WO1999063084A1 - T cell antigen receptor sequences specific to arthrosis deformans - Google Patents

T cell antigen receptor sequences specific to arthrosis deformans Download PDF

Info

Publication number
WO1999063084A1
WO1999063084A1 PCT/JP1999/002814 JP9902814W WO9963084A1 WO 1999063084 A1 WO1999063084 A1 WO 1999063084A1 JP 9902814 W JP9902814 W JP 9902814W WO 9963084 A1 WO9963084 A1 WO 9963084A1
Authority
WO
WIPO (PCT)
Prior art keywords
gly
patient
osteoarthritis
amino acid
antigen
Prior art date
Application number
PCT/JP1999/002814
Other languages
French (fr)
Japanese (ja)
Inventor
Kusuki Nishioka
Shinichi Yoshino
Original Assignee
St. Marianna University School Of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St. Marianna University School Of Medicine filed Critical St. Marianna University School Of Medicine
Priority to AU40577/99A priority Critical patent/AU4057799A/en
Publication of WO1999063084A1 publication Critical patent/WO1999063084A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a substance useful for diagnosing, preventing and treating osteoarthritis. More specifically, the present invention relates to an amino acid sequence containing a CDR3 region of a T cell antigen receptor (TCR) chain of ⁇ cells present in synovial cells and / or synovial fluid of osteoarthritis patients.
  • TCR T cell antigen receptor
  • Osteoarthritis is a disease caused by aging or dysplasia of joints, preceding joint disease or trauma, and is particularly common in weight-bearing joints.
  • the cartilage matrix production decreases and the degeneration of the synovial fluid, which supplements the cartilage, becomes insufficient due to insufficient penetration of the synovial fluid into the cartilage matrix.
  • Articular cartilage is damaged by the mechanical load. Articular cartilage has poor self-healing capacity due to the absence of blood vessels, and once damage occurs, degenerative changes can easily develop.
  • various chemical mediators and cytokines are released from the destroyed chondrocytes and the like, and it has been thought that they cause chronic inflammation of the synovium (CI.
  • rheumatoid arthritis is classified as an autoimmune disease.
  • the immune system's response is a response that is originally triggered to destroy and eliminate foreign invaders, and is usually specific for non-self molecules (antigens). However, if for some reason the identification of self and non-self molecules is impaired, an autoimmune disease results.
  • rheumatoid arthritis rheumatic arthritis
  • abnormal proliferation of synovial tissue is observed.
  • accumulation of T cells is observed in the proliferated tissue.
  • T cells responsible for cellular immunity to recognize antigens, not only antigens but also antigen-presenting cells such as macrophages are required.
  • T cell antigen receptor recognizes the complex with the foreign antigen that has undergone immunization. ⁇ cells that have received an antigen signal from antigen presenting cells proliferate locally, release various cytokines, and develop inflammation.
  • the TCR is a heterodimer due to the disulfide bond of two polypeptide chains, ⁇ and ⁇ , or ⁇ and ⁇ , respectively.
  • ⁇ cell clones expressing a specific V region of TCR accumulate in the joints of patients with rheumatoid arthritis.
  • the mainstays of treatment for rheumatoid arthritis are non-steroidal anti-inflammatory drugs, immunomodulators, and steroid drugs.
  • Rheumatoid arthritis In patients with osteoarthritis, inflammation in synovial tissue is observed as in patients with rheumatoid arthritis, which is an autoimmune disease, but is weaker than in rheumatoid arthritis.
  • Rheumatoid arthritis is an autoimmune disease in which strong ⁇ ⁇ cells accumulate in the synovium and synovial fluid, but no ⁇ cells infiltrate into the synovium and synovial fluid in osteoarthritis.
  • immunocompetent cells such as CD4 and CD8 positive T cells, B cells, and monocytes Z macula phage infiltrate the synovial tissue
  • An object of the present invention is to provide a method for treating oligoarthritis present in the synovial membrane and synovial fluid, based on the idea that osteoarthritis, like rheumatoid arthritis, is based on chronic arthritis involving autoreactive T cells.
  • the purpose of the present invention is to clarify the accumulation and proliferation of null T cells, determine the amino acid sequence of the CDR3 region of the TCR, and provide a diagnostic, preventive and therapeutic agent for osteoarthritis.
  • the present inventors aimed to elucidate the ⁇ cells associated with the development of osteoarthritis, and investigated the TCR of pathogenic T cells oligoclonally proliferating in the synovial tissue of osteoarthritis patients.
  • the amino acid sequence containing the V] 3 chain CDR3 region was determined.
  • TCRV in the synovial tissue of patients with osteoarthritis, almost all TCRV from VjS1 to V
  • the CDR3 region of the TCR is said to be a site where the amino acid sequence is most different depending on individual T cells.
  • T cells having the amino acid sequence of the present invention in the CDR3 region of the TCRV chain are pathogenic ⁇ cells of osteoarthritis
  • the amino acid sequence of the present invention can be used as a tool for confirmatory diagnosis of osteoarthritis.
  • the treatment of osteoarthritis can be performed. It can greatly contribute to the development of diagnostic and prophylactic drugs.
  • the amino acid sequence of the present invention may be used for TCR protein, TCR DNA protein, activation of immunoregulatory cells or anti-TCR By using it to elicit antibodies, it can also be used for immunotherapy of osteoarthritis.
  • a peptide having homology to the peptide having the amino acid sequence of the present invention can produce T cells that cannot attack a patient's antigen and can block antigen-specific TCR by direct administration to a patient.
  • the antigen peptide or antigen protein which specifically reacts with the TCR having the amino acid sequence in the CDR3 region of the present invention is a therapeutic agent for osteoarthritis which induces antigen-specific immune tolerance and / or Useful as a prophylactic.
  • FIG. 1 is a photograph of an electrophoresis chromatogram showing the results of detecting samples obtained from peripheral venous blood and synovial membrane of a patient with human osteoarthritis by the SSCP method.
  • the partial sequence of the TCR involved in osteoarthritis has been elucidated, and the partial peptide involved in the recognition of osteoarthritis-related antigen using the sequence, DNA encoding the same, and By obtaining the DNA fragment and expressing it in a suitable host cell, it can be used for diagnosis of osteoarthritis. Diagnosis of osteoarthritis is based on clinical characteristics, but the amino acid sequence of the present invention is compared with the sequence of the TCRV
  • mRNA is removed from the body fluid of a patient suspected of having osteoarthritis, a cDNA library is created by RT-PCR, and each V] 3 chain subclass is expressed using a V] 3 chain probe.
  • the expressed T cell clone can also be observed by SSCP. Thereafter, the nucleotide sequence of the clonally expressed CDR3 portion of the TCRVj3 chain and the amino acid sequence derived therefrom are compared.
  • the body fluid here includes synovial fluid.
  • the antigen peptide can be used alone or in combination of two or more of these as a therapeutic or prophylactic agent for osteoarthritis.
  • These peptides can be used systemically, orally or topically in various commonly used dosage forms. Dosage form For example, oral preparations, injection preparations, nasal preparations and the like can be mentioned.
  • Preparations in these dosage forms can be prepared according to a conventional method, using commonly used carriers such as a vehicle, a binder, a solubilizer, an emulsifier, and a suspending agent.
  • the oral preparations include solid preparations such as tablets, granules and powders, and liquid preparations such as solutions, suspensions and emulsions.
  • the peptide of the present invention is not limited by genetic recombination, and generally employs a method as described in RB Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). However, other equivalent known chemical synthetic methods can be used.
  • Peripheral blood of an osteoarthritis patient 1 Om1 was collected by adding heparin to blood, and lymphocytes were isolated by specific gravity centrifugation using Fico 11 P aque (Pharmacia Biotech). MRNA was extracted from the lymphocytes according to the kit of ISOGEN (Nitsubon Gene Co., Ltd.). That is, 1. Oml of IS1GEN was added to the lymphocyte pellet, mixed for 1 minute, and allowed to stand at room temperature for 5 minutes. Further, 0.2 ml of black-mouthed form was added, mixed for 15 seconds, and allowed to stand at room temperature for 2 minutes. Next, the mixture was centrifuged at 15,000 rpm at 4 ° C. for 15 minutes to obtain a supernatant.
  • Isopropanol 500 1 was added to the supernatant, and mixed several times by inversion. Incubate at room temperature for 5 minutes, 15,000 rpm, 4. The mixture was centrifuged at C for 10 minutes, and the obtained RNA pellet was washed with 75% ethanol.
  • the degree of purification and the concentration of the obtained RNA was confirmed by agarose gel.
  • cDNA was synthesized using mo lecule (Amersham). That is, TE buffer RNA pellet obtained in 1) (1 0 mM Tris-HCl buffer, 1 mM E DT A) 1 00 ⁇ 1 was added and dissolved, the RNA solution 1 0 y ul, 5 X Li carbonochloridate 1 ⁇ l of RNase inhibitor, 1 ⁇ l of RNase inhibitor, 2 ⁇ m of lNTPl, 2 ⁇ l of transcriptase reaction mixture, and 2 ⁇ l of oligo dT (An chored dT25 ) And reverse transcriptase 11 at 42 °. For 1 hour to obtain cDNA.
  • a DNA fragment encoding the TCRj 3 chain V region and a DNA fragment encoding the 3 chain C region were used as PCR primers. (Base sequences shown in Table 2) were synthesized using a DNA synthesizer manufactured by Applied Biosystems. (Choi.Y.eta1 and Proc.Natl.
  • VVO 331 ⁇ ⁇ V VOX 303 ⁇ ⁇ V VOV
  • V V3I VOV 131 310 1DV JLV ⁇ ⁇ V VOV 131 ⁇ - ⁇ ⁇ £ / ⁇ V ⁇ VV 9V3 3X3 I9V OVV 313 IIV VOV 131 zm i ⁇ ⁇ OL VV3 330 VII ⁇ ⁇ ⁇ VOO ⁇ ⁇ V VOV 131 2- ⁇ ⁇ m
  • the target CDR3 region was amplified with a primer of each family of the TCRV] 3 chain in Table 1 and a C ⁇ primer (1 st PCR).
  • the target CDR3 region was further amplified with primers of each family of the CRVJ3 chain in Table 2 and Cj3 primer (2nd PCR).
  • this cDNA was used in the presence of each V region primer in Table 1 and 50 pmo 1 of each of the C-region 5, terminal-end biotinylated primers, and 10 nmo 1 of each of the four types of deoxynucleotide triphosphates.
  • lO XT aq (thermostable DNA) polymerase buffer 50 OmM KC 1, l O OmM Tris-HCl,
  • the PCR product obtained above was subjected to the SSCP method (single-strand 'conformation. Polymorphism) (Ori t a Me t a 1.
  • the obtained PCR product is diluted 1:20 in a denaturing solution (95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), and is then diluted to 90 ° C for 2 minutes. It was left still.
  • a denaturing solution 95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol
  • Synovial tissue which is a joint lesion of a patient with osteoarthritis, was taken, placed in 1 ml of ISOGEN, and homogenized, and then RNA was isolated in the same manner as 1) in Example].
  • RNA obtained in 1) above was detected according to 2) to 5) of Example 1.
  • the results are shown in FIG. As is clear from the figure, in the synovium of the local knee, several bands were detected in all V / 3 chain repertoires from V ⁇ 1 force to V ⁇ 20, and oligo-specific ⁇ It became clear that the cells had accumulated.
  • the sequence can be determined by analyzing the mRNA extracted from the synovial tissue of Example 2 as type II using the RT-PCR method using the entire cellular RNA.
  • Total cellular RNA is isolated from peripheral blood or synovial tissue as described in the manual (ISOGEN, Nippon Gene).
  • the single-stranded cDNA is reverse-transcribed using a cDNA synthesis kit (cDNA Synthes ismodel, Amersham).
  • the TCRV 3 gene segment is amplified by PCR using the primers shown in Table 1 above.
  • sequence is determined by using SequencIngReadyReaction-2 1M1 3, PEApPli (EdBiosystems). (Array No .:! ⁇ 376)
  • the amino acid sequence of the present invention is used as a tool for confirmatory diagnosis of osteoarthritis. be able to. Furthermore, since it is possible to isolate and identify an antigen that the TCR specifically recognizes using a cell having the amino acid sequence of the present invention in the CDR3 region of the TCR V 3 chain, osteoarthritis Can greatly contribute to the development of therapeutic, diagnostic and prophylactic drugs.
  • the amino acid sequence of the present invention can also be used for immunotherapy of osteoarthritis by using it for TCR cutination, activation of immune control cells or induction of anti-TCR antibodies.
  • the peptide having homology to the peptide having the amino acid sequence of the present invention can create cells that cannot attack the antigen of the patient and can block the antigen-specific TCR by direct administration to the patient. It is. Further, an antigen peptide or antigen protein to which TCR having the amino acid sequence of the present invention in the CDR3 region specifically reacts is a therapeutic and / or prophylactic agent for osteoarthritis that induces antigen-specific immune tolerance. Useful as

