JP6816876B2 - Epitopes recognized by autoantibodies to Ro52 / TRIM21 protein and their utilization - Google Patents
Epitopes recognized by autoantibodies to Ro52 / TRIM21 protein and their utilization Download PDFInfo
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- JP6816876B2 JP6816876B2 JP2016220793A JP2016220793A JP6816876B2 JP 6816876 B2 JP6816876 B2 JP 6816876B2 JP 2016220793 A JP2016220793 A JP 2016220793A JP 2016220793 A JP2016220793 A JP 2016220793A JP 6816876 B2 JP6816876 B2 JP 6816876B2
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- trim21
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- amino acid
- interstitial pneumonia
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Description
本発明は、間質性肺炎の発症、その重症度の診断・予測に関する。本発明は、ライフサイエンス、および医療の分野で有用である。 The present invention relates to the diagnosis and prediction of the onset of interstitial pneumonia and its severity. The present invention is useful in the fields of life science and medicine.
間質性肺炎は肺の間質組織の線維化が起こる疾患の総称である。進行して炎症組織が線維化したものは肺線維症と呼ばれる。間質性肺炎は、薬剤性肺炎や放射性肺炎など、医原性原因によるもの、粉塵の吸入や過敏性肺炎など、職業・環境性因子によるもの、膠原病(全身の膠原線維にフィブリノイド変性を来し、その原因として自己免疫現象が考えられている疾患群)やサルコイドーシスなどの全身性疾患に付随して発病するものなど、その原因は様々であることが知られている。その他、背景となる疾患がなく、原因も特定できない特発性間質性肺炎がある。 Interstitial pneumonia is a general term for diseases in which fibrosis of the interstitial tissue of the lung occurs. Progressive fibrosis of inflamed tissue is called pulmonary fibrosis. Interstitial pneumonitis is caused by iatrogenic causes such as drug-induced pneumonitis and radioactive pneumonitis, by occupational / environmental factors such as inhalation of dust and hypersensitivity pneumonitis, and collagen disease (fibrinoid degeneration in collagen fibers throughout the body). However, it is known that there are various causes such as (a group of diseases in which an autoimmune phenomenon is considered as the cause) and those associated with systemic diseases such as sarcoidosis. In addition, there is idiopathic interstitial pneumonia for which there is no background disease and the cause cannot be identified.
間質性肺炎の体外診断用医薬品として、1999年にKL-6が保険適用された。KL-6抗体は、II型肺胞上皮細胞上に発現するMUC1を認識する抗体である。すなわち、KL-6は、200 kDa以上の分子量をもつ巨大な糖タンパク質であるMUC1の一部と考えられている。血清KL-6値は、特発性肺線維症、膠原病関連間質性肺炎、過敏性肺炎、放射性肺炎などの間質性肺炎で高値を示し、健常者や細菌性肺炎、肺気腫などのその他の肺疾患ではほとんど上昇しない。 In 1999, KL-6 was insured as an in-vitro diagnostic drug for interstitial pneumonia. The KL-6 antibody is an antibody that recognizes MUC1 expressed on type II alveolar epithelial cells. That is, KL-6 is considered to be part of MUC1, a giant glycoprotein with a molecular weight of 200 kDa or more. Serum KL-6 levels are high in interstitial pneumonia such as idiopathic pulmonary fibrosis, collagen disease-related interstitial pneumonia, hypersensitivity pneumonitis, and radioactive pneumonia, and other healthy subjects and other patients such as bacterial pneumonia and emphysema. It hardly increases in lung disease.
一方、Ro52は、近年ではtripartite motif containing 21(TRIM21)とも表記されるが、分子量約52KDの細胞内タンパク質であり、Ring finger、B Box、Coiled Coil(C-C)、およびPRYSPRYRYの各ドメインを有する分子である。この分子に対する自己抗体は、シェーグレン症候群(SjS)、全身性エリテマトーデス(SLE)等の自己免疫疾患の患者の血清中に広く認められる。 On the other hand, Ro52, which is also described as tripartite motif containing 21 (TRIM21) in recent years, is an intracellular protein having a molecular weight of about 52 KD, and is a molecule having each domain of Ring finger, B Box, Coiled Coil (CC), and PRYSPRYRY. Is. Autoantibodies to this molecule are widely found in the sera of patients with autoimmune diseases such as Sjogren's syndrome (SjS) and systemic lupus erythematosus (SLE).
マウスにヒトのRo52タンパク質を免疫した後にハイブリドーマを作成し、ヒトRo52タンパク質に対するモノクローナル抗抗体を作成した報告がある(非特許文献1)。また、SjSやSLEの患者血清(ポリクローナル抗体)を用いたエピトープの検索が行われてきている(非特許文献2)。さらに抗Ro52抗体と自己免疫疾患との関係に関する報告(非特許文献3)、抗Ro52/TRIM21抗体が、膠原病の一つである強皮症患者における間質性肺炎の合併率と重症度に関連するという報告がある(非特許文献4) There is a report that a hybridoma was prepared after immunizing a mouse with a human Ro52 protein to prepare a monoclonal anti-antibody against the human Ro52 protein (Non-Patent Document 1). In addition, epitopes have been searched for using SjS and SLE patient sera (polyclonal antibodies) (Non-Patent Document 2). Furthermore, a report on the relationship between anti-Ro52 antibody and autoimmune disease (Non-Patent Document 3), anti-Ro52 / TRIM21 antibody was used for the complication rate and severity of interstitial pneumonia in scleroderma patients, which is one of the collagen diseases. There is a report that it is related (Non-Patent Document 4)
これまでのRo52抗体と自己免疫疾患との関連についての報告では、抗原としてRo52タンパク質全体を用いていたため、患者血清中の様々な抗体が結合可能であり、有用な情報が得られなかった。間質性肺炎に関連する自己抗体のエピトープが特定できれば、間質性肺炎の発症メカニズムの研究や間質性肺炎の診断において有用であり、また間質性肺炎の発症の予測や、重症度の予測・診断への適用が期待できる。 In previous reports on the relationship between Ro52 antibody and autoimmune disease, since the entire Ro52 protein was used as an antigen, various antibodies in patient serum could bind, and useful information could not be obtained. If the epitope of the autoantibody associated with interstitial pneumonia can be identified, it will be useful in studying the onset mechanism of interstitial pneumonia and in diagnosing interstitial pneumonia. Expected to be applied to prediction and diagnosis.
本発明者らは、間質性肺炎を有する自己免疫疾患の患者血液から、本発明者らの所属する研究室で確立された方法(Immunospot-array assay on a chip、ISSAC法、Jin et al, Nature Medicine 2009;15:1088-1092)を用いて、抗Ro52/TRIM21抗体を産生するリンパ球を複数特定し、各々の抗体cDNAをクローニングし、複数の抗Ro52/TRIM21抗体をヒトモノクローナル抗体として得ることに成功した。そして、得られた抗体の多くがRo52/TRIM21タンパク質のC-Cドメインに結合することを明らかにし、さらに詳細なエピトープの解析を試みた。その結果、得られたモノクローナル抗体が認識するペプチドを特定し、さらに当該ペプチドに結合する自己抗体を有する患者群において間質性肺炎の発症率や重症度を比較することにより、本願発明を完成した。 The present inventors have established a method (Immunospot-array assay on a chip, ISSAC method, Jin et al,) from the blood of a patient with an autoimmune disease having interstitial pneumonia in the laboratory to which the present inventors belong. Using Nature Medicine 2009; 15: 1088-1092), multiple lymphocytes producing anti-Ro52 / TRIM21 antibody are identified, each antibody cDNA is cloned, and multiple anti-Ro52 / TRIM21 antibodies are obtained as human monoclonal antibodies. I succeeded in doing so. Then, it was clarified that most of the obtained antibodies bind to the C-C domain of the Ro52 / TRIM21 protein, and a more detailed analysis of the epitope was attempted. As a result, the present invention was completed by identifying the peptide recognized by the obtained monoclonal antibody and comparing the incidence and severity of interstitial pneumonia in a group of patients having an autoantibody that binds to the peptide. ..
