JPWO2018034332A1 - EphA2 N-terminal fragment antibody - Google Patents
EphA2 N-terminal fragment antibody Download PDFInfo
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- JPWO2018034332A1 JPWO2018034332A1 JP2018534429A JP2018534429A JPWO2018034332A1 JP WO2018034332 A1 JPWO2018034332 A1 JP WO2018034332A1 JP 2018534429 A JP2018534429 A JP 2018534429A JP 2018534429 A JP2018534429 A JP 2018534429A JP WO2018034332 A1 JPWO2018034332 A1 JP WO2018034332A1
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- amino acid
- acid sequence
- epha2
- seq
- antibody
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- G01N33/531—Production of immunochemical test materials
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Abstract
本発明は、MT1-MMPによって切断されるEphA2のN末端側フラグメントと特異的に結合する抗体の提供を目的とするもので、以下の（ａ）のタンパク質と結合し、かつ（ｂ）および（ｃ）のタンパク質とは結合しない抗体である。（ａ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質The present invention aims to provide an antibody that specifically binds to the N-terminal fragment of EphA2 cleaved by MT1-MMP, which binds to the following protein (a), and (b) and (b) It is an antibody which does not bind to the protein of c). (A) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer from 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by 28th to Xth, (b) consisting of an amino acid sequence 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1 Amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence of 28th to 976th in the amino acid sequence represented by protein, SEQ ID NO: 1 Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1, (c) the amino acid represented by SEQ ID NO: 3 A protein consisting of a sequence, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 3 or 80% with the amino acid sequence represented by SEQ ID NO: 3 A protein consisting of an amino acid sequence having the above sequence identity
Description
本発明は、MT1-MMPによって切断されるEphA2のN末端側フラグメントと特異的に結合する抗体および該抗体の作製方法に関する。 The present invention relates to an antibody that specifically binds to the N-terminal fragment of EphA2 cleaved by MT1-MMP and a method for producing the antibody.
EphA2（Erythropoietin-producing hepatocellular receptor 2）は、哺乳類Eph受容体キナーゼファミリのメンバーで、乳がん、肝臓がん、すい臓がん、前立腺がん、胃がん、神経膠腫、黒色腫および卵巣腺がんなどの種々の腫瘍組織において過剰発現しており、がんの進行および転移に関係している（非特許文献１）。
EphA2のチロシンキナーゼ活性は、ephrin A1などのリガンドが結合すると活性化される。正常な上皮細胞では、EphA2のチロシン残基（Tyr594）のリガンド誘導的な自己リン酸化がErb-B受容体によりメディエートされる増殖シグナルを抑制し、細胞を正常な状態に維持する役割を果たしている。つまり、EphA2は正常細胞において腫瘍サプレッサー様活性を有しているが、がん細胞において過剰に発現するとがんの進行を促進する。
最近の研究により、EphA2は相反する２つの活性を有していることが明らかになってきた。リガンド依存的に機能するEphA2とは異なり、リガンド非存在下でEphA2は、ErbB受容体の刺激後Ras/MAPKによりセリン残基（Ser897）がリン酸化され（非特許文献２）、ephexin-4/RhoGとのドッキングサイトとして作用し、Rac1の活性化を誘導する（非特許文献３）。このように、がんの進行におけるEphA2の作用機序が解明されてきたことにより、EphA2は抗がん剤の分子標的として注目されている。EphA2 (Erythropoietin-Producing Heptocellular Receptor 2) is a member of the mammalian Eph receptor kinase family and includes breast cancer, liver cancer, pancreatic cancer, prostate cancer, gastric cancer, glioma, melanoma and ovarian adenocarcinoma etc. It is overexpressed in various tumor tissues and is associated with cancer progression and metastasis (Non-patent Document 1).
The tyrosine kinase activity of EphA2 is activated upon binding of a ligand such as ephrin A1. In normal epithelial cells, ligand-induced autophosphorylation of tyrosine residue (Tyr 594 ) of EphA2 suppresses the proliferation signal mediated by the Erb-B receptor and plays a role in maintaining the cells in a normal state. There is. Thus, EphA2 has a tumor suppressor-like activity in normal cells, but overexpression in cancer cells promotes cancer progression.
Recent studies have revealed that EphA2 has two opposing activities. Unlike EphA2, which functions in a ligand-dependent manner, in the absence of ligand, EphA2 is phosphorylated on serine residue (Ser 897 ) by Ras / MAPK after stimulation with ErbB receptor (non-patent document 2), ephexin-4 Acts as a docking site for RhoG and induces activation of Rac1 (Non-patent Document 3). Thus, EphA2 has attracted attention as a molecular target for anticancer agents, as the mechanism of action of EphA2 in cancer progression has been elucidated.
他方、MT1-MMP（membrane-type 1-matrix metalloproteinase 1）は、腫瘍細胞において過剰発現することが知られている膜メタロプロテアーゼである。MT1-MMPは、細胞周辺の生理活性タンパク質や細胞外マトリックスのプロセッシング、また、細胞質側末端を介したHIF転写因子の活性化を通じて、腫瘍の進行を制御している。EphA2はMT1-MMPの基質であり、MT1-MMPで切断されてEphA2リガンド結合ドメインが除去され（非特許文献４、非特許文献５）、リガンドが結合できない構造に変換される。発明者らは、がん組織においてMT1-MMPと共発現されるEphA2のほとんどがN末端側のリガンド結合ドメインを欠失しており、Ser897がリン酸化されていることを見いだした（非特許文献５）。MT1-MMPで切断されない変異体EphA2を上皮癌腫A431細胞株内で強制発現させると、細胞の形態が上皮細胞様の形態に変化し、腫瘍の増殖と肺転移が抑制された（非特許文献５）。従って、がん細胞中でのMT1-MMPによるEphA2のプロセッシングは、がんの進行と深く関係する事象である。そして、MT1-MMPによって切断されたEphA2のN末端側フラグメントが細胞から離れ血流中を循環していれば、そのN末端側フラグメントは、従来の血液テストを用いて早期に検出可能ながんマーカーとして使用することが可能である（特許文献１）。On the other hand, MT1-MMP (membrane-type 1-matrix metalloproteinase 1) is a membrane metalloprotease which is known to be overexpressed in tumor cells. MT1-MMP regulates tumor progression through processing of bioactive proteins and extracellular matrix around the cell and activation of HIF transcription factor via cytoplasmic end. EphA2 is a substrate of MT1-MMP, which is cleaved with MT1-MMP to remove the EphA2 ligand binding domain (Non-patent Document 4, Non-patent Document 5) and converted to a structure incapable of binding a ligand. The inventors found that most of EphA2 coexpressed with MT1-MMP in cancer tissues lacks the N-terminal ligand binding domain and that Ser 897 is phosphorylated (non-patented) Literature 5). When forced expression of mutant EphA2, which is not cleaved by MT1-MMP, in epithelial carcinoma A431 cell line, the cell morphology changes to an epithelial cell-like morphology, and tumor growth and lung metastasis are suppressed (Non-patent document 5) ). Thus, processing of EphA2 by MT1-MMP in cancer cells is an event that is closely linked to cancer progression. And, if the N-terminal fragment of EphA2 cleaved by MT1-MMP is separated from the cells and circulates in the bloodstream, the N-terminal fragment is an early detectable cancer using conventional blood tests. It can be used as a marker (Patent Document 1).
上述の通り、MT1-MMPで切断されたEphA2のN末端側フラグメントは、がんマーカーとして利用できることが示されている。しかしながら、そのようなEphA2フラグメントを特異的に検出する手段（たとえば、抗体など）および方法は未だに確立されていないのが現状である。
このような事情に鑑み、本発明者らは、MT1-MMPで切断されたEphA2のN末端側フラグメントのみを特異的に認識する抗体の作製を解決課題として研究を進めた。
すなわち、本発明は、MT1-MMPで切断されたEphA2のN末端側フラグメントと特異的に結合する抗体および該抗体の作製方法の提供を目的とする。
また、本発明は前記抗体を用いたがんの検査方法の提供を目的とする。
さらに、本発明は前記抗体を含むがんの検査用キットの提供を目的とする。As described above, it has been shown that the N-terminal fragment of EphA2 cleaved with MT1-MMP can be used as a cancer marker. However, at present, means (eg, antibodies etc.) and methods for specifically detecting such EphA2 fragments have not been established yet.
In view of these circumstances, the present inventors have made researches directed to the preparation of an antibody that specifically recognizes only the N-terminal fragment of EphA2 cleaved with MT1-MMP.
That is, the present invention aims to provide an antibody that specifically binds to the N-terminal fragment of EphA2 cleaved with MT1-MMP and a method for producing the antibody.
Another object of the present invention is to provide a method for testing cancer using the above antibody.
Furthermore, another object of the present invention is to provide a kit for testing a cancer comprising the antibody.
本発明者らは、上記課題を解決すべく、EphA2のN末端側フラグメントを抗原として、モノクローナル抗体の作製を試みた。
EphA2はエクソソーム（exosome）にも存在していることが報告されており、血中にMT1-MMPで切断されたEphA2のN末端側フラグメント（以下、「MT1-MMP切断EphA2-N末側フラグメント」とも記載する）以外にも、完全長の（無傷の）EphA2が存在していることが予想された。また、他のEphファミリー分子は膜プロテアーゼで切断されることが報告されており、EphA2についても、膜プロテアーゼによってプロセッシングを受ける可能性も否定できず、膜プロテアーゼでプロセッシングを受けたフラグメントが血中に存在していることも予想された。
すなわち、単に、MT1-MMP切断EphA2-N末側フラグメントを免疫して得られた抗体が、MT1-MMP切断EphA2-N末側フラグメントのみを特異的に認識する保証はなく、抗体のスクリーニングの段階で新たな知見等に基づいた方法を検討する必要があった。In order to solve the above problems, the present inventors attempted to produce a monoclonal antibody using an N-terminal fragment of EphA2 as an antigen.
It is reported that EphA2 is also present in exosomes, and an N-terminal fragment of EphA2 cleaved by MT1-MMP in blood (hereinafter referred to as “MT1-MMP cleaved EphA2-N end fragment”) Besides (described also), it was predicted that full-length (intact) EphA2 was present. In addition, other Eph family molecules have been reported to be cleaved by membrane proteases, and EphA2 can not be denied the possibility of being processed by membrane proteases, and fragments processed by membrane proteases are in blood. It was also expected to exist.
That is, there is no guarantee that the antibody obtained by immunizing the MT1-MMP-cleaved EphA2-N end fragment specifically recognizes only the MT1-MMP-cleaved EphA2-N end fragment, and the antibody screening stage Need to consider methods based on new findings.
