JP2010286279A - Biomarker for inspecting autoimmune disease, and inspection method - Google Patents
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Abstract
Description
本発明は、自己免疫疾患検査用バイオマーカーに関する。より詳しくは、自己免疫疾患において、疾患活動性を判断しうるバイオマーカー、さらに詳しくは治療薬の有効性を判断しうるバイオマーカーに関し、さらに該バイオマーカーを検出又は定量することによる自己免疫疾患の検査方法に関する。 The present invention relates to a biomarker for testing autoimmune diseases. More particularly, in an autoimmune disease, a biomarker capable of determining disease activity, more specifically, a biomarker capable of determining the effectiveness of a therapeutic agent, and further detecting or quantifying the biomarker It relates to the inspection method.
ベーチェット病、関節リウマチ、クローン病、キャッスルマン氏病などの自己免疫疾患の治療薬として、キメラ型モノクローナル抗体製剤であるインフリキシマブ(Infliximab)や可溶性TNF受容体であるエタネルセプト(Etanercept)などのTNF-αを阻害する生物学的製剤が用いられている。しかし、TNF-α阻害療法に抵抗性を示す患者が一部存在し、このような患者は他の薬剤の投与やその他の治療方法に切り替える必要がある。 TNF-α such as Infliximab, a chimeric monoclonal antibody preparation, and Etanercept, a soluble TNF receptor, for the treatment of autoimmune diseases such as Behcet's disease, rheumatoid arthritis, Crohn's disease, and Castleman's disease Biological preparations that inhibit the above have been used. However, there are some patients who are resistant to TNF-α inhibition therapy and such patients need to switch to other medications or other treatments.
上記例示したような自己免疫疾患の炎症マーカーとして、C-reactive protein (CRP) が臨床上よく使われている（非特許文献１）。CRPは、炎症に伴って上昇するインターロイキン-6（IL-6）などの炎症性サイトカインによって肝臓で合成される急性期タンパク質のひとつである。急性期タンパク質には、CRPの他にα1-アシドグリコプロテイン、ハプトグロビン、シアル酸などがあり、血清中のこれらの物質の濃度増加は、炎症性疾患の診断や経過の推移の判定に用いられる。これらの炎症マーカーは、体内に炎症があればその程度に応じて異常値を示すが、炎症性疾患のみならず、炎症マーカーによっては、組織破壊や細胞壊死を伴う悪性腫瘍や、急性心筋梗塞、貧血、溶血、妊娠、外傷など様々な病態で異常値を示すことがあり、各マーカーにより各疾患に対する特異度や感度が異なる場合がある。 C-reactive protein (CRP) is frequently used clinically as an inflammation marker for autoimmune diseases as exemplified above (Non-patent Document 1). CRP is one of the acute phase proteins synthesized in the liver by inflammatory cytokines such as interleukin-6 (IL-6) that increases with inflammation. Acute phase proteins include α1-acid glycoprotein, haptoglobin, sialic acid and the like in addition to CRP, and the increase in the concentration of these substances in serum is used for diagnosis of inflammatory diseases and determination of progression. These inflammation markers show abnormal values depending on the degree of inflammation in the body, but depending not only on inflammatory diseases, depending on the inflammation marker, malignant tumors with tissue destruction and cell necrosis, acute myocardial infarction, Abnormal values may be shown in various pathologies such as anemia, hemolysis, pregnancy, trauma, and the specificity and sensitivity to each disease may differ depending on each marker.
上述のごとく、CRP値の確認だけでは、自己免疫疾患の疾患活動性を把握できず、例えばCRP値が正常レベルでも疾患活動性を認めたり、CRP値が正常レベルでも炎症を示す患者が存在する。このように、上記マーカーでは、必ずしも正確な疾患活動性を評価しうるとはいえず、例えば上述したように、TNF-αを阻害する生物学的製剤に対して抵抗性を有する患者の見極めや新たな治療方法の選択において、十分とはいえない。そこで、自己免疫疾患患者について、診断を行い、治療方針を決定するために、より正確に評価できるバイオマーカーが臨床上必要とされている。 As described above, the disease activity of autoimmune disease cannot be grasped only by confirming the CRP value. For example, there are patients who show disease activity even when the CRP value is normal, or who have inflammation even if the CRP value is normal. . Thus, it is not always possible to accurately evaluate disease activity with the above-described markers. For example, as described above, it is possible to identify patients with resistance to a biological agent that inhibits TNF-α. It is not enough to select a new treatment method. Therefore, there is a clinical need for biomarkers that can be more accurately evaluated in order to diagnose and determine treatment strategies for patients with autoimmune diseases.
ロイシンリッチα２グリコプロテイン（LRG）、フィブリノーゲンガンマ、C4a及びアルブミン前駆体からなる群から選択される少なくともひとつのタンパク質を含むことを特徴とする腹膜機能マーカーについて報告がある（特許文献１）。LRGは血清タンパク質のひとつで、約50kDaの糖タンパク質であり、好中球から分泌されることが報告されている（非特許文献２）。しかしながら、これらのマーカーと自己免疫疾患との関係については、報告はない。 There is a report on a peritoneal function marker characterized by containing at least one protein selected from the group consisting of leucine-rich α2 glycoprotein (LRG), fibrinogen gamma, C4a and albumin precursor (Patent Document 1). LRG is a serum protein, a glycoprotein of about 50 kDa, and is reported to be secreted from neutrophils (Non-patent Document 2). However, there is no report on the relationship between these markers and autoimmune diseases.
本発明は、自己免疫疾患において、疾患活動性を判断しうる自己免疫疾患検査用バイオマーカー、さらに詳しくは治療薬の有効性を判断しうるバイオマーカーを提供することを課題とし、並びに検査方法を提供することを課題とする。 An object of the present invention is to provide a biomarker for testing an autoimmune disease that can determine disease activity in an autoimmune disease, and more specifically, to provide a biomarker that can determine the effectiveness of a therapeutic agent. The issue is to provide.
本発明者らは上記課題を解決するために、プロテオミクス手法を用いて鋭意研究を重ねた結果、自己免疫疾患の活動性と相関しうる新規バイオマーカーとしてロイシンリッチα２グリコプロテイン（leucine rich alpha 2 glycoprotein: LRG）を検出した（以下、単に「LRG」ともいう。）。自己免疫疾患患者にTNF-αを阻害する生物学的製剤を投与した場合に、生物学的製剤抵抗性患者ではLRGの発現が有意に高値を示したが、有効な患者ではLRGの発現が抵抗性患者に比べて低かったことから、LRGは自己免疫疾患のバイオマーカー及び薬剤投与に伴う疾患活動性の指標マーカーとなりうることを確認し、本発明を完成した。 In order to solve the above-mentioned problems, the present inventors have conducted extensive research using a proteomic method, and as a result, leucine rich alpha 2 glycoprotein (leucine rich alpha 2 glycoprotein) is a novel biomarker that can be correlated with the activity of autoimmune diseases. : LRG) was detected (hereinafter also simply referred to as “LRG”). When a biologic that inhibits TNF-α was administered to patients with autoimmune disease, LRG expression was significantly higher in bioresistant patients, but LRG expression was less effective in effective patients Therefore, the present invention was completed by confirming that LRG can be a biomarker for autoimmune diseases and a marker for disease activity associated with drug administration.