本願は、以下を提供する。
[1] 対象における間質性肺炎の、診断、発症の予測、重症度の診断、および重症化の予測からなる群より選択される少なくとも一つのための方法であって、Ro52/TRIM21タンパク質に対する抗体が対象から得られた試料中に存在するかどうかを決定する工程を含む、方法。
[2] 対象が、自己免疫疾患の患者である、1に記載の方法。
[3] 自己免疫疾患が、膠原病である、2に記載の方法。
[4] 膠原病が、関節リウマチ、全身性強皮症、皮膚筋炎、多発性筋炎、混合性結合組織病 (mixed connective tissue disease :MCTD)からなる群より選択されるいずれかである、3に記載の方法。
[5] Ro52/TRIM21タンパク質に対する抗体が、Ro52/TRIM21タンパク質のC-Cドメインに対するものである、1〜5のいずれか1項に記載の方法。
[6] Ro52/TRIM21タンパク質に対する抗体が、配列番号23のアミノ酸配列の全部、または少なくとも9アミノ酸長の一部からなるペプチドに対するものである、1〜5のいずれか1項に記載の方法。
[7] Ro52/TRIM21タンパク質を認識可能な、ヒトモノクローナル抗体。
[8] Ro52/TRIM21タンパク質のC-Cドメインを認識可能である、7に記載の抗体。
[9] 配列番号23のアミノ酸配列の全部、または少なくとも9アミノ酸長の一部からなるペプチド。
[10] 9に記載のペプチドに対する、抗体。
[11] 7〜10のいずれか1項に記載された抗体またはペプチドを含む、間質性肺炎の検査キット。
[12] 7〜10のいずれか1項に記載された抗体またはペプチドを含む、医薬組成物。
The present application provides the following.
[1] A method for at least one selected from the group consisting of diagnosis, prediction of onset, diagnosis of severity, and prediction of severity of interstitial pneumonia in a subject, which is an antibody against the Ro52 / TRIM21 protein. A method comprising the step of determining if is present in a sample obtained from a subject.
[2] The method according to 1, wherein the subject is a patient with an autoimmune disease.
[3] The method according to 2, wherein the autoimmune disease is collagen disease.
[4] Collagen disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, dermatomyositis, polymyositis, and mixed connective tissue disease (MCTD). The method described.
[5] The method according to any one of 1 to 5, wherein the antibody against the Ro52 / TRIM21 protein is against the CC domain of the Ro52 / TRIM21 protein.
[6] The method according to any one of 1 to 5, wherein the antibody against the Ro52 / TRIM21 protein is against a peptide consisting of the entire amino acid sequence of SEQ ID NO: 23 or a part of at least 9 amino acid lengths.
[7] A human monoclonal antibody that can recognize the Ro52 / TRIM21 protein.
[8] The antibody according to 7, which can recognize the CC domain of the Ro52 / TRIM21 protein.
[9] A peptide consisting of the entire amino acid sequence of SEQ ID NO: 23, or a part of at least 9 amino acid lengths.
[10] An antibody against the peptide according to 9.
[11] A test kit for interstitial pneumonia containing the antibody or peptide according to any one of 7 to 10.
[12] A pharmaceutical composition comprising the antibody or peptide according to any one of 7 to 10.
本発明により、間質性肺炎に関連した、Ro52/TRIM21タンパク質に対する自己抗体が認識するエピトープが提供される。
本発明により、間質性肺炎の診断、発症の予測、および/または重症度の予測のための方法が提供される。
The present invention provides epitopes recognized by autoantibodies to the Ro52 / TRIM21 protein associated with interstitial pneumonia.
The present invention provides methods for diagnosing interstitial pneumonia, predicting its onset, and / or predicting its severity.
本発明および明細書で「 - 」で範囲を表す場合は、特に記載した場合を除き、その範囲は両端の値を含む。疾患または状態に関連して「処置」というときは、特に記載した場合を除き、発症リスクの低減、予防、治療、および進行の抑制を含む。 When the range is represented by "-" in the present invention and the specification, the range includes the values at both ends unless otherwise specified. The term "treatment" in relation to a disease or condition includes reduction, prevention, treatment, and suppression of progression, unless otherwise stated.
[抗Ro52/TRIM21抗体のエピトープ]
本発明は、間質性肺炎の患者の血清に存在するRo52/TRIM21タンパク質に対する自己抗体が認識するエピトープ、すなわち抗Ro52/TRIM21抗体が認識可能なペプチドを提供する。このようなペプチドは、具体的には、配列番号23のアミノ酸配列の全部、または一部からなるペプチドである。
[Epitope of anti-Ro52 / TRIM21 antibody]
The present invention provides an epitope recognized by an autoantibody against the Ro52 / TRIM21 protein present in the serum of a patient with interstitial pneumonia, i.e. a peptide recognizable by an anti-Ro52 / TRIM21 antibody. Specifically, such a peptide is a peptide consisting of all or part of the amino acid sequence of SEQ ID NO: 23.
配列番号23のアミノ酸配列からなるペプチドは、ヒトRo52/TRIM21タンパク質のC-Cドメインの一部であり、抗Ro52/TRIM21抗体価強陽性である間質性肺炎を発症した皮膚筋炎患者の末梢血中の抗Ro52/TRIM21自己抗体を解析することにより、本発明者らにより初めて特定されたものである。 The peptide consisting of the amino acid sequence of SEQ ID NO: 23 is part of the CC domain of the human Ro52 / TRIM21 protein and is in the peripheral blood of patients with dermatomyositis who developed interstitial pneumonia with strong anti-Ro52 / TRIM21 antibody titers. It was first identified by the present inventors by analyzing anti-Ro52 / TRIM21 autoantibodies.
配列番号23のアミノ酸配列の一部からなるペプチドの長さは、自己抗体により認識される限り、特に限定されないが、例えば少なくとも9アミノ酸長であればよく、10アミノ酸長、11アミノ酸長、12アミノ酸長、13アミノ酸長、14アミノ酸長、15アミノ酸長であってもよい。 The length of the peptide consisting of a part of the amino acid sequence of SEQ ID NO: 23 is not particularly limited as long as it is recognized by the autoantibodies, but may be at least 9 amino acids, for example, 10 amino acids, 11 amino acids, and 12 amino acids. It may be long, 13 amino acids long, 14 amino acids long, or 15 amino acids long.
このようなペプチドは、当業者には周知の方法によって調製することができる。 Such peptides can be prepared by methods well known to those of skill in the art.
ペプチドは、後述するように、対象における間質性肺炎の、診断、発症の予測、重症度の診断、および重症化の予測からなる群より選択される少なくとも一つのための方法において、対象から得られた試料中に、Ro52/TRIM21タンパク質に対する抗体が存在するか否かを決定する際に、抗原として用いることができる。 Peptides are obtained from a subject in a method for at least one selected from the group consisting of diagnosis, prediction of onset, diagnosis of severity, and prediction of severity of interstitial pneumonia in a subject, as described below. It can be used as an antigen in determining whether or not an antibody against the Ro52 / TRIM21 protein is present in the sample.
