JP7406784B2 - How to detect ovarian cancer - Google Patents
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- JP7406784B2 JP7406784B2 JP2019078850A JP2019078850A JP7406784B2 JP 7406784 B2 JP7406784 B2 JP 7406784B2 JP 2019078850 A JP2019078850 A JP 2019078850A JP 2019078850 A JP2019078850 A JP 2019078850A JP 7406784 B2 JP7406784 B2 JP 7406784B2
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Description
本発明は、被験体における卵巣癌の検出方法を提供するものであり、より具体的には卵巣癌の転移又は再発の予測方法、及び卵巣癌の治療効果のモニタリング方法を提供する。本発明はまた、卵巣癌の腹膜浸潤機構に関する新たな知見に基づく卵巣癌治療剤を提供する。 The present invention provides a method for detecting ovarian cancer in a subject, and more specifically provides a method for predicting metastasis or recurrence of ovarian cancer, and a method for monitoring the therapeutic effect of ovarian cancer. The present invention also provides a therapeutic agent for ovarian cancer based on new findings regarding the mechanism of peritoneal invasion of ovarian cancer.
卵巣癌は女性性器の悪性腫瘍のうち最も死亡率が高いが、その主要因は腹腔内播種性転移という、腹腔内全体に癌細胞が広がり微小な転移を形成する進展形式にある。卵巣は骨盤内臓器であるため、腫瘍が発生しても自覚症状に乏しく、40～50％の症例がこの腹膜播種性転移を起こした進行症例で発見される。 Ovarian cancer has the highest mortality rate among malignant tumors of the female genital organs, and the main reason for this is intraperitoneal disseminated metastasis, a type of progression in which cancer cells spread throughout the abdominal cavity and form minute metastases. Since the ovary is a pelvic organ, there are few symptoms even if a tumor develops, and 40-50% of cases are discovered in advanced cases with peritoneal disseminated metastasis.
卵巣癌の臨床経過としては、腹膜播種による胸腹水貯留のため、呼吸苦や腹部膨満感を引き起こすのみならず、治療上でも腹膜播種の制御が困難であるため、最終的に生命維持の恒常性が破綻して落命することが多い。従って、卵巣癌の治療方針や患者の予後を決める上で、腹膜播種形成の分子機構の解明が重要である。 The clinical course of ovarian cancer is that peritoneal dissemination causes thoracic and ascitic effusion, which not only causes breathing difficulties and abdominal distension, but it is also difficult to control peritoneal dissemination during treatment, and ultimately homeostasis on life support is required. often go bankrupt and die. Therefore, elucidation of the molecular mechanism of peritoneal dissemination formation is important in determining treatment strategies and patient prognosis for ovarian cancer.
従来、「癌細胞がまず腹膜中皮に直接接着した後、上皮間葉転換を介して腹膜の間質内に浸潤する」という機序が腹膜播種の主たる成立機構として認識されてきた（非特許文献１及び２）。 Conventionally, the mechanism in which cancer cells first adhere directly to the peritoneal mesothelium and then infiltrate into the peritoneal stroma through epithelial-to-mesenchymal transition has been recognized as the main mechanism for peritoneal dissemination (non-patent References 1 and 2).
一方、組織因子（Tissue Factor, TF）は分子量47,000の膜貫通型糖タンパク質であり、通常は血管外組織や血管内皮下の線維芽細胞等に発現しており、血管傷害により血液が流出した際に血中の第VII因子や第VIIa因子と結合して血液凝固カスケードを開始させる因子であることが知られている。TFは遺伝子発現の制御、細胞遊走・細胞死の抑制、炎症、創傷治癒等に関与する他、癌の進行との関連についても報告されている（非特許文献３）。 On the other hand, tissue factor (TF) is a transmembrane glycoprotein with a molecular weight of 47,000, and is normally expressed in extravascular tissues and fibroblasts under the vascular endothelium, and when blood flows out due to vascular injury. It is known that it is a factor that binds to factor VII and factor VIIa in the blood and initiates the blood coagulation cascade. TF is involved in regulating gene expression, suppressing cell migration/cell death, inflammation, wound healing, etc., and has also been reported to be associated with cancer progression (Non-Patent Document 3).
しかしながら、卵巣癌の転移について上記の機序を説明し得る実際の転移巣の形態的な提示はほとんど示されておらず、従って、卵巣癌腹膜播種の分子機構については不明な点が多く残されていた。そして、卵巣癌の転移及び再発に対する効果的な予測方法及び治療剤が望まれていた。 However, the morphological presentation of actual metastatic foci that could explain the above-mentioned mechanism of ovarian cancer metastasis has hardly been demonstrated, and therefore, many points remain unclear regarding the molecular mechanism of peritoneal dissemination of ovarian cancer. was. Therefore, effective prediction methods and therapeutic agents for metastasis and recurrence of ovarian cancer have been desired.
そこで、本発明者等は、卵巣癌患者の腹膜転移部分を形態学的に検討する目的で、卵巣癌患者の腹膜転移部分を詳細に観察した。 Therefore, the present inventors closely observed the peritoneal metastasis of an ovarian cancer patient in order to morphologically examine the peritoneal metastasis of the patient.
その結果、本発明者等は、「癌細胞集塊が腹膜上皮に接着することなく、周囲にフィブリン網を形成して腹腔内に存在したまま腹膜上皮に対峙し、宿主の間質から自身の周囲に線維芽細胞や血管内皮細胞の遊走およびそれに伴う新生血管網の形成を誘導し、それらを足場に成長した後に血管新生誘導経路を利用して腹膜間質へ浸潤する」という新しい卵巣癌の腹膜浸潤機構を発見した。そして、腹膜播種患者の腹膜上皮腹腔側で発育する卵巣癌細胞の周囲にTFが強発現しており、血液中と比較して有意に多量のTFが腹水中で検出されることを見出し、これらの知見に基づき、本発明に到った。 As a result, the present inventors found that ``cancer cell clusters do not adhere to the peritoneal epithelium, form a fibrin network around them, and confront the peritoneal epithelium while remaining in the peritoneal cavity, and release themselves from the host's stroma. A new type of ovarian cancer that induces the migration of fibroblasts and vascular endothelial cells and the formation of a new vascular network in the surrounding area, grows using them as a scaffold, and then invades the peritoneal stroma using the angiogenesis-inducing pathway. We discovered the mechanism of peritoneal invasion. We found that TF was strongly expressed around ovarian cancer cells growing on the peritoneal side of the peritoneal epithelium of patients with peritoneal dissemination, and that significantly higher amounts of TF were detected in ascites than in blood. Based on this knowledge, we have arrived at the present invention.