すなわち、本発明は以下よりなる。
１．LRGからなる、自己免疫疾患検査用バイオマーカー。
２．自己免疫性疾患が、ベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチである、前項１に記載の自己免疫疾患検査用バイオマーカー。
３．生体から分離した生体検体について、前項１又は２に記載のバイオマーカーを検出または定量することを特徴とする自己免疫疾患検査方法。
４．生体検体が血清である、前項３に記載の自己免疫疾患検査方法。
５．バイオマーカーの検出または定量を、免疫学的手法若しくはプロテオーム解析により行う、前項３又は４に記載の自己免疫疾患検査方法。
６．生体から分離した生体検体について、前項１又は２に記載のバイオマーカーを検出または定量することを特徴とする、自己免疫疾患に対するTNF-α阻害療法の奏功性の予測方法。
７．抗LRG抗体を含む、前項３〜６のいずれか１に記載の自己免疫疾患検査用キット。
That is, this invention consists of the following.
1. A biomarker for testing autoimmune diseases consisting of LRG.
2. 2. The biomarker for testing an autoimmune disease according to item 1, wherein the autoimmune disease is Behcet's disease, Castleman's disease, Crohn's disease, or rheumatoid arthritis.
3. An autoimmune disease test method comprising detecting or quantifying the biomarker according to item 1 or 2 on a biological specimen separated from a living body.
4). 4. The autoimmune disease test method according to item 3, wherein the biological specimen is serum.
5). 5. The method for examining an autoimmune disease according to item 3 or 4 above, wherein the biomarker is detected or quantified by an immunological technique or proteome analysis.
6). A method for predicting the efficacy of TNF-α inhibition therapy for an autoimmune disease, comprising detecting or quantifying the biomarker according to item 1 or 2 for a biological specimen separated from a living body.
7). 7. The autoimmune disease test kit according to any one of items 3 to 6, comprising an anti-LRG antibody.
本発明の新規自己免疫疾患検査用バイオマーカーであるLRGは、自己免疫疾患、とりわけベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチの検査用バイオマーカーとなりうる。LRGをバイオマーカーとして検出又は定量することにより、上記自己免疫疾患の検査を行うことができ、疾患活動性を評価することができる。また、生物学的製剤などの自己免疫疾患治療薬を投与したときの治療効果を評価しうるバイオマーカーともなる。具体的には、インフリキシマブ(Infliximab)及び／又はエタネルセプト(Etanercept)などのTNF-αを阻害する生物学的製剤を投与した場合の疾患活動性を、本発明のバイオマーカーにより評価することができる。これにより、上記生物学的製剤の有効性を確認したり、薬剤抵抗性を有する患者を早期に発見することができる。その結果、例えば薬剤投与終了時の判断や投与薬剤や治療方針の変更などに対して、最も効果的な方法を選択することができる。 LRG, which is a novel biomarker for testing autoimmune diseases of the present invention, can be a biomarker for testing autoimmune diseases, particularly Behcet's disease, Castleman's disease, Crohn's disease or rheumatoid arthritis. By detecting or quantifying LRG as a biomarker, the autoimmune disease can be examined, and disease activity can be evaluated. Moreover, it becomes a biomarker which can evaluate the therapeutic effect when administering an autoimmune disease therapeutic agent, such as a biologic. Specifically, disease activity when a biological preparation that inhibits TNF-α such as Infliximab and / or Etanercept can be evaluated by the biomarker of the present invention. Thereby, the effectiveness of the said biological preparation can be confirmed, or the patient who has drug resistance can be discovered at an early stage. As a result, for example, the most effective method can be selected with respect to the determination at the end of drug administration, the change of the administered drug and the treatment policy, and the like.
本発明においてはLGRが自己免疫疾患検査用バイオマーカーとして用いられる。本発明のバイオマーカーにより検査しうる自己免疫疾患は、好ましくはベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチである。ここでLRGとは、上記のごとくロイシンリッチα２グリコプロテインで、約50kDaの糖タンパク質であり、健常人血清では約3.0μg/mL濃度含まれていることが知られている。またLRGは好中球から分泌されることが報告されている（非特許文献２）。また、LRGは顆粒球で発現することも報告されているが、LRGの発現はIL-6の刺激により誘導されるものではないことが本発明者らにより確認された。一方、自己免疫疾患のマーカーとして汎用されていたCRPは、背景技術の欄で説明したように、炎症に伴って上昇するIL-6などの炎症性サイトカインによって肝臓で合成される急性期タンパク質のひとつであるので、LRGはCRPとは異なるメカニズムにより発現するバイオマーカーということができる。即ち、LRGはIL-6の影響を受けにくいことから、より正確な自己免疫疾患検査用バイオマーカーとして機能しうる。例えば、自己免疫疾患治療薬の投与によりIL-6の発現を抑制した場合でも、疾患活動性が抑制されない場合もあるが、このような場合であっても、LRGは薬剤の影響を受けることなく正確なバイオマーカーとなりうる。 In the present invention, LGR is used as a biomarker for testing autoimmune diseases. The autoimmune disease that can be examined by the biomarker of the present invention is preferably Behcet's disease, Castleman's disease, Crohn's disease or rheumatoid arthritis. Here, LRG is a leucine-rich α2 glycoprotein as described above, which is a glycoprotein of about 50 kDa, and is known to contain a concentration of about 3.0 μg / mL in healthy human serum. It has also been reported that LRG is secreted from neutrophils (Non-patent Document 2). In addition, although it has been reported that LRG is expressed in granulocytes, the present inventors have confirmed that LRG expression is not induced by IL-6 stimulation. On the other hand, CRP, which has been widely used as a marker for autoimmune diseases, is one of the acute phase proteins synthesized in the liver by inflammatory cytokines such as IL-6 that increase with inflammation, as explained in the background section. Therefore, LRG can be said to be a biomarker expressed by a mechanism different from CRP. That is, since LRG is not easily affected by IL-6, it can function as a more accurate biomarker for autoimmune disease testing. For example, even when IL-6 expression is suppressed by administration of a therapeutic drug for autoimmune disease, disease activity may not be suppressed, but even in such a case, LRG is not affected by the drug. It can be an accurate biomarker.
本発明に用いられるLGRは哺乳動物由来である。哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ等の家畜、イヌ、ネコ等のペット、ヒト、サル、オランウータン、チンパンジーなどの霊長類を挙げることが出来る。哺乳動物は好ましくは霊長類、より好ましくはヒトである。 The LGR used in the present invention is derived from a mammal. Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, humans, monkeys, Primates such as orangutans and chimpanzees. The mammal is preferably a primate, more preferably a human.