[Ro52/TRIM21タンパク質またはその一部に対する抗体]
本発明はRo52/TRIM21タンパク質を認識可能な、ヒトモノクローナル抗体を提供する。Ro52/TRIM21は、単にRo52、またはTRIM21(tripartite motif containing 21)と表記されることもある。Ro52/TRIM21タンパク質は、分子量約52KDの細胞内タンパク質であり、Ring finger、B Box、Coiled Coil(C-C)、およびPRYSPRYRYの各ドメインを有する。このタンパク質に対する自己抗体は、シェーグレン症候群(SjS)、全身性エリテマトーデス(SLE)等の自己免疫疾患の患者の血清中に広く認められる。
[Antibodies to Ro52 / TRIM21 protein or a part thereof]
The present invention provides a human monoclonal antibody capable of recognizing the Ro52 / TRIM21 protein. Ro52 / TRIM21 may be simply referred to as Ro52 or TRIM21 (tripartite motif containing 21). The Ro52 / TRIM21 protein is an intracellular protein with a molecular weight of about 52 KD and has the Ring finger, B Box, Coiled Coil (CC), and PRYSPRYRY domains. Autoantibodies to this protein are widely found in the sera of patients with autoimmune diseases such as Sjogren's syndrome (SjS) and systemic lupus erythematosus (SLE).
ヒト抗体とは、ヒトから得た抗体、およびヒトから得た抗体と同じアミノ酸配列を有する抗体をいう。ヒトから得た抗体には、患者の血清から得た抗体、およびヒトリンパ球が産生した抗体が含まれる。ヒトから得た抗体と同じアミノ酸配列を有する抗体には、例えば、ヒトから得た抗体のcDNAをクローニングし、得られたcDNAを導入した継代培養可能な細胞が産生した抗体が含まれる。継代培養可能な細胞は、非ヒト細胞(例えば、非ヒト哺乳動物細胞、具体的にはマウス細胞、ラット細胞、ハムスター細胞等)であっても、ヒト細胞であってもよい。 The human antibody refers to an antibody obtained from a human and an antibody having the same amino acid sequence as an antibody obtained from a human. Antibodies obtained from humans include antibodies obtained from patient sera and antibodies produced by human lymphocytes. Antibodies having the same amino acid sequence as antibodies obtained from humans include, for example, antibodies produced by subcultureable cells obtained by cloning the cDNA of an antibody obtained from humans and introducing the obtained cDNA. The subcultureable cells may be non-human cells (eg, non-human mammalian cells, specifically mouse cells, rat cells, hamster cells, etc.) or human cells.
認識されるRo52/TRIM21タンパク質は、好ましくはヒト由来である。配列表には、配列番号60として、ヒトRo52/TRIM21タンパク質のアミノ酸配列を示した。 The recognized Ro52 / TRIM21 protein is preferably of human origin. In the sequence listing, the amino acid sequence of the human Ro52 / TRIM21 protein is shown as SEQ ID NO: 60.
前掲非特許文献1により、マウスにヒトのRo52/TRIM21タンパク質を免疫した後に、ハイブリドーマを作成してヒトのRo52/TRIM21タンパク質を認識可能なマウスモノクローナル抗体を作成したことが報告されている。しかしながら、Ro52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体を提供するのは、本願が初めてである。マウス抗体をヒトに投与する場合、アナフィラキシー反応や効果の減弱が懸念される。本願によって提供されるRo52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体は、このような懸念が少ない。この観点からは、対象における間質性肺炎の診断等のみならず、ヒトの疾患または状態の処置に応用する際にも有用だと考えられる。 According to Non-Patent Document 1 described above, it is reported that after immunizing a mouse with a human Ro52 / TRIM21 protein, a hybridoma was prepared to prepare a mouse monoclonal antibody capable of recognizing the human Ro52 / TRIM21 protein. However, this application is the first to provide a human monoclonal antibody that can recognize the Ro52 / TRIM21 protein. When mouse antibodies are administered to humans, there are concerns about anaphylactic reactions and diminished effects. Human monoclonal antibodies capable of recognizing the Ro52 / TRIM21 protein provided by the present application have less such concern. From this point of view, it is considered to be useful not only for the diagnosis of interstitial pneumonia in the subject but also for the treatment of human diseases or conditions.
Ro52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体は、好ましくは間質性肺炎患者のBリンパ球が産生する抗体、またはそれと同じアミノ酸配列を有する抗体である。このような抗体は、マウス等の実験動物を人工的に過剰の抗原で免疫して得られる抗体よりも、高親和性である可能性が高いからである。後述するように、ISSAC法を用いることにより、患者由来のリンパ球群から、目的の抗原に対する抗体を産生する細胞を特定し、抗体遺伝子をクローニングすることができる。 A human monoclonal antibody that can recognize the Ro52 / TRIM21 protein is preferably an antibody produced by B lymphocytes of a patient with interstitial pneumonia, or an antibody having the same amino acid sequence. This is because such an antibody is likely to have a higher affinity than an antibody obtained by artificially immunizing an experimental animal such as a mouse with an excess antigen. As will be described later, by using the ISSAC method, cells producing an antibody against a target antigen can be identified from a group of lymphocytes derived from a patient, and the antibody gene can be cloned.
Ro52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体のRo52/TRIM21タンパク質における結合部位は、抗体が目的のために有用である限り、特に限定されないが、Ro52/TRIM21タンパク質のC-Cドメインに結合することが好ましい。本発明者らの検討によると、間質性肺炎の患者またはそのリスクの高い者の血清由来の抗Ro52/TRIM21抗体の多くがC-Cドメインに結合可能であり、抗C-Cドメイン自己抗体が間質性肺炎と何らかの関わりがあると考えられるからである。配列表には、配列番号61として、ヒトRo52/TRIM21タンパク質のC-Cドメインのアミノ酸配列(配列番号60のRo52/TRIM21タンパク質のアミノ酸配列において、130番-255番の部分に相当する。)を示した。 The binding site of a human monoclonal antibody that can recognize the Ro52 / TRIM21 protein in the Ro52 / TRIM21 protein is not particularly limited as long as the antibody is useful for the purpose, but it is preferable to bind to the CC domain of the Ro52 / TRIM21 protein. .. According to our study, many of the serum-derived anti-Ro52 / TRIM21 antibodies of patients with interstitial pneumonia or those at high risk thereof can bind to the CC domain, and the anti-CC domain autoantibodies are interstitial. This is because it is thought to have something to do with pneumonia. In the sequence listing, the amino acid sequence of the CC domain of the human Ro52 / TRIM21 protein (corresponding to the part 130-255 in the amino acid sequence of the Ro52 / TRIM21 protein of SEQ ID NO: 60) is shown as SEQ ID NO: 61. ..