すなわち、本発明は以下を提供するものである。
１．被験体由来の腹水中の組織因子（Tissue Factor、TF）の発現を検出することを特徴とする、被験体における卵巣癌の転移又は再発の予測方法。
２．被験体由来の腹水中のTFの発現を検出することを特徴とする、卵巣癌の治療効果のモニタリング方法。
３．TFに特異的に結合する抗体を用いてTFの発現を検出する、上記１又は２記載の方法。
４．ウェスタンブロット法、免疫組織学的検出法、免疫沈降法、又はELISA法によってTFの発現を検出する、上記３記載の方法。
５．被験体由来の腹水中のTF発現を、正常の対照サンプルにおけるTF発現又は基準値と比較して、卵巣癌の転移若しくは再発の可能性がある、又は治療効果が低いと判定するステップを含む、上記１～４のいずれか記載の方法。
６．TFに特異的に結合する抗体を含有する、被験体における卵巣癌の転移又は再発の検出のためのキット。
７．TFに結合し得る抗体と、抗癌剤とのコンジュゲートを含む卵巣癌治療剤であって、腹腔内投与される、上記治療剤。
８．抗体が、TFに特異的に結合するモノクローナル抗体である、上記７記載の治療剤。
９．抗癌剤が、有糸分裂阻害剤（微小管作用薬）、アルキル化剤、抗癌性抗生物質、代謝拮抗剤、プラチナ製剤、トポイソメラーゼ阻害剤、分子標的薬、及び放射性同位元素から選択される、上記７又は８記載の治療剤。
１０．投与対象患者が、腫瘍摘出手術後の患者である、上記７～９のいずれか記載の治療剤。
That is, the present invention provides the following.
1. A method for predicting metastasis or recurrence of ovarian cancer in a subject, the method comprising detecting the expression of tissue factor (TF) in ascites derived from the subject.
2. A method for monitoring the therapeutic effect of ovarian cancer, the method comprising detecting the expression of TF in ascites derived from a subject.
3. 3. The method according to 1 or 2 above, wherein the expression of TF is detected using an antibody that specifically binds to TF.
4. 3. The method according to 3 above, wherein the expression of TF is detected by Western blotting, immunohistological detection, immunoprecipitation, or ELISA.
5. Comparing the TF expression in ascites derived from the subject with the TF expression in a normal control sample or a reference value, and determining that there is a possibility of metastasis or recurrence of ovarian cancer, or that the therapeutic effect is low. The method according to any one of 1 to 4 above.
6. A kit for detecting metastasis or recurrence of ovarian cancer in a subject, comprising an antibody that specifically binds to TF.
7. A therapeutic agent for ovarian cancer comprising a conjugate of an antibody capable of binding to TF and an anticancer drug, which is administered intraperitoneally.
8. 7. The therapeutic agent according to 7 above, wherein the antibody is a monoclonal antibody that specifically binds to TF.
9. The above, wherein the anticancer agent is selected from mitotic inhibitors (microtubule agents), alkylating agents, anticancer antibiotics, antimetabolites, platinum agents, topoisomerase inhibitors, molecular targeting agents, and radioisotopes. 8. The therapeutic agent according to 7 or 8.
10. 10. The therapeutic agent according to any one of 7 to 9 above, wherein the patient to be administered is a patient who has undergone tumor removal surgery.
本発明者等が見出した卵巣癌の浸潤機構は従来の常識を覆すものであり、新たな視点から卵巣癌の検出及び治療のための手段を提供することができる。 The ovarian cancer invasion mechanism discovered by the present inventors overturns conventional wisdom and can provide a means for detecting and treating ovarian cancer from a new perspective.
本発明者等は、腹膜上皮の腹腔側で発育する卵巣癌細胞集塊の周囲にフィブリン網形成に関わるTFが強発現していることを見出した。卵細胞癌の新たな腹膜浸潤機構を示す本発明者等の知見は、卵巣癌の転移又は再発の予測、卵巣癌の治療効果のモニタリングに利用することができ、また新たな卵巣癌治療剤を提供することができる。 The present inventors discovered that TFs involved in fibrin network formation are strongly expressed around ovarian cancer cell clusters growing on the peritoneal side of the peritoneal epithelium. The findings of the present inventors showing a new peritoneal invasion mechanism of egg cell carcinoma can be used to predict metastasis or recurrence of ovarian cancer, monitor the therapeutic effects of ovarian cancer, and provide a new therapeutic agent for ovarian cancer. can do.
本発明は、一態様において、被験体由来の腹水中の組織因子（Tissue Factor、TF）の発現を検出することを特徴とする、被験体における卵巣癌の転移又は再発の予測方法を提供する。
本発明はまた、別の態様において、被験体由来の腹水中のTFの発現を検出することを特徴とする、卵巣癌の治療効果のモニタリング方法を提供する。
In one embodiment, the present invention provides a method for predicting metastasis or recurrence of ovarian cancer in a subject, which comprises detecting the expression of tissue factor (TF) in ascites derived from the subject.
In another aspect, the present invention provides a method for monitoring the therapeutic effect of ovarian cancer, which comprises detecting the expression of TF in ascites derived from a subject.
本明細書において、「TFの発現」とは、特に組織因子（TF）タンパク質の発現を意図するものとし、特に示さない限り、「TF」とは「TFタンパク質」を意味するものとする。 As used herein, "expression of TF" specifically refers to the expression of tissue factor (TF) protein, and unless otherwise indicated, "TF" shall mean "TF protein."
上記の通り、通常、TFは膜貫通型のタンパク質として存在するが、本発明者等は、ステージI～IVと診断された卵巣癌患者の腹水中に血液中と比較して有意に多量のTFが存在することを見出した。 As mentioned above, TF normally exists as a transmembrane protein, but the present inventors found that a significantly higher amount of TF was found in the ascites of ovarian cancer patients diagnosed with stages I to IV compared to the blood. found that it exists.
本発明において、被験体は、哺乳動物であり、特に限定するものではないが、ヒトであることが好ましい。卵巣癌の転移又は再発を検出するため、被験体は、卵巣癌を有するとして診断された被験体、及び卵巣摘出手術等の治療を受けた被験体であり得る。あるいは、本発明の方法を治療薬の開発等の研究で利用するためには、被験体は、マウス、ラット、ウサギ、ブタ、サル等の非ヒト哺乳動物であり得る。 In the present invention, the subject is a mammal, and is preferably a human, although it is not particularly limited. To detect metastasis or recurrence of ovarian cancer, the subject can be a subject diagnosed as having ovarian cancer and a subject who has undergone treatment such as oophorectomy. Alternatively, in order to utilize the method of the present invention in research such as the development of therapeutic agents, the subject may be a non-human mammal such as a mouse, rat, rabbit, pig, monkey, etc.
ヒトTFのアミノ酸配列の情報は、米国国立生物工学情報センター（National Center for Biotechnology Information, NCBI）が管理するデータベースからGenBank: AAA61152.1として取得することができる。また、ヒトTFをコードする遺伝子の塩基配列については、同様にGene ID: 2152として取得することができる。非ヒト哺乳動物におけるTFのアミノ酸配列及びこれをコードする遺伝子の塩基配列等の情報についても、同様に取得することができる。 Information on the amino acid sequence of human TF can be obtained from the database managed by the US National Center for Biotechnology Information (NCBI) as GenBank: AAA61152.1. Furthermore, the nucleotide sequence of the gene encoding human TF can be similarly obtained as Gene ID: 2152. Information such as the amino acid sequence of TF in non-human mammals and the base sequence of the gene encoding the same can be similarly obtained.