本発明の自己免疫疾患検査用バイオマーカーとして、LRGが同定された経緯を以下に示す。本発明者らは、ベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチなどの自己免疫疾患患者からインフォームドコンセントにより得られた血清を、ハイスループットなタンパク質の発現又は定量解析が可能なiTRAQTM試薬（ABI社）及び質量分析計の組み合わせによりプロテオーム解析し、これらの疾患特異的に発現量が異なるタンパク質であるLRGを検出した。さらに、インフリキシマブ又はエタネルセプトなどのTNF-αを阻害する生物学的製剤を投与した場合の疾患活動性と、LRGの発現量との関係について解析した結果、自己免疫疾患、好適にはベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチの患者血清において、上記LRGが有意に高値を示したことが確認された。上記により、LRGはこれらの疾患の検出用バイオマーカー又は疾患活動性の指標マーカーとなりうる。 The history of LRG identification as a biomarker for testing autoimmune diseases of the present invention is shown below. The present inventors have used iTRAQ ™ that enables high-throughput protein expression or quantitative analysis of sera obtained by informed consent from patients with autoimmune diseases such as Behcet's disease, Castleman's disease, Crohn's disease, or rheumatoid arthritis. Proteome analysis was performed using a combination of a reagent (ABI) and a mass spectrometer, and LRG, which is a protein with a different expression level, was detected specifically for these diseases. Furthermore, as a result of analyzing the relationship between the activity of disease and the expression level of LRG when a biological preparation that inhibits TNF-α such as infliximab or etanercept is administered, autoimmune disease, preferably Behcet's disease, castle It was confirmed that the above-mentioned LRG showed a significantly high value in the serum of patients with Mann's disease, Crohn's disease or rheumatoid arthritis. Based on the above, LRG can be a biomarker for detecting these diseases or an index marker for disease activity.
本発明において、自己免疫疾患の検査は、被験者より採取した生体検体に含まれるLRGを検出又は定量することによる。本発明の自己免疫疾患検査方法において、生体検体は被験者から採取した血液が挙げられ、好ましくは該血液から分離取得した血清を検査用検体とすることができる。血清の取得方法は特に限定されず、自体公知の方法、例えば血液から臨床検査用検体として取得する血清の分離方法に準じることができる。具体的には、血液検体を静置した後の上清や、遠心分離したときの上清などから取得することができる。また検査用検体は、LRGの検出を容易にするために、予め発現量の高い血清タンパク質、例えばアルブミン、免疫グロブリンＧ（IgG）、トランスフェリン、免疫グロブリンＡ（IgA）、ハプトグロビン、α１アンチトリプシン、フィブリノゲン、α２マクログロブリン、免疫グロブリンＭ（IgM）、α１-アシドグリコプロテイン、補体 C3、アポリポタンパク質 AI、アポリポタンパク質 AII、トランスサイレチンなどを除去するための前処理を行っても良い。 In the present invention, an autoimmune disease test is performed by detecting or quantifying LRG contained in a biological sample collected from a subject. In the autoimmune disease test method of the present invention, the biological sample includes blood collected from a subject, and preferably serum obtained separately from the blood can be used as the test sample. The method for obtaining serum is not particularly limited, and can be based on a method known per se, for example, a method for separating serum obtained as a sample for clinical examination from blood. Specifically, it can be obtained from a supernatant after leaving a blood sample, a supernatant obtained by centrifugation, or the like. In addition, in order to facilitate the detection of LRG, the test sample is a serum protein having a high expression level, such as albumin, immunoglobulin G (IgG), transferrin, immunoglobulin A (IgA), haptoglobin, α1 antitrypsin, fibrinogen. , Α2 macroglobulin, immunoglobulin M (IgM), α1-acid glycoprotein, complement C3, apolipoprotein AI, apolipoprotein AII, transthyretin and the like may be pretreated.
本発明の検査方法において、LRGの検出又は定量の方法は、LRGタンパク質の検出又は定量が可能な方法であれば良く、特に限定されない。ここで定量とは、LRGタンパク質の量を定量することであり、具体的には免疫学的手法や上述のプロテオーム解析による方法を適用することができる。 In the test method of the present invention, the method for detecting or quantifying LRG is not particularly limited as long as it is a method capable of detecting or quantifying LRG protein. Here, quantification means quantifying the amount of LRG protein, and specifically, an immunological technique or the above-described method based on proteome analysis can be applied.
免疫学的な手法としては、例えば、抗LRG抗体を用いてLRGとの抗原抗体反応を利用して測定する方法が挙げられる。固相担体に抗LRG抗体を固着し、検査用検体を固相担体と接触させて検体中のLRGを抗LRG抗体で捕捉し、捕捉したLRGを、標識物質を用いて測定する方法が挙げられる。具体的には、酵素免疫定量法（Enzyme-linked immunosorbent assay: ELISA法）やイムノブロッティング法が挙げられる。また、検査用検体を電気泳動したものについて、抗LRG抗体によりLRGを検出又は定量するウエスタンブロッティング法により検査することもできる。 As an immunological technique, for example, a method of measuring using an antigen-antibody reaction with LRG using an anti-LRG antibody can be mentioned. Examples include a method in which an anti-LRG antibody is fixed to a solid phase carrier, a test sample is brought into contact with the solid phase carrier, LRG in the sample is captured with the anti-LRG antibody, and the captured LRG is measured using a labeling substance. . Specific examples include an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting method. In addition, electrophoretic samples of test specimens can be examined by Western blotting in which LRG is detected or quantified with an anti-LRG antibody.
プロテオーム解析による方法では、種々の電気泳動やクロマトグラフィーを組み合わせて分離された少量のタンパク質を断片化してその質量を測定し、得られたデータをゲノム解析から推定されるタンパク質のアミノ酸配列データと比較して同定したり、定量することなどにより行うことができる。 In the proteome analysis method, a small amount of protein separated by combining various electrophoresis and chromatography is fragmented and its mass is measured, and the obtained data is compared with the amino acid sequence data of the protein estimated from genomic analysis. Identification or quantification.
本発明の自己免疫疾患検査方法において、本発明のバイオマーカーの検出又は定量により、自己免疫疾患の検査を行うことができる。自己免疫疾患の検査には、自己免疫疾患の診断や、治療後の疾患活動性の評価を行うことも含まれる。自己免疫疾患としては、上述したように好ましくはベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチが挙げられる。 In the autoimmune disease test method of the present invention, an autoimmune disease test can be performed by detecting or quantifying the biomarker of the present invention. Testing for autoimmune diseases includes diagnosing autoimmune diseases and evaluating disease activity after treatment. As described above, the autoimmune disease is preferably Behcet's disease, Castleman's disease, Crohn's disease or rheumatoid arthritis.
後述の実施例に示されるように、健常人と比較して自己免疫疾患の患者においてLRG発現量が高い。自己免疫疾患の診断は、LRGの発現量と自己免疫疾患への罹患率との間のこのような正の相関に基づき行われる。 As shown in the Examples described later, the LRG expression level is higher in patients with autoimmune disease than in healthy individuals. The diagnosis of autoimmune disease is performed based on such a positive correlation between the expression level of LRG and the prevalence of autoimmune disease.