Ro52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体のRo52/TRIM21タンパク質における結合部位は、Ro52/TRIM21タンパク質のC-Cドメイン中の特定の部分、具体的には配列番号60において177番-192番のアミノ酸配列に相当する16アミノ酸長からなる部分の全部(本明細書においては、P23と称されることがある。)または一部からなる部分に結合することが好ましい。配列表には、配列番号23として、本発明者らが見出した、ヒトRo52/TRIM21タンパク質のC-Cドメインにおいて疾患と特に強く関連付けられる16アミノ酸長のアミノ酸配列(配列番号60において、177番-192番のアミノ酸配列に相当する。)を示した。配列番号23のアミノ酸配列の一部からなるペプチドの長さは、自己抗体により認識される限り、特に限定されないが、例えば少なくとも9アミノ酸長であってよく、10アミノ酸長、11アミノ酸長、12アミノ酸長、13アミノ酸長、14アミノ酸長、15アミノ酸長であってもよい。 The binding site of a human monoclonal antibody that can recognize the Ro52 / TRIM21 protein in the Ro52 / TRIM21 protein is a specific portion of the Ro52 / TRIM21 protein in the CC domain, specifically the amino acid sequence 177-192 in SEQ ID NO: 60. It is preferable to bind to the whole (sometimes referred to as P23 in the present specification) or part of the 16 amino acid length portion corresponding to. In the sequence listing, the 16 amino acid length amino acid sequence found by the present inventors as SEQ ID NO: 23, which is particularly strongly associated with the disease in the CC domain of the human Ro52 / TRIM21 protein (Nos. 177-192 in SEQ ID NO: 60). Corresponds to the amino acid sequence of.). The length of the peptide consisting of a part of the amino acid sequence of SEQ ID NO: 23 is not particularly limited as long as it is recognized by the autoantibody, but may be, for example, at least 9 amino acids, 10 amino acids, 11 amino acids, and 12 amino acids. It may be long, 13 amino acids long, 14 amino acids long, or 15 amino acids long.
ある抗体が、Ro52/TRIM21タンパク質、そのC-Cドメイン、またはP23もしくはその一部を認識可能かどうかは、当業者であれば適切な免疫アッセイ、例えばELISAを企画し、評価することができる。 Whether an antibody can recognize the Ro52 / TRIM21 protein, its C-C domain, or P23 or a portion thereof can be evaluated by one of ordinary skill in the art by planning and assessing an appropriate immunoassay, such as ELISA.
ヒトモノクローナル抗体は、当業者であれば、従来法で製造することができる。従来法には、ハイブリドーマ法、大腸菌を用いるファージディスプレイ法、EBウイルス法等があり、必要に応じ、マウス抗体からヒト抗体へのヒト化、ファージディスプレイ法を用いての高活性化を、組み合わせることができる。 The human monoclonal antibody can be produced by a person skilled in the art by a conventional method. Conventional methods include a hybridoma method, a phage display method using Escherichia coli, an EB virus method, etc., and if necessary, humanization from a mouse antibody to a human antibody and high activation using a phage display method are combined. Can be done.
ヒトモノクローナル抗体の製造のための好ましい方法の一つは、ISSAC(Immunospot-array assay on a chip)法を利用した方法である。ISSAC法であれば、頻度が高い抗原特異的リンパ球は勿論のこと、頻度の低い抗原特異的リンパ球であっても、簡便に目的の抗原に特異的なリンパ球を選択し、この選択された抗原特異的リンパ球から、抗体遺伝子を効率的にクローニングすることができるからである。 One of the preferred methods for producing a human monoclonal antibody is a method using the ISSAC (Immunospot-array assay on a chip) method. With the ISSAC method, not only frequently antigen-specific lymphocytes but also infrequent antigen-specific lymphocytes can be easily selected and selected. This is because the antibody gene can be efficiently cloned from the antigen-specific lymphocytes.
ISSAC法は、基体の一方の主表面に複数のウェルを有し、ウェルは1つのリンパ球のみが入る大きさであるマイクロウェルアレイを用いて行うものである。マイクロウェルアレイを用いる方法に関しては、本発明者らの先の出願を参照することができる。この方法においては、B細胞が抗原を認識したときに産生される抗体の少なくとも一部と結合性を有する物質(結合性物質)として、抗原を用いることができる。そして抗原特異的抗体の有無の検出は、抗体と特異的に結合する物質を用いる場合と、結合性物質と特異的に結合する物質を用いる場合とがある。検出のための物質の典型例は、抗原である。同定されたB細胞は、キャピラリーにて回収し、抗体遺伝子の増幅に供する。 The ISSAC method is performed using a microwell array having a plurality of wells on one main surface of the substrate, the wells having a size that can accommodate only one lymphocyte. For methods using microwell arrays, the prior applications of the present inventors can be referred to. In this method, an antigen can be used as a substance (binding substance) that has binding to at least a part of the antibody produced when B cells recognize the antigen. The presence or absence of an antigen-specific antibody may be detected by using a substance that specifically binds to the antibody or by using a substance that specifically binds to the binding substance. A typical example of a substance for detection is an antigen. The identified B cells are collected in capillaries and used for amplification of antibody genes.
特定された細胞は、増殖させてからPCR に供してもよいが、1個または数個の細胞を対象としてcDNAの増幅が行えるPCRの手法が種々知られており、本発明においてもそのような公知の手法を適用することができる。典型的には、まず回収した細胞を溶解し、γ、κおよびλ鎖のプライマーを用いた単一細胞5'-RACE法を用いてVHおよびVL断片をコードする抗体cDNAを増幅する。 次いで、これらの配列を、IgG1または軽鎖についての免疫グロブリン定常領域全体をコードするcDNAを含む発現ベクターに挿入する。そして、重鎖および軽鎖発現ベクターの両方で、抗体生産用の宿主細胞(例えば、293T細胞、Expi293F細胞、等)を、コトランスフェクションする。その際、市販のFreeStyleTM MAX 293T発現システム(Life Technologies)等を用いることができる。得られた形質転換細胞を培養して培養上清を回収し、抗体を精製する。抗体の精製には、市販のプロテインGカラムが使用できる。また、免疫グロブリン遺伝子レパートリーは、IMGT / V-Questツール(http://www.imgt.org/)を用いて分析できる。 The identified cells may be proliferated and then subjected to PCR, but various PCR methods capable of amplifying cDNA in one or several cells are known, and such a method is also known in the present invention. Known techniques can be applied. Typically, the recovered cells are first lysed and the antibody cDNA encoding the V H and VL fragments is amplified using the single cell 5'-RACE method with γ, κ and λ chain primers. These sequences are then inserted into an expression vector containing cDNA encoding the entire immunoglobulin constant region for IgG1 or light chain. Then, host cells for antibody production (eg, 293T cells, Expi293F cells, etc.) are cotransfected with both heavy and light chain expression vectors. At that time, a commercially available FreeStyleTM MAX 293T expression system (Life Technologies) or the like can be used. The obtained transformed cells are cultured, the culture supernatant is collected, and the antibody is purified. A commercially available protein G column can be used for purification of the antibody. The immunoglobulin gene repertoire can also be analyzed using the IMGT / V-Quest tool (http://www.imgt.org/).
ISSAC法の詳細は、Jin et al, Nature Medicine 2009;15:1088-1092、特開2004-187676に記載されている。 Details of the ISSAC method are described in Jin et al, Nature Medicine 2009; 15: 1088-1092, JP-A-2004-187676.
本発明はまた、配列番号23のアミノ酸配列の全部、または少なくとも9アミノ酸長の一部からなるペプチドに対する、抗体を提供する。ここでいう抗体は、ヒトモノクローナル抗体には限られず、ポリクローナル抗体であってもよく、マウス抗体であってもよい。このような抗体もまた、従来技術により製造することができる。 The present invention also provides antibodies against peptides consisting of the entire amino acid sequence of SEQ ID NO: 23, or part of at least 9 amino acid lengths. The antibody referred to here is not limited to a human monoclonal antibody, and may be a polyclonal antibody or a mouse antibody. Such antibodies can also be produced by prior art.