TFの発現は、特に限定するものではないが、一般的には、当分野で通常行われているように、TFに特異的に結合する抗体を用いて検出することができる。抗体の検出のためには、限定するものではないが、例えばウェスタンブロット法、免疫組織学的検出法、免疫沈降法、又はELISA法を利用することができる。 Expression of TF can be generally, but not limited to, detected using antibodies that specifically bind to TF, as is commonly practiced in the art. For antibody detection, for example, but not limited to, Western blotting, immunohistological detection, immunoprecipitation, or ELISA can be used.
本発明において用いる抗体は、TF、特に膜結合型TFにおける細胞外ドメインを特異的に認識して結合し得る抗体であって、他のタンパク質には結合しない抗体であることが好ましい。あるいはまた、抗体は、TF特有のマイクロパーティクルを認識する抗体であっても良い。 The antibody used in the present invention is preferably an antibody that can specifically recognize and bind to the extracellular domain of TF, particularly membrane-bound TF, and does not bind to other proteins. Alternatively, the antibody may be one that recognizes TF-specific microparticles.
本明細書において、「結合する」及び「特異的に結合する」とは、特に限定するものではないが、抗原と抗体との結合が10-8M以下、好ましくは10-9M以下、より好ましくは10-10M以下のKD値の結合親和性を有するものであることを意味し得る。 As used herein, "bind" and "specifically bind" are not particularly limited, but the binding between the antigen and the antibody is 10 -8 M or less, preferably 10 -9 M or less, or more. Preferably, it can mean having a binding affinity with a KD value of 10 −10 M or less.
あるいはまた、本明細書において「TFに特異的に結合する」とは、TFに対する結合が、一般的な結合アッセイで検出して、TF以外の物質に対する結合と比較して2倍以上、3倍以上、4倍以上である場合を意味し得る。例えば、当分野で通常使用される蛍光標識によって結合を検出する場合に、S/N比が2以上、3以上、4以上である場合を意味し得る。 Alternatively, as used herein, "specifically binds to TF" means that the binding to TF is 2 times or more, 3 times more than the binding to substances other than TF, as detected by a general binding assay. or more may mean four times or more. For example, when binding is detected by a fluorescent label commonly used in the art, this may mean a case where the S/N ratio is 2 or more, 3 or more, or 4 or more.
ウェスタンブロット法による検出は、TFを含み得るサンプルを、SDS-PAGEによって展開した後、タンパク質を疎水性膜に転写し、TFに特異的に結合し得る抗体を用いて検出することを含む。 Detection by Western blotting involves developing a sample that may contain TF by SDS-PAGE, followed by transferring the proteins to a hydrophobic membrane and detecting using an antibody that can specifically bind to TF.
免疫組織学的検出法は、被験体から採取した組織切片を固定した後、TFとこれに対する抗体との結合を可視化することによって行う。可視化のための方法としては、オートラジオグラフィー、金コロイド法、蛍光抗体法、酵素抗体法等が挙げられ、また、抗体の標識は、TFに対して結合する抗体を直接標識するものであっても、標識した二次抗体を使用するものであっても良い。 The immunohistological detection method is performed by fixing a tissue section taken from a subject and then visualizing the binding between TF and an antibody against it. Methods for visualization include autoradiography, colloidal gold method, fluorescent antibody method, enzyme antibody method, etc. Also, antibody labeling directly labels the antibody that binds to TF. Alternatively, a labeled secondary antibody may be used.
免疫沈降法は、サンプル中のTFと、これに対する抗体との反応で得られる複合体を形成させ、ビーズに固相化したプロテインA若しくはG又は二次抗体と結合させた後、未結合の物質と分離することを含む。 In the immunoprecipitation method, TF in a sample is reacted with an antibody against it to form a complex, which is bound to protein A or G immobilized on beads or a secondary antibody, and then unbound substances are removed. including separation.
ELISA法は、抗原抗体反応の後、酵素標識した一次抗体又は二次抗体による酵素活性を測定して数値化することを含み、直接法、間接法、サンドイッチ法、競合法が知られ、当分野において広く使用されている検出・定量方法である。 The ELISA method involves measuring and quantifying enzyme activity using an enzyme-labeled primary antibody or secondary antibody after an antigen-antibody reaction.Direct methods, indirect methods, sandwich methods, and competitive methods are known, and are well known in the art. This is a detection and quantification method widely used in Japan.
本発明において使用される抗体は、TFに特異的に結合するものである限り、ポリクローナル抗体でもモノクローナル抗体であっても良いが、モノクローナル抗体であることが好ましい。また、本発明の抗体は、卵巣癌の転移又は再発の検出のためにin vitroで使用することが意図され、従って、マウス、ウサギ、ヤギ等の非ヒト抗体、キメラ抗体、ヒト化抗体、ヒト抗体のいずれであっても良く、特に限定するものではない。 The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it specifically binds to TF, but a monoclonal antibody is preferable. The antibodies of the present invention are also intended to be used in vitro for the detection of metastasis or recurrence of ovarian cancer, and can therefore be used in non-human antibodies such as mouse, rabbit, goat, etc., chimeric antibodies, humanized antibodies, human antibodies, etc. It may be any antibody and is not particularly limited.
抗原が特定されている場合の抗体の作製方法は当分野において周知である。本発明において使用し得る抗体は、TF又はその断片を非ヒト哺乳動物に免疫して、公知の手法によってポリクローナル抗体として取得することができる。また、モノクローナル抗体は、TFに対する抗体を産生する抗体産生細胞をミエローマ細胞と融合させて得られるハイブリドーマから得ることができる。 Methods for making antibodies where the antigen has been identified are well known in the art. Antibodies that can be used in the present invention can be obtained as polyclonal antibodies by immunizing non-human mammals with TF or its fragments by known techniques. Furthermore, monoclonal antibodies can be obtained from hybridomas obtained by fusing antibody-producing cells that produce antibodies against TF with myeloma cells.
本発明において使用し得る抗体はまた、活性が実証された抗体のアミノ酸配列情報又は該抗体をコードするポリヌクレオチドの塩基配列情報に基づいて、遺伝子工学的手法を用い、あるいは化学合成手段を用いて、合成によって取得することもできる。活性が実証された抗体の配列情報に基づいて更なる抗体を作製する場合、特に元の抗体の相補性決定領域（CDR）の配列を考慮して、同一又は同等の結合親和性を有する抗体を作製することができ、また更に結合親和性の高い抗体を得ることもできる。 Antibodies that can be used in the present invention can also be produced using genetic engineering techniques or chemical synthesis techniques based on the amino acid sequence information of antibodies with proven activity or the base sequence information of polynucleotides encoding the antibodies. , can also be obtained by synthesis. When generating additional antibodies based on sequence information of antibodies with demonstrated activity, antibodies with the same or equivalent binding affinity may be generated, particularly considering the complementarity determining region (CDR) sequences of the original antibody. Furthermore, antibodies with high binding affinity can also be obtained.