発明者らは、MT1-MMP切断EphA2-N末側フラグメントの構造と、完全長EphA2のN末端側領域の構造およびEphA2が膜プロテアーゼで切断されるのであればその切断フラグメントの構造（EphA2細胞外ドメインフラグメント）が異なるのではないか、あるいは、各々に特異なエピトープが存在するのではないかとの作業仮説を立て、「MT1-MMP切断EphA2-N末側フラグメント」のみを認識する抗体の調製を試みた。
発明者らは、鋭意検討を行い抗体のスクリーニングを行った結果、完全長EphA2とEphA2細胞外ドメインフラグメントは認識せず、「MT1-MMP切断EphA2-N末側フラグメント」のみを特異的に認識する抗体の作製に成功し、本発明を完成させた。The present inventors structure of MT1-MMP-cleaved EphA2-N terminal fragment, structure of N-terminal region of full-length EphA2 and structure of its cleavage fragment if EphA2 is cleaved by membrane protease (EphA2 extracellular Create a working hypothesis that domain fragments differ or that there is a specific epitope for each, and prepare an antibody that recognizes only "MT1-MMP-cleaved EphA2-N end fragment" I tried.
As a result of intensive investigations and screening of antibodies, the inventors do not recognize full-length EphA2 and EphA2 extracellular domain fragments, but specifically recognize only "MT1-MMP cleaved EphA2-N end fragment" Successful production of antibodies completed the present invention.
すなわち、本発明は以下の（１）〜（１２）に関する。
（１）MT1-MMP切断EphA2-N末側フラグメントと特異的に結合し、完全長のEphA2とは結合せず、かつ、EphA2細胞外ドメインフラグメントにも結合しない抗体。
（２）以下の（ａ）のタンパク質と結合し、かつ（ｂ）および（ｃ）のタンパク質とは結合しないことを特徴とする上記（１）に記載の抗体。
（ａ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
前記タンパク質について具体的な一例として、配列番号２で表されるアミノ酸配列からなるタンパク質、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（３）前記（ａ）、（ｂ）および／または（ｃ）に記載のタンパク質が動物細胞で発現したものであることを特徴とする上記（２）に記載の抗体。
（４）被験者由来の試料中のMT1-MMP切断EphA2-N末側フラグメント量を、上記（１）ないし（３）のいずれかに記載の抗体を用いて測定する工程を含むがんの検査方法。
（５）前記がんが、EphA2を発現していることを特徴とする上記（４）に記載のがんの検査方法。
（６）前記がんが、すい臓がんおよび胃がんであることを特徴とする上記（４）または（５）に記載のがんの検査方法。
（７）前記試料が、血液、リンパ液、唾液、痰、尿または糞便から選択される少なくとも１つであることを特徴とする上記（４）ないし（６）のいずれかに記載のがんの検査方法。
（８）上記（１）ないし（３）のいずれかに記載の抗体を含むがんの検査用キット。
（９）試料中に存在するMT1-MMP切断EphA2-N末側フラグメントの量を上記（１）ないし（３）のいずれかに記載の抗体で測定する方法。
（１０）上記（９）の測定方法で測定したMT1-MMP切断EphA2-N末側フラグメントの量に基づいて、試料中のMT1-MMPプロテアーゼ活性を測定する方法。
（１１）前記試料が、MT1-MMPおよびEphA2を共発現する細胞を培養した培養液であることを特徴とする上記（１０）に記載の方法。
（１２）以下の工程（ｉ）および（ｉｉ）を含む、抗体の作製方法。
（ｉ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質のいずれかに結合する抗体を作製する工程、
前記タンパク質について具体的な一例として、配列番号２で表されるアミノ酸配列からなるタンパク質、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質のいずれかに結合する抗体を作製する工程、
（ｉｉ）工程（ｉ）で作製した抗体のうち、下記の（ｂ）および（ｃ）のタンパク質とは結合しない抗体を選択する工程
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質That is, the present invention relates to the following (1) to (12).
(1) An antibody that specifically binds to the MT1-MMP-cleaved EphA2-N-terminal fragment, does not bind to the full-length EphA2, and does not bind to the EphA2 extracellular domain fragment.
(2) The antibody according to (1) above, which binds to the following protein (a) and does not bind to the proteins (b) and (c):
(A) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer from 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by the 28th to Xth,
As a specific example of the above-mentioned protein, a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(B) A protein consisting of the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1, 1 or several amino acids in the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1 Is a protein consisting of an amino acid sequence having an addition, substitution, insertion or deletion, or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence shown in SEQ ID NO: 1 ,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3;
(3) The antibody according to (2) above, wherein the protein according to (a), (b) and / or (c) is expressed in animal cells.
(4) A method of examining cancer comprising the step of measuring the amount of MT1-MMP-cleaved EphA2-N end fragment in a sample derived from a subject using the antibody according to any one of the above (1) to (3) .
(5) The method of testing a cancer according to (4) above, wherein the cancer expresses EphA2.
(6) The method of testing a cancer according to (4) or (5) above, wherein the cancer is pancreatic cancer and gastric cancer.
(7) The test for cancer according to any one of the above (4) to (6), wherein the sample is at least one selected from blood, lymph, saliva, sputum, urine or feces. Method.
(8) A kit for testing a cancer, which comprises the antibody according to any one of (1) to (3) above.
(9) A method of measuring the amount of MT1-MMP-cleaved EphA2-N end fragment present in a sample with the antibody according to any of the above (1) to (3).
(10) A method of measuring MT1-MMP protease activity in a sample based on the amount of MT1-MMP cleaved EphA2-N end fragment measured by the measurement method of (9) above.
(11) The method according to (10) above, wherein the sample is a culture solution in which cells co-expressing MT1-MMP and EphA2 are cultured.
(12) A method for producing an antibody, which comprises the following steps (i) and (ii):
(I) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer of 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 Producing an antibody that binds to any of a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by 28th to Xth,
As a specific example of the above-mentioned protein, a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Producing an antibody which binds to any of a protein consisting of SEQ ID NO: 2 or a protein consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2,
(Ii) a step of selecting an antibody which does not bind to the proteins of the following (b) and (c) among the antibodies produced in the step (i) (b) 28th to 28th amino acids in the amino acid sequence represented by SEQ ID NO: 1 A protein consisting of the amino acid sequence 976, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the 28th to 976th amino acids in the amino acid sequence represented by SEQ ID NO: 1; Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3
本発明の抗体は、MT1-MMP切断EphA2-N末側フラグメントと特異的に結合することができる。すなわち、本発明の抗体は、MT1-MMP切断EphA2-N末側フラグメントのみを認識し、完全長EphA2およびEphA2細胞外ドメインフラグメントなどは認識しない。 The antibody of the present invention can specifically bind to the MT1-MMP cleaved EphA2-N terminal fragment. That is, the antibody of the present invention recognizes only the MT1-MMP-cleaved EphA2-N-terminal fragment, but not the full-length EphA2 and EphA2 extracellular domain fragments and the like.
本発明の抗体を使用することで、試料中に存在する、MT1-MMP切断EphA2-N末側フラグメントのみを検出することが可能となり、試料が由来する被験者のがんによる罹患の有無、がんの進行度、がん治療の効果などにつき、検査、診断することができる。 By using the antibody of the present invention, it is possible to detect only the MT1-MMP-cleaved EphA2-N-terminal fragment present in the sample, and whether or not the subject from which the sample is derived is affected by cancer, cancer You can examine and diagnose the degree of progression of the disease, the effects of cancer treatment, etc.
本発明の第１の実施形態は、MT1-MMP切断EphA2-N末側フラグメントと特異的に結合し、完全長の（インタクト（無傷）の）EphA2とは結合せず、かつ、EphA2細胞外ドメインフラグメントにも結合しない抗体である。
発明者らは、これまでに、MT1-MMP切断EphA2-N末側フラグメントが、がん細胞から離れ血流中を循環することを見いだし、このMT1-MMP切断EphA2-N末側フラグメントが、早期に検出可能ながんマーカーとして使用し得ることを明らかにしている（特許文献１）。しかしながら、EphA2のN末端側フラグメントを免疫原として作製した抗体は、EphA2のN末端側フラグメントを特異的に認識するものではあるが、同時に、完全長のEphA2をも認識する可能性が高かった。しかも、血中には、MT1-MMP切断EphA2-N末側フラグメントのみならず、完全長のEphA2も存在している可能性が示唆されており（Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA）、MT1-MMP切断EphA2-N末側フラグメントと完全長のEphA2を区別して認識する抗体を取得する必要があった。さらに、Ephファミリー分子は膜プロテアーゼで切断されることが報告されていることから（Bai G., Pfaff S. L. (2011). Protease regulation: the Yin and Yang of neural development and disease. Neuron 72, 9-21）、EphA2についても、膜プロテアーゼによってプロセッシングを受ける可能性が考えられ、単にN末端側フラグメントを免疫原として作製した抗体は、MT1-MMP切断EphA2-N末側フラグメント以外のフラグメントをも認識してしまう可能性があった。The first embodiment of the present invention specifically binds to the MT1-MMP-cleaved EphA2-N end fragment, does not bind to the full-length (intact) EphA2, and the EphA2 extracellular domain It is an antibody that also does not bind to fragments.
The inventors have so far found that the MT1-MMP cleaved EphA2-N end fragment circulates away from the cancer cells in the bloodstream, and this MT1-MMP cleaved EphA2-N end fragment is early It has been clarified that it can be used as a detectable cancer marker (Patent Document 1). However, although the antibody produced by using the N-terminal fragment of EphA2 as the immunogen specifically recognizes the N-terminal fragment of EphA2, it was also highly likely to recognize full-length EphA2. Moreover, it has been suggested that not only MT1-MMP-cleaved EphA2-N end fragments but also full-length EphA2 may be present in blood (Proceedings: AACR 106th Annual Meeting 2015; April 18-22 , 2015; Philadelphia, PA), it was necessary to obtain an MT1-MMP-cleaved EphA2-N terminal fragment and a full-length EphA2 differentially recognizing antibody. Furthermore, it has been reported that Eph family molecules are cleaved by membrane proteases (Bai G., Pfaff SL (2011). Protease regulation: the Yin and Yang of neural development and disease. Neuron 72, 9-21. And EphA2 may also be processed by membrane proteases, and an antibody produced solely by using an N-terminal fragment as an immunogen also recognizes a fragment other than the MT1-MMP cleaved EphA2-N terminal fragment. There was a possibility of
以上のような状況において、発明者らは、MT1-MMP切断EphA2-N末側フラグメントと、完全長のEphA2および膜プロテアーゼによってプロセッシングを受けた場合に生じる可能性のあるEphA2の細胞外ドメインの立体構造が、各々、相違する可能性を考え、MT1-MMP切断EphA2-N末側フラグメントを免疫原として取得した抗体の中から、完全長のEphA2およびEphA2の細胞外ドメインのいずれにも反応しない抗体のみを取得することを試みた。具体的には、MT1-MMP切断EphA2-N末側フラグメントの一部を動物細胞中で発現させ、これを免疫原とし、当該フラグメントを免疫沈降することのできる抗体をいくつか取得し、得られた抗体の中から、完全長のEphA2を発現する細胞の抽出物から完全長のEphA2を免疫沈降させず、かつ、動物細胞中で発現させたEphA2の細胞外ドメインも免疫沈降させない抗体を取得した。
上記のような方法で取得した抗体は、後述の実施例に示すように、がん患者由来の血液サンプルに特異的に反応することが確認され、がんマーカーとしての利用が期待されている、MT1-MMP切断EphA2-N末側フラグメントと特異的に結合するものである。Under the circumstances as described above, the inventors found that the MT1-MMP cleaved EphA2-N end fragment and the extracellular domain of EphA2 that may be generated when processed by full-length EphA2 and membrane proteases. Among the antibodies obtained using the MT1-MMP-cleaved EphA2-N end fragment as an immunogen, each of which has a different possibility of structure, an antibody that does not react with either the full-length EphA2 or EphA2 extracellular domain Tried to get only. Specifically, a part of the MT1-MMP-cleaved EphA2-N terminal fragment is expressed in animal cells and used as an immunogen to obtain several antibodies capable of immunoprecipitating the fragment. Among the antibodies obtained, an antibody was obtained which did not immunoprecipitate full-length EphA2 from extracts of cells expressing full-length EphA2 and also did not immunoprecipitate the extracellular domain of EphA2 expressed in animal cells. .