本発明により提供される、Ro52/TRIM21タンパク質を認識可能なヒトモノクローナル抗体、および配列番号23のアミノ酸配列の全部、または少なくとも9アミノ酸長の一部からなるペプチドに対する抗体は、後述するように、対象における間質性肺炎の、診断、発症の予測、重症度の診断、および重症化の予測からなる群より選択される少なくとも一つのための方法において、対象から得られた試料中に、Ro52/TRIM21タンパク質に対する抗体が存在するか否かを決定する際に、抗原として用いることができる。 Antibodies to human monoclonal antibodies recognizable by the Ro52 / TRIM21 protein provided by the present invention and peptides consisting of the entire amino acid sequence of SEQ ID NO: 23, or at least part of the amino acid length of 9 amino acids, are the subjects, as described below. In a method for at least one selected from the group consisting of diagnosis, prediction of onset, diagnosis of severity, and prediction of severity of interstitial pneumonia in, Ro52 / TRIM21 in samples obtained from the subject. It can be used as an antigen in determining the presence or absence of antibodies to a protein.
[診断、発症の予測、重症度の診断、および重症化の予測]
本発明は、対象における間質性肺炎の、診断、発症の予測、重症度の診断、および重症化の予測からなる群より選択される少なくとも一つのための方法であって、Ro52/TRIM21タンパク質に対する抗体が対象から得られた試料中に存在するかどうかを決定する工程を含む方法を提供する。
[Diagnosis, Prediction of Onset, Diagnosis of Severity, and Prediction of Severity]
The present invention is a method for at least one selected from the group consisting of diagnosis, prediction of onset, diagnosis of severity, and prediction of aggravation of interstitial pneumonia in a subject for the Ro52 / TRIM21 protein. Provided is a method comprising the step of determining whether an antibody is present in a sample obtained from a subject.
本発明者らの検討によると、血清抗Ro52/TRIM21抗体陽性である膠原病患者において、P23に結合する自己抗体を有する患者群(P23(+)群)と自己抗体を有さない(P23(-)群)それぞれの間質性肺炎の発症率には差がみられる。また、P23(+)群、P23(-)群それぞれのKL-6の陽性率(KL-6≧500U/mLを陽性とする)にも差がみられる。したがって、Ro52/TRIM21タンパク質に対する自己抗体が対象からの試料中に存在する場合、その対象を、間質性肺炎である、もしくは間質性肺炎を発症すると予測できる。またはその対象の間質性肺炎が重症である、もしくは間質性肺炎が重症化すると予測できる。 According to the study by the present inventors, among patients with collagen disease positive for serum anti-Ro52 / TRIM21 antibody, a group of patients having an autoantibody that binds to P23 (P23 (+) group) and a group without autoantibodies (P23 (P23 ()). -) Group) There is a difference in the incidence of interstitial pneumonia. There is also a difference in the positive rate of KL-6 (assuming KL-6 ≥ 500 U / mL is positive) in each of the P23 (+) group and the P23 (-) group. Therefore, if autoantibodies to the Ro52 / TRIM21 protein are present in a sample from a subject, the subject can be predicted to have interstitial pneumonia or develop interstitial pneumonia. Alternatively, it can be predicted that the subject's interstitial pneumonia is severe, or that the interstitial pneumonia becomes severe.
診断等の対象は、健常人、間質性肺炎の発症のリスクがある人、間質性肺炎の患者が含まれる。間質性肺炎の発症のリスクがある人には、自己免疫疾患の患者、高齢者が含まれる。本発明の方法の適用が特に好ましい例の一つは、膠原病の患者である。膠原病は、病理形態学的に全身の膠原線維にフィブリノイド変性を来し、その原因として自己免疫現象が考えられている疾患群をいう。これには、全身性エリテマトーデス(SLE)、全身性硬化症(PSS)、皮膚筋炎(DM)、関節リウマチ(RA)、リウマチ熱(RF)、結節性多発性動脈炎(PN)、リウマチ性多発筋痛症、側頭動脈炎(巨細胞性動脈炎)、シェーグレン症候群、混合性結合組織病(MCTD)、重複症候群、アレルギー性肉芽腫性血管炎、ウェゲナー肉芽腫症が含まれる。膠原病のうち、本発明の適用が特に好ましいもは、関節リウマチ、全身性強皮症、皮膚筋炎、多発性筋炎、および混合性結合組織病である。 Targets for diagnosis, etc. include healthy people, people at risk of developing interstitial pneumonia, and patients with interstitial pneumonia. Those at risk of developing interstitial pneumonia include patients with autoimmune diseases and the elderly. One example in which the application of the method of the present invention is particularly preferred is in patients with collagen disease. Collagen disease refers to a group of diseases in which fibrinoid degeneration occurs in collagen fibers throughout the body pathologically, and an autoimmune phenomenon is considered to be the cause. These include systemic lupus erythematosus (SLE), systemic sclerosis (PSS), dermatomyositis (DM), rheumatoid arthritis (RA), rheumatoid fever (RF), polyarteritis nodosa (PN), and rheumatoid arteritis (PN). Includes myopathy, temporal arteritis (giant cell arteritis), Schegren's syndrome, mixed arteritis (MCTD), duplication syndrome, allergic granulomatous vasitis, and Wegener's granulomatosis. Of the collagen diseases, the application of the present invention is particularly preferred for rheumatoid arthritis, systemic scleroderma, dermatomyositis, polymyositis, and mixed connective tissue disease.
対象からの試料の例として、末梢血の血清または血漿、尿、髄液、喀痰、病理組織、毛根、爪、口腔粘膜等が挙げられる。 Examples of samples from the subject include peripheral blood serum or plasma, urine, cerebrospinal fluid, sputum, histopathology, hair roots, nails, oral mucosa and the like.
試料中にRo52/TRIM21タンパク質に対する抗体が含まれているか否かは、適切な抗原と結合する抗体が含まれているか否かを、従来技術の免疫アッセイ(例えば、ELISA等)を利用して評価することができる。抗原としては、Ro52/TRIM21タンパク質のC-Cドメインの全部または一部、P23の全部または少なくとも9アミノ酸長であるその一部を用いることができる。 Whether or not the sample contains an antibody against the Ro52 / TRIM21 protein is evaluated by using a conventional immunoassay (for example, ELISA) to evaluate whether or not an antibody that binds to an appropriate antigen is contained. can do. As the antigen, all or part of the C-C domain of the Ro52 / TRIM21 protein, all or part of P23 or at least a part thereof having a length of at least 9 amino acids can be used.
本発明の方法は、間質性肺炎の診断等のための他のマーカーを用いた方法と併用することができる。他のマーカーの例としてKL-6、SP-A、SP-Dを挙げることができる。これらは、特発性肺線維症(IPF)や非特異性間質性肺炎(NSIP)などの特発性間質性肺炎で高い率で陽性となり、病態のモニタリング、治療反応性の評価に有用とされている。別の例として、乳酸脱水素酵素(LDH)を挙げることができる。これも間質性肺炎において高値となることが知られている。 The method of the present invention can be used in combination with a method using other markers for the diagnosis of interstitial pneumonia and the like. Examples of other markers include KL-6, SP-A, SP-D. These are highly positive in idiopathic interstitial pneumonia such as idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP), and are useful for monitoring pathological conditions and evaluating treatment responsiveness. ing. Another example is lactate dehydrogenase (LDH). This is also known to be high in interstitial pneumonia.