例えば、健常人及び対象患者からの生体検体におけるLRGの発現量を定量し、対象患者からの生体検体におけるLRGの発現量を、健常人からの生体検体におけるLRGの発現量と比較する。あるいは、LRGの発現量と自己免疫疾患の罹患の有無との相関図をあらかじめ作成しておき、対象患者におけるLRG発現量をその相関図と比較してもよい。発現量の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, the expression level of LRG in the biological sample from the healthy subject and the target patient is quantified, and the expression level of LRG in the biological sample from the target patient is compared with the expression level of LRG in the biological sample from the healthy person. Alternatively, a correlation diagram between the LRG expression level and the presence or absence of an autoimmune disease may be prepared in advance, and the LRG expression level in the target patient may be compared with the correlation diagram. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.
そして、対象患者においてLRGが、健常人に比べて高値で検出若しくは定量された場合には、上記のような自己免疫疾患に罹患している可能性が高いと判断することができる。 Then, when LRG is detected or quantified at a higher value in healthy subjects than in healthy subjects, it can be determined that there is a high possibility of suffering from the autoimmune disease as described above.
後述の実施例に示されるように、疾患活動性と、LRGの発現量との関係について解析した結果、自己免疫疾患、好ましくはベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチの患者血清において、疾患活動性の高い患者は、LRGが有意に高値を示したことが確認された。自己免疫疾患の疾患活動性の評価は、LRG発現レベルと疾患活動性との間のこのような正の相関に基づき行われる。 As shown in the examples described below, as a result of analyzing the relationship between the disease activity and the expression level of LRG, it was found that the patient serum of autoimmune disease, preferably Behcet's disease, Castleman's disease, Crohn's disease or rheumatoid arthritis It was confirmed that patients with high disease activity showed significantly high LRG. Assessment of disease activity of autoimmune disease is based on such positive correlation between LRG expression level and disease activity.
例えば、活動性の高い自己免疫疾患の患者及び、活動性の低い自己免疫疾患の患者から、生体検体を採取し、対象患者から採取された生体検体におけるLRG発現量が活動性の高い自己免疫疾患の患者及び活動性の低い自己免疫疾患のそれと比較される。あるいは、生体検体におけるLRG発現量と自己免疫疾患の疾患活動性との間の相関図をあらかじめ作成しておき、対象患者から採取された生体検体におけるLRG発現量をその相関図と比較してもよい。発現量の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, a biological sample is collected from a patient with high activity autoimmune disease and a patient with low activity autoimmune disease, and the LRG expression level in the biological sample collected from the target patient is high autoimmune disease Compared to that of patients with low-activity autoimmune diseases. Alternatively, a correlation diagram between the LRG expression level in a biological sample and the disease activity of an autoimmune disease may be prepared in advance, and the LRG expression level in a biological sample collected from a target patient may be compared with the correlation diagram. Good. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.
そして、LRG発現量の比較結果より、対象患者のLRG発現量が相対的に高い場合には、自己免疫疾患の疾患活動性が高いと判断することができる。 Then, from the comparison result of the LRG expression level, when the LRG expression level of the target patient is relatively high, it can be determined that the disease activity of the autoimmune disease is high.
したがって、自己免疫疾患治療薬投与、具体的にはインフリキシマブやエタネルセプトなどのTNF-αを阻害する生物学的製剤などの投与に伴い、患者の血清LRGを定量し、解析することで、自己免疫疾患の疾患活動性を評価することができ、投与した自己免疫疾患治療薬の有効性や、薬剤抵抗性などを客観的に判断することができる。 Therefore, autoimmune diseases can be determined by quantifying and analyzing the serum LRG of patients with the administration of therapeutic agents for autoimmune diseases, specifically biological products that inhibit TNF-α such as infliximab and etanercept. Therefore, the effectiveness of the administered autoimmune disease drug, drug resistance, etc. can be objectively determined.
ここで、投与した自己免疫疾患治療薬の有効性や、薬剤抵抗性などを客観的に判断するための検査は、自己免疫疾患治療薬の投与前後に採取して得た血清LRGを定量し、各LRG量を比較することにより、行うことができる。 Here, the test for objectively judging the effectiveness of the administered autoimmune disease drug, drug resistance, etc., quantifies the serum LRG collected before and after the administration of the autoimmune disease drug, This can be done by comparing the amounts of each LRG.
具体的には、以下の工程を含む方法による。
１）自己免疫疾患治療剤の投与前及び投与後に対象患者から生体検体（例、血清）を分離取得し、各検査用検体とする工程；
２）上記１）の検査用検体について、本発明の自己免疫疾患検査方法により検査を行う工程；
３）上記２）で得られた結果をもとに、自己免疫疾患治療剤の投与前及び投与後の各検査用検体中のLRG量比を算出する工程。
Specifically, the method includes the following steps.
1) A step of separating and obtaining a biological sample (eg, serum) from a target patient before and after administration of a therapeutic agent for autoimmune disease, and using it as a test sample
2) A step of examining the test specimen of 1) by the autoimmune disease test method of the present invention;
3) A step of calculating an LRG amount ratio in each test sample before and after administration of the therapeutic agent for autoimmune disease based on the result obtained in 2) above.
更に、後述の実施例に示されるように、自己免疫疾患の患者において、生体検体中のLRG発現量が高いほど、TNF-α阻害療法に対する抵抗性が高い。このような、LRG発現量とTNF-α阻害療法に対する抵抗性との間の正の相関に基づき、自己免疫疾患に対するTNF-α阻害療法の奏功性を予測することが出来る。 Furthermore, as shown in Examples described later, in patients with autoimmune disease, the higher the LRG expression level in the biological specimen, the higher the resistance to TNF-α inhibition therapy. Based on such a positive correlation between the expression level of LRG and resistance to TNF-α inhibition therapy, the efficacy of TNF-α inhibition therapy for autoimmune diseases can be predicted.
例えば、TNF-α阻害療法に対して抵抗性があり、該療法が無効な自己免疫疾患の患者（ポジティブコントロール）及び、TNF-α阻害療法に対して抵抗性がなく、該療法が有効な自己免疫疾患の患者（ネガティブコントロール）から、生体検体を採取し、対象患者から採取された生体検体におけるLRG発現量がポジティブコントロール及びネガティブコントロールのそれと比較される。あるいは、自己免疫疾患患者の生体検体におけるLRG発現量とTNF-α阻害療法の奏功性との間の相関図をあらかじめ作成しておき、対象患者から採取された生体検体におけるLRG発現量をその相関図と比較してもよい。発現量の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, patients with autoimmune diseases that are resistant to TNF-α inhibitory therapy and ineffective (the positive control), and those who are resistant to TNF-α inhibitory therapy A biological specimen is collected from a patient with an immune disease (negative control), and the LRG expression level in the biological specimen collected from the subject patient is compared with that of the positive control and the negative control. Alternatively, a correlation diagram between the LRG expression level in biological specimens of patients with autoimmune disease and the efficacy of TNF-α inhibition therapy is prepared in advance, and the correlation between LRG expression levels in biological specimens collected from the target patient You may compare with a figure. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.