The antibody obtained by the method as described above is confirmed to specifically react with a blood sample derived from a cancer patient, as shown in the examples described later, and is expected to be used as a cancer marker. It specifically binds to the MT1-MMP cleaved EphA2-N terminal fragment.
すなわち、本発明の第１の実施形態を具体的には、たとえば、以下の（ａ）のタンパク質と結合し、かつ（ｂ）および（ｃ）のタンパク質とは結合しない抗体である。
（ａ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
前記タンパク質について具体的な一例として、配列番号２で表されるアミノ酸配列からなるタンパク質、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質That is, specifically, the first embodiment of the present invention is, for example, an antibody that binds to the following protein (a) and does not bind to the proteins (b) and (c).
(A) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer from 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by the 28th to Xth,
As a specific example of the above-mentioned protein, a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(B) A protein consisting of the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1, 1 or several amino acids in the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1 Is a protein consisting of an amino acid sequence having an addition, substitution, insertion or deletion, or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence shown in SEQ ID NO: 1 ,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3
ここで、EphA2（なお、特記しない限り、EphA2タンパク質のことを表す）のMT1-MMP切断EphA2-N末側フラグメントとは、たとえば、配列番号１に示されるアミノ酸配列中、28番目〜385番目まで、28番目〜395番目まで、28番目〜432番目まで、または28番目〜435番目までのアミノ酸配列を含むフラグメントのことである。したがって、MT1-MMP切断EphA2-N末側フラグメントに結合する抗体を作製するための抗原としては、たとえば、配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質を用いることができる。前記抗原の一例として、配列番号２（配列番号１で表されるアミノ酸配列中、28番目〜328番目のアミノ酸配列）で表されるアミノ酸配列からなるタンパク質（以下、「Antigen #1」と記載する）、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質を挙げることができる。
MT1-MMP切断EphA2-N末側フラグメントに結合する抗体を作製するための抗原は、どのように調製したものであってもよいが、動物細胞内で発現したものが好ましい。Here, the MT1-MMP-cleaved EphA2-N-terminal fragment of EphA2 (note that unless otherwise specified, EphA2 protein) is, for example, the 28th to 385th in the amino acid sequence shown in SEQ ID NO: 1 , 28th to 395th, 28th to 432nd, or 28th to 435th amino acid sequence. Therefore, as an antigen for producing an antibody that binds to the MT1-MMP-cleaved EphA2-N-terminal fragment, for example, 28th to Xth in the amino acid sequence represented by SEQ ID NO: 1 (where X is 328 or more) A protein consisting of an amino acid sequence represented by an integer of 435 or less, the same applies hereinafter, 1 or several amino acids are added or substituted in the amino acid sequence represented by 28th to Xth in the amino acid sequence represented by A protein consisting of an inserted or deleted amino acid sequence, or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by positions 28 to X in the amino acid sequence represented by SEQ ID NO: 1 Can be used. As an example of the antigen, a protein consisting of an amino acid sequence represented by SEQ ID NO: 2 (amino acid sequence of 28th to 328th amino acids in the amino acid sequence represented by SEQ ID NO: 1) (hereinafter referred to as “Antigen # 1” ), A protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 or a sequence of 80% or more with the amino acid sequence represented by SEQ ID NO: 2 A protein consisting of an amino acid sequence having identity can be mentioned.
The antigen for producing an antibody that binds to the MT1-MMP-cleaved EphA2-N end fragment may be prepared in any way, but is preferably expressed in animal cells.
本明細書において完全長（インタクト（無傷）の）のEphA2とは、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質のことである。「完全長のEphA2」とは、EphA2のフラグメントに対して、細胞内で機能しているEphA2全長のことを指し、細胞内で発現している状態のEphA2、または、細胞内で発現しているEphA2の立体構造等を保持しているタンパク質のことである。「完全長のEphA2」とは、たとえば、動物細胞内で発現したものが好ましくは、たとえば、EphA2を発現させた細胞抽出液物中に存在するものを挙げることができる。 In the present specification, full-length (intact) EphA2 is a protein consisting of the 28th to 976th amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1, and in the amino acid sequence shown in SEQ ID NO: 1 A protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the 28th to 976th amino acid sequences, or an amino acid sequence of 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1 And a protein consisting of an amino acid sequence having 80% or more of sequence identity. "Full-length EphA2" refers to a full-length EphA2 functioning in cells, with respect to a fragment of EphA2, and is expressed in EphA2 in a state of being expressed in cells, or in cells It is a protein that retains the steric structure of EphA2. Examples of the "full-length EphA2" preferably include those expressed in animal cells, preferably those present in a cell extract in which EphA2 is expressed, for example.
また、EphA2細胞外ドメインとは、EphA2の細胞外に突出した領域のことであり、たとえば、配列番号３（配列番号１で表されるアミノ酸配列中、28番目〜537番目のアミノ酸配列）で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質のことである。EphA2の細胞外ドメインとは、どのように調製したものであってもよいが、動物細胞内で発現したものが好ましい。 The EphA2 extracellular domain is a region of EphA2 that protrudes extracellularly, and is, for example, shown in SEQ ID NO: 3 (the 28th to 537th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1). , A protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence represented by SEQ ID NO: 3 And a protein consisting of an amino acid sequence having 80% or more of sequence identity. The extracellular domain of EphA2 may be prepared in any way, but is preferably expressed in animal cells.
本明細書において、「１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配」と記す場合、付加、置換、挿入または欠失したアミノ酸の数は、特に限定はしないが、たとえば、1、2、3、4、5、6、7、8、9または10個程度が好ましい。また、本明細書において、「80％以上の配列同一性を有するアミノ酸配列」は80％以上の配列同一性を有するアミノ酸配列であれば、何％であってもよいが、たとえば、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上または99％以上であってもよい。 In the present specification, when referring to "an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted", the number of amino acids added, substituted, inserted or deleted is not particularly limited. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or so is preferable. In the present specification, “amino acid sequence having 80% or more sequence identity” may be any amino acid sequence having 80% or more sequence identity, for example, 90% or more 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
本発明の第１の実施形態にかかる抗体（「本発明の抗体」と記載する場合もある）は、たとえば、「配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質」、より具体的には、「配列番号２で表されるアミノ酸配列からなるタンパク質、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質」を免疫原として、いくつかの抗体を作製し、作製した抗体の中から、「配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質」および「配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質」のいずれにも結合しない抗体のみを選択して、本発明の抗体とすることができる。
抗体と抗原タンパク質との結合の有無を確認する方法としては、特に限定されず、当業者において公知の方法を採用すればよいが、たとえば、免疫沈降法、ウエスタンブロッティング法により抗原タンパク質との結合の有無を確認する方法を挙げることができる。The antibody according to the first embodiment of the present invention (sometimes referred to as "the antibody of the present invention") is, for example, the 28th to Xth (here, X) in the amino acid sequence represented by SEQ ID NO: 1 Is a protein consisting of an amino acid sequence represented by an integer of 328 to 435, and the same applies hereinafter, and one or several amino acids in the amino acid sequence represented by 28th to Xth in the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence added, substituted, inserted or deleted, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by 28th to Xth in the amino acid sequence represented by SEQ ID NO: 1 More specifically, “a protein consisting of the amino acid sequence represented by SEQ ID NO: 2”, one or several amino acids are added to the amino acid sequence represented by SEQ ID NO: 2 Several antibodies are prepared using as a immunogen a protein consisting of a substituted, inserted or deleted amino acid sequence, or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 Among the prepared antibodies, “a protein consisting of the 28th to 976th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence from the 28th to 976th amino acid sequence represented by SEQ ID NO: 1 A protein consisting of an amino acid sequence in which one or several amino acids have been added, substituted, inserted or deleted in SEQ ID NO: 1, or 80% or more sequence identity with the 28th to 976th amino acid sequence in the amino acid sequence shown in SEQ ID NO: 1 And a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, a table in SEQ ID NO: 3 Consisting of an amino acid sequence in which one or several amino acids have been added, substituted, inserted or deleted in the amino acid sequence, or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 Only antibodies that do not bind to any of the "proteins" can be selected to be antibodies of the invention.
The method for confirming the presence or absence of the binding between the antibody and the antigen protein is not particularly limited, and any method known to those skilled in the art may be employed. For example, the binding with the antigen protein is carried out by immunoprecipitation or western blotting. A method of confirming the presence or absence can be mentioned.
本発明の抗体には、モノクローナル抗体、ポリクローナル抗体またはそれら抗体の断片が含まれる。また、遺伝子工学的に修飾した、いわゆる、キメラ抗体も含まれる。
本発明の抗体断片は、本発明の抗体の一部分の領域を意味し、たとえば、Fab、Fab’、F(ab’)2、Fv（variable fragment of antibody）、一本鎖抗体（重鎖、軽鎖、重鎖可変領域、及び軽鎖可変領域等）、scFv、diabody（scFv二量体）、dsFv（ジスルフィド安定化可変領域）、ならびに、CDRを少なくとも一部に含むペプチド等が挙げられる。
ここで、「モノクローナル」というのは、実質的に均一な抗体の集団より得られた抗体の特性を示唆するものであって、抗体が特定の方法により製造されることを限定するものではない。たとえば、本発明において用いられるモノクローナル抗体を、たとえばハイブリドーマ法（Kohler and Milstein,Nature256:495(1975)）または組換え法（米国特許第4,816,567号）により製造してもよい。また、本発明のモノクローナル抗体は、ファージ抗体ライブラリーから単離してもよい（Clackson et al., Nature 352:624-628(1991);Marks et al.,J.Mol.Biol.222:581-597(1991)）。The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies or fragments of those antibodies. Also included are so-called chimeric antibodies that have been genetically engineered.