Abstract

Amino acid sequences of the Vβchain CDR3 region of an antigen receptor of oligoclonal pathogenic T cells which are accumulated and proliferate in the articular synovial membranes of patients with arthrosis deformans. Use of these amino acid sequences enables the diagnosis of arthrosis deformans. Moreover, self-antigen peptides or derivatives thereof to which the T cells containing these amino acid sequences bond specifically and self-antigen proteins from which these self-antigen peptides originate are useful as remedies or preventives capable of inducing antigen-specific immunological tolerance to arthrosis deformans.

Description

明 細 書 変形性関節炎に特異的な τ細胞抗原レセプター配列 技術分野  Description τ cell antigen receptor sequence specific for osteoarthritis
本発明は、 変形性関節炎の診断、 予防及び治療に有用な物質に関する。 さらに 詳しくは、 変形性関節炎患者の滑膜細胞及び/又は滑液中に存在する τ細胞の T 細胞抗原レセプター （TCR) 鎖の CDR 3領域を含むアミノ酸配列に関す る。  The present invention relates to a substance useful for diagnosing, preventing and treating osteoarthritis. More specifically, the present invention relates to an amino acid sequence containing a CDR3 region of a T cell antigen receptor (TCR) chain of τ cells present in synovial cells and / or synovial fluid of osteoarthritis patients.
背景技術 Background art
変形性関節炎は、 老化あるいは関節の形成異常、 先行する関節疾患または外傷 などを要因として起こる疾患で、 特に荷重関節に多い。 加齢に伴い軟骨基質産生 の低下や軟骨に栄養を補給する関節液の軟骨基質内への侵入が不十分となる退行 性変化をきたし、 また軟骨への肥満などによる荷重負荷や関節運動などによる機 械的負荷が加わることで関節軟骨は損傷される。 関節軟骨は血管が存在しないた め自己修復能力に乏しく、 ひとたび損傷が起こると退行性変化は容易に進展する。 これに伴い、 破壊された軟骨細胞等より様々な化学伝達物質やサイ トカインが放 出され、 滑膜の慢性炎症を引き起こすと考えられてきた （C I .  Osteoarthritis is a disease caused by aging or dysplasia of joints, preceding joint disease or trauma, and is particularly common in weight-bearing joints. As the body ages, the cartilage matrix production decreases and the degeneration of the synovial fluid, which supplements the cartilage, becomes insufficient due to insufficient penetration of the synovial fluid into the cartilage matrix. Articular cartilage is damaged by the mechanical load. Articular cartilage has poor self-healing capacity due to the absence of blood vessels, and once damage occurs, degenerative changes can easily develop. Along with this, various chemical mediators and cytokines are released from the destroyed chondrocytes and the like, and it has been thought that they cause chronic inflammation of the synovium (CI.
We s t a c o t t e t a 1. , s e m i n a r s i n We s t a c o t t e t a 1., s e m i n a r s i n
A r t h r i t i s a n d Rh e uma t i s m, 2 5 : 2 54— 2 72 : 1 996) 。 A r t h r i t i s a n d Rh e uma t i s m, 25: 2 54— 2 72: 1 996).
一方、 変形性関節炎とは異なり、 慢性関節リゥマチは自己免疫疾患として分類 されている。 免疫系の応答は、 本来、 外部から侵入する異物を破壊し、 排除する ために惹起される反応であり、 通常は非自己分子 （抗原） に対して特異的である。 しかし、 何らかの原因で自己分子と非自己分子の識別に障害が起きると自己免疫 疾患が生じる。 慢性関節リウマチ （リウマチ性関節炎） においては滑膜組織の異 常な増殖が認められる。 また、 増殖した組織には T細胞の集積が観察される。 細 胞性免疫を担う T細胞が抗原を認識するには抗原のみならず、 マクロファージな どの抗原呈示細胞が必要である。 組織適合性抗原と抗原呈示細胞内でプロセッシ ングを受けた外来抗原との複合体を T細胞抗原レセプター （TCR) が認識する。 抗原呈示細胞からの抗原シグナルを受けた Τ細胞は局所で増殖し、 各種サイ トカ インを放出し、 炎症を進展させる。 TCRはひおよび ]3、 あるいは γおよび δの それぞれ 2本のポリべプチド鎖のジスルフィ ド結合によるへテロダイマ一である。 近年、 慢性関節リウマチ患者の関節中には TCRのある特定の V領域を発現す る Τ細胞クローンが蓄積することが報告されている。 慢性関節リゥマチの治療の 中心は非ステロイ ド系抗炎症剤、 免疫調節剤およびステロイ ド剤であるが、 近年、 病因である特定の自己抗原に反応性を有する Τ細胞の生体からの除去 （例、 Τ細 胞ヮクチネーシヨン、 TCRヮクチネーシヨンの使用） や、 該抗原に対する自己 免疫寛容の誘導が行われるようになった。 慢性関節リゥマチに上記の方法を適用 するためには、 慢性関節リゥマチの発症に関与する TCRとそれをコードする遺 伝子について分析し、 該 T C Rが特異的に認識する抗原を解明することが重要で あるとされている。 On the other hand, unlike osteoarthritis, rheumatoid arthritis is classified as an autoimmune disease. The immune system's response is a response that is originally triggered to destroy and eliminate foreign invaders, and is usually specific for non-self molecules (antigens). However, if for some reason the identification of self and non-self molecules is impaired, an autoimmune disease results. In rheumatoid arthritis (rheumatic arthritis), abnormal proliferation of synovial tissue is observed. In addition, accumulation of T cells is observed in the proliferated tissue. In order for T cells responsible for cellular immunity to recognize antigens, not only antigens but also antigen-presenting cells such as macrophages are required. Processing in histocompatible antigens and antigen-presenting cells The T cell antigen receptor (TCR) recognizes the complex with the foreign antigen that has undergone immunization. Τ cells that have received an antigen signal from antigen presenting cells proliferate locally, release various cytokines, and develop inflammation. The TCR is a heterodimer due to the disulfide bond of two polypeptide chains, γ and δ, or γ and δ, respectively. Recently, it has been reported that 関節 cell clones expressing a specific V region of TCR accumulate in the joints of patients with rheumatoid arthritis. The mainstays of treatment for rheumatoid arthritis are non-steroidal anti-inflammatory drugs, immunomodulators, and steroid drugs. Recently, the removal of cells that are reactive to specific pathogenic autoantigens (eg, And the use of cell culture and TCR culture) and induction of autoimmune tolerance to the antigen. In order to apply the above method to rheumatoid arthritis, it is important to analyze the TCRs involved in the onset of rheumatoid arthritis and the genes encoding them, and to clarify the antigens that the TCRs specifically recognize It is said to be.
変形性関節炎患者においても、 自己免疫疾患である慢性関節リゥマチ患者同様 に滑膜組織での炎症は認められるが、 慢性関節リウマチに比較して弱い。 また、 慢性関節リゥマチは滑膜及び滑液中に強い Τ細胞の集積が認められる自己免疫疾 患であるが、 変形性関節炎では関節滑膜及び滑液への τ細胞の浸潤は認められず In patients with osteoarthritis, inflammation in synovial tissue is observed as in patients with rheumatoid arthritis, which is an autoimmune disease, but is weaker than in rheumatoid arthritis. Rheumatoid arthritis is an autoimmune disease in which strong 強 い cells accumulate in the synovium and synovial fluid, but no τ cells infiltrate into the synovium and synovial fluid in osteoarthritis.
{C I . We s t a c o t t e t a 1 . , S e m i n a r s i n A r t h r i t i s a n d Rh e um a t i s m, 2 5 ： 254— 2 7 2 ： 1 9 96, O Du k e e t a 1. , C 1 i E x p I mm u n o 1 4 9 : 22 : 1 9 8 2) 、 自己免疫疾患ではないという考え方が主流である。 We stacotteta 1., S eminarsin A rthritisand Rh eum atism, 25: 254—27 2: 1 96, O Du keeta 1., C 1 iE xp I mm uno 1 4 9: 22: 1 9 8 2) The main idea is that it is not an autoimmune disease.
しかし、 変形性関節炎患者では、 CD 4そして CD 8陽性 T細胞、 B細胞、 単 球 Zマク口ファージのような免疫担当細胞が滑液組織に浸潤してくる  However, in patients with osteoarthritis, immunocompetent cells such as CD4 and CD8 positive T cells, B cells, and monocytes Z macula phage infiltrate the synovial tissue
