugation under reduced pressure, added with 30 μl of trypsin solution (50 mM ammonium bicarbonate solution containing 0.5 μg of trypsin), and incubated at 37 ° C. for 18 hours. Then, 5 μl of the reaction solution was analyzed.
For treatment with two kinds of enzymes, trypsin and V8 protease, 30 μl of trypsin solution is used instead of 30 μl of trypsin / V8 protease mixed solution (50 mM ammonium bicarbonate solution containing 1 μg of trypsin and 1 μg of V8 protease). It was.
トリプシンのみで処理を行ったペプチド断片の分析結果を表２及び図７に示す。その結果、ｐｒｏＮＴ／ＮＭＮの一部であるペプチド断片が検出された。同様にトリプシン・Ｖ８プロテアーゼ混合液で処理を行ったペプチド断片の分析結果を表３及び図８に示す。図９のスキーム中に下線で示したものが二種類の質量分析計を用いて検出されたペプチドである。ｐｒｏＮＴ／ＮＭＮのＮ末端およびＣ末端を含むほとんどの配列を検出することができた。 The analysis results of peptide fragments treated with trypsin alone are shown in Table 2 and FIG. As a result, a peptide fragment that was a part of proNT / NMN was detected. Similarly, analysis results of peptide fragments treated with trypsin / V8 protease mixed solution are shown in Table 3 and FIG. The peptide underlined in the scheme of FIG. 9 is a peptide detected using two types of mass spectrometers. Most sequences including the N-terminus and C-terminus of proNT / NMN could be detected.
（Neurotensin及びLarge Neuromedin Nに対する抗体を用いたウエスタンブロット）
ｐｒｏＮＴ／ＮＭＮは分子内にNeurotensin（ＮＴ）、Neuromedin N（ＮＭＮ）、Large Neuromedin N（Ｌａｒｇｅ ＮＭＮ）の配列を有する（図１０）。そこで、ＳＢＣ３を無血清培養した培養上清、ＳＢＣ−３を血清培養した培養上清、ＳＢＣ３の細胞破砕液を泳動し、ＮＴ、Ｌａｒｇｅ ＮＭＮに対する抗体を用いてウエスタンブロットを行った（図１１）。
(Western blot using antibodies against Neurotensin and Large Neuromedin N)
proNT / NMN has a sequence of Neurotensin (NT), Neuromedin N (NMN), and Large Neuromedin N (Large NMN) in the molecule (FIG. 10). Therefore, the culture supernatant obtained by serum-free culture of SBC3, the culture supernatant obtained by serum culture of SBC-3, and the cell disruption solution of SBC3 were electrophoresed, and Western blotting was performed using antibodies against NT and Large NMN (FIG. 11). .
その結果、ＳＢＣ−３を血清培養した培養上清及びＳＢＣ−３の細胞破砕液について、ｐｒｏＮＴ／ＮＭＮ（分子量１７２２５）前後に陽性バンドが検出された（赤矢印）。ＮＴ、Ｌａｒｇｅ ＮＭＮの分子量はこれと大きく異なることから、検出できたバンドはｐｒｏＮＴ／ＮＭＮであると言える。また細胞内でも検出できたことから細胞がｐｒｏＮＴ／ＮＭＮを生産し上清に放出したと言える。
抗体を使ってｐｒｏＮＴ／ＮＭＮが検出できたため、今後ＥＬＩＳＡなど汎用の臨床検査を用いてもｐｒｏＮＴ／ＮＭＮが検出できる可能性を示している。
As a result, a positive band was detected before and after proNT / NMN (molecular weight 17225) in the culture supernatant obtained by serum-culturing SBC-3 and the cell disruption solution of SBC-3 (red arrow). Since the molecular weights of NT and Large NMN are greatly different from these, it can be said that the detected band is proNT / NMN. Moreover, since it could be detected in the cells, it can be said that the cells produced proNT / NMN and released it into the supernatant.
Since proNT / NMN could be detected using an antibody, this indicates the possibility that proNT / NMN can be detected using general-purpose clinical tests such as ELISA in the future.
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