そして、LRG発現量の比較結果より、対象患者のLRG発現量が相対的に高い場合には、TNF-α阻害療法に抵抗性であり、該療法の奏功性が低い可能性が高いと判断することができる。 From the comparison result of the LRG expression level, when the LRG expression level of the target patient is relatively high, it is judged that the therapy is resistant to TNF-α inhibitory therapy and the effectiveness of the therapy is low. be able to.
ここで、TNF-α阻害療法とは、TNF-αやその受容体に対する特異的抗体や、可溶性TNF-α受容体等により、TNF-αとその受容体との結合を阻害することを含む治療法を意味する。 Here, TNF-α inhibition therapy includes treatment including inhibiting binding of TNF-α to its receptor with TNF-α and its specific antibody, soluble TNF-α receptor, etc. Means the law.
本発明は、自己免疫疾患検査用のキット（診断剤）にも及ぶ。本発明の自己免疫疾患検査用キットは、上述の本発明の検査方法（TNF-α阻害療法の奏功性の予測方法を含む）を簡便に実施するためのキットであればよく、特に限定されない。免疫学的手法による場合は、本発明の自己免疫疾患検査用キットには、キットの構成として抗LRG抗体を含むことができる。さらには、抗体の固相用担体、標識抗体、緩衝液など必要な試薬又は器具等を適宜含めることができる。プロテオーム解析による場合は、市販の試薬、例えばiTRAQTM試薬やICATTM試薬（いずれもABI社）などを用いることができる。 The present invention extends to a kit (diagnostic agent) for testing an autoimmune disease. The autoimmune disease test kit of the present invention is not particularly limited as long as it is a kit for simply carrying out the above-described test method of the present invention (including a method for predicting the efficacy of TNF-α inhibition therapy). When using an immunological technique, the kit for testing an autoimmune disease of the present invention can contain an anti-LRG antibody as a component of the kit. Furthermore, necessary reagents or instruments such as an antibody solid phase carrier, a labeled antibody, and a buffer can be appropriately included. In the case of proteome analysis, commercially available reagents such as iTRAQ ™ reagent and ICAT ™ reagent (both from ABI) can be used.
以下に、実施例を示して本発明をさらに具体的に説明するが、本発明はこれらにより限定されるものではないことは明らかである。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, it is obvious that the present invention is not limited to these examples.
（実施例１）自己免疫疾患患者血清のプロテオーム解析
１）生物学的製剤による治療
インフリキシマブ(抗TNF-抗体製剤)治療を受けているベーチェット病患者より採血し、生体検体を得た。採血は、インフリキシマブ３回目投与直前及びインフリキシマブ８回目投与直前に行った。インフリキシマブによる治療は１回目から３回目までが２週間間隔、それ以降は８週間間隔の投与で行った。
CRP値及び眼炎症、口内炎、結節性紅斑、発熱、関節炎などの症状より炎症所見を確認した結果、インフリキシマブ投与３回目直前は、著明な疾患活動性の低下が見られたが、インフリキシマブ投与８回目直前において、炎症所見の上昇など疾患活動性が高いことが確認された。CRP値はインフリキシマブ３回目投与直前(<0.2)からインフリキシマブ８回目投与直前(4.45)にかけて上昇していた。ここで、CRP値は<0.2が正常値と判断される。
(Example 1) Proteome analysis of serum of autoimmune disease patient 1) Treatment with biological preparation Blood was collected from a patient with Behcet's disease who received infliximab (anti-TNF-antibody preparation) treatment, and a biological specimen was obtained. Blood collection was performed immediately before the third administration of infliximab and immediately before the eighth administration of infliximab. Treatment with infliximab was administered at intervals of 2 weeks from the 1st to the 3rd and thereafter at intervals of 8 weeks.
As a result of confirming inflammatory findings from symptoms such as CRP value and ocular inflammation, stomatitis, erythema nodosum, fever, arthritis, etc., there was a marked decrease in disease activity immediately before the third infliximab administration, but infliximab administration 8 Immediately before the second round, it was confirmed that the disease activity such as an increase in inflammatory findings was high. CRP values increased from just before the third dose of infliximab (<0.2) to just before the eighth dose of infliximab (4.45). Here, a CRP value of <0.2 is determined as a normal value.
２）検査用試料の調製
上記のベーチェット病患者２名より得た生体検体を、各々血清分離用試験管に採り、室温で10分間3500rpm遠心分離して血清を得た。得られた血清中のタンパク質の発現変動をiTRAQTM(isobaric tags for relative and absolute quantitation)法にて効率よく定量解析するため、血清を以下の方法にて前処理し、検査用試料とした。健常人５名の血清をプールし、同様の処理で得た検査用試料を対照とした。
2) Preparation of test sample Biological specimens obtained from the above two Behcet's disease patients were each taken in a test tube for serum separation and centrifuged at 3500 rpm for 10 minutes at room temperature to obtain serum. In order to efficiently quantitatively analyze protein expression fluctuations in the obtained serum by the iTRAQ ™ (isobaric tags for relative and absolute quantitation) method, the serum was pretreated by the following method and used as a test sample. Sera from five healthy individuals were pooled, and a test sample obtained by the same treatment was used as a control.
マルチプルアフィニティ除去Hu-14カラム(Agilent社 4.6 x 100 mm)を用いて血清中の発現量の高い14種類の血清タンパク質 （アルブミン、免疫グロブリンＧ（IgG）、トランスフェリン、免疫グロブリンＡ（IgA）、ハプトグロビン、α１アンチトリプシン、フィブリノゲン、α２マクログロブリン、免疫グロブリンＭ（IgM）、α１-アシドグリコプロテイン、補体 C3、アポリポタンパク質 AI、アポリポタンパク質 AII、トランスサイレチン）をカラムに吸着させることで除去し、血清の素通り画分を回収し、検査用試料として以降の解析に用いた。 14 serum proteins (albumin, immunoglobulin G (IgG), transferrin, immunoglobulin A (IgA), haptoglobin) with high expression in serum using multiple affinity-removed Hu-14 column (Agilent 4.6 x 100 mm) , Α1 antitrypsin, fibrinogen, α2 macroglobulin, immunoglobulin M (IgM), α1-acid glycoprotein, complement C3, apolipoprotein AI, apolipoprotein AII, transthyretin) The serum flow-through fraction was collected and used as a test sample for subsequent analysis.