(R e v e l l PA, e t a 1. An n. Rh e um. D i s . 4_7_ 300- 3 0 7 (1 9 8 8) ) という報告があり、 免疫システムが変形 性関節炎発症の原因であることを示唆していると考えることができる。 また、 変 形性関節炎患者の滑膜から T細胞を精製し、 ]3鎖の遺伝子をサザンプロッティン グにより解析すると、 ]3鎖の遺伝子再構成はオリゴクローナルなパターンを示し、 このことは、 変形性関節炎患者においては限定された数の T細胞クローンが活性 ィ匕されていることを示している （S t ame n k o v i c I， P r o c. Na t l . Ac a d. S c i . USA 8_5_ 1 1 79— 1 1 83 ( 1(R evell PA, eta 1. Ann. Rh eum. D is. 4_7_ 300- 3 0 7 (1 988)) suggesting that the immune system is the cause of the development of osteoarthritis You can think that you are. In addition, when T cells were purified from the synovium of a patient with osteoarthritis and the three-chain gene was analyzed by Southern blotting, the rearrangement of the three-chain gene showed an oligoclonal pattern. Limited number of T cell clones active in osteoarthritis patients United States 8_5_ 1 1 79—1 1 83 (1
988) ) という報告もある。 988)).
発明の開示 Disclosure of the invention
本発明の目的は、 変形性関節炎も慢性関節リウマチ同様自己反応性 T細胞が関 与する慢性関節炎をその基礎病態とするとの考え方のもとに、 関節滑膜及び滑液 中に存在するオリゴクロ一ナルな T細胞の集積 ·増殖を明らかにし、 さらに、 T C Rの C D R 3領域のァミノ酸配列を決定し、 変形性関節炎の診断、 予防及び治 療薬を提供することに有る。  An object of the present invention is to provide a method for treating oligoarthritis present in the synovial membrane and synovial fluid, based on the idea that osteoarthritis, like rheumatoid arthritis, is based on chronic arthritis involving autoreactive T cells. The purpose of the present invention is to clarify the accumulation and proliferation of null T cells, determine the amino acid sequence of the CDR3 region of the TCR, and provide a diagnostic, preventive and therapeutic agent for osteoarthritis.
本発明者らは、 変形性関節炎の発症に関連する τ細胞を明らかにすることを目 的とし、 変形性関節炎患者の滑膜組織中において、 オリゴクローナルに増殖して いる病因性 T細胞の TCRの V ]3鎖 CD R 3領域を含むァミノ酸配列を決定した。 その結果、 変形性関節炎患者の滑膜組織中には、 VjS 1から V|320までのほと んど全ての TCRV ]3鎖レパトァに属する CD R 3領域を有する T細胞が集積し、 各サブタイプにおいて更に幾つかのクローンが存在していることを見出した。 更 に、 TCRの CDR3領域は、 個々の T細胞によってアミノ酸配列が最も異なる 部位であるとされているが、 本発明においては、 例えば、 Gln-Val - Gly (患者 C ：配列番号 1 75の 6〜8番目及び患者 A：配列番号 1 77の6〜8番目） ゃ、 Lys-Arg-Gly-Ser (患者 B ：配列番号 1 79の 6〜 9番目及び患者 C ：配列番号 1 81の 6〜 9番目） のように、 異なる患者間で CDR 3領域が完全に一致した 配列が認められた。 また、 Arg- Ala- Gly (患者 A ：配列番号 43の 1 2〜14番 目、 患者 B ：配列番号 91の 6〜 8番目、 患者 C ：配列番号 105の 3〜 5番目 及び患者 G ：配列番号 343の 5〜 7番目の配列） や、 Arg- Gin- Gly (患者 A ： 配列番号 43の 6〜 8番目、 患者 B ：配列番号 87の 7〜 9番目、 患者 C ：配列 番号 1 09の 6〜8番目、 患者 D ：配列番号 1 53の 7〜 9番目及び患者 G ：配 列番号 3 25の 3番目〜 5番目の配列） 、 Gly-Thr- Gly (患者 A：配列番号 1 9 1の 5〜 7番目、 患者 C ：配列番号 1 93の 8〜 1 0番目、 患者 D ：配列番号 1 51の 6〜 8番目、 患者 E ：配列番号 275の 3〜5番目、 患者 G ：配列番号 3 37の 4〜 6番目及び患者 H：配列番号 367の 2〜4番目の配列） 、 Gly- Ser Ala (患者 A ：配列番号 1 97の8〜1 0番目及び患者 B ：配列番号 61の 6〜 8番目） 、 Ser-Gly- Ser (患者 F ：配列番号 257の :！〜 3番目及び患者 G ：配 列番号 303の 2〜4番目の配列） 、 Arg-Arg- Ala (患者 E ：配列番号 20 1の 2〜 4番目及び患者 G ：配列番号 343の 4〜 6番目の配列） 、 Gin- Gly- Val (患者 F ：配列番号 267の 2〜4番目及び患者 G ：配列番号 35 1の 3〜5番 目の配列） 、 Pro- Gly- Thr (患者 F ：配列番号 275の 2〜4番目及び患者 G ： 配列番号 3 1 1の 2〜4番目の配列） 、 Thr-Gly-Leu (患者 F ：配列番号 275 の 4番目〜 6番目、 患者 F ：配列番号 233の 2〜4番目及び患者 G ：配列番号 33 7の 5〜 7番目の配列） 、 Ser- Gly- Ala (患者 F ：配列番号 273の 2〜 4 番目及び患者 G ：配列番号 3 1 7の 2〜 4番目の配列） 、 Glu-Gly-Pro (患者 F ：配列番号 29 1の 4〜6番目、 患者 F ：配列番号 255の 2〜4番目及び患 者 G ：配列番号 333の 2〜4番目の配列） 、 Leu-Thr- Gly (患者 F ：配列番号 24 1の 2〜4番目、 患者 F ：配列番号 229の 1〜 3、 患者 F ：配列番号 23 3の 1〜 3番目及び患者 G ：配列番号 34 7の 3〜 5番目の配列） 、 Leu- Ser- Pro (患者 F ：配列番号 23 1の 1〜 3番目及び患者 H：配列番号 3 59の 3〜 5番目の配列） 、 Gly- Gly- Gly (患者 F ：配列番号 23 1の 5〜7番目及び患者 G ：配列番号 33 5の 2〜4番目の配列） 、 Thr-Gly- Ala (患者 F ：配列番号 2 29の 2〜4番目及び患者 G ：配列番号 34 1の 2〜4番目の配列） 、 Thr- Gly- Lys (患者 F ：配列番号 249の 3〜 5番目及び患者 G ：配列番号 307の 3〜 5番目の配列） 、 Gin- Gly- Leu (患者 F ：配列番号 263の 3〜 5番目及び患者 G ：配列番号 33 1の:！〜 3番目の配列） のように、 異なる患者間で C D R 3領 域のァミノ酸配列の一部が保存された配列も認められた。 The present inventors aimed to elucidate the τ cells associated with the development of osteoarthritis, and investigated the TCR of pathogenic T cells oligoclonally proliferating in the synovial tissue of osteoarthritis patients. The amino acid sequence containing the V] 3 chain CDR3 region was determined. As a result, in the synovial tissue of patients with osteoarthritis, almost all TCRV from VjS1 to V | 320, T cells having the CDR3 region belonging to the three-chain repertoire accumulate, and It was found that several more clones were present in the type. Furthermore, the CDR3 region of the TCR is said to be a site where the amino acid sequence is most different depending on individual T cells. In the present invention, for example, Gln-Val-Gly (patient C: 6 of SEQ ID NO: 175) 8, Lys-Arg-Gly-Ser (Patient B: 6th to 9th of SEQ ID NO: 179 and Patient C: 6th to 8th of SEQ ID NO: 181) As shown in Fig. 9), the CDR3 region was completely identical between different patients. Arg-Ala-Gly (Patient A: 12-14th of SEQ ID NO: 43, Patient B: 6th-8th of SEQ ID NO: 91, Patient C: 3-5th of SEQ ID NO: 105 and Patient G: Sequence No. 343 5th to 7th sequence) and Arg-Gin-Gly (Patient A: 6th to 8th of SEQ ID NO: 43, Patient B: 7th to 9th of SEQ ID NO: 87, Patient C: SEQ ID NO: 109 6th to 8th, Patient D: 7th to 9th of SEQ ID NO: 153 and Patient G: 3rd to 5th sequence of SEQ ID NO: 325, Gly-Thr-Gly (Patient A: SEQ ID NO: 191) 5th to 7th, Patient C: 8th to 10th of SEQ ID NO: 193, Patient D: 6th to 8th of SEQ ID NO: 151, Patient E: 3rd to 5th of SEQ ID NO: 275, Patient G: SEQ ID NO: 337 4th to 6th and patient H: SEQ ID NO: 367 2nd to 4th sequence), Gly-Ser Ala (Patient A: 8 to 10th of SEQ ID NO: 197 and Patient B: 6th to 8th of SEQ ID NO: 61), Ser-Gly- Ser (Patient F::! To 3rd of SEQ ID NO: 257 and Patient G : Arg-Arg-Ala (patient E: 2nd to 4th of SEQ ID NO: 201 and patient G: 4th to 6th sequence of SEQ ID NO: 343), Gin- Gly-Val (Patient F: 2nd to 4th of SEQ ID NO: 267 and Patient G: SEQ ID NO: 35 to 3rd to 5th sequence), Pro-Gly-Thr (Patient F: 2nd to 4th of SEQ ID NO: 275) And patient G: SEQ ID NO: 311 2nd to 4th sequence), Thr-Gly-Leu (patient F: SEQ ID NO: 275 4th to 6th, patient F: SEQ ID NO: 233 2nd to 4th and patient G: 5th to 7th sequence of SEQ ID NO: 337), Ser-Gly-Ala (patient F: 2nd to 4th sequence of SEQ ID NO: 273 and patient G: 2nd to 4th sequence of SEQ ID NO: 317), Glu-Gly-Pro (Patient F: 4th-6th of SEQ ID NO: 29, Patient F: No. 255, 2nd to 4th and patient G: SEQ ID NO: 333, 2nd to 4th sequence), Leu-Thr-Gly (patient F: 2nd to 4th of SEQ ID NO: 24, patient F: SEQ ID NO: 229) 1 to 3, Patient F: 1st to 3rd of SEQ ID NO: 233 and Patient G: 3rd to 5th sequence of SEQ ID NO: 347), Leu- Ser-Pro (Patient F: 1 to 3 of SEQ ID NO: 231) And patient H: 3rd to 5th sequence of SEQ ID NO: 359), Gly-Gly-Gly (patient F: 5th to 7th of SEQ ID NO: 231 and patient G: 2nd to 4th of SEQ ID NO: 335) Sequence), Thr-Gly-Ala (patient F: 2nd to 4th sequence of SEQ ID NO: 229 and patient G: 2nd to 4th sequence of SEQ ID NO: 34 1), Thr-Gly-Lys (patient F: SEQ ID NO: 249) 3rd to 5th and patient G: 3rd to 5th sequence of SEQ ID NO: 307), Gin-Gly-Leu (patient F: 3rd to 5th of SEQ ID NO: 263 and patient G: SEQ ID NO. (3rd sequence), a sequence in which part of the amino acid sequence in the CDR3 region was conserved among different patients was also observed.