[キット、医薬組成物]
本発明のペプチドおよび抗体は、間質性肺炎の検査のためのキットの構成要素として用いることができる。キットは、少なくとも抗原としての本発明のペプチドを含み、他に、コントロールとして用いるための本発明の抗体、対象から試料を採取するための器具、試料を保存するための容器、試料希釈液、免疫アッセイを行うための器具(例えばELISAのためのプレート)および試薬、免疫アッセイのためのインストラクションを記録した媒体(例えば、文書、CD)等を含んでいてもよい。また、キットには、キットの使用目的、例えば対象における間質性肺炎の、診断、発症の予測、重症度の診断、および重症化の予測からなる群より選択される少なくとも一つのためのキットである旨の表示がされていてもよい。
[Kit, pharmaceutical composition]
The peptides and antibodies of the present invention can be used as components of a kit for testing interstitial pneumonia. The kit contains at least the peptides of the invention as antigens, as well as the antibodies of the invention for use as controls, instruments for collecting samples from subjects, containers for storing samples, sample diluents, immunity. It may include instruments and reagents for performing the assay (eg, plates for ELISA) and reagents, media recording instructions for immunoassays (eg, documents, CDs), and the like. The kit also includes a kit for at least one selected from the group consisting of the purpose of use of the kit, for example, diagnosis of interstitial pneumonia in a subject, prediction of onset, diagnosis of severity, and prediction of severity. It may be displayed to that effect.
本発明のペプチドおよび抗体は、医薬として許容し得る担体とともに、医薬組成物として用いることができる。医薬組成物は、間質性肺炎に関連した、検査、診断、または処置のためのものであり得る。 The peptides and antibodies of the present invention can be used as pharmaceutical compositions together with pharmaceutically acceptable carriers. The pharmaceutical composition may be for testing, diagnosis, or treatment associated with interstitial pneumonia.
医薬として許容される担体としては、有効成分であるペプチドまたは抗体の機能を損なわない限り特に限定されないが、例えば、賦形剤、溶剤(分散剤)、溶解補助剤、懸濁化剤、安定化剤、等張化剤、緩衝剤、pH調節剤、無痛化剤、保存剤、抗酸化剤等が挙げられる。賦形剤の好適な例としては、乳糖、白糖、D-マンニトール、D-ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、アラビアゴム、プルラン、軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウムなどが挙げられる。溶剤の好適な例としては、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、ゴマ油、トウモロコシ油、オリーブ油、綿実油などが挙げられる。溶解補助剤の好適な例としては、ポリエチレングリコール、プロピレングリコール、D-マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウムなどが挙げられる。懸濁化剤の好適な例としては、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリンなどの界面活性剤;例えばポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロースなどの親水性高分子;ポリソルベート類、ポリオキシエチレン硬化ヒマシ油などが挙げられる。安定化剤の好適な例としては、ヒト血清アルブミン(HSA)、ピロ亜硫酸ナトリウム、ロンガリット、メタ亜硫酸水素ナトリウムなどが挙げられる。等張化剤の好適な例としては、塩化ナトリウム、グリセリン、D-マンニトール、D-ソルビトール、ブドウ糖などが挙げられる。緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などが挙げられる。pH調節剤の好適な例としては、塩酸、水酸化ナトリウムなどの酸または塩基が上げられる。無痛化剤の好適な例としては、ベンジルアルコールなどが挙げられる。保存剤の好適な例としては、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸などが挙げられる。抗酸化剤の好適な例としては、亜硫酸塩、アスコルビン酸塩などが挙げられる。 The pharmaceutically acceptable carrier is not particularly limited as long as it does not impair the function of the peptide or antibody as the active ingredient, and is, for example, an excipient, a solvent (dispersant), a lysis aid, a suspending agent, and a stabilizer. Examples thereof include agents, tonicity agents, buffers, pH regulators, soothing agents, preservatives, antioxidants and the like. Suitable examples of excipients are lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, light Examples thereof include silicate silicate, synthetic aluminum silicate, and magnesium aluminometasilicate. Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like. Preferable examples of solubilizers are polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. And so on. Preferable examples of suspending agents are surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; eg polyvinyl alcohol, polyvinyl Hydrophilic polymers such as pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose; polysorbates, polyoxyethylene hydrogenated castor oil and the like. Preferable examples of stabilizers include human serum albumin (HSA), sodium pyrosulfite, longalit, sodium metabisulfite and the like. Preferable examples of the tonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like. Preferable examples of the buffer include buffers such as phosphates, acetates, carbonates and citrates. Preferable examples of pH regulators include acids or bases such as hydrochloric acid, sodium hydroxide and the like. Preferable examples of the soothing agent include benzyl alcohol and the like. Preferable examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of antioxidants include sulfites, ascorbic acid salts and the like.
医薬組成物の剤形は、特に限定されないが、検査のためには、各種の液体または固体の剤(例えば、乳剤、懸濁剤、液剤、顆粒剤、粉末剤等)としてもよく、対象への投与のためには、例えば注射剤(皮下注射剤、静脈内注射剤、筋肉内注射剤、腹腔内注射剤、動脈内注射剤を含む。)、点滴剤等の注入型製剤としてもよい。医薬組成物中の有効成分の含量は、剤形、投与量などにより異なるが、例えば約0.1-100重量%とすることができる。医薬組成物は、従来法により製造することができる。 The dosage form of the pharmaceutical composition is not particularly limited, but for inspection, various liquid or solid agents (for example, emulsions, suspensions, liquids, granules, powders, etc.) may be used. For the administration of, for example, an injectable preparation such as an injection (including a subcutaneous injection, an intravenous injection, an intramuscular injection, an intraperitoneal injection, and an intraarterial injection), a drip infusion, or the like may be used. The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, dosage and the like, but can be, for example, about 0.1-100% by weight. The pharmaceutical composition can be produced by a conventional method.
以下、本発明を実施例により説明する。 Hereinafter, the present invention will be described with reference to Examples.
[ペプチド23の同定]
4名の抗Ro52/TRIM21抗体価強陽性である、間質性肺炎を発症した皮膚筋炎患者4名の末梢血リンパ球より、ISAAC (Immunospot-array assay on a chip) 法(Jin et al, Nature Medicine 2009;15:1088-1092)を用いて、Ro52/TRIM21タンパク質に結合する抗体を産生するリンパ球を特定し、抗体cDNAをクローニングし、遺伝子導入により抗体産生細胞を得て、ヒトモノクローナル抗体を13種類作製した。得られた抗体の名称を下表にまとめた。なお、表中、「100, 112」は、100番の患者と112番の患者のリンパ球を混合したものから抗体を得たという趣旨である。100番と112番からは、単独ではISAAC法を行うのに十分なリンパ球数を確保できなかったため、混合してISSAC法に供した。
[Identification of peptide 23]
ISAAC (Immunospot-array assay on a chip) method (Jin et al, Nature) from peripheral blood lymphocytes of 4 patients with dermatitis who developed interstitial pneumonia who were strongly positive for anti-Ro52 / TRIM21 antibody titer. Using Medicine 2009; 15: 1088-1092), lymphocytes that produce antibodies that bind to the Ro52 / TRIM21 protein were identified, antibody cDNAs were cloned, and antibody-producing cells were obtained by gene transfer to obtain human monoclonal antibodies. 13 types were prepared. The names of the obtained antibodies are summarized in the table below. In the table, "100, 112" means that the antibody was obtained from a mixture of lymphocytes of the 100th patient and the 112th patient. From No. 100 and No. 112, since sufficient lymphocyte counts could not be secured for the ISAAC method alone, they were mixed and used for the ISSAC method.
また、作製した13種の抗体が、Ro52/TRIM21タンパク質のC-Cドメインに結合するか否かを確認したところ、11種類(26、29、37、8、14、15、42、44、45、46、60)がRo52/TRIM21タンパク質のC-Cドメインに結合することが明らかになった。 In addition, when it was confirmed whether or not the 13 types of antibodies produced bind to the CC domain of the Ro52 / TRIM21 protein, 11 types (26, 29, 37, 8, 14, 15, 42, 44, 45, 46) were confirmed. , 60) were found to bind to the CC domain of the Ro52 / TRIM21 protein.