遺伝子工学的手法によって抗体を作製する場合、重鎖及び軽鎖をコードするポリヌクレオチドを適切な宿主細胞に導入して発現させ、組換え蛋白質として得ることができる。この場合、ポリヌクレオチドはDNAであってもRNAであっても良く、また宿主細胞への導入手段は当分野で使用されているものを適宜利用することができる。ポリヌクレオチドを宿主細胞に導入するためのベクターとして、ウイルスベクター、プラスミドベクター、ファージベクター等を適宜使用することができる。宿主細胞としては、例えば大腸菌等の細菌、酵母、昆虫細胞、動物細胞等を利用することができる。ここで、重鎖及び軽鎖をコードするポリヌクレオチドは、別個のベクターに導入しても、同一のベクターに連結して導入しても良い。 When producing antibodies by genetic engineering techniques, polynucleotides encoding heavy and light chains can be introduced into appropriate host cells and expressed to obtain recombinant proteins. In this case, the polynucleotide may be DNA or RNA, and any means used in the art can be used as appropriate for introduction into the host cell. Viral vectors, plasmid vectors, phage vectors, etc. can be used as appropriate as vectors for introducing polynucleotides into host cells. As host cells, for example, bacteria such as Escherichia coli, yeast, insect cells, animal cells, etc. can be used. Here, the polynucleotides encoding the heavy chain and light chain may be introduced into separate vectors, or may be introduced by being linked to the same vector.
例えば、本発明において使用し得る抗体の重鎖及び軽鎖をコードするcDNAを、場合によってシグナル配列、ポリA配列、更にプロモーター配列等の調節配列、選択マーカーと共に含むベクターに組み込んで、適切な宿主細胞中に導入して培養することで、TFを特異的に認識し得る抗体を組換えタンパク質として取得することができる。 For example, cDNA encoding the heavy chain and light chain of an antibody that can be used in the present invention is inserted into a vector containing a signal sequence, a polyA sequence, a regulatory sequence such as a promoter sequence, and a selection marker as the case may be, and then By introducing it into cells and culturing it, antibodies that can specifically recognize TF can be obtained as recombinant proteins.
従って、本発明において使用し得る抗体は、上記のようにして作製された抗体であり、例えば遺伝子工学的手法を用いて取得された組換えタンパク質であるか、あるいは化学合成手段を用いて合成されたタンパク質であり得る。 Therefore, antibodies that can be used in the present invention are antibodies produced as described above, and are, for example, recombinant proteins obtained using genetic engineering techniques, or synthesized using chemical synthesis means. It can be a protein.
本発明において使用し得る抗体をモノクローナル抗体として使用する場合、抗体は、IgG抗体分子、IgM抗体分子、又はそれらの抗原結合性断片及び抗原結合性誘導体であり得る。例えば、抗体は、完全抗体、Fab、Fab'、F(ab')2断片、また重鎖可変領域（VH）及び軽鎖可変領域（VL）をリンカーで連結した一本鎖抗体（scFv）断片、scFv-Fc、sc(Fv)2、Fv、ダイアボディー等であり得る。 When the antibodies that can be used in the present invention are used as monoclonal antibodies, the antibodies can be IgG antibody molecules, IgM antibody molecules, or antigen-binding fragments and antigen-binding derivatives thereof. For example, antibodies include complete antibodies, Fab, Fab', F(ab') 2 fragments, and single chain antibody (scFv) fragments in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker. , scFv-Fc, sc(Fv) 2 , Fv, diabody, etc.
scFv、scFv-Fc、及びsc(Fv)2はリンカーで可変領域を連結した合成ポリペプチドである。リンカーとしては、当分野で通常使用されるものであればいずれでも良く、特に限定するものではないが、例えば5～25個、好ましくは10～20個のアミノ酸残基からなるペプチドリンカー、例えばGSリンカー等を好適に使用することができる。 scFv, scFv-Fc, and sc(Fv) 2 are synthetic polypeptides in which variable regions are connected with a linker. The linker may be any linker commonly used in the art, and is not particularly limited, such as a peptide linker consisting of 5 to 25, preferably 10 to 20 amino acid residues, such as GS. Linkers and the like can be suitably used.
本発明において使用し得る抗体には更に、抗原結合性に影響しない範囲で当業者に理解され得る誘導体、例えば抗体精製を容易にしたり安定性を高めたりするための修飾が施された誘導体も含まれる。本明細書においては、TFとの結合性を保持する断片及び誘導体を、文脈に矛盾のない限り、便宜的に「抗体」に含めることが意図される。 Antibodies that can be used in the present invention also include derivatives that can be understood by those skilled in the art without affecting antigen binding, such as derivatives that have been modified to facilitate antibody purification or increase stability. It will be done. As used herein, fragments and derivatives that retain TF-binding properties are intended to be included in the term "antibody" for convenience, unless the context contradicts.
本発明において使用し得る抗体はまた、二量体、三量体、四量体等の多量体として合成することもできる。更に、本発明において使用し得る抗体は、TFに結合する第1の特異性と、他の抗原に対して結合する第2の特異性とを有する二重特異性抗体であっても良い。当業者であれば、本明細書の記載、及び当分野における技術常識に基づいて、本発明の抗体を用途に応じた適切な形態のものとして取得することができる。 Antibodies that can be used in the present invention can also be synthesized as multimers such as dimers, trimers, and tetramers. Furthermore, antibodies that can be used in the present invention may be bispecific antibodies that have a first specificity that binds to TF and a second specificity that binds to other antigens. Those skilled in the art can obtain the antibody of the present invention in an appropriate form depending on the intended use, based on the description herein and common general knowledge in the field.
本発明において使用し得る抗体として、TFに対する結合特異性を有する抗体として市販されているものを使用しても良く、またそのような抗体の合成を依頼することもできる。更に、本発明において使用し得る抗体は、検出のために標識された抗体であっても良く、標識は、特に限定するものではないが、例えば蛍光色素標識、酵素標識、放射性標識等であって良い。 As antibodies that can be used in the present invention, commercially available antibodies with binding specificity for TF may be used, or the synthesis of such antibodies may be requested. Furthermore, the antibody that can be used in the present invention may be an antibody labeled for detection, and the label is not particularly limited, but may include, for example, a fluorescent dye label, an enzyme label, a radioactive label, etc. good.
TFの発現の検出は、サンプル中のTFの存在又は増減（変動）を検出することを含む。更に、本発明の方法は、例えば、被験体由来の腹水中のTF発現を検出するステップに加えて、卵巣癌を有さない正常の対照サンプルにおけるTF発現、又は予め作成した基準値と比較して卵巣癌の転移若しくは再発の可能性があると判定するステップを含み得る。あるいは、本発明の方法は、被験体由来の腹水中のTF発現を検出するステップに加えて、卵巣癌を有さない正常の対照サンプルにおけるTF発現、又は予め作成した基準値と比較して、被験体に対して行われた手術等の卵巣癌に対する治療の効果が低いと判定するステップを含み得る。 Detecting the expression of TF includes detecting the presence or increase or decrease (variation) of TF in a sample. Furthermore, the method of the present invention includes, for example, in addition to the step of detecting TF expression in ascites from a subject, comparing TF expression in a normal control sample without ovarian cancer or with a pre-defined reference value. and determining that there is a possibility of metastasis or recurrence of ovarian cancer. Alternatively, the method of the present invention, in addition to the step of detecting TF expression in ascites from a subject, may also include comparing TF expression in a normal control sample without ovarian cancer or with a predefined reference value. The method may include determining that a treatment for ovarian cancer, such as a surgery, performed on the subject is less effective.