The antibody fragment of the present invention means a region of a part of the antibody of the present invention, and for example, Fab, Fab ′, F (ab ′) 2 , Fv (variable fragment of antibody), single chain antibody (heavy chain, light) Chain, heavy chain variable region, light chain variable region and the like), scFv, diabody (scFv dimer), dsFv (disulfide stabilized variable region), and a peptide containing at least a part of CDR and the like.
Here, "monoclonal" indicates the properties of the antibody obtained from a substantially homogeneous population of antibodies, and does not limit that the antibody is produced by a particular method. For example, the monoclonal antibodies used in the present invention may be produced, for example, by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) or a recombinant method (US Patent No. 4,816, 567). The monoclonal antibodies of the invention may also be isolated from phage antibody libraries (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-). 597 (1991)).
本発明の抗体がポリクローナル抗体の場合、たとえば、哺乳類宿主動物に対して、免疫原およびアジュバントの混合物をインジェクトすることにより調製することができる。通常は、免疫原としての抗原および／またはアジュバントを宿主動物の皮下または腹腔内へ複数回インジェクトする。アジュバントの例には、完全フロイトおよびモノホスホリル脂質Ａ合成−トレハロースジコリノミコレート（MPL-TDM）が含まれる。 When the antibody of the present invention is a polyclonal antibody, it can be prepared, for example, by injecting a mixture of an immunogen and an adjuvant into a mammalian host animal. Usually, the antigen as an immunogen and / or the adjuvant are injected into the subcutaneous or intraperitoneal multiple times of the host animal. Examples of adjuvants include complete Freud and monophosphoryl lipid A synthetic-trehalose dicorynomycolate (MPL-TDM).
また、本発明の抗体がモノクローナル抗体の場合、たとえば、ハイブリドーマ法を用いて調製することができる。
この方法には以下に示す４つの工程が含まれる：（1）宿主動物に免疫原を免疫する、（2）モノクローナル抗体分泌性（または潜在的に分泌性）のリンパ球を回収する、（3）リンパ球を不死化細胞に融合させる、（4）所望のモノクローナル抗体を分泌する細胞を選択する。
マウス、ラット、モルモット、ハムスターまたは他の適当な宿主動物が、免疫動物として選択され免疫原がインジェクトされる。
免疫後、宿主動物から得られたリンパ球はハイブリドーマ細胞を樹立するために、ポリエチレングリコールなどの融合剤を用いて不死化細胞株と融合する。融合細胞としては、たとえば、ラットまたはマウスのミエローマ細胞株が使用される。細胞融合を行った後、融合しなかったリンパ球および不死化細胞株の成長または生存を阻害する１または複数の基質を含む適切な培地中で細胞を生育させる。通常の技術では、酵素のヒポキサンチン−グアニンホスホリボシルトランスフェラーゼ（HGPRTまたはHPRT）を欠く親細胞を使用する。この場合、ヒポキサンチン、アミノプテリン及びチミジンがHGPRT欠損細胞の成長を阻害し、ハイブリドーマの成長を許容する培地（HAT培地）に添加される。このようにして得られたハイブリドーマから、所望の抗体を産生するハイブリドーマを選択し、該ハイブリドーマが生育する培地から、常法に従い、目的のモノクローナル抗体を取得することができる。
このようにして調製したハイブリドーマをインビトロ培養し、または、マウス、ラット、モルモット、ハムスターなどの腹水中でインビボ培養し、目的の抗体を培養上清、また、腹水から調製することができる。In addition, when the antibody of the present invention is a monoclonal antibody, it can be prepared, for example, using a hybridoma method.
The method comprises the following four steps: (1) immunizing the host animal with an immunogen, (2) recovering monoclonal antibody secreting (or potentially secreting) lymphocytes, (3 B) fusing lymphocytes to immortalized cells, (4) selecting cells secreting the desired monoclonal antibody.
Mice, rats, guinea pigs, hamsters or other suitable host animals are selected as immunized animals and the immunogen injected.
After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hybridoma cells. As a fusion cell, for example, a rat or mouse myeloma cell line is used. After performing cell fusion, cells are grown in an appropriate medium containing one or more substrates that inhibit the growth or survival of unfused lymphocytes and immortalized cell lines. The usual technique uses parent cells that lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine inhibit the growth of HGPRT deficient cells, and are added to a medium (HAT medium) that allows the growth of hybridomas. From the hybridomas thus obtained, hybridomas that produce a desired antibody can be selected, and from the medium in which the hybridomas grow, the desired monoclonal antibody can be obtained according to a conventional method.
The thus prepared hybridomas can be cultured in vitro or in vivo in ascites fluid of mouse, rat, guinea pig, hamster or the like, and an antibody of interest can be prepared from culture supernatant or ascites fluid.
本発明の第２の実施形態は、被験者由来の試料中に存在するMT1-MMP切断EphA2-N末側フラグメントの量を、本発明の第１の実施形態にかかる抗体を用いて測定する工程を含む、がんの検査方法である。
本発明の第２の実施形態（「本発明のがんの検査方法」とも記載する）において、「MT1-MMP切断EphA2-N末側フラグメント」の量の抗体による測定は、被験者由来の試料中に存在する当該フラグメントを検出し得る、当業者において公知のあらゆる方法により実施することができ、たとえば、免疫学的手法を利用した方法により行うことができる。免疫学的手法としては、本発明の抗体、場合によっては、検出可能に標識した本発明の抗体を用いて、たとえば、免疫クロマトグラフィー法、ウエスタンブロッティング法、表面プラズモン法、ELISA法（たとえば、直接競合ELISA、間接競合ELISA、サンドイッチELISAなど）、放射免疫測定法（RIA）、蛍光免疫測定法（FIA）、水晶振動子マイクロバランス法（Quartz crystal microbalance; QCM）および磁性ビーズを使用した方法などを挙げることができる。In the second embodiment of the present invention, the step of measuring the amount of MT1-MMP-cleaved EphA2-N end fragment present in a sample derived from a subject using the antibody according to the first embodiment of the present invention It is an examination method of cancer including.
In the second embodiment of the present invention (also described in the “test method of cancer of the present invention”), measurement of the amount of “MT1-MMP-cleaved EphA2-N end fragment” by the antibody is carried out in a sample from a subject The fragment can be carried out by any method known to those skilled in the art that can detect the fragment present in, for example, a method utilizing immunological techniques. Immunological methods include, for example, immunochromatography, western blotting, surface plasmon method, ELISA (for example, direct analysis) using the antibody of the present invention, and optionally, the detectably labeled antibody of the present invention Competitive ELISA, indirect competitive ELISA, sandwich ELISA etc), radioimmunoassay (RIA), fluorescence immunoassay (FIA), quartz crystal microbalance (QCM) and methods using magnetic beads etc. It can be mentioned.
免疫学的手法の中でも、特に、サンドイッチELISA法が簡便かつ速やかに測定することができるため好ましい。サンドイッチELISA法は、たとえば、本発明の抗体（捕捉抗体として使用）を固相担体（たとえば、ガラス製、金属製または樹脂製のマイクロタイタープレート、基板、樹脂または磁気ビーズ、ナイロン、PVDF製のメンブレンなど）に吸着させ、抗体が吸着していない担体部分をブロッキングした後、抗原を含む試料を担体に添加して反応させ、その後担体を洗浄し、さらに、抗原を認識する別の抗体（検出抗体；別のエピトープに結合する抗体）を担体に添加して反応させる。反応後、抗体に結合させた標識からのシグナルなどを検出し、試料中の抗原の存在量を測定する。
捕捉抗体と結合した抗原の検出は、非標識の１次抗体（検出抗体）を使用して、酵素標識した２次抗体由来のシグナルを、たとえば、吸光度として検出し、標準曲線に基づいて、試料中の抗原量を定量することもできる。標識としては、HRPなどを例示することができ、この場合、HRPの基質として、3,3’-ジアミノベンジジン（DAB）、3,3’,5,5’-テトラメチルベンジジン（TMB）、o-フェニレンジアミン（OPD）などを使用することができる。また、アルカリホスファターゼで標識する場合には、基質としてp-ニトロフェニルホスフェート（NPP）などを使用することができる。
なお、抗体の標識は、ビオチン−アビジン結合を介して行ってもよい（すなわち、ビオチン化した抗体にアビジン-HRPまたはアビジン-アルカリホスファターゼを添加する）。Among immunological techniques, sandwich ELISA is particularly preferable because it can be measured conveniently and rapidly. In the sandwich ELISA method, for example, the antibody of the present invention (used as a capture antibody) is used as a solid phase carrier (for example, a glass, metal or resin microtiter plate, a substrate, resin or magnetic beads, nylon, PVDF membrane) Etc.) and blocking the non-adsorbed part of the carrier, a sample containing the antigen is added to the carrier and reacted, and then the carrier is washed, and another antibody that recognizes the antigen (detection antibody An antibody that binds to another epitope) is added to the carrier and reacted. After the reaction, the signal from the label bound to the antibody is detected to measure the amount of antigen present in the sample.
The detection of the antigen bound to the capture antibody is carried out using a non-labeled primary antibody (detection antibody) to detect a signal derived from the enzyme-labeled secondary antibody as, for example, absorbance, based on a standard curve. The amount of antigen in it can also be quantified. As the label, HRP and the like can be exemplified, and in this case, 3,3'-diaminobenzidine (DAB), 3,3 ', 5,5'-tetramethylbenzidine (TMB), o as a substrate of HRP. Phenylenediamine (OPD) etc. can be used. In addition, when labeling with alkaline phosphatase, p-nitrophenyl phosphate (NPP) or the like can be used as a substrate.
Furthermore, labeling of the antibody may be performed via biotin-avidin binding (ie, avidin-HRP or avidin-alkaline phosphatase is added to the biotinylated antibody).
本発明の抗体が、がんの検査に使用できる抗体であるかどうかは、既知のがん患者由来の試料と既知の健康者（検査対象のがんに罹患していないことが明らかな者）由来の試料を用いて、これらの試料に対する抗体の反応性に基づいたROC解析（Receiver operating Characteristic analysis）を実施することで、判断することができる。 Whether or not the antibody of the present invention is an antibody that can be used for a cancer test, a sample from a known cancer patient and a known healthy person (a person who is clearly not suffering from the cancer to be examined) The determination can be made by performing ROC analysis (Receiver operating Characteristic analysis) based on the reactivity of the antibody to these samples using the derived samples.