本発明のァミノ酸配列を T C R V 鎖 C D R 3領域に有する T細胞は、 変形性 関節炎の病因性 Τ細胞であるため、 本発明のアミノ酸配列は変形性関節炎の確認 的診断の道具として用いることができる。 更に、 本発明のアミノ酸配列を TCR Vj3鎖の CDR3領域に有する Τ細胞を使用して該 TCRが特異的に認識する抗 原を単離、 同定することが可能となるため、 変形性関節炎の治療、 診断及び予防 薬の開発に大きく寄与し得る。 本発明のアミノ酸配列は、 TCRヮクチネーショ ン、 TCR DNAヮクチネーシヨン、 免疫制御細胞の活性化あるいは抗 TCR 抗体の誘発にそれを用いることにより、 変形性関節炎の免疫治療のために用いる こともできる。 本発明のアミノ酸配列を有するペプチドと相同性のあるペプチド は、 直接患者に投与することにより、 患者の抗原を攻撃できない T細胞を作り出 し、 抗原特異的な TCRをブロックすることが可能である。 また、 本発明のアミ ノ酸配列を C D R 3領域に有する T C Rが特異的に反応する抗原ぺプチド若しく は抗原蛋白質は、 抗原特異的な免疫寛容を誘導する変形性関節炎治療薬及び/又 は予防薬として有用である。 Since T cells having the amino acid sequence of the present invention in the CDR3 region of the TCRV chain are pathogenic Τ cells of osteoarthritis, the amino acid sequence of the present invention can be used as a tool for confirmatory diagnosis of osteoarthritis. . Furthermore, since it is possible to isolate and identify an antigen that the TCR specifically recognizes using a cell having the amino acid sequence of the present invention in the CDR3 region of the TCR Vj3 chain, the treatment of osteoarthritis can be performed. It can greatly contribute to the development of diagnostic and prophylactic drugs. The amino acid sequence of the present invention may be used for TCR protein, TCR DNA protein, activation of immunoregulatory cells or anti-TCR By using it to elicit antibodies, it can also be used for immunotherapy of osteoarthritis. A peptide having homology to the peptide having the amino acid sequence of the present invention can produce T cells that cannot attack a patient's antigen and can block antigen-specific TCR by direct administration to a patient. . In addition, the antigen peptide or antigen protein which specifically reacts with the TCR having the amino acid sequence in the CDR3 region of the present invention is a therapeutic agent for osteoarthritis which induces antigen-specific immune tolerance and / or Useful as a prophylactic.
図面の簡単な説明 BRIEF DESCRIPTION OF THE FIGURES
図 1はヒ ト変形性関節炎患者の末梢静脈血及び関節滑膜から得た試料を S S C P法により検出した結果を示す電気泳動クロマトグラムの写真である。  FIG. 1 is a photograph of an electrophoresis chromatogram showing the results of detecting samples obtained from peripheral venous blood and synovial membrane of a patient with human osteoarthritis by the SSCP method.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明により、 変形性関節炎に関与する T C Rの部分配列が明らかにされたの で、 該配列を用いて変形性関節炎関連抗原の認識に関与する部分ペプチド、 およ びそれをコードする DN Aおよび DN A断片を得、 適当な宿主細胞に発現させる ことにより、 変形性関節炎の診断に用いることができる。 変形性関節炎の診断は 臨床的特徴に基づいてなされるが、 本発明のアミノ酸配列と、 変形性関節炎と疑 われる患者の T C R V |3鎖の配列を比較し、 相同性を指標にして変形性関節炎に 罹患しているかどうかを診断する。 たとえば、 変形性関節炎の疑いのある患者の 体液から mRNAを取り出し、 RT— PCR法により c DNAライブラリーを作 成し V ]3鎖のプローブを用いて各 V ]3鎖サブクラスを発現させる。 この発現させ た T細胞クロ一ンを S S C Pにより観察することもできる。 その後クローナルに 発現した TCRVj3鎖の CDR3部分の塩基配列並びにこれから由来するァミノ 酸配列を比較する。 ここでいう体液には滑液も含まれる。  According to the present invention, the partial sequence of the TCR involved in osteoarthritis has been elucidated, and the partial peptide involved in the recognition of osteoarthritis-related antigen using the sequence, DNA encoding the same, and By obtaining the DNA fragment and expressing it in a suitable host cell, it can be used for diagnosis of osteoarthritis. Diagnosis of osteoarthritis is based on clinical characteristics, but the amino acid sequence of the present invention is compared with the sequence of the TCRV | 3 chain of a patient suspected of having osteoarthritis. Diagnose if you are suffering from For example, mRNA is removed from the body fluid of a patient suspected of having osteoarthritis, a cDNA library is created by RT-PCR, and each V] 3 chain subclass is expressed using a V] 3 chain probe. The expressed T cell clone can also be observed by SSCP. Thereafter, the nucleotide sequence of the clonally expressed CDR3 portion of the TCRVj3 chain and the amino acid sequence derived therefrom are compared. The body fluid here includes synovial fluid.
本発明のァミノ酸配列を有するぺプチドと相同性を有するぺプチド若しくはそ れらの誘導体、 あるいは TCRV/3鎖 CDR3領域に本発明のアミノ酸配列を有 する T細胞と特異的に結合する抗原蛋白質若しくは抗原ぺプチドは、 変形性関節 炎の治療もしくは予防薬として単独で、 あるいは、 これらの 2種以上を組み合わ せて、 使用することができる。 また、 これらのペプチドは、 通常、 一般に使用さ れる各種剤形で、 全身的、 経口あるいは局所に使用することができる。 剤形とし ては、 たとえば、 経口剤、 注射剤、 経鼻剤等が挙げられる。 これらの剤形の製剤 は、 常法に従い、 陚形剤、 結合剤、 溶解剤、 乳化剤、 懸濁化剤などの通常使用す ることができる担体を用いて調製することができる。 例えば、 経口剤としては、 錠剤、 顆粒剤、 散剤等の固形剤、 溶液剤、 懸濁剤、 乳剤等の液剤等が挙げられる。 本発明のペプチドは遺伝子組換えにより限定されるものではなく、 一般に、 R. B. Me r r i f i e l d、 J . Am. C h e m. S o c. 85， 2149 (1 963) に記載されているような方法を使用して調製することができるが、 他の 同等の既知の化学的合成法を用いる事もできる。 A peptide having homology to the peptide having the amino acid sequence of the present invention or a derivative thereof, or an antigen protein which specifically binds to a T cell having the amino acid sequence of the present invention in the TCRV / 3 chain CDR3 region Alternatively, the antigen peptide can be used alone or in combination of two or more of these as a therapeutic or prophylactic agent for osteoarthritis. These peptides can be used systemically, orally or topically in various commonly used dosage forms. Dosage form For example, oral preparations, injection preparations, nasal preparations and the like can be mentioned. Preparations in these dosage forms can be prepared according to a conventional method, using commonly used carriers such as a vehicle, a binder, a solubilizer, an emulsifier, and a suspending agent. For example, the oral preparations include solid preparations such as tablets, granules and powders, and liquid preparations such as solutions, suspensions and emulsions. The peptide of the present invention is not limited by genetic recombination, and generally employs a method as described in RB Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). However, other equivalent known chemical synthetic methods can be used.
実施例 Example
以下に本発明を実施例によりさらに詳細に説明するが、 これらの実施例は本発 明の具体例を説明するものであって、 本発明はこれらの実施例に限定されるもの ではない。  Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples illustrate specific examples of the present invention, and the present invention is not limited to these examples.