「基準値」は、例えばサンプル中のTF濃度、TFに結合する抗体量（絶対量若しくは標識に由来する蛍光強度等）等として得ることができる。あるいは、「基準値」は、ウェスタンブロットで検出されたバンド強度から算出することもできる。 The "reference value" can be obtained as, for example, the TF concentration in the sample, the amount of antibody that binds to TF (absolute amount, fluorescence intensity derived from a label, etc.), and the like. Alternatively, the "reference value" can also be calculated from the band intensity detected by Western blotting.
例えば、卵巣癌患者と健常者、あるいは卵巣癌の各ステージにおける臨床的症状との相関性を考慮して2群（高発現群及び低発現群）に分けた数値を多数の被験体由来のデータからそれぞれ取得し、統計学的に得られるカットオフ値を基準値として使用することができる。 For example, data derived from a large number of subjects can be used to calculate numbers divided into two groups (high expression group and low expression group) considering the correlation between ovarian cancer patients and healthy subjects, or the clinical symptoms of each stage of ovarian cancer. The statistically obtained cutoff values can be used as reference values.
上記の基準値を使用して、被験体由来の腹水サンプルからの検出結果に基づいて、その被験体の状態、例えば卵巣癌の転移又は再発の可能性を予測することが可能となる。上記の基準値と比較して被験体由来のTFの検出結果が高い場合に、その被験体が卵巣癌の転移又は再発の可能性を予測することが可能である。
一実施形態において、本発明の方法は、卵巣癌の転移及び／又は再発を予測するための方法であり得る。
Using the above reference values, it is possible to predict the condition of a subject, for example the possibility of metastasis or recurrence of ovarian cancer, based on the detection results from an ascites sample derived from the subject. When the detection result of TF derived from a subject is high compared to the above reference value, it is possible to predict the possibility of metastasis or recurrence of ovarian cancer in that subject.
In one embodiment, the method of the invention can be a method for predicting metastasis and/or recurrence of ovarian cancer.
本発明の方法は、被験体由来の腹水中のTFを新たなバイオマーカーとして検出するものである。被験体由来の検出結果から、被験体由来の腹水サンプル中でTFが高発現している場合に、被験体において卵巣癌が転移又は再発をしている可能性が示される。 The method of the present invention detects TF in ascites fluid derived from a subject as a new biomarker. Based on the detection results derived from the subject, when TF is highly expressed in the ascites sample derived from the subject, the possibility that ovarian cancer has metastasized or recurred in the subject is indicated.
＜キット＞
本発明はまた、TFに特異的に結合する抗体を含有する、被験体における卵巣癌の転移又は再発の検出のためのキットを提供する。本キットは、上記の本発明の方法において好適に使用することができる。
<Kit>
The invention also provides a kit for the detection of ovarian cancer metastasis or recurrence in a subject, containing an antibody that specifically binds to TF. This kit can be suitably used in the above-described method of the present invention.
本発明のキットにより、TFに特異的に結合する抗体を被験体に由来する腹水サンプルと接触させ、サンプル中の上記抗体と結合する抗原、すなわちTFの発現の有無及び／又は発現量を検出することができる。例えば上記抗体、又は上記抗体に対する二次抗体を蛍光試薬や発色試薬等で標識し、蛍光又は発色を検出することで確認することができる。
更に、本発明のキットは、抗体とTFとの結合反応のための反応液、反応容器、検出のための標識試薬、二次抗体、緩衝剤、使用説明書等を含むことができる。
Using the kit of the present invention, an antibody that specifically binds to TF is brought into contact with an ascites sample derived from a subject, and the presence or absence and/or expression level of the antigen that binds to the antibody, that is, TF, in the sample is detected. be able to. For example, confirmation can be made by labeling the above-mentioned antibody or a secondary antibody against the above-mentioned antibody with a fluorescent reagent, a coloring reagent, etc., and detecting fluorescence or coloring.
Furthermore, the kit of the present invention can include a reaction solution for the binding reaction between the antibody and TF, a reaction container, a labeling reagent for detection, a secondary antibody, a buffer, instructions for use, and the like.
＜卵巣癌治療剤＞
本発明はまた、TFに結合し得る抗体と、抗癌剤とのコンジュゲートを含む卵巣癌治療剤であって、腹腔内投与される、上記治療剤を提供する。
<Ovarian cancer therapeutic agent>
The present invention also provides an ovarian cancer therapeutic agent comprising a conjugate of an antibody capable of binding to TF and an anticancer drug, which is administered intraperitoneally.
抗TF抗体を用いた抗癌剤のデリバリーシステムが開発されつつあるが、それらは血中投与を念頭においている（例えばYamamoto et al., Cancer Sci, 2015; 106: 627-634；Koga et al., Int J. Cancer, 2015; 137: 1457-1466; Sugaya et al., Cancer Sci, 2016; 107: 335-340）。一方、卵巣癌の抗癌剤投与法の一つに腹腔内投与が挙げられているが（Karam et al., Ann Oncol. 2017; 28: 711-717）、これまで周囲にTFを強発現させたまま卵巣癌細胞集塊が腹腔内で発育するという概念がなかったため、抗TF抗体を用いた抗癌剤においては腹腔内投与という選択肢はなかった。本発明により、卵巣癌の腹膜播種症例には腹腔内投与のほうが有利である理論的根拠が示された。 Delivery systems for anticancer drugs using anti-TF antibodies are being developed, but they are intended for blood administration (e.g. Yamamoto et al., Cancer Sci, 2015; 106: 627-634; Koga et al., Int J. Cancer, 2015; 137: 1457-1466; Sugaya et al., Cancer Sci, 2016; 107: 335-340). On the other hand, intraperitoneal administration is one of the methods of administering anticancer drugs for ovarian cancer (Karam et al., Ann Oncol. 2017; 28: 711-717); Since there was no concept that ovarian cancer cell clusters grow intraperitoneally, intraperitoneal administration was not an option for anticancer drugs using anti-TF antibodies. The present invention has demonstrated the rationale that intraperitoneal administration is more advantageous in cases of peritoneal dissemination of ovarian cancer.
TFに結合し得る抗体は、本明細書中で上記した通りのものを好適に使用することができるが、ヒトへの投与が意図される場合、好ましくはヒトTFに特異的に結合するモノクローナル抗体である。また、ヒトに投与する場合、ヒト化抗体又はヒト抗体であることが好ましい。
本発明の治療剤は、上記の抗体を、細胞増殖抑制活性及び／又は細胞毒性を有する抗癌剤とコンジュゲートとして作製される。
As the antibody capable of binding to TF, those as described above can be suitably used, but when administration to humans is intended, monoclonal antibodies that specifically bind to human TF are preferably used. It is. Furthermore, when administered to humans, humanized antibodies or human antibodies are preferred.
The therapeutic agent of the present invention is produced by conjugating the above-mentioned antibody with an anticancer drug having cell growth-inhibiting activity and/or cytotoxicity.