本発明のがんの検査方法は、被験者由来の試料中に存在する、MT1-MMP切断EphA2-N末側フラグメントの量と健康者の試料中に存在する当該N末端側フラグメントの量を比較し、被験者由来の試料中に存在するフラグメントの量が有意に多い場合に、当該被験者ががんに罹患している可能性があると判断することができる。
また、発明者らは、MT1-MMPの発現が亢進するとEphA2の切断頻度が高まり、がん細胞の増殖、生存および運動が促進され、がん細胞の悪性形質への進行が進むことを見いだしている（特許文献１など）。したがって、患者由来の試料について、経時的に本発明のがんの検査方法を適用することにより、がんの進行度またはがん治療の治療効果の判断資料を得ることも可能である。The test method for cancer of the present invention compares the amount of MT1-MMP-cleaved EphA2-N-terminal fragment present in a sample derived from a subject with the amount of the N-terminal fragment present in a sample of a healthy subject. If the amount of fragments present in the sample derived from the subject is significantly high, it can be judged that the subject is possibly suffering from cancer.
In addition, the inventors have found that when the expression of MT1-MMP is enhanced, the frequency of cleavage of EphA2 is increased, the growth, survival and exercise of cancer cells are promoted, and the progression of cancer cells to malignant characteristics is advanced. (E.g., Patent Document 1). Therefore, it is also possible to obtain data for judging the degree of progression of cancer or the therapeutic effect of cancer treatment by applying the inspection method of the present invention to the sample derived from the patient sequentially.
本発明のがんの検査方法に使用される資料は、たとえば、血液、リンパ液、尿、唾液、痰などの体液、糞便、細胞や組織の抽出物などを挙げることができる。
また、本発明のがんの検査方法の対象となるがんは、EphA2が発現している細胞ががん化したものであれば、いかなるものであってもよく、たとえば、すい臓がん、胃がん、食道がん、大腸がん、肝臓がん、肺がん、乳がん、卵巣がん、甲状腺がん、子宮がん、前立腺がん、膀胱がん、腎がん、黒色腫またはグリオーマなどを挙げることができる。Examples of the material used in the method of testing a cancer of the present invention include blood, lymph fluid, urine, saliva, body fluid such as saliva, sputum, feces, extracts of cells and tissues, and the like.
Further, any cancer may be used as long as the cells expressing EphA2 become cancerous, for example, pancreatic cancer, gastric cancer, etc. Esophagus cancer, colon cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, thyroid cancer, uterine cancer, prostate cancer, bladder cancer, renal cancer, melanoma or glioma etc. it can.
本発明の第３の実施形態は、がん検査用キットである（「本発明のがん検査用キット」とも記載する）。本発明の第３の実施形態には、本発明の第１の実施形態にかかる抗体が含まれる。また、本発明の抗体の他、本発明のがんの検査方法に使用する試薬、器具などが含まれていてもよい。たとえば、本発明のがんの検査方法が、サンドイッチELISA法によって実施される場合、本発明のがん検査用キットには、本発明の抗体の他、たとえば、MT1-MMP切断EphA2-N末側フラグメントと結合する抗体（検出抗体；固相担体に吸着させる抗体とは異なるエピトープを認識する抗体であって、市販の抗体を含む）、固相担体（たとえば、ガラス製、金属製または樹脂製のマイクロタイタープレート、基板、樹脂または磁気性ビーズ、ナイロン、PVDF製のメンブレンなど）、固相担体のブロッキング用試薬、標識済み2次抗体または抗体標識のために使用する標識（たとえば、HRP、アルカリホスファターゼ）や試薬類、標識を発色させるための基質（たとえば、3,3’-ジアミノベンジジン（DAB）、3,3’,5,5’-テトラメチルベンジジン（TMB）、o-フェニレンジアミン（OPD）、p-ニトロフェニルホスフェート（NPP）など）が含まれていてもよい。 The third embodiment of the present invention is a kit for cancer testing (also described as "a kit for cancer testing of the present invention"). The third embodiment of the present invention includes the antibody according to the first embodiment of the present invention. In addition to the antibody of the present invention, reagents, devices, and the like used in the method of testing a cancer of the present invention may be included. For example, when the cancer inspection method of the present invention is carried out by a sandwich ELISA method, the cancer test kit of the present invention contains, in addition to the antibody of the present invention, for example, the MT1-MMP cleaved EphA2-N end An antibody that binds to a fragment (detection antibody; an antibody that recognizes an epitope different from the antibody adsorbed to the solid phase carrier and includes a commercially available antibody), a solid phase carrier (for example, glass, metal or resin) Microtiter plate, substrate, resin or magnetic beads, nylon, PVDF membrane, etc., blocking reagent for solid phase carrier, labeled secondary antibody or label used for antibody labeling (eg HRP, alkaline phosphatase) And reagents, substrates for color development of labels (eg, 3,3'-diaminobenzidine (DAB), 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-phenylene) Diamine (OPD), p-nitrophenyl phosphate (NPP), etc.) may be included.
本発明の第４の実施形態は、試料中に存在するMT1-MMP切断EphA2-N末側フラグメントの量を本発明の第１の実施形態にかかる抗体を使用して測定する方法である。上述のとおり、試料中に存在する当該EphA2タンパク質のN末端側フラグメントの量は、本発明の抗体を使用して、免疫学的方法（たとえば、ELISA法など）により測定することができる。
MT1-MMPの活性はがんの発症およびがんの進行と密接な関連性を有しており、MT1-MMPは抗がん剤の標的分子としても重要であると考えられている。これまで、インビボにおけるMT1-MMP（プロテアーゼ）活性を測定する手段はなかったが、本発明の抗体を使用して、被験者由来の試料（たとえば、体液など）中に存在するMT1-MMP切断EphA2-N末側フラグメントの量を測定し、その量に基づいて被験者の生体内におけるMT1-MMP活性を評価することが可能である。
また、抗がん剤の候補分子をスクリーニングする目的で、抗がん剤候補分子存在下におけるMT1-MMP活性を上述の方法でモニターすることにより、抗がん剤候補分子がMT1-MMP活性に与える影響を評価することができる。より具体的には、MT1-MMPおよびEphA2を共発現する細胞をMT1-MMPを標的とする候補分子の存在下で培養し、その培養液を試料としてMT1-MMPの活性を評価すれば、その候補分子のMT1-MMPに対する影響を調べることができる。したがって、本発明の第４の実施形態にかかる方法は、抗がん剤候補分子のスクリーニングにも使用することができる。The fourth embodiment of the present invention is a method of measuring the amount of MT1-MMP cleaved EphA2-N end fragment present in a sample using the antibody according to the first embodiment of the present invention. As described above, the amount of the N-terminal fragment of the EphA2 protein present in a sample can be measured by an immunological method (eg, ELISA method, etc.) using the antibody of the present invention.
The activity of MT1-MMP is closely related to the onset of cancer and the progression of cancer, and MT1-MMP is considered to be important as a target molecule for anticancer agents. So far, there has been no means to measure MT1-MMP (protease) activity in vivo, but using the antibody of the present invention, MT1-MMP cleaved EphA2-present in a sample from a subject (eg, body fluid etc.) It is possible to measure the amount of the N-terminal fragment and to evaluate MT1-MMP activity in the living body of the subject based on the amount.
In addition, for the purpose of screening candidate molecules for anti-cancer agents, monitoring for MT1-MMP activity in the presence of anti-cancer agent candidate molecules by the above-mentioned method allows the anti-cancer agent candidate molecules to become MT1-MMP activities. Impacts can be assessed. More specifically, cells co-expressing MT1-MMP and EphA2 are cultured in the presence of a candidate molecule targeting MT1-MMP, and the culture solution is used as a sample to evaluate the activity of MT1-MMP. The effects of candidate molecules on MT1-MMP can be examined. Therefore, the method according to the fourth embodiment of the present invention can also be used to screen for anticancer drug candidate molecules.
本発明の第５の実施形態は、MT1-MMP切断EphA2-N末側フラグメントと特異的に結合する抗体の製造方法である。
上述の通り、発明者らは、MT1-MMP切断EphA2-N末側フラグメントと、完全長のEphA2および膜プロテアーゼによってプロセッシングを受けた場合に生じる可能性のあるEphA2の細胞外ドメインの立体構造が、各々、相違すると予想した。そして、MT1-MMP切断EphA2-N末側フラグメントを免疫原として作製したいくつかの抗体の中から、下記の（ｉｉ）の工程を含む抗体作製方法を行うことにより、完全長（インタクト（無傷））のEphA2およびEphA2の細胞外ドメインのいずれにも反応しない抗体を調製することに初めて成功した。
すなわち、本発明の第５の実施形態は、以下の工程（ｉ）および（ｉｉ）を含む、抗体の作製方法である。
（ｉ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質のいずれかに結合する抗体を作製する工程、
前記タンパク質について具体的な一例として、配列番号２で表されるアミノ酸配列からなるタンパク質、配列番号２で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号２で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質のいずれかに結合する抗体を作製する工程、
（ｉｉ）工程（ｉ）で作製した抗体のうち、下記の（ｂ）および（ｃ）のタンパク質とは結合しない抗体を選択する工程
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質
第５の実施形態の抗体（ｉｉ）に記載される（ｂ）および（ｃ）のタンパク質は、第１の実施形態の（ｂ）および（ｃ）のタンパク質と、各々、同一である。
上記第５の実施形態により得られた抗体は、MT1-MMP切断EphA2-N末側フラグメントのみと特異的に結合し、がんの診断において非常に有用であることが明らかとなった（実施例を参照のこと）。
本発明の第５の実施形態に含まれる工程（ｉ）および（ｉｉ）は、当業者において過度の試行錯誤が必要となる工程ではなく、当該技術分野において通常の実施の範囲内において、容易に行うことが可能である。The fifth embodiment of the present invention is a method for producing an antibody that specifically binds to an MT1-MMP cleaved EphA2-N terminal fragment.
As mentioned above, we found that the conformation of the MT1-MMP-cleaved EphA2-N end fragment and the extracellular domain of EphA2 that may occur when processed by full-length EphA2 and membrane protease, Each was expected to be different. Then, among several antibodies prepared using the MT1-MMP-cleaved EphA2-N end fragment as an immunogen, full-length (intact (intact) by performing an antibody preparation method including the following step (ii) For the first time, it has succeeded in preparing an antibody that does not react with either EphA2 or the extracellular domain of EphA2.
That is, the fifth embodiment of the present invention is a method for producing an antibody, which comprises the following steps (i) and (ii).
(I) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer of 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 Producing an antibody that binds to any of a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by 28th to Xth,
As a specific example of the above-mentioned protein, a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2 Producing an antibody which binds to any of a protein consisting of SEQ ID NO: 2 or a protein consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2,
(Ii) a step of selecting an antibody which does not bind to the proteins of the following (b) and (c) among the antibodies produced in the step (i) (b) 28th to 28th amino acids in the amino acid sequence represented by SEQ ID NO: 1 A protein consisting of the amino acid sequence 976, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the 28th to 976th amino acids in the amino acid sequence represented by SEQ ID NO: 1; Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3 The proteins of (b) and (c) described in the antibody (ii) of the fifth embodiment Each of the proteins of (b) and (c) of the embodiment of is identical.
The antibody obtained according to the fifth embodiment specifically binds only to the MT1-MMP-cleaved EphA2-N-terminal fragment and has proved to be very useful in the diagnosis of cancer (Example) checking).