実施例 1 変形性関節炎患者の末梢静脈血由来の T C R V β鎖の C D R 3領域の 検出 Example 1 Detection of CDR3 Region of TCRV β Chain from Peripheral Venous Blood of Osteoarthritis Patient
1 ) 変形性関節炎患者の末梢静脈血のリンパ球からの R Ν Αの単離  1) Isolation of R Ν リ ン パ from lymphocytes of peripheral venous blood of patients with osteoarthritis
変形性関節炎患者の末梢血 1 Om 1をへパリンを加えて採血し F i c o 1 1 P a q u e (Ph a rma c i a B i o t e c h) を用いて、 比重遠心法によ りリンパ球を単離し、 得られたリンパ球から、 I SOGEN (株式会社二ツボン ジーン） のキットに従い、 mRNAを抽出した。 すなわち、 リンパ球ペレッ トに I S〇GENを1. Om l加え、 1分間混和し、 室温で 5分間静置した。 さらに クロ口ホルムを 0. 2m l加え、 1 5秒間混和し、 室温で 2分間静置した。 次に、 1 5， 000 r pm、 4°Cで 1 5分間遠心し上清を得た。 上清に、 イソプロパノ ール 500 1を加え、 数回転倒混和した。 室温に 5分間静置し、 1 5， 000 r p m、 4。Cで 1 0分間遠心し、 得られた R N Aぺレッ トを 75 %ェタノールで 洗浄した。  Peripheral blood of an osteoarthritis patient 1 Om1 was collected by adding heparin to blood, and lymphocytes were isolated by specific gravity centrifugation using Fico 11 P aque (Pharmacia Biotech). MRNA was extracted from the lymphocytes according to the kit of ISOGEN (Nitsubon Gene Co., Ltd.). That is, 1. Oml of IS1GEN was added to the lymphocyte pellet, mixed for 1 minute, and allowed to stand at room temperature for 5 minutes. Further, 0.2 ml of black-mouthed form was added, mixed for 15 seconds, and allowed to stand at room temperature for 2 minutes. Next, the mixture was centrifuged at 15,000 rpm at 4 ° C. for 15 minutes to obtain a supernatant. Isopropanol 500 1 was added to the supernatant, and mixed several times by inversion. Incubate at room temperature for 5 minutes, 15,000 rpm, 4. The mixture was centrifuged at C for 10 minutes, and the obtained RNA pellet was washed with 75% ethanol.
得られた R N Aの精製度及び濃度はァガロースゲルにより確認した。  The degree of purification and the concentration of the obtained RNA was confirmed by agarose gel.
2) c DNAの合成  2) cDNA synthesis
上記 1 ) で得られた m RNAから c DNA s y n t h e s i s  From the mRNA obtained in 1) above, cDNA synthesis
mo l e c u l e (アマシャム） を用い、 c DNAを合成した。 すなわち、 上記 1 ) で得られた R N Aペレッ トに T E緩衝液 （1 0 mMトリス塩酸緩衝液、 1 mM E DT A) を 1 00 μ 1加え溶解し、 RNA溶液1 0 yu l に、 5 Xリ ノく ーストランスクリプターゼ反応液を 4 μ N a P P i (S o d i um p y r o p h o s p h a t e) を l /i l、 RNa s eインヒビタ一 1 μ 1、 d N TPを l O nmo lを 2 μ し オリゴ dT (An c h o r e d dT25) を 1 μ ΐ加え、 リバーストランスクリプターゼ 1 1を加え、 42°。で1時間ィンキ ュペートし、 c DNAを得た。 cDNA was synthesized using mo lecule (Amersham). That is, TE buffer RNA pellet obtained in 1) (1 0 mM Tris-HCl buffer, 1 mM E DT A) 1 00 μ 1 was added and dissolved, the RNA solution 1 0 y ul, 5 X Li carbonochloridate 1 μl of RNase inhibitor, 1 μl of RNase inhibitor, 2 μm of lNTPl, 2 μl of transcriptase reaction mixture, and 2 μl of oligo dT (An chored dT25 ) And reverse transcriptase 11 at 42 °. For 1 hour to obtain cDNA.
3) RT— PCRプライマーの合成 3) RT—PCR primer synthesis
上記 2) で得られた c DNA混合物を RT— PCR法に付すため、 PCRプラ イマ一として、 TCRj3鎖 V領域をコードする DNA断片及び ]3鎖 C領域をコー ドする DNA断片 （表 1および表 2に示す塩基配列） をアプライ ドバイオシステ ム （Ap p l i e d B i o s y s t em s) 社製 D N Aシンセサイザ一にて合 成した。 （Ch o i . Y. e t a 1 , P r o c. Na t l .  In order to subject the cDNA mixture obtained in 2) to the RT-PCR method, a DNA fragment encoding the TCRj 3 chain V region and a DNA fragment encoding the 3 chain C region (Tables 1 and 2) were used as PCR primers. (Base sequences shown in Table 2) were synthesized using a DNA synthesizer manufactured by Applied Biosystems. (Choi.Y.eta1 and Proc.Natl.
Ac a d. S c i . USA 86, 894 1 -8945 (1 989) ) Ac a d. S ci. USA 86, 894 1 -8945 (1 989))
マ— iia 基 配 列 Ma-iia base array
V]3 1 5 -TCT AGA ATT CCA AAA GGA AAC ATT CTT GAA C-3' V] 3 1 5 -TCT AGA ATT CCA AAA GGA AAC ATT CTT GAA C-3 '
2 5 -TCT AGA ATT CCC ACA TAG GAG CAA GGC GTC G-3' β 3 5 -TCT AGA ATT CGA AAA AGG AGA TAT TCC TGA G-3'25-TCT AGA ATT CCC ACA TAG GAG CAA GGC GTC G-3'β 35-TCT AGA ATT CGA AAA AGG AGA TAT TCC TGA G-3 '
4 5 -TCT AGA ATT CCA TAT GAG AGT GGA TTT GTC A— 3，4 5 -TCT AGA ATT CCA TAT GAG AGT GGA TTT GTC A—3,
Vi3 5 5 -TCT AGA ATT CAA AGG AAA CTT CCC TGG TCG A— 3' Vj3 6 5 -TCT AGA ATT CAG ATG ACT CAG GGC TGC CCA A— 3' V|3 7 5 -TCT AGA ATT CAG TGT GCC AAC TCG CTT CTC A— 3， β 8 5 -TCT AGA ATT CAT AGA TGA TTC AGG GAT GCC C-3' Vj3 9 5 -TCT AGA ATT CTG AAA CAG TTC CAA ATC GCT T-3' Vi3 10 5 -TCT AGA ATT CAA AGC AGA AAT AAT CAA TGA G-3' V|3 11 5 -TCT AGA ATT CAA GGG AGA TCT TTC CTC TGA G-3' VjS 12 5 -TCT AGA ATT CAA AGG AGA AGT CTC AGA TGG C-3' V|3 13 5 -TCT AGA ATT CAT GGC TAC AAT GTC TCC AGA T-3' V]3 14 5 -TCT AGA ATT CAA GGG AGA TGT TCC TGA AGG G-3' Vi3 15 5 -TCT AGA ATT CAA AGG AGA GAT CTC TGA TGG A— 3' V]3 16 5 - TCT AGA ATT CCT TTA TCG ACG TGT TAT GGG A— 3， V|3 17 5 -TCT AGA ATT CGG AGA TAT AGC TGA AGG GTA C-3' V|3 18 5 -TCT AGA ATT CGG AAT GCC AAA GGA ACG ATT T-3' Vj3 19 5 -TCT AGA ATT CGA GAT GCA CAA GAA GCG ATT C-3' Vi3 20 5 -TCT AGA ATT CCT CCT GGC AGG GGC CTC CAG C-3' Ci3 -TCT AGA ATT CTT CTG ATG GCT CAA ACA C-3' 3' 一 C]S 5 -CAT AGA CGA TGG TGG CAG-3' Vi3 5 5 -TCT AGA ATT CAA AGG AAA CTT CCC TGG TCG A— 3 'Vj3 65 -TCT AGA ATT CAG ATG ACT CAG GGC TGC CCA A— 3' V | 3 75 -TCT AGA ATT CAG TGT GCC AAC TCG CTT CTC A—3, β85 -TCT AGA ATT CAT AGA TGA TTC AGG GAT GCC C-3 'Vj3 95 -TCT AGA ATT CTG AAA CAG TTC CAA ATC GCT T-3' Vi3 105 -TCT AGA ATT CAA AGC AGA AAT AAT CAA TGA G-3 'V | 3 11 5 -TCT AGA ATT CAA GGG AGA TCT TTC CTC TGA G-3' VjS 12 5 -TCT AGA ATT CAA AGG AGA AGT CTC AGA TGG C-3 'V | 3 13 5 -TCT AGA ATT CAT GGC TAC AAT GTC TCC AGA T-3 'V] 3 14 5 -TCT AGA ATT CAA GGG AGA TGT TCC TGA AGG G-3' Vi3 15 5 -TCT AGA ATT CAA AGG AGA GAT CTC TGA TGG A—3 'V] 3 16 5-TCT AGA ATT CCT TTA TCG ACG TGT TAT GGG A—3, V | 3 17 5 -TCT AGA ATT CGG AGA TAT AGC TGA AGG GTA C-3' V | 3 18 5 -TCT AGA ATT CGG AAT GCC AAA GGA ACG ATT T-3 'Vj3 19 5 -TCT AGA ATT CGA GAT GCA CAA GAA GCG ATT C-3' Vi3 205 -TCT AGA ATT CCT CCT GGC AGG GGC CTC CAG C- 3 'Ci3 -TCT AGA ATT CTT CTG ATG GCT CAA ACA C-3' 3 'one C] S 5 -CAT AGA CGA TGG TGG CAG-3'
V voo 010 003 133 VOD 0V3 丄丄 V VOV 131 Ζ-Έ. HV voo 010 003 133 VOD 0V3 丄 丄 V VOV 131 Ζ-Έ. H
D VOO V33 3DV 0V3 313 303 丄丄 V VOV 131 z-mz ϋ κηD VOO V33 3DV 0V3 313 303 丄 丄 V VOV 131 z-mz ϋ κη
3 WD wo cm oiv VOX 313 1IV vov 丄 )丄 z- \ ϋ οζ3 WD wo cm oiv VOX 313 1IV vov 丄) 丄 z- \ ϋ οζ
0 VOV VVO 331 丄丄 V VOX 303 丄丄 V VOV 丄っ丄 0 VOV VVO 331 丄 丄 V VOX 303 丄 丄 V VOV
D VVO VOO 031 313 103 OVD IIV vov 丄:)丄  D VVO VOO 031 313 103 OVD IIV vov 丄 :) 丄
0 VOV 3 丄 )丄:） 丄；)丄 0V3 V13 丄丄 V VOV 丄：)丄 2-39 τ ϋ m 0 VOV 3 丄) 丄 :) 丄;) 丄 0V3 V13 丄 丄 V VOV 丄 :) 丄 2-39 τ ϋ m