抗癌剤としては、特に限定するものではないが、例えば有糸分裂阻害剤（微小管作用薬）、アルキル化剤、抗癌性抗生物質、代謝拮抗剤、プラチナ製剤、トポイソメラーゼ阻害剤、分子標的薬、放射性同位元素等から選択されるものを好適に使用することができる。 Anticancer agents include, but are not limited to, mitotic inhibitors (microtubule agents), alkylating agents, anticancer antibiotics, antimetabolites, platinum agents, topoisomerase inhibitors, molecular target drugs, Those selected from radioactive isotopes and the like can be suitably used.
有糸分裂阻害剤（微小管作用薬）としては、例えば、ビンクリスチン、ビンブラスチン、パクリタキセル、ドセタキセル等が挙げられる。
アルキル化剤としては、例えば、ナイトロジェンマスタード、ニトロソウレア、シクロホスファミド、ダカルバジン、テモゾロミド等が挙げられる。
Examples of mitosis inhibitors (microtubule agents) include vincristine, vinblastine, paclitaxel, and docetaxel.
Examples of the alkylating agent include nitrogen mustard, nitrosourea, cyclophosphamide, dacarbazine, and temozolomide.
抗癌性抗生物質としては、例えば、ブレオマイシン、マイトマイシンC、アクチノマイシン、ドキソルビシン、エピルビシン、アムルビシン等が挙げられる。
代謝拮抗剤としては、例えば、フルオロウラシル（5-FU）、シタラビン、ゲムシタビン、カペシタビン、フルダラビン、メルカプトプリン、アザシチジン、メトトレキサート等が挙げられる。
Examples of anticancer antibiotics include bleomycin, mitomycin C, actinomycin, doxorubicin, epirubicin, amrubicin, and the like.
Examples of antimetabolites include fluorouracil (5-FU), cytarabine, gemcitabine, capecitabine, fludarabine, mercaptopurine, azacitidine, methotrexate, and the like.
プラチナ製剤としては、例えば、シスプラチン、カルボプラチン、オキサリプラチン等が挙げられる。
トポイソメラーゼ阻害剤としては、例えば、エトポシド、ドキソルビシン、ミトキサントロン、テニポシド、イリノテカン等が挙げられる。
Examples of platinum preparations include cisplatin, carboplatin, oxaliplatin, and the like.
Examples of topoisomerase inhibitors include etoposide, doxorubicin, mitoxantrone, teniposide, irinotecan, and the like.
分子標的薬としては、例えば、トラスツズマブ、ベバシズマブ、リツキシマブ、イマチニブ、ゲフィチニブ、エルロチニブ等が挙げられる。
放射性同位元素としては、例えば、89Sr、223Ra、131I、90Y等が挙げられる。
Examples of molecular target drugs include trastuzumab, bevacizumab, rituximab, imatinib, gefitinib, erlotinib, and the like.
Examples of radioactive isotopes include 89 Sr, 223 Ra, 131 I, 90 Y, and the like.
TFに結合し得る抗体と、抗癌剤とのコンジュゲートの作製は、特に限定するものではなく、当分野で通常用いられる手段により適宜達成することができる。 The preparation of a conjugate between an antibody capable of binding to TF and an anticancer drug is not particularly limited, and can be appropriately achieved by means commonly used in the art.
本発明の治療剤は、更に他の薬剤と組み合わせて使用することができる。また、本発明の治療剤は、単独、又は他の有効成分と組み合わせて上記の治療剤を有効成分として含有する医薬組成物として使用することもできる。医薬組成物には、本発明の治療剤及び他の有効成分の他に、投与形態に応じて、当分野で通常使用される担体、賦形剤、緩衝剤、安定化剤等を含めることができる。 The therapeutic agent of the present invention can further be used in combination with other drugs. Furthermore, the therapeutic agent of the present invention can be used alone or in combination with other active ingredients as a pharmaceutical composition containing the above-mentioned therapeutic agent as an active ingredient. In addition to the therapeutic agent of the present invention and other active ingredients, the pharmaceutical composition may contain carriers, excipients, buffers, stabilizers, etc. commonly used in the art, depending on the dosage form. can.
本発明の治療剤の投与対象患者は、特に限定するものではないが、手術又は化学療法による卵巣癌治療後の患者であり、特に腫瘍摘出手術後の患者とすることが好ましい。 Patients to whom the therapeutic agent of the present invention is administered are not particularly limited, but are patients who have undergone ovarian cancer treatment by surgery or chemotherapy, and are particularly preferably patients who have undergone tumor removal surgery.
本発明の治療剤又は医薬組成物は、被験体に対して腹腔内投与により投与されることが意図される。腹腔内投与により、癌細胞集塊に強発現するTFに対して抗体が結合することで治療剤濃度が局所的に高くなり、癌細胞を標的とした抗癌剤による攻撃が非常に有効に達成され得る。 The therapeutic agent or pharmaceutical composition of the present invention is intended to be administered to a subject by intraperitoneal administration. When administered intraperitoneally, the antibody binds to TF that is strongly expressed in cancer cell clusters, increasing the concentration of the therapeutic agent locally, making it possible to very effectively attack cancer cells with the anticancer agent. .
本発明の治療剤の投与量は、患者の体重、年齢、疾患の重篤度等に応じて変動するものであり、特に限定するものではないが、例えば0.0001～100mg/kg体重の範囲の有効成分を１日１回～数回、２日毎、３日毎、１週間毎、２週間毎、毎月、２カ月毎、３カ月毎に投与することが可能である。 The dosage of the therapeutic agent of the present invention varies depending on the patient's weight, age, severity of disease, etc., and is not particularly limited, but for example, an effective dose in the range of 0.0001 to 100 mg/kg body weight may be used. The components can be administered once to several times a day, every two days, every three days, every week, every two weeks, every month, every two months, every three months.
以下、実施例を用いて本発明をより詳細に説明するが、これらの実施例は本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be explained in more detail using Examples, but these Examples do not limit the scope of the present invention.
［実施例１ 卵巣癌組織におけるTFの発現及び卵巣癌の腹膜浸潤機構］
倫理委員会の承認のもとに、金沢大学附属病院産婦人科で手術施行した卵巣癌患者から採取した腹膜組織の一部を解析に用いた。
腹膜播種のある20名の卵巣癌患者から採取した腹膜を、その表面を傷つけないよう留意してゴム板に貼り付けて、10%中性緩衝ホルマリンで固定した。固定後に5mm間隔で腹膜組織を切開し、垂直に立ててカセット内に並べてパラフィン包埋した。包埋したパラフィンブロックから3μmの厚さで切片を作成し、それらをプレパラートに貼り付けてHE染色や免疫染色に供した(Obata T. et al., PLOS one. 2017; 12(11): e0188641)。
取得したホルマリン固定切片に対する免疫組織染色は下記の要領でABC法を用いて行った。
[Example 1 TF expression in ovarian cancer tissue and peritoneal invasion mechanism of ovarian cancer]
With the approval of the Ethics Committee, a portion of peritoneal tissue collected from an ovarian cancer patient who underwent surgery at the Department of Obstetrics and Gynecology, Kanazawa University Hospital was used for analysis.