Steps (i) and (ii) included in the fifth embodiment of the present invention are not steps requiring excessive trial and error in the art, and are easily performed within the scope of ordinary practice in the art. It is possible to do.
本明細書において引用されたすべての文献の開示内容は、全体として明細書に参照により組み込まれる。また、本明細書全体において、単数形の「a」、「an」、および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものを含むものとする。
以下に実施例を示してさらに本発明の説明を行うが、実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。The disclosure content of all the documents cited in the present specification is incorporated by reference in the specification in its entirety. Also, where the singular form “a,” “an,” and “the” are included throughout the specification, unless the context clearly indicates otherwise, not only the singular but also the plural will be used. Shall be included.
EXAMPLES The present invention will be further described with reference to the following examples, but the examples are merely examples of the embodiments of the present invention, and do not limit the scope of the present invention.
１．方法
EphA2抗体
抗-EphA2モノクローナル抗体（mAb）96-1およびポリクローナル抗体（pAb）C309は、第一三共株式会社から供与された（Tokyo, Japan）。mAb 96-1およびpAb C309のエピトープは、各々、リガンド結合ドメインおよびシステインリッチドメイン（CRD; cysteine-rich domain）に存在することが確認されている。
EphA2に対する他の抗体、pAbs 371805およびpAbs C20は、各々、R&D Systems（Minneapolis, MN, USA）およびSanta Cruz Biotechnology（Dallas, TX, USA）から購入した。pAbs 371805およびpAbs C20抗体のエピトープは、各々、EphA2リガンド結合ドメインおよび細胞質ドメインに存在する。
pAb 309以外のすべての抗体は、免疫沈降に使用することができる。1. Method
EphA2 antibody anti- EphA2 monoclonal antibody (mAb) 96-1 and polyclonal antibody (pAb) C309 were provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). The epitopes of mAb 96-1 and pAb C309 have been identified to be present in the ligand binding domain and in the cysteine-rich domain (CRD; cysteine-rich domain), respectively.
Other antibodies to EphA2, pAbs 371805 and pAbs C20, were purchased from R & D Systems (Minneapolis, MN, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The epitopes of pAbs 371805 and pAbs C20 antibodies are present in the EphA2 ligand binding domain and the cytoplasmic domain, respectively.
All antibodies except pAb 309 can be used for immunoprecipitation.
組換え抗原
ヒトEphA2 cDNAはOpen Biosystems（Huntsville, AL, USA）から購入した。コード領域の1-981番目までのヌクレオチドおよび1-1578番目までのヌクレオチド（各々、Antigen #1およびAntigen #5）に相当するフラグメントの3’末端にFLAGタグのコード領域を連結させ、PCRで増幅し、Gateway System（Invitrogen, Carlsbad, CA, USA）を使用して、pcDNA3.2 DEST mammalian-expression vectorにサブクローニングした（図１Ａ）。Ephrin結合ドメイン＋CRD（Antigen #1）またはEphrin結合ドメイン＋CRD＋stem領域（Antigen #5；EphA2細胞外ドメイン）の発現ベクターは、血清フリーの条件下でHEK293細胞に一過的にトランスフェクトした。発現した組換え抗原は、血清フリー条件培地中に分泌されたものを回収し、抗FLAGアガロースアフィニティークロマトグラフィーを用いて精製した。組換え抗原の純度は、SDS-PAGE後CBB染色を行い確認した。
抗体のエピトープマッピングを行うために、EphA2の28番目〜125番目 (Antigen #2)、101番目〜250番目 (Antigen #3)および226番目〜328番目 (Antigen #4)のアミノ酸配列に対応する塩基配列のC末端にFLG配列を付してPCRで増幅し、Gateway System を用いてpET160ベクターにサブクローニングした（図１Ａ）。組換え抗原は、大腸菌中で発現させ、抗FLAGモノクローナル抗体結合アガロースクロマトグラフィーで精製した。 Recombinant antigen human EphA2 cDNA was purchased from Open Biosystems (Huntsville, AL, USA). The coding region of the FLAG tag is ligated to the 3 'end of the fragment corresponding to the nucleotides 1 to 981 and the nucleotides 1 to 1578 of the coding region (Antigen # 1 and Antigen # 5 respectively) and amplified by PCR And subcloned into pcDNA 3.2 DEST mammalian-expression vector using the Gateway System (Invitrogen, Carlsbad, CA, USA) (FIG. 1A). Expression vectors for Ephrin binding domain + CRD (Antigen # 1) or Ephrin binding domain + CRD + stem region (Antigen # 5; EphA2 extracellular domain) were transiently transfected into HEK 293 cells under serum free conditions. The recombinant antigens expressed were recovered in serum free conditioned medium and recovered and purified using anti-FLAG agarose affinity chromatography. The purity of the recombinant antigen was confirmed by CBB staining after SDS-PAGE.
Bases corresponding to the amino acid sequences of 28th to 125th (Antigen # 2), 101st to 250th (Antigen # 3) and 226th to 328th (Antigen # 4) of EphA2 for performing epitope mapping of antibodies The C-terminal of the sequence was added with FLG sequence, amplified by PCR, and subcloned into pET160 vector using Gateway System (FIG. 1A). The recombinant antigens were expressed in E. coli and purified by anti-FLAG monoclonal antibody coupled agarose chromatography.
免疫、細胞融合およびハイブリドーマのスクリーニング
リガンド結合ドメインとAntigen #1を等量の完全フロイントアジュバント（Difco; Diagnostic Systems, Sparks, MD, USA）でエマルジョン化し、50μgをBALB/cマウスの腹腔内にインジェクトした。その後、不完全フロイントアジュバントでエマルジョン化した等量のAntigen #1で、２週間毎に２回ブーストした。３回目の免疫から６週間後、アジュバント無しで、同量のラミニン‐γ2タンパク質をマウスの血管内に注射した。血管内注射から3日後、採血した血液を、室温にて3,000rpmで遠心し血清を得た。得られたリガンド結合ドメインに対する特異性を示す血清は、ハイブリドーマのスクリーニングの間、ポジティブコントロールとして使用した。
マウスミエローマ細胞株（P3U1）と脾臓細胞との細胞融合は、IBL Co. Ltd.（Gunma, Japan）から報告されている方法に従って行った。調製したハイブリドーマ細胞はHAT選択培地中、10日間培養し、その後、96ウェルプレートにまき直した。増殖している細胞を含む各ウェルの上清は、Antigen #1を抗原とするELISA法により、生産された抗体の確認に使用した。細胞の限界希釈およびクローニングを２回繰り返し、Antigen #1に対する抗体を生産する３つのハイブリドーマ（mAb 46A1、62A1および76A1）を得た。得られたモノクローナル抗体のエピトープ解析行うために、組換え抗原（Antigens #2、#3および#4）を用い、非還元条件および還元条件でウエスタンブロッティングを行った。 Immunization, cell fusion and screening of hybridomas The ligand binding domain of the hybridoma and Antigen # 1 are emulsified with an equal volume of complete Freund's adjuvant (Difco; Diagnostic Systems, Sparks, MD, USA), and 50 μg is injected into the peritoneal cavity of BALB / c mice. did. It was then boosted twice every two weeks with an equal volume of Antigen # 1 emulsified with incomplete Freund's adjuvant. Six weeks after the third immunization, the same amount of laminin-γ2 protein was injected into the blood vessels of mice without adjuvant. Three days after intravascular injection, the collected blood was centrifuged at 3,000 rpm at room temperature to obtain serum. Sera showing specificity for the resulting ligand binding domain were used as positive controls during hybridoma screening.
Cell fusion between mouse myeloma cell line (P3U1) and spleen cells was performed according to the method reported from IBL Co. Ltd. (Gunma, Japan). The prepared hybridoma cells were cultured in HAT selection medium for 10 days, and then replated on a 96-well plate. The supernatant of each well containing proliferating cells was used to confirm the antibody produced by the ELISA method using Antigen # 1 as an antigen. Limited dilution of cells and cloning were repeated twice to obtain three hybridomas (mAb 46A1, 62A1 and 76A1) producing antibodies against Antigen # 1. In order to perform epitope analysis of the obtained monoclonal antibodies, western blotting was performed under non-reducing conditions and reducing conditions using recombinant antigens (Antigens # 2, # 3 and # 4).
免疫沈降
放射免疫沈降アッセイ（RIPA ; radioimmunoprecipitation assay）バッファー（最終体積；200μL）中、A431細胞抽出物に含まれるAntigen #1:（1μg）、Antigen #5:（1μg）または完全長のEphA2タンパク質を、モノクローナル抗体またはポリクローナル抗体と共に、4℃で一晩インキュベートした。次に、懸濁したProtein-G磁気ビーズを40μLのインキュベーション混合物に添加し、撹拌しながら4℃で1時間インキュベートした。Protein-G磁気ビーズをRIPAバッファーで3回洗浄し、抗原／抗体複合体を含むビーズを磁気ラックで除去した。沈降させた抗原／抗体複合体は、5 % β2-メルカプトエタノールを含む2 x SDSサンプルバッファーを添加し、磁気ビーズから遊離させた。 Immunoprecipitation Radioimmunoprecipitation assay (RIPA; radioimmunoprecipitation assay) buffer (final volume; 200 μL), Antigen # 1: (1 μg), Antigen # 5: (1 μg) or full-length EphA2 protein contained in A431 cell extract Incubate overnight at 4 ° C. with monoclonal or polyclonal antibodies. Next, suspended Protein-G magnetic beads were added to 40 μL of the incubation mixture and incubated for 1 hour at 4 ° C. with agitation. The Protein-G magnetic beads were washed three times with RIPA buffer and the beads containing the antigen / antibody complex were removed with a magnetic rack. The precipitated antigen / antibody complex was released from the magnetic beads by addition of 2 × SDS sample buffer containing 5% β2-mercaptoethanol.