そこで我々はモノクローナル抗体での解析でさらに詳細なエピトープが同定できると考え、下表に示したように、16アミノ酸長ずつ、計59個のRo52/TRIM21タンパク質のペプチドライブラリーを作製し、解析した。 Therefore, we thought that more detailed epitopes could be identified by analysis with monoclonal antibodies, and as shown in the table below, we prepared and analyzed a total of 59 Ro52 / TRIM21 protein peptide libraries with 16 amino acid lengths each. ..
その結果、2種類の抗体(Ab55-37、およびAb150-61)がC-Cドメインに属する1個のペプチド(ペプチド23, P23)に結合することが明らかとなった。また、このP23以外にこれらのモノクローナル抗体と結合性を示すペプチドは同定されなかった。 As a result, it was clarified that two kinds of antibodies (Ab55-37 and Ab150-61) bind to one peptide (peptides 23, P23) belonging to the C-C domain. In addition to this P23, no peptide showing binding to these monoclonal antibodies was identified.
[抗Ro52/TRIM21抗体の各々の結合部位の相同性解析]
C-Cドメインに結合する11種の抗体(うち2抗体がP23と結合する抗体)のRo52/TRIM21タンパク質内での結合部位の相同性を確認するため、抗原としてリコンビナントRo52/TRIM21タンパク質(Prospec-Tany TechnoGene社のRo52/SS-A, Human, Recombinant、商品コード:PRO-328)を用い、異なる2抗体を競合させてELISAを行い、これらの抗体の結合部位に相同性があるかどうかを確認した。
[Homology analysis of each binding site of anti-Ro52 / TRIM21 antibody]
Recombinant Ro52 / TRIM21 protein (Prospec-Tany TechnoGene) as an antigen to confirm the homology of the binding site within the Ro52 / TRIM21 protein of 11 antibodies that bind to the CC domain (of which 2 antibodies bind to P23) Using Ro52 / SS-A, Human, Recombinant, product code: PRO-328) of the same company, ELISA was performed by competing two different antibodies, and it was confirmed whether or not there was homology in the binding sites of these antibodies.
ELISAではmaxisorp plate を使用し、リコンビナントRo52/TRIM21タンパク質(0.275μg/mL/well)を抗原として使用した。ブロッキング液は3%BSA/PBST(1x)を用い、一次抗体は1種のモノクローナル抗Ro52抗体(10μg/mL)を1時間反応させた後にPBSTで洗浄し、二次抗体は一次抗体とは別のモノクローナル抗Ro52抗体をペルオキシダーゼ標識したもの(100μg/mL)を1時間反応させた。各抗体につき、抗原であるRo52/TRIM21タンパク質をそれぞれ3wellずつ使用した。検出はTMBを用いて450nmでAbsを測定した。 For ELISA, a maxisorp plate was used, and recombinant Ro52 / TRIM21 protein (0.275 μg / mL / well) was used as an antigen. 3% BSA / PBST (1x) was used as the blocking solution, the primary antibody was washed with PBST after reacting one type of monoclonal anti-Ro52 antibody (10 μg / mL) for 1 hour, and the secondary antibody was separated from the primary antibody. Peroxidase-labeled monoclonal anti-Ro52 antibody (100 μg / mL) was reacted for 1 hour. For each antibody, 3 wells of the antigen Ro52 / TRIM21 protein were used. For detection, Abs was measured at 450 nm using TMB.
<ELISAのプロトコール>
1. Maxisorp plate に5μg/mL/wellのリコンビナントRo52/TRIM21タンパク質を50μL添加し、4℃で一晩静置し、ペプチドをウェルにコートした。
2. ペプチドが入った液を除去後、PBST(1%Tween20入りPBS)で2回ウェルを洗浄した。
3. 3% BSA/PBSTを350μLずつウェルに添加し、室温で1時間静置し、ブロッキングを行った。
4. 3% BSA/PBSTを除去後、PBSTで2回ウェルを洗浄した。
5. 1μg/mLに調整したモノクローナル抗Ro52抗体を50 μLずつウェルに添加し、室温で1時間静置した。
6. 5.の抗体を除去後、PBSTで2回ウェルを洗浄した。
7. 1μg/mLに調整した、5.とは異なるモノクローナル抗Ro52抗体をペルオキシダーゼ標識したものを50μLずつウェルに添加し、室温で1時間静置した。
8. 7.の抗体を除去後、PBSTで2回ウェルを洗浄した。
9. TMB solutionを50μLずつウェルに添加し、30分静置した。
10. Stop solutionを50μLずつウウェルに添加し、450nmの吸収を測定した。
<ELISA protocol>
1. 50 μL of 5 μg / mL / well recombinant Ro52 / TRIM21 protein was added to a Maxisorp plate and allowed to stand overnight at 4 ° C. to coat the wells with peptides.
2. After removing the solution containing the peptide, the wells were washed twice with PBST (PBS containing 1% Tween 20).
3.3 3% BSA / PBST was added to each well in an amount of 350 μL, and the mixture was allowed to stand at room temperature for 1 hour for blocking.
4.3 After removing 3% BSA / PBST, the wells were washed twice with PBST.
5. Monoclonal anti-Ro52 antibody adjusted to 1 μg / mL was added to each well in an amount of 50 μL, and the mixture was allowed to stand at room temperature for 1 hour.
6. After removing the antibody from 5., the wells were washed twice with PBST.
7. Peroxidase-labeled monoclonal anti-Ro52 antibody different from 5. adjusted to 1 μg / mL was added to each well in an amount of 50 μL, and the mixture was allowed to stand at room temperature for 1 hour.
8. After removing the antibody from 7., the wells were washed twice with PBST.
9. 50 μL of TMB solution was added to the wells and allowed to stand for 30 minutes.
10. 50 μL of Stop solution was added to the wellell, and the absorption at 450 nm was measured.
結果は下表に示したように、C-Cドメインで結合する11種の各抗体のうち異なる2抗体を競合させると吸光度(Abs)が低下したことから、各々の抗体が競合して結合性が減弱したと考える。これは、各々の抗体のRo52/TRIM21内での結合部位が近いために競合していると考えられる。このことから、P23と結合しない他の9抗体もP23付近で結合する可能性が示唆された。 As shown in the table below, when two different antibodies out of the 11 antibodies that bind in the CC domain compete with each other, the absorbance (Abs) decreases, so that each antibody competes and the binding property is weakened. I think I did. This is thought to be due to the close binding sites of each antibody within Ro52 / TRIM21. This suggests that the other 9 antibodies that do not bind to P23 may also bind near P23.
[抗Ro52/TRIM21抗体強陽性患者の血清とP23と結合性の解析]
ペプチド23(P23)に結合する自己抗体を有する患者群(P23(+)群)と抗体を有さない(P23(-)群)で、間質性肺炎の発症率や重症度を比較した。
[Analysis of serum and P23 binding in patients strongly positive for anti-Ro52 / TRIM21 antibody]
The incidence and severity of interstitial pneumonia were compared between the group of patients with autoantibodies that bind to peptide 23 (P23) (P23 (+) group) and the group without antibodies (P23 (-) group).