The peritoneum collected from 20 ovarian cancer patients with peritoneal dissemination was attached to a rubber plate, taking care not to damage the surface, and fixed in 10% neutral buffered formalin. After fixation, the peritoneal tissue was incised at 5 mm intervals, placed vertically in a cassette, and embedded in paraffin. Sections with a thickness of 3 μm were created from embedded paraffin blocks, and the sections were pasted onto slides and subjected to HE staining and immunostaining (Obata T. et al., PLOS one. 2017; 12(11): e0188641 ).
Immunohistochemical staining of the obtained formalin-fixed sections was performed using the ABC method as described below.
＜抗原賦活化＞
まず、各プレパラートをpH6またはpH9のクエン酸緩衝液で96℃で20分間処理した。
一次抗体として下記の抗体を用い、プレパラートを4℃で一晩処理した。
1. TF (clone:2K1, Abcam, Cambridge, UK) マウスモノクローナル抗体（1:2002希釈）
2. フィブリノゲンα鎖 (clone:UC45, Abcam, Cambridge, UK) マウスモノクローナル抗体（1:100希釈）
3. CD34 (clone:QBEnd-10, Dako, Santa Clara, US) マウスモノクローナル抗体（1:100希釈）
4. ポドプラニン (clone:D2-40, Dako, Santa Clara, US) マウスモノクローナル抗体（1:100希釈）
5. αSMA (clone:1A4, Dako, Santa Clara, US) マウスモノクローナル抗体（1:500希釈）
6. 血管内皮細胞増殖因子-A (VEGF-A, clone:VG1, Dako, Santa Clara, US) マウスモノクローナル抗体（1:50希釈）
7. CD31 (clone:EPR3094, Abcam, Cambridge, UK) ウサギモノクローナル抗体（1:25希釈）
8. 間質細胞由来因子-1/CXCL12 (SDF-1/CXCL12, Abcam, Cambridge, UK) ウサギポリクローナル抗体（1:1000希釈）
<Antigen retrieval>
First, each preparation was treated with pH 6 or pH 9 citrate buffer at 96°C for 20 minutes.
The preparations were treated overnight at 4°C using the following antibody as the primary antibody.
1. TF (clone:2K1, Abcam, Cambridge, UK) mouse monoclonal antibody (1:2002 dilution)
2. Fibrinogen α chain (clone:UC45, Abcam, Cambridge, UK) mouse monoclonal antibody (1:100 dilution)
3. CD34 (clone:QBEnd-10, Dako, Santa Clara, US) mouse monoclonal antibody (1:100 dilution)
4. Podoplanin (clone:D2-40, Dako, Santa Clara, US) mouse monoclonal antibody (1:100 dilution)
5. αSMA (clone:1A4, Dako, Santa Clara, US) mouse monoclonal antibody (1:500 dilution)
6. Vascular endothelial growth factor-A (VEGF-A, clone:VG1, Dako, Santa Clara, US) mouse monoclonal antibody (1:50 dilution)
7. CD31 (clone:EPR3094, Abcam, Cambridge, UK) rabbit monoclonal antibody (1:25 dilution)
8. Stromal cell-derived factor-1/CXCL12 (SDF-1/CXCL12, Abcam, Cambridge, UK) rabbit polyclonal antibody (1:1000 dilution)
次いで、二次抗体として下記の抗体を用い、室温でプレパラートを30分間処理した。
1. ビオチン標識ウマ抗マウスIgG
2. ビオチン標識ヤギ抗ウサギIgG
Next, the preparations were treated at room temperature for 30 minutes using the following antibody as a secondary antibody.
1. Biotinylated horse anti-mouse IgG
2. Biotinylated goat anti-rabbit IgG
ABC染色は下記のキットを用いて施行し、顕微鏡下に観察した。
1. アビジン-ビオチン複合体 (VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA, USA)
2. ジアミノベンジジン(Liquid DAB+ Substrate Chromogen System; Dako, Carpinteria, CA, USA)
ABC staining was performed using the kit below and observed under a microscope.
1. Avidin-biotin complex (VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA, USA)
2. Diaminobenzidine (Liquid DAB+ Substrate Chromogen System; Dako, Carpinteria, CA, USA)
その結果、20名中9名が高異型度漿液性腺癌症例であったが、そのうちの4名に形態的に下記の播種形成所見が観察された。
ポドプラニンで認識される腹膜中皮細胞層はインタクトに保たれた状態で、それに対峙する癌細胞集塊の周囲にはTFやフィブリノゲンの発現を認めるフィブリン網が形成されていた。また癌細胞集塊を含めたフィブリン網内には遊走してきたCD31、CD34およびVEGF-A陽性の血管内皮細胞が観察され、さらに血管腔が形成されると血管内に宿主側の間質との間を循環する赤血球が確認された（図１Ａ－Ｂ）。
As a result, 9 out of 20 patients had high-grade serous adenocarcinoma, and the following morphological findings of dissemination formation were observed in 4 of them.
The peritoneal mesothelial cell layer, which is recognized by podoplanin, remained intact, and a fibrin network was formed around the cancer cell cluster facing it, where TF and fibrinogen were expressed. In addition, migrating CD31-, CD34-, and VEGF-A-positive vascular endothelial cells were observed within the fibrin network, including cancer cell aggregates, and when a vascular cavity was formed, there was a connection between the host interstitium within the blood vessel. Red blood cells were observed circulating between the cells (Figures 1A-B).
一方、遊走してきた線維芽細胞により癌細胞集塊の周囲に形成された間質組織にはαSMAおよびポドプラニン発現陽性のがん間質線維芽細胞(CAF)が観察された。さらに進行すると癌細胞集塊と腹膜間質組織を結ぶ栄養血管の走行ルート周囲で中皮細胞層の消失が拡大され、この部位において癌細胞集塊の腹膜間質組織内への浸潤・進展像が観察された。またこれらの一連の過程で癌細胞集塊周囲にTFが強発現していることが観察された（図２Ａ－Ｄ）。 On the other hand, cancer stromal fibroblasts (CAF) positive for αSMA and podoplanin expression were observed in the stromal tissue formed around cancer cell clusters by migrating fibroblasts. As it progresses further, the disappearance of the mesothelial cell layer expands around the route of the feeding blood vessels connecting the cancer cell cluster and the peritoneal stromal tissue, and the cancer cell cluster infiltrates and spreads into the peritoneal stromal tissue at this site. was observed. In addition, strong expression of TF was observed around cancer cell clusters during these series of steps (FIGS. 2A to 2D).
上記の結果から、下記の新しい卵巣癌の腹膜浸潤機構が推測される（図３Ａ－Ｄ）。
すなわち、図３Ａに示すように、癌細胞が腹膜組織に炎症を誘導し、透過性を亢進した毛細血管からフィブリノゲンが析出されて癌細胞集塊周囲にフィブリン網を形成し、癌細胞集塊が腹膜上皮に接着することなくこれに対峙する（図３Ａ）。
From the above results, the following new mechanism of peritoneal invasion of ovarian cancer is inferred (FIGS. 3A-D).