サンドイッチELISA
96ウェルプレートをmAb 76A1またはmAb 46A1（PBS中、20μg/mL）、ネガティブコントロールとしての免疫前血清で、4℃で一晩処理した。次に、室温にて、ウェルをタンパク質フリーのlocking buffer（Pierce Biotechnology, Rockford, IL, USA）で1時間ブロッキングし、その後40分間乾燥させた。各ウェルは、PBS/0.05% Tween-20 トリスで洗浄後、MT1-MMP切断フラグメントの量を増加させながら（Antigen #1: 0-1,000 pg/mL）各ウェルに添加し、次いで、ビオチン化mAb 62A1と37℃で1時間反応させた。その後ウェルを洗浄した後、結合した抗原をアビジン-D-HRP (0.1μg/mL)で検出した。1 N H2SO4で反応を停止させた後、3,3',5,5'-テトラメチルベンジジンを添加し、ウェルの吸光度／リファレンス（590 nm/620 nm）をBio-Rad spectrophotometer (Bio-Rad, Hercules, CA, USA)を使用して測定した。 Sandwich ELISA
96 well plates were treated overnight at 4 ° C. with mAb 76A1 or mAb 46A1 (20 μg / mL in PBS), preimmune serum as a negative control. The wells were then blocked with protein free locking buffer (Pierce Biotechnology, Rockford, Ill., USA) for 1 hour at room temperature and then allowed to dry for 40 minutes. Each well is washed with PBS / 0.05% Tween-20 Tris, added to each well with increasing amount of MT1-MMP cleavage fragment (Antigen # 1: 0-1,000 pg / mL), then biotinylated mAb The reaction was allowed to proceed with 62A1 at 37 ° C for 1 hour. The wells were then washed and bound antigen was detected with avidin-D-HRP (0.1 μg / mL). After quenching the reaction with 1 NH 2 SO 4 , add 3,3 ', 5,5'-Tetramethylbenzidine, and absorb the absorbance / reference (590 nm / 620 nm) of the wells to a Bio-Rad spectrophotometer (Bio-Rad It measured using Rad, Hercules, CA, USA).
臨床試料
すい臓がん患者由来の血清（10ケース）をProMedDx（Norton, MA, USA）から入手した。胃（17ケース）、食道（8ケース）、胃食道（1ケース）および頭頸部（9ケース）の各がん患者由来の血清、および健康者由来の血清（50ケース）は、Kokusai Bio, Inc.（Tokyo, Japan）から入手した。また、コントロールとして、すい臓がん患者および健康者由来の血清試料に対して、消化器系がんマーカーであるCA19-9の解析を行った。 Clinical samples Sera from pancreatic cancer patients (10 cases) were obtained from ProMedDx (Norton, Mass., USA). Sera from stomach (17 cases), esophagus (8 cases), gastroesophagus (1 case) and head and neck (9 cases) cancer patients and sera from healthy people (50 cases) are available from Kokusai Bio, Inc. Obtained from (Tokyo, Japan). In addition, as a control, analysis of digestive tract cancer marker CA19-9 was performed on serum samples from pancreatic cancer patients and healthy people.
２．結果
組換え抗原の精製
SP（シグナルペプチド、アミノ酸1-27；配列番号１）に続いて存在するEphA2細胞外ドメインは、N末側から順に、リガンド（Ephrin）結合ドメイン（アミノ酸28-201）、CRD（システインリッチドメイン、アミノ酸202-328）およびステム領域（アミノ酸329-537）から構成されている（図１Ａ）。発明者らおよび他の研究者によりマッピングされたMT1-MMP切断サイトを矢印で示した。
抗体の調製に使用する抗原として、アミノ酸28-328（Antigen #1）およびアミノ酸28-537（Antigen #5）の各領域のC末端にFLAGタグを結合したものを、HKE293細胞中で発現させた（図１Ａ）。各抗原は、抗FLAG mAbを使用して精製し、抗FLAG mAbでウエスタンブロッティングを行った（図１Ｂ）。さらに、C末端にFLAGタグを、N末端にGSTタグを付した3種類の抗原（Antigen #2、#3および#4）を大腸菌中で発現させて調製した。各抗原は、抗FLAG mAbを用いて精製し、その純度は抗FLAG mAbを使用してウエスタンブロッティングを行い確認した（図１Ｂ）。2. result
Purification of recombinant antigen
The EphA2 extracellular domain present following SP (signal peptide, amino acids 1 to 27; SEQ ID NO: 1) is a ligand (Ephrin) binding domain (amino acids 28 to 201), CRD (cysteine rich domain, It is composed of amino acids 202-328) and stem region (amino acids 329-537) (FIG. 1A). Arrows indicate MT1-MMP cleavage sites mapped by the inventors and other researchers.
As an antigen used for preparation of antibodies, one obtained by binding a FLAG tag to the C-terminus of each region of amino acids 28-328 (Antigen # 1) and amino acids 28-537 (Antigen # 5) was expressed in HKE293 cells (Figure 1A). Each antigen was purified using anti-FLAG mAb and western blotted with anti-FLAG mAb (FIG. 1B). Furthermore, three types of antigens (Antigen # 2, # 3 and # 4) with a FLAG tag at the C-terminus and a GST tag at the N-terminus were expressed in E. coli. Each antigen was purified using anti-FLAG mAb, and its purity was confirmed by western blotting using anti-FLAG mAb (FIG. 1B).
EphA2の切断フラグメントに対するモノクローナル抗体
マウスをAntigen #1（5μg/マウス）で免疫し、400個のハイブリドーマ細胞を得た。Antigen #1（0.5μg/プレート）でコートしたプレートを用いて、各ハイブリドーマから分泌される抗体をELISA法でスクリーニングした。その結果、IgG1クラスを産生する3つのハイブリドーマクローン（46A1、62A1および76A1）を取得し、さらに解析を行った。抗体の反応性を確認するために、Antigen #1を取得した抗体で免疫沈降させ、沈降物を還元条件でPAGEを行い、その後、抗FLAG mAbを用いてウエスタンブロッティングを行った（図２Ａ）。異なるハイブリドーマクローン由来の3種類のモノクローナル抗体は、ポジティブコントロール抗体（371805および96-1）を用いた結果と同様にAntigen #1を沈降させたが、ネガティブコントロール抗体（C309、マウスIgG、抗FLAG mAb）はAntigen #1を沈降させなかった（図２Ａ）。 Monoclonal antibody mice against the cleaved fragment of EphA2 were immunized with Antigen # 1 (5 μg / mouse) to obtain 400 hybridoma cells. The antibody secreted from each hybridoma was screened by ELISA using a plate coated with Antigen # 1 (0.5 μg / plate). As a result, three hybridoma clones (46A1, 62A1 and 76A1) producing IgG1 class were obtained and further analyzed. In order to confirm antibody reactivity, immunoprecipitation was performed with the antibody for which Antigen # 1 was obtained, the precipitate was subjected to PAGE under reducing conditions, and then western blotting was performed using an anti-FLAG mAb (FIG. 2A). The three monoclonal antibodies from different hybridoma clones precipitated Antigen # 1 as well as the results with positive control antibodies (371805 and 96-1) but negative control antibodies (C309, mouse IgG, anti-FLAG mAb) ) Did not precipitate Antigen # 1 (FIG. 2A).
エピトープマッピングを行うために、4つの組換えEphA2フラグメント（Antigens #1、#2、#3および#4）を還元および非還元条件下でPAGEを行い、ウエスタンブロッティングにより解析した（図３）。すべてのモノクローナル抗体（46A1、62A1および76A1）は非還元条件下でAntigen #1を検出した（図２Ａ）が、mAb 46A1は還元条件下ではAntigen #1を検出することができなかった。62A1はAntigen #3に反応したが、46A1と76A1は、Antigen #2、#3および#4と反応しなかった。したがって、46A1と76A1に対するAntigen #1のエピトープは、Antigens #2、#3または#4上にオーバーラップして存在しないと考えられる。特に、46A1はエピトープの構造を認識している可能性がある。 To perform epitope mapping, four recombinant EphA2 fragments (Antigens # 1, # 2, # 3 and # 4) were subjected to PAGE under reducing and nonreducing conditions and analyzed by western blotting (FIG. 3). While all monoclonal antibodies (46A1, 62A1 and 76A1) detected Antigen # 1 under non-reducing conditions (FIG. 2A), mAb 46A1 could not detect Antigen # 1 under reducing conditions. 62A1 reacted to Antigen # 3, but 46A1 and 76A1 did not react to Antigen # 2, # 3 and # 4. Thus, the epitope of Antigen # 1 against 46A1 and 76A1 is considered to be non-overlapping on Antigens # 2, # 3 or # 4. In particular, 46A1 may recognize the structure of the epitope.
完全長EphA2およびEphA2の細胞外ドメイン（Antigen #5）に対する調製したモノクローナル抗体の反応性を確認するために、内在性EphA2を発現するA431細胞およびC末端側にFLAG標識したEphA2の細胞外ドメイン（Antigen #5）を発現・分泌するA431細胞の無血清培養液を作製した。作製した細胞抽出物に対し、ここで作製したモノクローナル抗体を使用して免疫沈降実験を行い、沈降物を非還元条件下でPAGEを行った。ポジティブコントロール抗体（96-1、371805およびC20）は、完全長のEphA2を免疫沈降させ、C20抗体を用いたウエスタンブロッティングにより検出された（図２Ｂ）。興味深いことに、46A1は完全長EphA2を免疫沈降させたが、62A1および76A1の完全長EphA2に対する反応性は非常に弱かった。これに対し、また、62A1はEphA2の細胞外ドメイン（Antigen #5）を免疫沈降させたが、76A1のEphA2の細胞外ドメイン（Antigen #5）を免疫沈降させなかった（図２Ｃ）。したがって、76A1は、完全長EphA2および細胞外ドメイン（Antigen #5）とは結合せずに、MT1-MMPによる切断フォーム（Antigen #1）と特異的に結合する。他方、 46A1は、MT1-MMPによる切断フォーム（Antigen #1）以外にも完全長EphA2および細胞外ドメイン（Antigen #5）の両方と結合し、62A1はMT1-MMPによる切断フォーム（Antigen #1）の他に細胞外ドメイン（Antigen #5）と結合することが分かった。
以上のことから、MT1-MMPによるEphA2の切断フラグメント（Antigen #1）を免疫原として得られた抗体のうち、完全長EphA2およびEphA2の細胞外ドメイン（Antigen #5）に結合しない抗体をスクリーニングすることで、MT1-MMPによるEphA2の切断フラグメントのみを特異的に認識する抗体の取得が可能であることが分かった。In order to confirm the reactivity of the prepared monoclonal antibody to the extracellular domain (Antigen # 5) of full-length EphA2 and EphA2, A431 cells expressing endogenous EphA2 and the extracellular domain of EphA2 FLAG-tagged at the C-terminal side ( A serum-free medium of A431 cells expressing and secreting Antigen # 5 was prepared. Immunoprecipitation experiments were performed on the cell extract prepared using the monoclonal antibody prepared herein, and the precipitate was subjected to PAGE under non-reducing conditions. Positive control antibodies (96-1, 371805 and C20) immunoprecipitated full length EphA2 and were detected by Western blotting using C20 antibody (FIG. 2B). Interestingly, 46A1 immunoprecipitated full-length EphA2, but the reactivity of 62A1 and 76A1 to full-length EphA2 was very weak. In contrast, 62A1 also immunoprecipitated the extracellular domain of EphA2 (Antigen # 5), but not the extracellular domain of EphA2 of 76A1 (Antigen # 5) (FIG. 2C). Thus, 76A1 binds specifically to the MT1-MMP cleaved form (Antigen # 1) without binding to full-length EphA2 and the extracellular domain (Antigen # 5). On the other hand, 46A1 binds to both full-length EphA2 and extracellular domain (Antigen # 5) besides MT1-MMP cleaved form (Antigen # 1), and 62A1 forms MT1-MMP cleaved form (Antigen # 1) In addition, it was found to bind to the extracellular domain (Antigen # 5).