３）iTRAQTM解析
ベーチェット病患者１におけるインフリキシマブ３回目投与直前とインフリキシマブ８回目投与直前の検査用試料のタンパク質の発現変動を、iTRAQTM法を用いて解析した。iTRAQTM試薬(ABI社)により、各試料中のペプチドを修飾し、114標識（インフリキシマブ３回目投与直前）、115標識（インフリキシマブ８回目投与直前）及び117標識（対照）とした。標識した全ての検査用試料を混合した。
ベーチェット病患者２におけるインフリキシマブ３回目投与直前とインフリキシマブ６回目投与直前の検査用試料のタンパク質の発現変動についても同様に、iTRAQTM法を用いて解析した。iTRAQTM試薬(ABI社)により、各試料中のペプチドを修飾し、114標識（インフリキシマブ３回目投与直前）、115標識（インフリキシマブ６回目投与直前）及び117標識（対照）とした。標識した全ての検査用試料を混合した。
3) iTRAQ TM analysis In the Behcet's disease patient 1, protein expression fluctuations in the test samples immediately before the third administration of infliximab and immediately before the eighth administration of infliximab were analyzed using the iTRAQ TM method. Peptides in each sample were modified with iTRAQ ™ reagent (ABI) to give 114 labels (immediately before the third administration of infliximab), 115 labels (immediately before the eighth administration of infliximab), and 117 labels (control). All labeled test samples were mixed.
Similarly, changes in protein expression in test samples immediately before the third administration of infliximab and immediately before the sixth administration of infliximab in Behcet's disease patient 2 were also analyzed using the iTRAQ TM method. Peptides in each sample were modified with iTRAQ ™ reagent (ABI) to give 114 labels (immediately before the third administration of infliximab), 115 labels (immediately before the sixth administration of infliximab), and 117 labels (control). All labeled test samples were mixed.
混合試料中の多種類のペプチドを含む溶液に対し、LC/MSによるタンパク質の同定、定量を効率よく行うため、オフラインでの強陽イオン交換HPLCによりペプチド溶液を23分画した。それぞれのペプチドをナノLC-MS/MSシステムQSTAR(R)-XL（アプライドバイオシステムズ社）にて測定した。得られた23回分の測定データをProtein Pilot 2.0 (ABI社)にてSwiss Protに対してデータベースサーチを行い、タンパク質の同定と定量を行った。タンパク質の同定はデータベースサーチにより95%以上の信頼性を示すものを採用した。カットオフ値は、1.3倍以上、0.7倍以下とした。 In order to efficiently identify and quantify proteins by LC / MS for a solution containing many kinds of peptides in a mixed sample, the peptide solution was fractionated into 23 fractions by off-line strong cation exchange HPLC. Each peptide was measured by nano LC-MS / MS system QSTAR (R) -XL (Applied Biosystems). A database search was performed on Swiss Prot using Protein Pilot 2.0 (ABI) for the measurement data obtained 23 times, and protein identification and quantification were performed. For protein identification, a database search with a reliability of 95% or more was adopted. The cut-off value was 1.3 times or more and 0.7 times or less.
タンパク質は、混合試料のMS/MSスペクトルについて114標識, 115標識及び117標識された面積を算出して定量した。114標識（インフリキシマブ３回目投与直前、即ちベーチェット病患者血清で生物学的製剤投与により疾患活動性が低い状態）、又は117標識（対照）を分母にし、115標識（インフリキシマブ８回目投与直前、即ち114と同じベーチェット病患者で疾患活動性が高い状態）を分子にして比率をタンパク質毎に示した。 The protein was quantified by calculating the areas labeled 114, 115 and 117 for the MS / MS spectrum of the mixed sample. 114 label (immediately before the third dose of infliximab, i.e., low disease activity due to biologic administration in Behcet's disease patient serum), or 117 label (control) as the denominator, and 115 label (immediately before the eighth dose of infliximab, ie 114 The ratio is shown for each protein using the same Behcet's disease patient in a state of high disease activity) as a molecule.
４）結果
iTRAQTM解析結果を表１〜３に示した。混合した検査用試料中に88種類のタンパク質が同定された（表１）。同定されたタンパク質について解析した結果、ベーチェット病患者１について、インフリキシマブ３回目投与直前、即ち生物学的製剤投与により疾患活動性が低い状態のときに比べて、同剤８回目投与直前、即ち疾患活動性が高い状態では、LRGの量が、6.55倍に増加したことが確認された（表２：解析１）。また、同様にベーチェット病患者２について、インフリキシマブ３回目投与直前、即ち生物学的製剤投与により疾患活動性が低い状態のときに比べて、同剤６回目投与直前、即ち疾患活動性が高い状態では、LRG量が、4.79倍に増加したことが確認された（表３：解析２）。解析１及び解析２で用いた８回目投与直前及び６回目投与直前の検査用試料のLRG量は、対照と比較した結果、各々8.36倍及び8.10倍であった。
4) Results
The iTRAQ TM analysis results are shown in Tables 1-3. 88 types of proteins were identified in the mixed test samples (Table 1). As a result of the analysis of the identified protein, it was found that in Behcet's disease patient 1, immediately before the third administration of infliximab, that is, before the eighth administration of the same agent, that is, the disease activity, compared with the case where the disease activity is low due to the administration of the biologic agent It was confirmed that the amount of LRG increased 6.55 times in the state with high property (Table 2: Analysis 1). Similarly, in the case of Behcet's disease patient 2, in the state immediately before the third administration of infliximab, that is, when the disease activity is low due to the administration of the biological preparation, in the state where disease activity is high immediately before the sixth administration of the same agent. It was confirmed that the amount of LRG increased 4.79 times (Table 3: Analysis 2). As a result of comparison with the control, the amount of LRG in the test sample immediately before the eighth administration and immediately before the sixth administration used in Analysis 1 and Analysis 2 was 8.36 times and 8.10 times, respectively.
（実施例２）ベーチェット病患者の血清LRGの評価１（ウエスタンブロット法）
実施例１のインフリキシマブ治療を受けているベーチェット病患者２名及び健常人より採血し、実施例１の２）に示す方法と同手法にて検査用試料を調製した。各検査用試料を10μgずつ用いて、ウエスタンブロット法により血清LRG発現を評価した。一次抗体は抗LRG抗体(ウサギポリクローナル抗体、Protein Tech Group, INC)、二次抗体は抗ウサギIgG-抗体ＨＲＰ標識(ラクダポリクローナル抗体、GE healthcare社)を用いた。
(Example 2) Evaluation 1 of serum LRG of patients with Behcet's disease (Western blotting)
Blood samples were collected from two Behcet's disease patients who received infliximab treatment in Example 1 and healthy subjects, and a test sample was prepared by the same method as in Example 1 2). Serum LRG expression was evaluated by Western blotting using 10 μg of each test sample. The primary antibody was an anti-LRG antibody (rabbit polyclonal antibody, Protein Tech Group, INC), and the secondary antibody was an anti-rabbit IgG-antibody HRP label (camel polyclonal antibody, GE healthcare).