That is, as shown in FIG. 3A, cancer cells induce inflammation in the peritoneal tissue, and fibrinogen is precipitated from capillaries with increased permeability to form a fibrin network around the cancer cell cluster. It confronts the peritoneal epithelium without adhering to it (Fig. 3A).
次いで、図３Ｂに示すように、癌細胞集塊周囲のフィブリン網内に線維芽細胞や血管内皮細胞の遊走およびそれに伴う新生血管網の形成が誘導される（図３Ｂ）。
新生された血管や間質組織を足場に癌細胞集塊が発育する（図３Ｃ）。
発育した癌細胞集塊が血管誘導経路を利用して腹膜中皮細胞層の欠損を拡大しつつ腹膜間質内に浸潤する（図３Ｄ）。
Next, as shown in FIG. 3B, the migration of fibroblasts and vascular endothelial cells into the fibrin network surrounding the cancer cell cluster and the accompanying formation of a new vascular network are induced (FIG. 3B).
Cancer cell clusters grow using the newly generated blood vessels and interstitial tissue as scaffolds (Figure 3C).
The developed cancer cell cluster infiltrates into the peritoneal stroma while expanding the defect in the peritoneal mesothelial cell layer using the vascular guidance route (FIG. 3D).
［実施例２ 卵巣癌患者の血中及び腹水中TF発現量の測定］
33例のステージI～IVの卵巣癌患者における腹水中また血中のTF濃度の値を、市販のキット（Quantikine Human Coagulation Factor III/Tissue Factor Immunoassay, R&D Systems, Minneapolis, MN, USA）を用いてELISAで測定した。
[Example 2 Measurement of TF expression level in blood and ascites of ovarian cancer patients]
Ascitic fluid and blood TF concentrations in 33 stage I-IV ovarian cancer patients were determined using a commercially available kit (Quantikine Human Coagulation Factor III/Tissue Factor Immunoassay, R&D Systems, Minneapolis, MN, USA). Measured by ELISA.
その結果、腹水サンプルを用いて測定した25例の患者のうちの21例は、TF濃度が血漿中での正常範囲とされる22.6 - 53.6 pg/mlより高値を示していた。また、腹水中TF (n=25)と血液中TF (n=20) を比較すると、腹水中のTFが著明に高値であった（図４Ａ）。さらに同一患者 (n=12)の腹水及び血中のTF濃度を比較すると、全ての患者において腹水中の方が血中より顕著に高い値であった（図４Ｂ）。 As a result, 21 of the 25 patients whose ascitic fluid samples were measured had TF concentrations higher than the normal plasma range of 22.6 - 53.6 pg/ml. Furthermore, when comparing TF in ascites (n=25) and TF in blood (n=20), TF in ascites was significantly higher (Figure 4A). Furthermore, when the TF concentrations in ascites and blood of the same patient (n=12) were compared, the concentration of TF in ascites was significantly higher than in blood in all patients (Fig. 4B).
［実施例３ ステージIII～IVの卵巣癌患者における血中及び腹水中TF発現量の測定］
また、腹膜播種のあるステージIII～IVの20例の卵巣癌患者における血中及び腹水中TF濃度（pg/ml）を表１に示す。「－」はサンプルの取得ができなかったため測定していないことを示す。
[Example 3 Measurement of TF expression level in blood and ascites in stage III to IV ovarian cancer patients]
Table 1 also shows the TF concentrations (pg/ml) in blood and ascites in 20 stage III to IV ovarian cancer patients with peritoneal dissemination. "-" indicates that the sample was not measured because it could not be obtained.
その結果、いずれの患者においても測定されたTF濃度は血中よりも腹水中で顕著に高いことが確認された。また、同一患者において、パクリタキセル、シスプラチン、又はハイカムチンを用いた化学療法による治療前後での腹水中のTF濃度を比較したところ、治療後に腹水中TF値が著明に減少していた。 As a result, it was confirmed that the TF concentration measured in all patients was significantly higher in ascites than in blood. Furthermore, in the same patient, when we compared the TF concentration in ascites before and after chemotherapy using paclitaxel, cisplatin, or hycamtin, we found that the TF value in ascites decreased markedly after treatment.
［実施例４ RT-PCRによるTFの検出］
卵巣癌組織におけるTFの存在を更に確認するために、患者組織におけるTFの発現についてRT-PCRを行った。
RNeasy Mini Kit（Qiagen, Hilden, Germany）を使用して、卵巣癌患者由来の一次病変組織及び腹膜転移組織の凍結腫瘍サンプルからRNAを抽出した。DNase（Ambion Diagnostics Inc., Austin, TX）処理の後、全RNA 1μgをSuperScript II（Invitrogen Carlsbad, CA）を使用して逆転写した。
[Example 4 Detection of TF by RT-PCR]
To further confirm the presence of TF in ovarian cancer tissues, RT-PCR was performed for the expression of TF in patient tissues.
RNA was extracted from frozen tumor samples of primary lesion tissue and peritoneal metastasis tissue from ovarian cancer patients using the RNeasy Mini Kit (Qiagen, Hilden, Germany). After DNase (Ambion Diagnostics Inc., Austin, TX) treatment, 1 μg of total RNA was reverse transcribed using SuperScript II (Invitrogen Carlsbad, CA).
TFの増幅のためには以下の配列を有するプライマー：
5’-GCCAGGAGAAAGGGGAAT-3’（配列番号１）及び
5’-CAGTGCAATATAGCATTTGCAGTAGC-3’（配列番号２）
を使用し、対照としてのGAPDHの増幅のためには以下の配列を有するプライマー：
5’-GCACCGTCAAGGCTGAGAAC-3’（配列番号３）及び
5’-TGGTGAAGACGCCAGTGGA-3’（配列番号４）
を使用した。
For amplification of TF, primers with the following sequences:
5'-GCCAGGAGAAAGGGGAAT-3' (SEQ ID NO: 1) and
5'-CAGTGCAATATAGCATTTGCAGTAGC-3' (SEQ ID NO: 2)
For amplification of GAPDH as a control use primers with the following sequences:
5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 3) and
5'-TGGTGAAGACGCCAGTGGA-3' (SEQ ID NO: 4)
It was used.
PCRは、95℃で30秒間、60℃で30秒間、及び72℃で30秒間を50サイクル実施した。増幅後、得られたPCR産物をMinElute Gel Extraction Kit（Qiagen）を使用して精製した。精製したPCR産物の配列をDNAシーケンサー（3730xl DNA Analyzer; Thermo Fisher Scientific）を使用して解析し、検出した。
その結果、図４Ｃに示すように、一次病変（Pri）及び腹膜転移（Met）組織の双方で、TF mRNAの発現が確認された。
PCR was performed for 50 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. After amplification, the resulting PCR product was purified using MinElute Gel Extraction Kit (Qiagen). The purified PCR product was sequenced and detected using a DNA sequencer (3730xl DNA Analyzer; Thermo Fisher Scientific).
As a result, as shown in FIG. 4C, expression of TF mRNA was confirmed in both the primary lesion (Pri) and peritoneal metastasis (Met) tissues.
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