From the above, among the antibodies obtained using MT1-MMP-cleaved fragment of EphA2 (Antigen # 1) as an immunogen, screening for antibodies that do not bind to full-length EphA2 and extracellular domain (Antigen # 5) of EphA2 This indicates that it is possible to obtain an antibody that specifically recognizes only the cleavage fragment of EphA2 by MT1-MMP.
がん患者の血清中に存在する可溶性EphA2フラグメント（MT1-MMPによるEphA2の切断フラグメント）の解析
サンドイッチELISA法の実施にあたり、捕捉抗体として76A1を、検出抗体として62A1を使用した。抗原量を増大させた場合のELISAの定量的反応性の程度を確認するために、Antigen #1を血清に添加（0-250 pg/mL）し、その血清をアッセイに供した。標準曲線は用量依存的な直線性を示し（図４Ａ）、10 ng/mLのAntigen #1を添加しても飽和しなかった。そこで、胃がん患者17人、すい臓がん患者9人、食道がん患者8人、胃食道がん患者1人、頭頸部のがん患者9人に由来する血清サンプル、および50人（男性25人、女性25人）の健康な被験者由来の血清サンプルに対し、ELISAを行った（図４ＢおよびＣ）。健康な被験者のサンプル中の可溶性EphA2の平均濃度は、95.6±13.04 pg/mLであったので、カットオフ値を350 pg/mL（平均値±2SDs）に設定した。興味深いことに、より高い可溶性EphA2濃度が、胃がんサンプル（460.0±379.2 pg/mL）とすい臓がんサンプル（950.7±316.9 pg/mL）において検出された。特に、すい臓がんの9患者のうち8患者のサンプルにおいて、可溶性EphA2濃度がカットオフ値を超えていた（図４ＢおよびＣ）。 Analysis of Soluble EphA2 Fragment (Cleavage Fragment of EphA2 by MT1-MMP) Present in Serum of Cancer Patient For the implementation of sandwich ELISA, 76A1 was used as a capture antibody and 62A1 as a detection antibody. In order to confirm the degree of quantitative reactivity of ELISA when the amount of antigen was increased, Antigen # 1 was added to serum (0-250 pg / mL), and the serum was subjected to assay. The standard curve showed a dose dependent linearity (FIG. 4A), which was not saturated with the addition of 10 ng / mL of Antigen # 1. Therefore, serum samples from 17 stomach cancer patients, 9 pancreatic cancer patients, 8 esophageal cancer patients, 1 gastro-oesophageal cancer patients, 9 head and neck cancer patients, and 50 (25 males) ELISA was performed on serum samples from healthy subjects (25 females) (Figures 4B and C). Since the average concentration of soluble EphA2 in the samples of healthy subjects was 95.6 ± 13.04 pg / mL, the cut-off value was set to 350 pg / mL (mean ± 2 SDs). Interestingly, higher soluble EphA2 concentrations were detected in gastric cancer samples (460.0 ± 379.2 pg / mL) and pancreatic cancer samples (950.7 ± 316.9 pg / mL). In particular, soluble EphA2 concentrations exceeded the cut-off value in samples of 8 out of 9 patients with pancreatic cancer (FIGS. 4B and C).
予備的なROC解析の結果から、すい臓がんおよび胃がん患者の血清中の可溶性EphA2フラグメントを検出した場合のAUC（area under the curve）は、各々、0.89および0.88であった。健康な被験者由来の血清サンプルに対するすい臓がん患者由来の血清サンプルの感度および特異性は、各々、89.0 %および90.0 %であった（図５Ａ）。また、健康な被験者由来の血清サンプルに対する胃がん患者由来の血清サンプルの感度および特異性は、各々、88.2 %および84.0 %であった（図５Ｂ）。
比較のために、既知の消化器系腫瘍（すい臓がんを含む）マーカーであるCA19-9のレベルを測定した（図５Ｃ）。すい臓がん患者由来の血清に対するCA19-9の検出感度および特異性は、各々、88.9%および72.0%であった。また、CA19-9のAUCは、0.83であった。この結果から、可溶性EphA2フラグメントの量を本発明の抗体を用いて測定した結果に基づくすい臓がんの診断精度は、CA19-9を検出することに基づく診断精度よりも優れていることが明らかとなった（図５ＡおよびＣ）。From the results of preliminary ROC analysis, the AUC (area under the curve) when detecting soluble EphA2 fragments in serum of pancreatic cancer and gastric cancer patients was 0.89 and 0.88, respectively. The sensitivity and specificity of serum samples from pancreatic cancer patients to serum samples from healthy subjects were 89.0% and 90.0%, respectively (FIG. 5A). Also, the sensitivity and specificity of serum samples from gastric cancer patients to serum samples from healthy subjects were 88.2% and 84.0%, respectively (FIG. 5B).
For comparison, the level of CA19-9, a known digestive system tumor (including pancreatic cancer) marker, was measured (FIG. 5C). The detection sensitivity and specificity of CA19-9 to serum from pancreatic cancer patients were 88.9% and 72.0%, respectively. In addition, the AUC of CA19-9 was 0.83. From this result, it is clear that the diagnostic accuracy of pancreatic cancer based on the result of measuring the amount of soluble EphA2 fragment using the antibody of the present invention is superior to the diagnostic accuracy based on detection of CA19-9. (Figures 5A and C).
本発明の抗体の血中可溶性EphA2フラグメントの検出精度の検討
可溶性EphA2フラグメントの検出を行う際に、本発明の抗体（すなわち、MT1-MMPによるEphA2の切断フラグメントのみを特異的に認識する抗体）を用いた場合と、野生型EphA2も認識する既存の抗体を用いた場合とで、血中の可溶性EphA2フラグメントの検出精度にどのような違いが生じるか検討を行った。
捕捉抗体として46A1（野生型EphA2と可溶性EphA2の両方に反応する）と76A1（可溶性EphA2フラグメントのみに反応する）を、検出抗体として62A1を使用し、健康な被験者由来の血清サンプルに対してサンドイッチELISAを行った。その結果、捕捉抗体として46A1を用いた測定結果から、健常人血清の幾つかのサンプルで高値のEphA2を含むことが明らかとなった（図６左）。捕捉抗体として76A1を用いたEphA2遊離断片特異的なELISAでは、これらのサンプルは陽性を示さなかった（図６右）。
これらの結果から、既存の汎用抗体（野生型EphA2と可溶性EphA2の両方に反応する）を用いると、エクソソーム等に含まれる野生型EphA2を検出する可能性があり、がんの検査において、可溶性EphA2フラグメントを特異的に検出する抗体（すなわち、本発明の抗体）を使用することの重要性が確認できた。 Examination of Detection Accuracy of Soluble EphA2 Fragment in Blood of Antibody of the Present Invention When detecting soluble EphA2 fragment, the antibody of the present invention (that is, an antibody that specifically recognizes only the cleaved fragment of EphA2 by MT1-MMP) is used. It was examined what difference would occur in the detection accuracy of soluble EphA2 fragments in blood between the case where it was used and the case where an existing antibody that also recognizes wild type EphA2 was used.
A sandwich ELISA against serum samples from healthy subjects using 46A1 (responsive to both wild type EphA2 and soluble EphA2) and 76A1 (responsive to only soluble EphA2 fragments) as capture antibodies and 62A1 as a detection antibody Did. As a result, from the measurement results using 46A1 as a capture antibody, it became clear that some samples of healthy human serum contain high EphA2 (FIG. 6, left). In EphA2 free fragment specific ELISA using 76A1 as capture antibody, these samples did not show positive (FIG. 6 right).
From these results, it is possible to detect wild-type EphA2 contained in exosomes etc. by using the existing general-purpose antibody (which reacts to both wild-type EphA2 and soluble EphA2). The importance of using an antibody that specifically detects a fragment (ie, the antibody of the present invention) has been confirmed.
本発明の抗体は、がんマーカーとして有用なEphA2のN末端側フラグメントと特異的に結合する。本発明の抗体を使用することで、これまで早期発見が困難であったがん（たとえば、すい臓がんなど）の診断も簡便かつ迅速に行い得るため、がんの診断および治療の分野において大きな効果をもたらすことが期待される。 The antibody of the present invention specifically binds to an N-terminal fragment of EphA2 useful as a cancer marker. By using the antibody of the present invention, it is possible to easily and quickly diagnose cancers (eg pancreatic cancer etc.) for which early detection was difficult so far, which is a major field in the field of cancer diagnosis and treatment. Expected to bring effects.
Claims (12)
（ａ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、An antibody which binds to the following protein (a) and does not bind to the proteins (b) and (c):
(A) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer from 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by the 28th to Xth,
(B) A protein consisting of the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1, 1 or several amino acids in the 28th to 976th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1 Is a protein consisting of an amino acid sequence having an addition, substitution, insertion or deletion, or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence shown in SEQ ID NO: 1 ,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3;
（ｉ）配列番号１で表されるアミノ酸配列中28番目〜X番目（ここで、Xは328以上435以下の整数、以下同じ）で表されるアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または、配列番号１で表されるアミノ酸配列中28番目〜X番目で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質のいずれかに結合する抗体を作製する工程、
（ｉｉ）工程（ｉ）で作製した抗体のうち、下記の（ｂ）および（ｃ）のタンパク質とは結合しない抗体を選択する工程
（ｂ）配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列からなるタンパク質、配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号１で表されるアミノ酸配列中28番目〜976番目のアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質、
（ｃ）配列番号３で表されるアミノ酸配列からなるタンパク質、配列番号３で表されるアミノ酸配列において１もしくは数個のアミノ酸が付加、置換、挿入もしくは欠失したアミノ酸配列からなるタンパク質、または配列番号３で表されるアミノ酸配列と80％以上の配列同一性を有するアミノ酸配列からなるタンパク質A method for producing an antibody, comprising the following steps (i) and (ii):
(I) A protein consisting of an amino acid sequence represented by 28th to Xth (where X is an integer of 328 to 435, hereinafter the same) in the amino acid sequence represented by SEQ ID NO: 1, represented by SEQ ID NO: 1 In the amino acid sequence represented by 28th to Xth in the amino acid sequence, or a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted, or in the amino acid sequence represented by SEQ ID NO: 1 Producing an antibody that binds to any of a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by 28th to Xth,
(Ii) a step of selecting an antibody which does not bind to the proteins of the following (b) and (c) among the antibodies produced in the step (i) (b) 28th to 28th amino acids in the amino acid sequence represented by SEQ ID NO: 1 A protein consisting of the amino acid sequence 976, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the 28th to 976th amino acids in the amino acid sequence represented by SEQ ID NO: 1; Or a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of 28th to 976th in the amino acid sequence represented by SEQ ID NO: 1,
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of an amino acid sequence in which one or several amino acids are added, substituted, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3 A protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by No. 3
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