その結果、患者１では薬剤投与６回目及び８回目直前の検査用試料で、投与前及び３回目投与直前に比べてLRGの濃いバンドが認められ、患者２についても薬剤投与６回目〜８回目直前の検査用試料では、投与前及び３回目投与直前に比べてLRGの濃いバンドが認められた（図１）。各々についてCRPの検査も並行して行った。これにより、LRG発現量はCRP値及び疾患活動性とほぼ同様の動態を示すことが確認された。 As a result, in patient 1, the test sample immediately before the 6th and 8th administration of the drug showed a band with a higher LRG than before the administration and just before the 3rd administration, and for patient 2, the 6th to 8th immediately before the administration of the drug. In the test sample, a band with a deeper LRG was observed than before administration and immediately before the third administration (FIG. 1). Each was also tested for CRP. As a result, it was confirmed that the LRG expression level showed almost the same kinetics as the CRP value and disease activity.
（実施例３）ベーチェット病患者の血清LRGの評価２（ウエスタンブロット法）
インフリキシマブ治療を受けているベーチェット病患者５名及び健常人より採血し、実施例１の２）に示す方法と同手法にて検査用試料を調製した。各検査用試料を10μgずつ用いて、実施例２と同手法によりウエスタンブロット法により血清LRG発現を評価した。
(Example 3) Evaluation of serum LRG of Behcet's disease patients 2 (Western blotting)
Blood samples were collected from 5 patients with Behcet's disease who received infliximab treatment and healthy individuals, and test samples were prepared by the same method as shown in 2) of Example 1. Serum LRG expression was evaluated by Western blotting in the same manner as in Example 2 using 10 μg of each test sample.
患者３〜７について評価した結果、患者３では薬剤投与６回目及び８回目直前の検査用試料で、投与前及び３回目投与直前に比べてLRGの濃いバンドが認められ、患者４では薬剤投与３回目〜６回目直前の検査用試料で、投与前に比べてLRGの濃いバンドが認められた。また、患者５〜７では、LRGの濃いバンドは認められなかった（図２）。各々CRPの検査も並行して行った。これにより、LRG発現量はCRP値及び疾患活動性とほぼ同様の動態を示すことが確認された。 As a result of evaluation for patients 3 to 7, in patient 3, a test sample immediately before the sixth and eighth administrations of the drug showed a band with a higher LRG than before administration and immediately before the third administration. In the test sample immediately before the 6th time, a band with a deeper LRG was observed than before the administration. In patients 5 to 7, no dense band of LRG was observed (FIG. 2). Each CRP was also tested in parallel. As a result, it was confirmed that the LRG expression level showed almost the same kinetics as the CRP value and disease activity.
（実施例４）関節リウマチ患者の血清LRGの評価１（ウエスタンブロット法）
インフリキシマブ治療を受けている関節リウマチ患者（RA患者）４名及び健常人より採血し、実施例１の２）に示す方法と同手法にて検査用試料を調製した。採血は、インフリキシマブ投与直後、及び投与後１年目に行った。各検査用試料を10μgずつ用いて、実施例２と同手法によりウエスタンブロット法により血清LRG発現を評価した。
(Example 4) Evaluation 1 of serum LRG of patients with rheumatoid arthritis (Western blotting)
Blood samples were collected from 4 rheumatoid arthritis patients (RA patients) who received infliximab treatment and healthy subjects, and test samples were prepared by the same method as shown in 2) of Example 1. Blood collection was performed immediately after administration of infliximab and one year after the administration. Serum LRG expression was evaluated by Western blotting in the same manner as in Example 2 using 10 μg of each test sample.
RA患者１〜４について評価した結果、インフリキシマブ治療が有効であった患者１及び２では、いずれの場合もLRGの濃いバンドは認めなかったのに対し、インフリキシマブ治療が無効のRA患者３及び４ではLRGの濃いバンドを認めた（図３）。各々CRP及び慢性関節リウマチで早期から上昇する血中の関節破壊バイオマーカーであるマトリックスメタロプロテアーゼ-3(matrix metalloproteinase-3: MMP-3)の検査も並行して行った。RA患者４は、インフリキシマブ治療が無効で疾患活動性が高いにもかかわらず、CRP及びMMP-3の発現量は低く抑えられていたが、LRG発現量は、疾患活動性とほぼ同様の動態を示した。これにより、LRGはインフリキシマブを投与したときの治療効果を評価しうる優れたバイオマーカーともなり得ることが確認された。疾患活動性は、関節症状、CRP値とMMP3値などの評価により行った。ここで、CRP値は<0.2が正常値、MMP-3は男性で<120、女性で<60が正常範囲と判断した。 As a result of evaluating RA patients 1 to 4, patients 1 and 2 in which infliximab treatment was effective did not show a strong band of LRG in either case, whereas in RA patients 3 and 4 in which infliximab treatment was ineffective A dark band of LRG was observed (FIG. 3). In parallel, tests of matrix metalloproteinase-3 (MMP-3), which are biomarkers of joint destruction in blood, which are elevated from early in each case of CRP and rheumatoid arthritis, were also performed. Although RA patient 4 was ineffective with infliximab treatment and had high disease activity, the expression level of CRP and MMP-3 was kept low, but the expression level of LRG was almost the same as that of disease activity. Indicated. Thus, it was confirmed that LRG can be an excellent biomarker that can evaluate the therapeutic effect when infliximab is administered. Disease activity was evaluated by evaluating joint symptoms, CRP value and MMP3 value. Here, the CRP value was judged to be <0.2 for normal, MMP-3 for male <120, and female <60 for normal range.
（実施例５）ウエスタンブロット法による血清LRGの評価２（ウエスタンブロット法）
エタネルセプト（可溶性TNF受容体製剤）治療を受けている関節リウマチ患者（RA患者）５名及び健常人より採血し、実施例１の２）に示す方法と同手法にて検査用試料を調製した。採血は、インフリキシマブ投与直後、及び投与後１年目に行った。各検査用試料を10μgずつ用いて、実施例４と同手法によりウエスタンブロット法により血清LRG発現を評価した。
(Example 5) Evaluation of serum LRG by Western blotting 2 (Western blotting)
Blood samples were collected from 5 patients with rheumatoid arthritis (RA patients) who received etanercept (soluble TNF receptor preparation) treatment and healthy subjects, and a test sample was prepared by the same method as shown in 2) of Example 1. Blood collection was performed immediately after administration of infliximab and one year after the administration. Serum LRG expression was evaluated by Western blotting in the same manner as in Example 4 using 10 μg of each test sample.