V vvv 3 丄 3丄:） 丄!) V WO VXD 丄丄 V VOV 131 V vvv 3 丄 3 丄 :) 丄!) V WO VXD 丄 丄 V VOV 131
丄 300 DDI XIV ODV DVD VOO IIV VDV 丄:)丄 £ ΛΗ ST 丄 300 DDI XIV ODV DVD VOO IIV VDV 丄 :) 丄 £ ΛΗ ST
V V3I VOV 131 310 1DV JLVつ 丄丄 V VOV 131 Ζ-^ΖΙ £/ ΛΗ V 丄 VV 9V3 3X3 I9V OVV 313 IIV VOV 131 z-m i ϋ 丄 OL VV3 330 VII 丄丄丄 VOO 丄丄 V VOV 131 2-ΗΟΪ ΰ mV V3I VOV 131 310 1DV JLV 丄 丄 V VOV 131 Ζ- ^ ΖΙ £ / ΛΗ V 丄 VV 9V3 3X3 I9V OVV 313 IIV VOV 131 zm i ϋ 丄 OL VV3 330 VII 丄 丄 丄 VOO 丄 丄 V VOV 131 2- ΗΟΪ ΰ m
V 0V3 313 XVV VXD DVO 1DD 丄丄 V VOV 131 2-36 8ΆΗ 3 DXV 。W 丄っ。 VOX Oil V03 IIV VOV 101 z-m ϋ οτ 3 0V3 VV3 ODD 91V VOX 333 IIV VOV 131 Ζ- Ι £/ ΛΗ 0 0V3 10V 301 1X3 1X0 ODD 丄丄 V vov 丄:丄 s-39 d m 3 丄丄 3 VOO 030 00V IIV VOV 131 V 0V3 313 XVV VXD DVO 1DD 丄 丄 V VOV 131 2-36 8ΆΗ 3 DXV. W Po. VOX Oil V03 IIV VOV 101 zm ϋ οτ 3 0V3 VV3 ODD 91V VOX 333 IIV VOV 131 Ζ- Ι £ / ΛΗ 0 0V3 10V 301 1X3 1X0 ODD 丄 丄 V vov 丄: 丄 s-39 dm 3 丄 丄 3 VOO 030 00V IIV VOV 131
V 31V 333 III 9VV 3V0 丄丄：） 丄丄 V VOV 131  V 31V 333 III 9VV 3V0 丄 丄 :) 丄 丄 V VOV 131
V OVX 313 101 0V3 VIO 000 丄丄 V VOV 131  V OVX 313 101 0V3 VIO 000 丄 丄 V VOV 131
V 313 111 OVV 3V0 OVV ovo XIV VOV 丄っ丄 Ζ-ΆΖ £/ ΛΗ 1 DVD VVO VOO 331 Oil V03 IIV VOV 131  V 313 111 OVV 3V0 OVV ovo XIV VOV Pop 丄 -ΆΖ £ / ΛΗ 1 DVD VVO VOO 331 Oil V03 IIV VOV 131
fI8Z0/66df/X3d 80£9/66 Ο 4) RT-PCR fI8Z0 / 66df / X3d 80 £ 9/66 Ο 4) RT-PCR
上記 2) で得られた c DNAを铸型として、 表 1の TCRV ]3鎖の各ファミリ 一のプライマー及び C βプライマーにより、 目的の CDR 3領域を増幅させた (1 s t PCR) 。 1 s t PCRで得られた生成物を鍩型として、 表 2の Τ CRVJ3鎖の各ファミリーのプライマー及び C j3プライマーにより、 目的の CD R 3領域をさらに増幅させた （2 n d PCR) 。  Using the cDNA obtained in 2) above as type III, the target CDR3 region was amplified with a primer of each family of the TCRV] 3 chain in Table 1 and a Cβ primer (1 st PCR). Using the product obtained by 1 st PCR as type I, the target CDR3 region was further amplified with primers of each family of the CRVJ3 chain in Table 2 and Cj3 primer (2nd PCR).
すなわち、 この c DN Aを表 1の各 V領域のプライマー及び C領域の 5， 一末 端ピオチン化プライマー各 50 p mo 1の存在下で、 並びに 4種のデォキシヌク レオチドトリホスフェート各 1 0 n m o 1 の存在下で、 l O XT a q (耐熱性 DNA) ポリメラーゼ緩衝液 （50 OmM KC 1 、 l O OmMトリス一塩酸、 That is, this cDNA was used in the presence of each V region primer in Table 1 and 50 pmo 1 of each of the C-region 5, terminal-end biotinylated primers, and 10 nmo 1 of each of the four types of deoxynucleotide triphosphates. In the presence of lO XT aq (thermostable DNA) polymerase buffer (50 OmM KC 1, l O OmM Tris-HCl,
1 5 mM Mg C 1 2、 1 %T r i t ο η X— 1 00) を 5 1用レ、、 50 μ 1の容量で T a qポリメラ一ゼ （C e t u s ) 0. 5単位で増幅させた。 この ために、 この反応混合物をミネラルオイル 1 0 / 1で被覆し、 温度プロダラミン グ可能なブロック （DNAサーマルサイクラ一） 中で 94°C、 1. 5分； 60°C、 1. 5分；及び 72°C、 1分からなる温度循環 3 0回をこれに行ない、 目的とす る DNAを増幅した。 15 mM MgCl 2 , 1% Trit ο η X-100) was amplified in a volume of 51, 50 μl, and 0.5 units of Taq polymerase (Cetus). For this purpose, the reaction mixture is coated with mineral oil 10/1 and placed in a temperature programmable block (DNA thermal cycler) at 94 ° C, 1.5 minutes; 60 ° C, 1.5 minutes; This was followed by 30 cycles of temperature cycling consisting of 1 minute at 72 ° C. to amplify the target DNA.
5 ) 増幅した D N Aの非変性ゲルでの電気泳動  5) Electrophoresis of amplified DNA on non-denaturing gel
上記により得られた PCR生成物を S S C P法 （シングル · ストランド 'コン フオメーション . ポリモルフィズム） （O r i t a M e t a 1.  The PCR product obtained above was subjected to the SSCP method (single-strand 'conformation. Polymorphism) (Ori t a Me t a 1.
G e n om i c s 5 : 8 74— 8 79 : 1 9 8 9) により解析した。 The analysis was performed according to G enom ics 5: 874-87: 1989).
得られた PCR生成物を変性液 （9 5%ホルムアミ ド、 1 0mM EDTA、 0. 1 %ブロモフエノールブルー、 0. 1 %キシレンシァノール） に 1 ： 20に希釈 し、 90°Cに 2分間静置した。 The obtained PCR product is diluted 1:20 in a denaturing solution (95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), and is then diluted to 90 ° C for 2 minutes. It was left still.
この希釈液 2 μ 1を非変性ポリアクリルアミ ドゲル （ 4 %アクリルアミ ド /ビ ス （4 9 : 1 ) ： 1 0 ΧΤΒ Ε (0. 5 Μトリス、 2 0 mM N a , EDT A, 0. 9 7M B o r i c A c i d) p H 8. 3 5m l : 5%グリセ ロール： 1 0%AP S (アンモニゥムバーサルフェイ ト） 2 50 / 1 : ΤΕΜΕ D (Ν， Ν, Ν', Ν'—テトラメチレンジァミン） 50 μ 1 ) にのせ、 2時間 3 0Wで電気泳動を行なった。 泳動後、 ゲル中の DNAをィモビロン S (ミリジェン /バイオサーチ） に転 写し、 ストレプトアビジンピオチン標識アルカリホスファターゼ、 P PD 2 μl of this diluted solution was added to a non-denaturing polyacrylamide gel (4% acrylamide / vis (49: 1)): 10% (0.5% Tris, 20 mM Na, EDTA, 0%). 9 7M Boric A cid) pH 8.35 ml: 5% glycerol: 10% APS (Ammonium Versulfate) 2 50/1: ΤΕΜΕ D (Ν, Ν, Ν ', Ν) '-Tetramethylenediamine) and electrophoresed at 30 W for 2 hours. After electrophoresis, transfer the DNA in the gel to Immobilon S (Milligen / Biosearch), and use streptavidin-pyotin-labeled alkaline phosphatase, PPD
(Ch e m i l um i n e s c e n t s u b s t r a t e s y s t e m : P 1 e x TM Lum i n e s c e n t K i t , M i 1 1 i p o r e / B i o s e a c h) により、 検出した。  (Chemiluminescentssubbstratatesystem: P1exTM LuminesescentKit, Mi11ipore / Bioseach).
結果は図 1に示した。  The results are shown in FIG.
図 1から明らかなように、 変形性関節炎患者 （A) の末梢血においては、 バン ドはスメァ状になり、 これは、 ある特別の T細胞クローンが増加した状態でなく、 無数のクローンの集まりを示していると考えられる。  As is evident from Figure 1, in the peripheral blood of the osteoarthritis patient (A), the band became smeared, indicating that a large number of clones were collected instead of an increased number of certain special T cell clones. It is considered to indicate.
実施例 2 変形性関節炎患者の滑膜由来の TCRV ]3鎖の CD R 3領域の検出Example 2 Detection of CDR3 region of TCRV] 3 chain derived from synovium of osteoarthritis patient
1 ) 変形性関節炎患者の関節病変部である滑膜組織をとり、 I S O G E N 1 m 1に入れホモゲナイズした後、 実施例 ]の 1 ) と同様に RN Aを単離した。1) Synovial tissue, which is a joint lesion of a patient with osteoarthritis, was taken, placed in 1 ml of ISOGEN, and homogenized, and then RNA was isolated in the same manner as 1) in Example].
2) 上記 1 ) で得られた RNAを実施例 1の 2) から 5) に従い検出した。 結果 は図 1に示した。 図から明らかなように、 局所膝関節滑膜では、 V β 1力 ら V β 20までのすベての V/3鎖レパトァにおいて、 数本のバンドが検出され、 オリゴ ク口一ナルな Τ細胞が集積していることが明らかとなった。 2) The RNA obtained in 1) above was detected according to 2) to 5) of Example 1. The results are shown in FIG. As is clear from the figure, in the synovium of the local knee, several bands were detected in all V / 3 chain repertoires from Vβ1 force to Vβ20, and oligo-specific 口It became clear that the cells had accumulated.
実施例 3 配列の決定 Example 3 Determination of Sequence
1 ) 実施例 2で関節滑膜組織から抽出した mRNAを铸型として、 細胞性 RNA の全体を用いた RT— P C R法を用いて分析することにより配列を決定すること ができる。  1) The sequence can be determined by analyzing the mRNA extracted from the synovial tissue of Example 2 as type II using the RT-PCR method using the entire cellular RNA.