血清は、ユーロライン検査(筋炎・強皮症)で血清中の抗Ro52抗体が強陽性(3+)と判定された皮膚筋炎、強皮症等の膠原病患者の血清を用いた。ELISAではmaxisorp plateを使用し、P23(RIHAEFVQQKNFLVEE 配列番号23)、および陰性コントロールとしてHIVペプチド(RYLRDQQLL 配列番号62)を抗原として使用した。ブロッキング液は3%BSA/PBST(1x)を用い、一次抗体は患者血清(x200希釈)、二次抗体はアルカリフォスファターゼ標識抗ヒトIgGFc (α-hIgGFc-ALP, x1000希釈)を用いた。各血清検体につき、P23とHIVペプチドをそれぞれ3wellずつ使用した。検出はpNNで行い405nmでAbsを測定した。 As the serum, the serum of patients with collagen diseases such as dermatomyositis and scleroderma, whose anti-Ro52 antibody in the serum was determined to be strongly positive (3+) by the Euroline test (myositis / scleroderma), was used. For ELISA, a maxisorp plate was used, and P23 (RIHAEFVQQKNFLVEE SEQ ID NO: 23) and HIV peptide (RYLRDQQLL SEQ ID NO: 62) were used as antigens as a negative control. 3% BSA / PBST (1x) was used as the blocking solution, patient serum (x200 dilution) was used as the primary antibody, and alkaline phosphatase-labeled anti-human IgGFc (α-hIgGFc-ALP, x1000 dilution) was used as the secondary antibody. For each serum sample, 3 wells of P23 and 3 wells of HIV peptide were used. The detection was performed by pNN and Abs was measured at 405 nm.
<ELISAのプロトコール>
1. Maxisorp plate に5μg/mL/wellのP23(RIHAEFVQQKNFLVEE 配列番号23)、あるいは5μg/mL/wellのHIVペプチド(RYLRDQQLL 配列番号62)を50μL添加し、4℃で一晩静置し、ペプチドをウェルにコートした。
2. ペプチドが入った液を除去後、PBST(1%Tween20入りPBS)で2回ウェルを洗浄した。
3. 3% BSA/PBSTを350μLずつウェルに添加し、室温で1時間静置し、ブロッキングを行った。
4. 3% BSA/PBSTを除去後、PBSTで2回ウェルを洗浄した。
5. 200倍希釈した患者血清を50μLずつウェルに添加し、室温で1時間静置した。
6. 患者血清を除去後、PBSTで2回ウェルを洗浄した。
7. アルカリフォスファターゼ標識抗ヒトIgGFc(1000倍希釈)を50μLずつウェルに添加し、室温で1時間静置した。
8. アルカリフォスファターゼ標識抗ヒトIgGFcを除去後、PBSTで2回ウェルを洗浄した。
9. アルカリフォスファターゼ基質をバッファーに1 mg/mLになるように溶解し、50μLずつウェルに添加した。
10. 室温で30分静置し、405nmの吸収を測定した。
<ELISA protocol>
1. Add 50 μL of 5 μg / mL / well P23 (RIHAEFVQQKNFLVEE SEQ ID NO: 23) or 5 μg / mL / well HIV peptide (RYLRDQQLL SEQ ID NO: 62) to a Maxisorp plate and allow to stand overnight at 4 ° C. Coated well.
2. After removing the solution containing the peptide, the wells were washed twice with PBST (PBS containing 1% Tween 20).
3.3 3% BSA / PBST was added to each well in an amount of 350 μL, and the mixture was allowed to stand at room temperature for 1 hour for blocking.
4.3 After removing 3% BSA / PBST, the wells were washed twice with PBST.
5. 50 μL of patient serum diluted 200-fold was added to the wells and allowed to stand at room temperature for 1 hour.
6. After removing patient serum, the wells were washed twice with PBST.
7. 50 μL of alkaline phosphatase-labeled anti-human IgGFc (diluted 1000-fold) was added to the wells, and the mixture was allowed to stand at room temperature for 1 hour.
8. After removing the alkaline phosphatase-labeled anti-human IgGFc, the wells were washed twice with PBST.
9. The alkaline phosphatase substrate was dissolved in buffer at 1 mg / mL and added 50 μL to each well.
10. The mixture was allowed to stand at room temperature for 30 minutes, and the absorption at 405 nm was measured.
P23のAbsの平均値が、[(HIVペプチドへのAbsの平均値)+(HIVペプチドのAbsの標準偏差×3]より大きくなった場合にP23と結合したと判定した。このELISAで3回中3回ともP23との結合がみられた患者をP23(+)群、3回ともP23との結合をみられなかった患者をP23(-)群とした。また3回のうち1回または2回陰性となった患者は対象外とした。 When the mean value of Abs of P23 was larger than [(mean value of Abs to HIV peptide) + (standard deviation of Abs of HIV peptide x 3], it was judged that it bound to P23. This ELISA was used 3 times. Patients who had P23 binding in all three times were in the P23 (+) group, and patients who did not have P23 binding in all three times were in the P23 (-) group. Patients who were negative twice were excluded.
1) ペプチド23(P23)を用いた患者血清中の自己抗体の結合と間質性肺炎(IP)の発症率の比較
ユーロライン検査で血清抗Ro52抗体陽性の皮膚筋炎、強皮症等の膠原病患者のうち、P23(+)群、P23(-)群それぞれの間質性肺炎(IP)の発症率をFisher検定で比較したところ、両群の間に有意差がみられた(p=0.008)。
1) Comparison of autoantibody binding in patient serum using peptide 23 (P23) and incidence of interstitial pneumonia (IP) Serum anti-Ro52 antibody-positive dermatomyositis, scleroderma, etc. in Euroline test When the incidence of interstitial pneumonia (IP) in each of the P23 (+) group and the P23 (-) group was compared by the Fisher test among the diseased patients, a significant difference was observed between the two groups (p =). 0.008).
2) P23を用いた患者血清中の自己抗体の結合と間質性肺炎の重症度の比較
上記2群につき、間質性肺炎の重症度の指標として汎用されているKL-6の値について、KL-6の測定歴のある患者を対象にしてその2群について比較した。
2) Comparison of autoantibody binding in patient serum and severity of interstitial pneumonia using P23 Regarding the value of KL-6, which is widely used as an index of severity of interstitial pneumonia in the above two groups. The two groups were compared in patients who had previously measured KL-6.
対象となる患者はP23(+)群34名、P23(-)群36名であり、それぞれのKL-6の陽性率(KL-6≧500U/mLを陽性とする)をFisher検定で比較したところ、両群の間に有意差がみられた(p=0.001)。 The target patients were 34 in the P23 (+) group and 36 in the P23 (-) group, and their KL-6 positive rates (KL-6 ≥ 500 U / mL were positive) were compared by Fisher's exact test. However, there was a significant difference between the two groups (p = 0.001).
本発明のペプチドおよびそれに対する抗体は、間質性肺炎を発症するか否かの予測およびその重症度等を予測する検査キットへの応用が期待できる。 The peptide of the present invention and an antibody against it can be expected to be applied to a test kit for predicting whether or not interstitial pneumonia develops and its severity.
SEQ ID NOs.:1-59 Ro52/TRIM21タンパク質のC-Cドメインに由来するペプチドのアミノ酸配列
SEQ ID NO.:60 Ro52/TRIM21タンパク質のアミノ酸配列
SEQ ID NO.:61 Ro52/TRIM21タンパク質のC-Cドメインのアミノ酸配列
SEQ ID NO.:62 HIV由来のペプチドのアミノ酸配列
SEQ ID NOs .: 1-59 Amino acid sequence of peptide derived from CC domain of Ro52 / TRIM21 protein
SEQ ID NO .: 60 Amino acid sequence of Ro52 / TRIM21 protein
SEQ ID NO .: 61 Amino acid sequence of CC domain of Ro52 / TRIM21 protein
SEQ ID NO .: 62 Amino acid sequence of HIV-derived peptide
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