RA患者５〜９について評価した結果、エタネルセプト治療が有効であった患者５〜６では、いずれの場合もLRGの濃いバンドは認められなかったのに対し、エタネルセプト治療が無効の患者７〜９ではLRGの濃いバンドを認めた（図４）。各々CRP及びMMP-3の検査も並行して行った。LRG発現量は、各種マーカー及び疾患活動性とほぼ同様の動態を示し、エタネルセプト抵抗性関節リウマチ患者では、LRGが高発現することが確認された。これにより、LRG発現量の検査は、エタネルセプトを投与したときの治療効果を評価しうる優れたバイオマーカーともなり得ることが確認された。疾患活動性は、関節症状、CRP値とMMP3値などの評価により行った。ここで、CRP値は<0.2が正常値、MMP-3は男性で<120、女性で<60が正常範囲と判断した。 As a result of evaluating RA patients 5 to 9, patients 5 to 6 in which etanercept treatment was effective showed no LRG-dense band in all cases, whereas patients 7 to 9 in whom etanercept treatment was ineffective A dark band of LRG was observed (FIG. 4). Each of CRP and MMP-3 was also tested in parallel. The expression level of LRG showed almost the same kinetics as various markers and disease activity, and it was confirmed that LRG is highly expressed in patients with etanercept-resistant rheumatoid arthritis. Thus, it was confirmed that the LRG expression level test can be an excellent biomarker that can evaluate the therapeutic effect when etanercept is administered. Disease activity was evaluated by evaluating joint symptoms, CRP value and MMP3 value. Here, the CRP value was determined to be <0.2 for normal, MMP-3 for male <120, and female <60 for normal range.
（実施例６）ELISA法による血清LRGの評価（ELISA法）
インフリキシマブ治療前のクローン病患者（CD患者）12名（治療有効例8名、治療無効例4名）より採血し、血清を調製した。血清は500倍に希釈し、血清中LRG濃度をELISA法により測定した。ELISAにはHuman LRG Assay Kit (IBL社)を使用した。
(Example 6) Evaluation of serum LRG by ELISA method (ELISA method)
Serum was collected from 12 patients with Crohn's disease (CD patients) before treatment with infliximab (8 effective patients and 4 ineffective patients). Serum was diluted 500 times and the serum LRG concentration was measured by ELISA. Human LRG Assay Kit (IBL) was used for ELISA.
その結果、血清中LRG濃度はインフリキシマブ治療有効例に比べ、インフリキシマブ治療無効例で有意に高値を示した（図５）。これにより、LRG発現量の検査は、クローン病においてインフリキシマブ治療奏功性を評価（又は予測）し得るバイオマーカーともなり得ることが確認された。 As a result, the serum LRG concentration was significantly higher in the ineffective patients treated with infliximab than in the treated patients treated with infliximab (FIG. 5). Thereby, it was confirmed that the test | inspection of the expression level of LRG can also become a biomarker which can evaluate (or predict) the efficacy of infliximab treatment in Crohn's disease.
（参考例１）IL-6刺激の有無による顆粒球中のLRG量
健常人より10 mL採血し、密度勾配遠心法により顆粒球を精製した。20 ng/mL IL-6(PeproTech社)による刺激の有無で顆粒球を2時間培養した。培養後、顆粒球よりRneasy mini kit(キアゲン社)にてRNAを抽出し、QuantitectTM (キアゲン社)を用いてRT-PCR法によりLRGの発現を確認した。
Reference Example 1 Amount of LRG in granulocytes with or without IL-6 stimulation 10 mL of blood was collected from healthy individuals, and granulocytes were purified by density gradient centrifugation. Granulocytes were cultured for 2 hours with or without stimulation with 20 ng / mL IL-6 (PeproTech). After culturing, RNA was extracted from granulocytes with an Rneasy mini kit (Qiagen), and the expression of LRG was confirmed by RT-PCR using Quantitect ™ (Qiagen).
その結果、LRGは顆粒球で発現することも報告されているが、LRGの発現はIL-6刺激による影響はないことが確認された（図６）。自己免疫疾患のバイオマーカーとして公知のCRPは、炎症に伴って上昇するIL-6などの炎症性サイトカインによって肝臓で合成される急性期タンパク質のひとつであるので、LRGはCRPとは異なるメカニズムにより発現するバイオマーカーであることが確認された。即ち、LRGはIL-6の影響を受けにくいことから、より正確な自己免疫疾患検査用バイオマーカーとして機能しうる。例えばインフリキシマブやエタネルセプト等の生物学的製剤の投与により、IL-6が抑制され、その結果CRPの発現が抑制されるような場合でも、LRGはより正確に疾患活動性を評価しうるバイオマーカーとなりうることが示唆された。 As a result, LRG was also reported to be expressed in granulocytes, but it was confirmed that the expression of LRG was not affected by IL-6 stimulation (FIG. 6). CRP, known as a biomarker for autoimmune diseases, is one of the acute phase proteins synthesized in the liver by inflammatory cytokines such as IL-6 that increase with inflammation, so LRG is expressed by a mechanism different from CRP. It was confirmed that this is a biomarker. That is, since LRG is not easily affected by IL-6, it can function as a more accurate biomarker for autoimmune disease testing. For example, even when biological preparations such as infliximab and etanercept suppress IL-6 and consequently suppress CRP expression, LRG is a biomarker that can more accurately assess disease activity. It was suggested that
以上詳述したように、本発明の自己免疫疾患検査用バイオマーカーであるLRGは、自己免疫疾患、とりわけベーチェット病、キャッスルマン氏病、クローン病又は関節リウマチの検査用バイオマーカーとなりうる。LRGをバイオマーカーとして、検出又は定量することにより、上記自己免疫疾患の検査を行うことができ、疾患活動性を評価することができる。
また生物学的製剤などの自己免疫疾患治療薬を投与したときの治療効果を評価しうるバイオマーカーともなる。具体的には、インフリキシマブ及び／又はエタネルセプトなどのTNF-αを阻害する生物学的製剤を投与した場合の疾患活動性を、本発明のバイオマーカーにより評価することができる。これにより、上記生物学的製剤に対して、薬剤の有効性を確認したり、薬剤抵抗性を有する患者を早期に発見することができる。その結果、例えば薬剤投与終了時の判断や投与薬剤の変更などに対して、最も効果的な方法を選択することができる。例えば生物学的製剤などの高価な治療薬を投与しても、治療効果が認められない場合などは、他の治療方法を早期に模索することができ、患者自身にとっても有用のみならず、医療経済を考慮しても優れた効果を有する。
As described in detail above, LRG, which is a biomarker for testing autoimmune diseases of the present invention, can be a biomarker for testing autoimmune diseases, particularly Behcet's disease, Castleman's disease, Crohn's disease, or rheumatoid arthritis. By detecting or quantifying LRG as a biomarker, the autoimmune disease can be examined, and disease activity can be evaluated.
It is also a biomarker that can evaluate the therapeutic effect when a therapeutic drug for an autoimmune disease such as a biologic is administered. Specifically, disease activity when a biological preparation that inhibits TNF-α such as infliximab and / or etanercept is administered can be evaluated by the biomarker of the present invention. Thereby, the effectiveness of a drug can be confirmed against the above-mentioned biological preparation, or a patient having drug resistance can be discovered at an early stage. As a result, for example, it is possible to select the most effective method for determination at the end of drug administration, change of administered drug, and the like. For example, if a therapeutic effect is not observed even after administration of an expensive therapeutic agent such as a biologic, other treatment methods can be sought at an early stage. Even if economy is considered, it has an excellent effect.
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