全体の細胞性 RNAは、 マニュアル （ I S OGEN、 株式会社ニッボンジー ン） に記載されているように、 末梢血液または滑膜組織から単離する。 一本鎖 c DNAを c DNA合成キット （c DNA S y n t h e s i s mo d e l、 ァ マシャム） を用いて逆転写する。 TCRV ]3遺伝子セグメントを、 上記表 1に示 すプライマーを用いて PCR法により増幅する。  Total cellular RNA is isolated from peripheral blood or synovial tissue as described in the manual (ISOGEN, Nippon Gene). The single-stranded cDNA is reverse-transcribed using a cDNA synthesis kit (cDNA Synthes ismodel, Amersham). The TCRV 3 gene segment is amplified by PCR using the primers shown in Table 1 above.
2) その後、 S S CP法により、 明瞭なバンドを切り出し、 この DN Aを上記表 2に示すプライマーにより増幅する。 あるいは、 上記 1 ) で得られる PCR産物 を 2 %ァガロースゲルにて電気泳動し、 増幅された P C R産物をゲルから回収し、 精製する。 3) 上記 2) で得られる PC R生成物あるいは PC R産物の精製物を 2) Then, a clear band is cut out by the SSCP method, and this DNA is amplified with the primers shown in Table 2 above. Alternatively, the PCR product obtained in 1) above is electrophoresed on a 2% agarose gel, and the amplified PCR product is recovered from the gel and purified. 3) Purify the PCR product or the purified PCR product obtained in 2) above.
I n v i t r o g e n, S a n D i e g o, C Aから提供されている指導 マニュアルによって T/Aクローニングベクターにサブクローン化する。 ライゲ ーシヨン混合物を、 適格の I NVa F' を形質転換するために用いる。 プラスミ ド DNAサンプルを調製し、 シークェンシングキット （DNA Subclone into the T / A cloning vector according to the instruction manual provided by Invitrogen, SanDigo, CA. The ligation mixture is used to transform competent I NVa F '. Prepare a plasmid DNA sample and use a sequencing kit (DNA
S e q u e n c i n g K i t , Dy e p r i me r し y c l e  S e q u e n c i n g K i t, Dy e p r i me r then y c l e
S e q u e n c i n g R e a d y R e a c t i o n-2 1M1 3, P E Ap p l i e d B i o s y s t e m s) を用いて配列を決定する。 （配列 N o . ：!〜 3 76) The sequence is determined by using SequencIngReadyReaction-2 1M1 3, PEApPli (EdBiosystems). (Array No .:! ~ 376)
4) なお、 本発明において開示するアミノ酸配列および塩基配列の由来は次の通 りである。 4) The origins of the amino acid sequence and base sequence disclosed in the present invention are as follows.
変形性関節炎患者 レパトァ 配列番号  Osteoarthritis patient repertoire SEQ ID NO:
Δ V fi 2 1 8ヽ 1丄 2 1 1 26  Δ V fi 2 1 8 ヽ 1 丄 2 1 1 26
A V j34 9 1 6、 1 2 7 1 2 8、  A V j34 9 1 6, 1 2 7 1 2 8,
1 9 1 ― 1 9 2  1 9 1 ― 1 9 2
A V j3 5 1 7 26  A V j3 5 1 7 26
A V β 6 2 7 28 、 1 2 9一 1 3 2 A V β 6 2 7 28, 1 2 9 1 1 3 2
A V β 7 2 9 30 、 1 9 5一 1 9 6A V β 7 2 9 30, 1 9 5 1 1 9 6
A V β 8 3 1 3 2 、 1 9 7一 1 98A V β 8 3 1 3 2, 1 9 7 1 1 98
A V β 9 1 7 7一 1 78 A V β 9 1 7 7 1 1 78
A V β 1 0 3 3 5 8 、 1 7 1一 1 72 A V β 1 0 3 3 5 8, 1 7 1 1 1 72
A V β 1 3 5 9 60 A V β 1 3 5 9 60
B V β 4 6 1 6 6 、 1 8 9一 1 90 B V β 4 6 1 6 6, 1 8 9 1 1 90
B V β 5 1 7 9一 1 80 B V β 5 1 7 9 1 1 80
B V β 7 6 7 72  B V β 7 6 7 72
B V β 8 7 3 74  B V β 8 7 3 74
B V β 9 1 7 3一 1 74  B V β 9 1 7 3 1 1 74
B V β 1 2 7 5 78  B V β 1 2 7 5 78
B V β 1 9 7 9 86 V T i c T I ε p d Λ し 6 z マ、 A [\ BV β 1 9 7 9 86 VT ic TI ε pd Λ 6 z Ma, A [\
q 7 T 8 Z ノ I Λ a q 7 T 8 Z no I Λ a
Figure imgf000015_0001
Figure imgf000015_0001
o Gコ 9 z i? T cf Λ J S2 ? 7 し V Z 7 T cf Λ J  o G KO 9 z i? T cf Λ J S2? 7 V V Z 7 T cf Λ J
Q 7 Ό ε z j J  Q 7 Ό ε z j J
o O p G T L z z Q o d σ j Λ v J o O p G T L z z Q o d σ j Λ v J
Figure imgf000015_0002
J Π 0 z a ci A 02
Figure imgf000015_0002
J Π 0 za ci A 02
7し fりj 7 Ό 6 I 7 cf Λ リ ム し D 9 I Q T d Λ ΓΤ 7 f 6 j 7 Ό 6 I 7 cf Λ rim D 9 I Q T d Λ ΓΤ
o  o
L g I ry  L g I ry
T T cf Λ Γ v_ΤL  T T cf Λ Γ v_ΤL
フ P T し I 6 L y ノ LI SI  F P T I 6 L y NO LI SI
G I n j d Τ  G I n j d Τ
T p o I a Ό d o \ v_L\  T p o I a Ό do \ v_L \
T o d Λ  T o d Λ
O V I L I π
Figure imgf000015_0003
OVILI π
Figure imgf000015_0003
8 8 I - 8 Π d Λ  8 8 I-8 Π d Λ
り T L p  R T L p
o ε I V a 1\ 0ΐ
Figure imgf000015_0004
o ε IV a 1 \ 0ΐ
Figure imgf000015_0004
O O T L T し I I Π T Λ  O O T L T shi I I Π T Λ
^ 6 I - G 6 I Q P T Q 8 I f\ fj A  ^ 6 I-G 6 I Q P T Q 8 I f \ fj A
7 g T T 8 I o / Λ  7 g T T 8 I o / Λ
G L I / d Λ  G L I / d Λ
し a i Then a i
Figure imgf000015_0005
Figure imgf000015_0005
Z I I 6 0 I £ ΰ A D  Z I I 6 0 I £ ΰ A D
8 0 I e 0 I z ϋ A D  8 0 I e 0 I z ϋ A D
0 I L 8 o z d A a  0 I L 8 o z d A a
8ΐ  8ΐ
f l8Z0/66df/13d 刚 £9/66 ΟΛΛ G V β 4 3 1 5 -3 28 f l8Z0 / 66df / 13d 刚 £ 9/66 ΟΛΛ GV β 4 3 1 5 -3 28
G V β 9 3 2 9 -3 3 6  G V β 9 3 2 9 -3 3 6
G V β 1 3 3 3 7 - 34 6  G V β 1 3 3 3 7-34 6
G V β 1 4 34 7 - 3 50  G V β 1 4 34 7-3 50
G V β 1 8 3 5 1 - 3 5 8  G V β 1 8 3 5 1-3 5 8
H V β 7 3 5 9 - 3 64  H V β 7 3 5 9-3 64
H V β 1 3 3 6 5 - 3 6 6  H V β 1 3 3 6 5-3 6 6
H V β 1 4 3 6 7 - 3 70  H V β 1 4 3 6 7-3 70
H V β 1 5 3 7 1 - 3 74  H V β 1 5 3 7 1-3 74
H V β 1 7 3 7 5 - 3 7 6 産業上の利用の可能性  H V β 1 7 3 7 5-3 7 6 Industrial potential
本発明のァミノ酸配列を T C R V /3鎖 C D R 3領域に有する Τ細胞は、 変形性 関節炎の病因性 Τ細胞であるため、 本発明のァミノ酸配列は変形性関節炎の確認 的診断の道具として用いることができる。 更に、 本発明のアミノ酸配列を TCR V 3鎖の CDR 3領域に有する Τ細胞を使用して該 TCRが特異的に認識する抗 原を単離、 同定することが可能となるため、 変形性関節炎の治療、 診断及び予防 薬の開発に大きく寄与し得る。 本発明のアミノ酸配列は、 TCRヮクチネーショ ン、 免疫制御細胞の活性化あるいは抗 T C R抗体の誘発にそれを用いることによ り、 変形性関節炎の免疫治療のために用いることもできる。 本発明のアミノ酸配 列を有するぺプチドと相同性のあるぺプチドは、 直接患者に投与することにより、 患者の抗原を攻撃できない Τ細胞を作り出し、 抗原特異的な TCRをブロックす ることが可能である。 また、 本発明のアミノ酸配列を CD R 3領域に有する TC Rが特異的に反応する抗原べプチド若しくは抗原蛋白質は、 抗原特異的な免疫寛 容を誘導する変形性関節炎治療薬及び/又は予防薬として有用である。  Since the ミ ノ cells having the amino acid sequence of the present invention in the TCRV / 3 chain CDR3 region are pathogenic to osteoarthritis, the amino acid sequence of the present invention is used as a tool for confirmatory diagnosis of osteoarthritis. be able to. Furthermore, since it is possible to isolate and identify an antigen that the TCR specifically recognizes using a cell having the amino acid sequence of the present invention in the CDR3 region of the TCR V 3 chain, osteoarthritis Can greatly contribute to the development of therapeutic, diagnostic and prophylactic drugs. The amino acid sequence of the present invention can also be used for immunotherapy of osteoarthritis by using it for TCR cutination, activation of immune control cells or induction of anti-TCR antibodies. The peptide having homology to the peptide having the amino acid sequence of the present invention can create cells that cannot attack the antigen of the patient and can block the antigen-specific TCR by direct administration to the patient. It is. Further, an antigen peptide or antigen protein to which TCR having the amino acid sequence of the present invention in the CDR3 region specifically reacts is a therapeutic and / or prophylactic agent for osteoarthritis that induces antigen-specific immune tolerance. Useful as

Claims

請求の範囲 i . 変形性関節炎患者の滑液及び Z又は滑膜に存在する τ細胞抗原レセプタ 一 V 鎖の C D R 3領域をコードする D N A配列を含む D N A。  Claims i. A DNA comprising a DNA sequence encoding the CDR3 region of the V chain of a τ cell antigen receptor present in synovial fluid and Z or synovium of a patient with osteoarthritis.
2 . 変形性関節炎患者の滑液及び Z又は滑膜に存在する T細胞抗原レセプタ 一 V ]3鎖の C D R 3領域を含むアミノ酸配列を含むぺプチド。  2. A peptide comprising an amino acid sequence containing the CDR3 region of the T cell antigen receptor [V] 3 chain present in synovial fluid and Z or synovium of a patient with osteoarthritis.
3 . ァミノ酸配歹 IjGln- Val-Glyまたは Lys- Arg - Gly-Serを有する請求項 2に記 載のぺプチド。  3. The peptide according to claim 2, comprising an amino acid system IjGln-Val-Gly or Lys-Arg-Gly-Ser.
4 . アミノ酸配列 Arg - Ala-Gly、 Arg- Gin- Gly、 Gly-Thr - Gly、 Gly- Ser - Ala、 Ser - Gly_Ser、 Arg- Arg Ala、 Gin - Gly_Val、 Pro_Gly Thr、 Thr - Gly -し eu、 Ser - 4. Amino acid sequence Arg-Ala-Gly, Arg-Gin-Gly, Gly-Thr-Gly, Gly-Ser-Ala, Ser-Gly_Ser, Arg-Arg Ala, Gin-Gly_Val, Pro_Gly Thr, Thr-Gly-Eu , Ser-
Gly- Alaゝ Glu-Gly-Pro, Leu- Thr- Gly、 Leu- Ser- Pro、 Gly-Gly- Gly、 Thr- Gly- Alaヽ Thr-Gly-Lys及び Zまたは Gln-Gly- Leuを有する請求項 2に記載のぺプチド。 Claim with Gly-Ala ゝ Glu-Gly-Pro, Leu-Thr-Gly, Leu- Ser-Pro, Gly-Gly-Gly, Thr-Gly-Ala ヽ Thr-Gly-Lys and Z or Gln-Gly-Leu Item 2. The peptide according to item 2.
5 . 請求項 2に記載のァミノ酸配列を指標とする変形性関節炎診断薬。  5. A diagnostic agent for osteoarthritis using the amino acid sequence according to claim 2 as an index.
6 . 請求項 2に記載のアミノ酸配列を有する T細胞が特異的に反応する抗原 蛋白質、 抗原ペプチド若しくはその誘導体を有効成分とする変形性関節炎に対す る抗原特異的な免疫寛容誘導薬。  6. An antigen-specific immune-tolerant drug against osteoarthritis against an osteoarthritis, comprising an antigen protein, an antigen peptide or a derivative thereof to which T cells having the amino acid sequence according to claim 2 react specifically.
PCT/JP1999/002814 1998-05-29 1999-05-28 T cell antigen receptor sequences specific to arthrosis deformans WO1999063084A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU40577/99A AU4057799A (en) 1998-05-29 1999-05-28 T cell antigen receptor sequences specific to arthrosis deformans

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP14985598 1998-05-29
JP10/149855 1998-05-29
JP10/328761 1998-10-14
JP32876198 1998-10-14

Publications (1)

Publication Number Publication Date
WO1999063084A1 true WO1999063084A1 (en) 1999-12-09

Family

ID=26479624

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1999/002814 WO1999063084A1 (en) 1998-05-29 1999-05-28 T cell antigen receptor sequences specific to arthrosis deformans

Country Status (2)

Country Link
AU (1) AU4057799A (en)
WO (1) WO1999063084A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020751A2 (en) * 2001-09-05 2003-03-13 King's College London Homing peptides
JP2013510863A (en) * 2009-11-10 2013-03-28 アレグロ ファーマシューティカルズ インコーポレイテッド Compositions and methods for inhibiting cell adhesion or delivering diagnostic or therapeutic agents to an RGD binding site
US9896480B2 (en) 2009-11-10 2018-02-20 Allegro Pharmaceuticals, Inc. Integrin receptor antagonists and their methods of use
US11673914B2 (en) 2009-11-10 2023-06-13 Allegro Pharmaceuticals, LLC Peptide therapies for reduction of macular thickening

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DOHERTY M, PATTRICK M, POWELL R: "NODAL GENERALISED OSTEOARTHRITIS IS AN AUTOIMMUNE DISEASE", ANNALS OF THE RHEUMATIC DISEASES, BRITISH MEDICAL ASSOCIATION, LONDON, GB, vol. 49, no. 12, 1 January 1990 (1990-01-01), GB, pages 1017 - 1020, XP002926585, ISSN: 0003-4967 *
EPPLEN C, ET AL.: "IMMUNOPRINTING REVEALS DIFFERENT GENETIC BASES FOR (AUTO)IMMUNE DISEASES", ELECTROPHORESIS, WILEY INTERSCIENCE, DE, vol. 16, no. 09, 1 January 1995 (1995-01-01), DE, pages 1693 - 1697, XP002926586, ISSN: 0173-0835, DOI: 10.1002/elps.11501601281 *
Heisei 7 Nendo Kouseishou Chouki Mansei Shikkan Sougou Kenkyuu Jigyou Kenkyuu Houkokusho, Ministry of Healt and Welfare, (17-07-96), pages 69-71, XP002926582 *
Hiroshi Kudoo, Heisei 9 Nendo Kousei Kagaku Kenkyuuhi Hojokin Men'eki Allergy Kenkyuu Jigyou Kenkyuu Houkokusho, (03-98), pahes 37-38, XP002926580 *
KIEBER-EMMONS T, ET AL.: "STRUCTURAL MOTIFS IN RHEUMATOID T-CELL RECEPTORS", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 17, no. 02, 1 January 1998 (1998-01-01), US, pages 133 - 149, XP002926583, ISSN: 1044-5498 *
KIYOSHI TAKATSU, "Men'ekibyou no Bunshi Kikou to Sono Shuufuku", MOMBUSHOU KAGAKU KENKYUUJI HOJOKIN JUUTEN RYOUIKI KENKYUU HEISEI 9 NENDO KENKYUU SEIKA HOUKOKUSHO, (03-98), pages 120-121, XP002926581 *
Kouseishou Tokutei Shikkan ni Kansuru Men'eki Kenkyuu Han-Heisei 8 Nendo Kenkyuu Houkokusho, Kouseishou Tokutei Shikkan ni Kansuru Men'eki Kenkyuuhan Jimukyoku, (31-03-97), pages 17-20, XP002926578 *
Kouseishou Tokutei Shikkan ni Kansuru Men'eki Kenkyuu Han-Heisei 9 Nendo Kenkyuu Houkokusho, Kouseishou Tokutei Shikkan ni Kansuru Men'eki Kenjyuuhan Jimukyoku, (31-03-98), pages 12-16, XP002926579 *
ZWILLICH S H, ET AL.: "V ALPHA GENE USAGE IN RHEUMATOID COMPARED WITH OSTEOARTHRITIC SYNOVIAL TISSUE T CELLS", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 13, no. 09, 1 January 1994 (1994-01-01), US, pages 923 - 931, XP002926584, ISSN: 1044-5498 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020751A2 (en) * 2001-09-05 2003-03-13 King's College London Homing peptides
WO2003020751A3 (en) * 2001-09-05 2003-08-21 King S College London Homing peptides
JP2013510863A (en) * 2009-11-10 2013-03-28 アレグロ ファーマシューティカルズ インコーポレイテッド Compositions and methods for inhibiting cell adhesion or delivering diagnostic or therapeutic agents to an RGD binding site
US9872886B2 (en) 2009-11-10 2018-01-23 Allegro Pharmaceuticals, Inc. Compositions and methods for inhibiting cellular adhesion or directing diagnostic or therapeutic agents to RGD binding sites
US9896480B2 (en) 2009-11-10 2018-02-20 Allegro Pharmaceuticals, Inc. Integrin receptor antagonists and their methods of use
US10307460B2 (en) 2009-11-10 2019-06-04 Allegro Pharmaceuticals, LLC Compositions and methods for inhibiting cellular adhesion or directing diagnostic or therapeutic agents to RGD binding sites
US10590166B2 (en) 2009-11-10 2020-03-17 Allegro Pharmaceuticals, LLC Peptides useable for treating cancer
US10639347B2 (en) 2009-11-10 2020-05-05 Allegro Pharmaceuticals, LLC Peptides useable for treatment of disorders of the eye
US11666625B2 (en) 2009-11-10 2023-06-06 Allegro Pharmaceuticals, LLC Pharmaceutical compositions and preparations for administration to the eye
US11673914B2 (en) 2009-11-10 2023-06-13 Allegro Pharmaceuticals, LLC Peptide therapies for reduction of macular thickening

Also Published As

Publication number Publication date
AU4057799A (en) 1999-12-20

Similar Documents

Publication Publication Date Title
JP6483071B2 (en) Peptide cancer antigen-specific T cell receptor gene
JP5832572B2 (en) Receptor gene of cancer antigen-specific T cell, peptide encoded thereby and use thereof
Kramer Purification of splicing factor SF1, a heat-stable protein that functions in the assembly of a presplicing complex
JP6941561B2 (en) Multiple variable IL-2 dose regimens for treating immune disorders
Lallena et al. Functional association of nuclear protein 4.1 with pre-mRNA splicing factors
AU2020104307A4 (en) Bispecific chimeric antigen receptor for treating hematological tumor complicated with HIV infection
Fazou et al. Epitope specificity of clonally expanded populations of CD8+ T cells found within the joints of patients with inflammatory arthritis
WO2018081624A1 (en) Microrna in t cell activation
Takei et al. Decreased expression of signaling lymphocytic-activation molecule-associated protein (SAP) transcripts in T cells from patients with rheumatoid arthritis
Masuko et al. Dynamic changes of accumulated T cell clonotypes during antigenic stimulation in vivo and in vitro
KR20000010561A (en) Novel peptides suitable for use in antigen specific immunosuppressive therapy
JP4322504B2 (en) Method for modulating the binding activity of novel ICAM-3 binding receptors on sinusoidal endothelial cells in liver and lymph nodes
WO1999063084A1 (en) T cell antigen receptor sequences specific to arthrosis deformans
WO2005070964A1 (en) Method of isolating monocytes
CN109182511A (en) It is a kind of for detecting the SNP site of abo blood group anomaly
EP3055426B1 (en) Detection of hepatitis delta virus (hdv) for the diagnosis and treatment of sjögren's syndrome and lymphoma
WO1996000300A1 (en) Anergy genes
JPH05509233A (en) Nucleotide and amino acid sequences of Mycobacterium tuberculosis protein MTP40 and synthetic peptides derived therefrom
Treviño et al. CD8+ T cells oligoclonally expanded in synovial fluid at onset of spondyloarthropathy selectively proliferate in response to self-antigens: characterization of cell specificities in nonclonal populations.
WO2023018782A1 (en) Methods to determine cd4+ memory t cell responses to sars-cov-2 infection or vaccination
Pinelli Inflammatory Bowel Diseases: genetic and functional approaches in the NGS era
CA3209360A1 (en) Immune enhancers
Garcia-Batres Exploration of methods for sequence based HLA typing and application to patients with hair dye allergy
WO2001030851A2 (en) Idiotype vaccines
Freimanis The Detection and Role of Human Endogenous Retrovirus K (HML-2) In Rheumatoid Arthritis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase