JP2004506743A - T cell receptor Vβ-Dβ-Jβ sequence and its detection method - Google Patents

Abstract

In one embodiment, the invention relates to a first oligonucleotide or a nucleic acid sequence complementary thereto comprising or derived from the sequence 5'-CTAGGGGCGGGCGGACTCACCTAC-3 '. The first oligonucleotide can be used with a nucleic acid of between 15 and 30 nucleotides to amplify a portion of the Vβ13.1 gene, which nucleotide does not include the sequence of the first oligonucleotide; It is then found in the region from Vβ to Jβ of the Vβ13.1 gene in Vβ13.1 T cells, where the oligonucleotide and nucleic acid sequences are not found on the same strand of the Vβ13.1 gene pair. Alternatively, the first oligonucleotide can be used with a labeling group in a method for detecting the LGRALTY motif found in the T cell receptor of Vβ13.1 T cells.

Description

（表２：ＭＢＰ８３−９９ペプチドに特異的なＴＣＲ　Ｖβ遺伝子配列）
【００６９】
【表２】

ＶαおよびＶβの再配列は、個々のＭＢＰ８３−９９　Ｔ細胞クローンの間で変化したが、所定の個体由来の、これらの非依存性Ｔ細胞クローンの多くが、同じＶα−ＪαおよびＶβ−Ｄβ−Ｊβ接合部領域配列と、同一のＶα鎖およびＶβ鎖を共有した。この知見は、以前に報告されたように（Ｖａｎｄｅｖｙｖｅｒ　１９９５，Ｗｕｃｈｅｒｐｆｅｎｎｉｎｇ　１９９４）、ＭＳを有する所定の患者におけるＭＢＰ８３−９９特異的Ｔ細胞のインビボでのクローン増殖と一致する。
【００７０】
興味深いことに、表１および表２に示されるように、１人の患者（ＭＳ−１）由来の非依存性Ｔ細胞クローン（クローンＥ２．６）は、別の患者（ＭＳ−２）から得られた４つのＴ細胞クローンのうちの３つのクローン（クローンＣ３．１、Ｄ７．１６およびＦ３．４）と、同じＶβ１３．１およびＶα１７を共有した。これらのＴ細胞クローンのＶβ１３．１は、Ｖβ−Ｄβ−Ｊβ接合部領域内に同一のＤＮＡ配列を共有した。
【００７１】
（実施例２：Ｖβ−Ｄβ−Ｊβ特異的オリゴヌクレオチドプライマーは、もとのＭＢＰ８３−９９　Ｔ細胞クローン、およびもとのＭＢＰ８３−９９　Ｔ細胞を含有するＰＢＭＣ中に存在する、対応するＤＮＡ配列を検出する際に、高度に特異的であり、かつ高感受性であった）
１４のオリゴヌクレオチドプライマーのセットを、非依存性ＭＢＰ８３−９９　Ｔ細胞クローンのＶβ−Ｄβ−Ｊβ接合部領域内のＤＮＡ配列に従って合成し、続いて、ＲＴ−ＰＣＲにおいて、これらの特異性について試験した。これらのオリゴヌクレオチドプライマーのＤＮＡ配列を、表３に示す。
【００７２】
（表３：Ｖβ−Ｄβ−Ｊβ特異的オリゴヌクレオチドプライマーのＤＮＡ配列）
【００７３】
【表３】

これらのＶβ−Ｄβ−Ｊβ特異的プライマーは、もとのＭＢＰ８３−９９　Ｔ細胞クローン内に存在するＤＮＡ配列に排他的に結合したが、関連していないＭＢＰ８３−９９　Ｔ細胞クローン由来の配列には結合しなかった（図２）。このことは、もとのＶβ−Ｄβ−Ｊβ　ＤＮＡ配列に対する、それらの高度な特異性を示唆する。クローンＭＳ１−Ｅ２．６およびクローンＭＳ２−Ｃ３．１についての例外にのみ注意した（これらにおいて、同じプライマーが、Ｔ細胞クローンによって共有されるＶβ−Ｄβ−Ｊβ接合部のＤＮＡ配列に結合した）。
【００７４】
Ｖβ−Ｄβ−Ｊβオリゴヌクレオチドプライマーの特異性、およびＰＣＲ検出システムの高感度性を考慮して、本発明者らは、５’ＶβプライマーおよびＶβ−Ｄβ−Ｊβ特異的オリゴヌクレオチドプライマーを使用する、この二段階ＰＣＲ検出システムを使用して、ＭＢＰ８３−９９　Ｔ細胞クローンが由来する末梢血単核細胞（ＰＢＭＣ）検体中に存在する、対応するＶβ−Ｄβ−Ｊβ　ＤＮＡ配列を検出するか否かを求めた。２つの別々の実験の結果は、もとのＰＢＭＣ検体におけるＶβ−Ｄβ−Ｊβ配列のポジティブな検出を示した。従って、Ｖβ−Ｄβ−Ｊβ配列がフィンガープリントとして働くＰＣＲ検出システムは、同一のＤＮＡ配列を探索することによって、末梢血単核細胞内に存在するＭＢＰ８３−９９　Ｔ細胞を追跡する際に、特異的でありかつ感受性であるという知見が実証された。
【００７５】
（実施例３：ＭＳを有する異なる患者および健康な個体に由来するＰＢＭＣ標本中の共通のＶβ−Ｄβ−Ｊβ　ＤＮＡ配列の検出）
次いで、本発明者らは、ＭＢＰ８３−９９　Ｔ細胞クローンのＶβ−Ｄβ−Ｊβ接合領域に対応するＤＮＡ配列が、ＭＳを有する患者の群および健康な個体の群から無作為に選択したＰＢＭＣ標本中に検出され得るか否かを調べた。（第１のＰＣＲにおいて）対応するＶβファミリーに対する特異的なプライマーおよび（第２の半ネストティドＰＣＲにおいて）Ｖβ−Ｄβ−Ｊβ配列に対する特異的なプライマーを使用する同一のＰＣＲ増幅系を使用した。これと、対応するＶβ−Ｄβ−Ｊβプローブを用いるサザンブロット分析を併用し、ハイブリダイゼーションを実施した。この２工程ＰＣＲ検出系の特定の必要条件ならびにＶβ−Ｄβ−Ｊβプライマーおよびプローブの特異性を与える場合、同定されたＤＮＡ配列は特定のＴＣＲ　Ｖβ鎖由来であり、そして、このＤＮＡ配列は、目的のＶβ−Ｄβ−Ｊβ配列に対して、同一性または類似性のいずれかを表す。
【００７６】
これらの結果によって、１つのＶβ−Ｄβ−Ｊβオリゴヌクレオチドプライマー（ＭＳＩ−Ｅ２．６，Ｖβ１３．１−ＬＧＲＡＧＬＴＹ）のみが、ＭＳを有する異なった患者から得た４８個のＰＢＭＣ標本のうち１５個のＰＢＭＣ標本（３１％）において、相補的ＴＣＲ　Ｖβ１３．１　ＤＮＡ配列を検出したことが示された。従って、この知見によって、ＭＳを有するこれらの患者においてＶβ１３．１−ＬＧＲＡＧＬＴＹを発現するＭＢＰ８３−９９　Ｔ細胞の存在が示される。類似の実験条件下で、同じプライマーはまた、健康な個体から得た２０個のＰＢＭＣ標本のうち５個のＰＢＭＣ標本（２５％）において対応するＤＮＡ配列を検出した。残りの１３個のＶβ−Ｄβ−Ｊβプライマーは、ＰＢＭＣ標本の同じパネルにおいていずれの配列シグナルも同定できなかった。これらの結果は、３つの別個の実験において再現可能であった。増幅のための第１のＰＣＲにおいて１３．１特異的プライマーを使用したので、Ｅ２．６プライマーによって増幅された同定されたＤＮＡ産物は、Ｖβ１３．１を発現するＴ細胞に由来する。
【００７７】
さらに、この同定されたＶβ１３．１−ＬＧＲＡＧＬＴＹ配列はまた、ＭＳ（ＭＳ−３５，ＭＳ３６およびＭＳ３９）を有する５人の患者から生成された２４個の短期（ｓｈｏｒｔ−ｔｅｒｍ）ＭＢＰ８３−９９　Ｔ細胞株のうち１３個の細胞株（５４％）において増幅され、これらの患者のＰＢＭＣ標本は、Ｖβ１３．１−ＬＧＲＡＧＬＴＹ配列を含むことが示された。従って、これらの結果によって、これらのＰＢＭＣ標本において検出されたＶβ１３．１−ＬＧＲＡＧＬＴＹ　ＤＮＡ配列が、ＭＢＰ８３−９９を認識する数人のＴ細胞に由来することを確認した。この知見はまた、Ｖβ１３．１−ＬＧＲＡＧＬＴＹ配列を発現するＭＢＰ８３−９９　Ｔ細胞は、ＭＳを有する患者において見出されたＭＢＰ８３−９９　Ｔ細胞株のうちの全てまたは大部分のＭＢＰ８３−９９　Ｔ細胞株を表すことを示唆する。
【００７８】
併用したＰＣＲ−ＤＮＡハイブリダイゼーション検出系（ここで、Ｖβ−Ｄβ−Ｊβ配列をフィンガープリントとして使用した）によって、同一のＶβ−Ｄβ−Ｊβ接合部配列の検出による抗原特異的Ｔ細胞の追跡における強力なツールを提供した。この検出系の高い特異性および感度によって、末梢血Ｔ細胞における特定のＶβ−Ｄβ−Ｊβ配列の同定を可能にした。この研究によって、ＭＢＰの免疫優性の８３−９９のペプチドを認識し、同一のＶβ−Ｄβ−Ｊβ配列を一様に発現する、Ｖβ１３．１　Ｔ細胞の共通のサブセットが、ＭＳを有する患者のうち約３０％に存在するということを初めて実証した。この結果を、本明細書中に記載される段階的な実験に基づいて得た。第一に、同一のＤＮＡ配列（Ｖβ１３．１−ＬＧＲＡＧＬＴＹ）を、ＭＳを有する異なる患者に由来する独立したＭＢＰ８３−９９　Ｔ細胞クローンにおいて見出した。第二に、この配列を、ＭＳを有する異なる患者から得たＰＢＭＣ標本のＴＣＲ　Ｖβ１３．１から増幅したｃＤＮＡ産物中で同定した。第三に、このＤＮＡ配列を、Ｖβ１３．１−ＬＧＲＡＧＬＴＹ配列を含むことが示されているＰＢＭＣ標本から生成した短期の独立したＭＢＰ８３−９９　Ｔ細胞株において検出した。Ｖβ１３．１−ＬＧＲＡＧＬＴＹ配列を発現するＭＢＰ８３−９９　Ｔ細胞は、ＭＳを有する数人の患者から生じたＭＢＰ８３−９９　Ｔ細胞株のうち全てまたは大部分を示すようである。最後に、ＰＢＭＣ標本におけるＶβ１３．１−ＬＧＲＡＧＬＴＹ配列の存在を、組換えＤＮＡクローニングおよび直接的なＤＮＡ配列決定によって確認した。
【００７９】
さらに、共通のＶβ１３．１−ＬＧＲＡＧＬＴＹ配列を発現するＭＢＰ８３−９９　Ｔ細胞がまた、数人の健康な個体においても存在することは驚くべきことではない。これまで報告された研究は、ＭＢＰ反応性Ｔ細胞（これは、免疫優性の８３−９９ペプチドを認識するＴ細胞を含む）がまた、数人の健康な個体においても存在することを示す（Ｚｈａｎｇ　１９９４，Ｏｔａ　１９９０）。しかし、これらのＴ細胞は、健康な個体とは対照的に、ＭＳを有する患者においてインビボ活性化およびクローン性増殖を受けるという機能的な違いが存在する（Ｚｈａｎｇ　１９９４）。
【００８０】
共通のＶβ−Ｄβ−Ｊβ配列を共有するこれらのＶβ１３．１　ＭＢＰ８３−９９　Ｔ細胞は、ＭＳを有する数人の患者において見出されるＭＢＰ８３−９９　Ｔ細胞のうちの有意な部分を表し得る。この可能性は、Ｖβ１３．１−ＬＧＲＡＧＬＴＹ配列が、２回の刺激サイクル後にＭＳを有する患者から生じた短期ＭＢＰ８３−９９　Ｔ細胞株のうちの４０％で存在したという観察によって支持される。
【００８１】
同定された共通のＶβ−Ｄβ−Ｊβ配列を、定量的ＰＣＲ検出系における特異的なマーカーとして使用して、この疾患に潜在的に関連するインビボクローン増殖およびインビボの活性をモニターする目的のために、ＭＳ患者の大きな群において血液および脳脊髄液におけるＭＢＰ８３−９９　Ｔ細胞の共通のサブセットを検出し得る。この方法は、従来の細胞培養に基づくアッセイよりも優れており、これは、ＭＢＰ反応性Ｔ細胞のインビトロの選択および増殖が、しばしば、細胞培養に特有の種々の阻害性因子によって阻まれるからである。これは、直接的なエキソビボ分析を使用してＭＢＰ反応性Ｔ細胞を定量する場合に、ＭＢＰ反応性Ｔ細胞の頻度がＭＳを有する患者において驚く程高いことが見出された最近の研究（Ｈａｆｌｅｒ（最後に列挙された著者）ＪＥＭ　１９９７）と合致する。
【００８２】
さらに、このＴＣＲに対応する合成ペプチドは、ＭＳを有する患者において、ＭＢＰ反応性Ｔ細胞に対する抗イディオタイプＴ細胞の応答を誘導することが示された（Ｃｈｏｕら，Ｊ．Ｉ．）。従って、共通のＣＤＲ配列を含有するＴＣＲペプチドは、そのＭＢＰ８３−９９　Ｔ細胞が共通のＣＤＲ３配列モチーフを保有する患者の群においてＭＢＰ反応性Ｔ細胞の特定のサブセットを抑制するために抗イディオタイプＴ細胞を誘引する、大変大きな潜在能力のあるものであり得る。このような共通のＣＤＲ３ペプチドで免疫することは、ＭＳを有する患者における潜在的な処置手順としてＣＤＲ２ペプチドまたは個体依存性ＣＤＲ３ペプチドよりも有益である（Ｖａｎｄｅｎｂａｒｋ　１９９６）。
【００８３】
（実施例４：ＭＳを有する患者におけるＴ細胞ワクチン接種による免疫ＭＢＰ反応性Ｔ細胞クローンおよび２０マーのＴＣＲペプチドに対する免疫応答の誘導ならびに免疫された患者由来の共通のＣＤＲ３配列を組み込むＴＣＲペプチドに対する特異的な抗体を産生するＢ細胞株の生成）
照射を受けた自己ＭＢＰ反応性Ｔ細胞クローンで皮下接種することによって、ＭＳを有する患者における実質的な抗イディオタイプＴ細胞応答が誘導されることが実証され、これはワクチン接種に使用される循環ＭＢＰ反応性Ｔ細胞の進行性の枯渇と相関した（Ｍｅｄａｅｒら，１９９５）。
【００８４】
（材料および方法）
（試薬およびペプチド）
細胞培養に使用した培地は、Ａｉｍ−Ｖ無血清培地（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｇｒａｎｄ　Ｉｓｌａｎｄ，ＮＹ）であった。ＭＢＰの免疫優性ペプチド（残基８３〜９９）および２０アミノ酸の２つのＴＣＲペプチドを、Ｃｈｉｒｏｎ　Ｍｉｍｏｔｏｐｅ（Ｓａｎ　Ｄｉｅｇｏ，ＣＡ）によって合成した。このペプチドの純度は、９５％を超えた。
【００８５】
（ＭＢＰ反応性Ｔ細胞の前駆体頻度の評価）
ＰＢＭＣを、ＭＢＰ（４０μｇ／ｍＬ）の存在下にて（全部で９６ウェルに対して）２００，０００細胞／ウェルでプレートした。７日後、全ての培養物を、照射した自己ＰＢＭＣの存在下にてＭＢＰで再刺激した。さらに１週間後、各ウェルを４つのアリコート（１アリコート当たり約１０^４細胞）に分割し、そして、ＭＢＰの存在下およびＭＢＰの非存在下で、１０^５の照射した自己ＰＭＢＣを用いて二連で培養した。この培養を、３日間維持し、そして最後の１６時間の培養の間、１ウェル当たり１μＣｉにて［^３Ｈ］チミジン（Ｎｙｃｏｍｅｄ　Ａｍｅｒｓｈａｍ，Ａｒｌｉｎｇｔｏｎ　Ｈｅｉｇｈｔｓ，ＩＬ）でパルスした。次いで、細胞を、自動細胞収集機を使用して収集し、そして［^３Ｈ］チミジンの取り込みを測定した。
【００８６】
１分当たりの計数（ｃｏｕｎｔｓ　ｐｅｒ　ｍｉｎｕｔｅ）（ＣＰＭ）は、１０００を超え、そして、少なくとも３倍分、（ＭＢＰの非存在下で）参照ＣＰＭを超過した場合、ウェル／培養物を、ＭＢＰまたはＭＢＰのペプチドについて特異的であると規定した。次いで、ＭＢＰ反応性Ｔ細胞の前駆体頻度を、初期培養において播種されたＰＢＭＣの総数（１９．２×１０^６細胞）で特定のウェルの数を除算することによって評価した。
【００８７】
（ミエリン反応性Ｔ細胞クローン）
陽性同定されたＴ細胞株を、２μｇ／ｍｌのフィトヘマグルチニン−タンパク質（ＰＨＡ−Ｐ）の存在下で限界希釈アッセイを使用してクローン化した。培養物に、３〜４日毎に新たな培地を供給した。増殖陽性ウェルを、増殖アッセイでのＭＢＰ８３−９９ペプチドに対する特異的反応性について試験した。得られたＭＢＰ８３−９９特異的Ｔ細胞クローンを、さらに特徴づけ、そしてこれをＴ細胞ワクチン接種のために使用した。
【００８８】
（ＴＣＲ　Ｖ遺伝子分析およびＤＮＡ配列決定）
免疫ＭＢＰ反応性Ｔ細胞クローンのＴ細胞レセプターＶ遺伝子再編成を、逆転写ＰＣＲを使用して分析した。ＴＣＲα鎖およびＴＣＲβ鎖の遺伝子を増幅し、そして他に記載されるように直接的に配列決定した（Ｖａｎｄｅｖｙｅｒら，１９９５；Ｚｈａｎｇ，Ｙ．Ｃ．Ｑ．ら、１９９８）。簡潔には、総ＲＮＡを、ＲＮｅａｓｙミニキット（ＱＩＡＧＥＮ，Ｓａｎｔａ　Ｃｌａｒｉｔａ，ＣＡ）を使用して１０^６細胞の各ＭＢＰ８３−９９反応性Ｔ細胞クローンから抽出した。総ＲＮＡから逆転写された第一鎖ｃＤＮＡを、ＴＣＲ　ＶαおよびＶβの遺伝子ファミリー（この配列は、刊行された（Ｖａｎｄｅｖｙｅｒら，１９９５；Ｚｈａｎｇ，Ｙ．Ｃ．Ｑ．ら、１９９８））に特異的な１セットのプライマーを用いるＰＣＲ増幅に供した。増幅されたＰＣＲ産物を、電気泳動によって１％アガロースゲル中で分離し、エチジウムブロマイドで染色した。この視覚化したＰＣＲ産物を切出し、その後、これを、配列分析前にＱＩＡｑｕｉｃｋ（登録商標）ゲル抽出キット（ＱＩＡＧＥＮ，Ｓａｎｔａ　Ｃｌａｒｉｔａ，ＣＡ）を使用して精製した。精製したＰＣＲ産物を、Ｔ７配列決定キット（Ｐｈａｒｍａｃｉａ，Ｕｐｐｓａｌａ，Ｓｗｅｄｅｎ）で直接的に配列決定した。１．５μｇのテンプレートを、ジデオキシ鎖終止法を使用して２ｐｍｏｌの対応するＶ遺伝子プライマーを用いて配列決定した。
【００８９】
（照射された自己ＭＢＰ反応性Ｔ細胞クローンを用いるＭＳ患者の免疫）
磁気共鳴画像法で確認した臨床的に明確なＭＳを有する２人の患者を、この研究に含めた。彼らを、二年を超える再発弛張性（ｒｅｌａｐｓｉｎｇ−ｒｅｍｉｔｔｉｎｇ）ＭＳを有するとして診断した。これらの患者は、この研究前の少なくとも三ヶ月間にいずれの免疫調製薬物も服用していなかった。照射された自己ＭＢＰ８３−９９反応性Ｔ細胞クローンでの免疫を、以前に記載したように実施した（Ｊ．Ｚｈａｎｇら、１９９２，１９９３）。簡潔には、ＭＢＰ８３−９９反応性Ｔ細胞クローンを、活性化し、そしてＰＨＡの存在下にて注射前に７日間増殖させた。次いで、Ｔ細胞に、（^６０Ｃｏ源を使用して）１０，０００　ｒａｄで照射し、滅菌生理食塩水で完全に洗浄した。２つの自己Ｔ細胞クローン由来の全部で４×１０^７の細胞を、２ｍｌの無菌生理食塩水に再懸濁し、これを腕の皮下に注射した。各患者は、二ヶ月の間隔で総計４回の注射を受け、免疫Ｔ細胞クローンに対するＰＢＭＣの増殖で定義されるような、十分な免疫応答を達成した。このプロトコルは、ｔｈｅ　Ｉｎｓｔｉｔｕｔｉｏｎａｌ　Ｈｕｍａｎ　Ｓｕｂｊｅｃｔｓ　Ｃｏｍｍｉｔｔｅｅ　ａｔ　Ｂａｙｌｏｒ　Ｃｏｌｌｅｇｅ　ｏｆ　Ｍｅｄｉｃｉｎｅにより承認を得た。同意書を、実験手順を説明した後に患者から得た。これらの患者を、各免疫の前後に、有害事象および障害スコア（Ｅｘｐａｎｄｅｄ　Ｄｉｓａｂｉｌｉｔｙ　Ｓｃａｌｅ　Ｓｃｏｒｅ）に対して評価した。ガドリニウム増強ＭＲＩスキャンを、免疫前、および免疫後の異なる時点において実施した。この臨床的評価および放射線写真的評価は、別個の臨床研究の部分である。
【００９０】
（ＥＢＶ形質転換による抗体産生Ｂ細胞株の生成）
この方法は、他のところに記載されている（Ｊ．Ｚｈａｎｇ，１９８９，１９９１）。簡潔には、ＰＢＭＣを、ＥＢＶを産生するＢ９５．８株（ＡＴＣＣ，Ｂｅｔｈｅｓｄａ，ＭＤ）の無細胞上清および０．５μｇ／ｍｌのＣｙｃｌｏｓｐｏｒｉｎ　Ａ（Ｓａｎｄｏｚ，Ｂａｓｅｌ，Ｓｗｉｔｚｅｒｌａｎｄ）の存在下でマイクロタイタープレート（Ｃｏｓｔａｒ，Ｃａｍｂｒｉｄｇｅ，ＭＡ）中に２０，０００　細胞／ウェルでプレートし、Ｔ細胞増殖を選択的に阻害した。細胞を、３〜４日毎に培地を変えつつ１４日間培養した。第１４日目に、この増殖陽性ウェルを視覚化し、そして培養物上清を、試験のために収集した。特定の抗体を産生するＢ細胞の前駆体頻度を、プレートされたＰＢＭＣの総数で陽性ウェルの数を除算することによって評価した。陽性Ｂ細胞株を、その後、増殖のために２４ウェルプレート（Ｃｏｓｔａｒ，Ｃａｍｂｒｉｄｇｅ，ＭＡ）に移した。このＢ細胞株は、代表的に、２〜１０μｇ／ｍｌの比較的純粋な抗体を産生した。
【００９１】
（抗ＴＣＲ抗体のＥＬＩＳＡによる検出）
培養物上清を、個々のＢ細胞株から収集し、そしてこれらを、ＥＬＩＳＡを使用して抗イディオタイプ抗体の存在について試験した。簡潔には、マイクロタイタープレートを、４℃にて、このモチーフ陽性ＴＣＲペプチドまたはコントロールＴＣＲペプチドでそれぞれ１μｇ／ｍｌの濃度で一晩被覆した。ウェルを、次いで、３７℃にて２時間、２％ウシ血清アルブミン（ＢＳＡ）（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）含有ＰＢＳでブロックし、そしてこれらを、０．９％　ＮａＣｌ溶液中の０．０２％　Ｔｗｅｅｎ（登録商標）２０（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）にて４回洗浄した。各サンプルおよびそのコントロールを、隣のウェルに添加し、２時間インキュベートした。プレートを、４回洗浄し、そして１：１５００希釈率で西洋ワサビペルオキシダーゼに結合体化したヤギ抗ヒトＩｇＧ／ＩｇＭ抗体（Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）と共に３０分間インキュベートした。クエン酸緩衝液（ｐＨ＝５．０）中の０．０１２５％テトラメチルベンジジン／０．００８％Ｈ_２Ｏ_２を基質として使用し、そして発色を２Ｎ　Ｈ_２ＳＯ_４を使用して停止した。光学密度を、ＥＬＩＳＡリーダー（Ｂｉｏｒａｄ，Ｈｅｒｃｕｌｅｓ，ＣＡ）を使用して測定した。培地のみを含むウェルを、バックグラウンドコントロールとして供給した。
【００９２】
（免疫ブロット分析）
溶解物を、他のところに記載される標準的な方法（Ｈｊｅｌｍｅｌａｎｄら，１９８４）を使用して、共通ＣＤＲ３配列を発現する代表的なＭＢＰ反応性Ｔ細胞クローン（ＭＳ７．Ｅ２．６）から調製した。簡潔には、５×１０^６のＴ細胞を、以下を含む１００μｌの溶解緩衝液中で溶解した：１５０Ｍ　ＮａＣｌ、５０ｍＭ　Ｔｒｉｓ（ｐＨ７．６）、０．５％Ｔｒｉｔｏｎ　Ｘ−１００、１ｍＭ　ＰＭＳＦ、１０μｇ／ｍｌアプロチニン、１０μｇ／ｍｌロイペプチン。細胞残屑を、１３，０００ｇにて、２０分間、４℃で遠心分離した。得られた溶解物を、１０％ＳＤＳ−ＰＡＧＥを使用して電気泳動した。ブロッティング後、ニトロセルロースメンブレンを、細片（ｓｔｒｉｐ）に切断し、次いで、０．１％　Ｔｗｅｅｎ（登録商標）２０を含むＴｒｉｓ緩衝化生理食塩水中の５％低脂肪粉乳（ｍｉｌｋ−ＴＢＳＴ）でブロックした。次いで、この細片を、室温にて、１時間、ミニインキュベーショントレイ中で希釈していない上清と共にインキュベートした。西洋ワサビペルオキシダーゼに結合したヤギ抗ヒトＩｇＧおよびＩｇＭ（重鎖および軽鎖）を二次抗体（２％　ｍｉｌｋ−ＴＢＳＴ中の１００ｎｇ／ｍｌ）として使用して、そしてこれを洗浄した細片と共に４５分間インキュベートし、その後、メンブレン（Ａｍｅｒｓｈａｍ，Ｐｉｓｃａｔａｗａｙ，ＮＪ）上のタンパク質の増強された化学発光視覚化を行った。非反応性抗体を産生するＥＢＶ形質転換Ｂ細胞株から得た上清を陰性コントロールとして使用した。ウサギポリクローナル抗ヒトＴＣＲβ鎖抗体（Ｓａｎｔａ　Ｃｒｕｚ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｓａｎｔａ　Ｃｒｕｚ，ＣＡ）を、陽性コントロールとして含めた。
【００９３】
（フローサイトメトリー）
共通ＣＤＲ３配列を発現する代表的なＭＢＰ反応性Ｔ細胞クローン（ＭＳ７−Ｅ２．６）を、４℃にて、３０分間、個々のＢ細胞株由来の抗イディオタイプ抗体と共にインキュベートした。非反応性抗体を産生するＥＢＶ形質転換Ｂ細胞株から得た上清をコントロールとして使用した。ＦＡＣＳ緩衝液（５％ウシ胎仔血清および０．０１％アジ化ナトリウムを含有するＰＢＳ）を用いて２３００ｒｐｍにて、２分間、４℃で遠心分離により洗浄した後、細胞を再懸濁し、そしてフルオレセインに結合体化したヤギ抗ヒトＩｇＧ／ＩｇＭ抗体（ＦＩＴＣ）で染色した。２回の洗浄後、この細胞を、３００μｌのＦＡＣＳ緩衝液中に再懸濁し、ＦＡＣＳｃａｎ（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ，Ｓａｎ　Ｊｏｓｅ，ＣＡ）を使用してフローサイトメトリーで分析した。ＦＩＴＣ結合体化抗ＩｇＧ_１を使用してバックグラウンド染色を検出した（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ，Ｓａｎ　Ｊｏｓｅ，ＣＡ）。
【００９４】
（阻害アッセイ）
２０，０００細胞の免疫ＭＢＰ反応性Ｔ細胞クローン（モチーフ陽性およびモチーフ陰性のＴ細胞クローン）を、ＭＢＰの８３〜９９ペプチド（２０μｇ／ｍｌ）の存在下およびこれの非存在下にて、照射された自己ＰＢＭＣ（１００，０００細胞／ウェル）と共に１５０μｌ中で培養した。５０μｌの希釈していない上清を各ウェルに添加した。細胞増殖を、［^３Ｈ］−チミジン取り込みアッセイで７２時間後に測定した。非反応性抗体を産生するＥＢＶ形質転換Ｂ細胞株から得た上清をコントロールとして使用した。
【００９５】
（結果）
（免疫ＭＢＰ反応性Ｔ細胞クローンの機能的および構造的な特徴）
４つのＭＢＰ反応性Ｔ細胞クローンのパネルを、再発性弛張性ＭＳを有する２人の患者から生成した。これらのＴ細胞クローンは、ＣＤ４表現型を示し、そしてＤＲ４分子またはＤＲ２（ＤＲＢ１^＊１５０１）分子に関してＭＢＰの８３〜９９免疫優性ペプチドを認識した。これらを、ＶαおよびＶβに特異的なプライマーを使用するＲＴ−ＰＣＲによってＴＣＲ　Ｖ遺伝子再編成について分析し、その後Ｖα−ＪαおよびＶβ−Ｄβ−Ｊβ接合領域について配列決定した。患者ＭＳ７由来の独立したＴ細胞クローン（Ｅ２．６）は、同一のＴＣＲ　Ｖα１７遺伝子およびＶβ１３．１遺伝子を、異なる患者（ＭＳ２７）から得た別のＴ細胞クローン（Ｃ３．１）と共有した。２つのＴ細胞クローンは、Ｖβ１３．１−Ｄβ−Ｊβ接合領域中に同一の配列（配列番号３）を有したが、一方で、これらのＶα１７鎖は、２つ別個のＶα−Ｊα接合領域配列を有した。上述したように、同定されたＬｕｅＧｌｙＡｒｇＡｌａＧｌｙＬｅｕＴｈｒＴｙｒ（配列番号３）という配列は、ＭＳを有する異なる患者（２３）におけるＭＢＰの８３−９９免疫優性領域を認識するＶβ１３．１Ｔ細胞において共通ＣＤＲ３モチーフに相当した。
【００９６】
（ＭＳに罹患した患者におけるＴ細胞ワクチン接種による、ＭＢＰ反応性Ｔ細胞クローンおよび２０マーＴＣＲペプチドの免疫化に対する免疫応答の誘導）
各患者は、合計４回の皮下接種を、２回照射した自家ＭＢＰ反応性Ｔ細胞クローン（２×１０^７細胞／クローン）で２ヶ月の間を空けて受けた。ＰＢＭＣの免疫化自家Ｔ細胞クローンに対する増殖性応答を、ベースラインおよび最後の免疫化から２ヶ月に対応する２つの時点で試験した。図８Ａに示すように、両方の照射した免疫化Ｔ細胞クローンに対する増殖性応答は、Ｔ細胞ワクチン接種後に、これらの患者において増加し、実質的にベースライン値を超えた。さらに、ＣＤＲ３共通配列を取りこんだＴＣＲペプチド（モチーフポジティブペプチド、配列番号３２のアミノ酸２〜２１からなる）に対する応答は、非免疫化Ｔ細胞クローンに由来するコントロールＣＤＲ３ペプチド（モチーフネガティブペプチド、配列番号４８のアミノ酸１〜２０からなる）と対照的に、ワクチン接種後に明白であった。しかし、モチーフポジティブペプチドに応答する特異的増殖の強度は、照射した免疫化Ｔ細胞による誘導よりかなり低かった（図８Ａ）。免疫化Ｔ細胞に対する増殖性応答は、免疫化された患者中の循環するＭＢＰ応答性Ｔ細胞の頻度の減少に逆相関した（図８Ｂ）。
【００９７】
（免疫化された患者由来のＣＥＲ３共通配列を取りこむＴＣＲペプチドに対する特異的抗体を産生するＢ細胞株の生成）
次いで、本発明者らは、照射したＴ細胞での免疫化が、患者における特定の抗イディオタイプ抗体応答を誘発するか否かを試験した。Ｔ細胞全体が、血清抗イディオタイプ抗体の検出を妨げ得る表面分子のアレイを発現したので、共通のＣＤＲ３配列を取りこんだＴＣＲペプチド（モチーフポジティブペプチド）を、スクリーニングに対する抗原として用いた。非免疫化ＭＢＰ応答性Ｔ細胞クローン由来の２０マーＴＣＲペプチド（モチーフネガティブペプチド）を、コントロールとして全ての実験に含んだ。コントロールペプチドのＣＤＲ３配列は、免疫化Ｔ細胞クローンにおいて検出されなかった。
【００９８】
２人の患者に由来する血清を用いて試験した場合に、ＴＣＲペプチドまたは元の免疫化Ｔ細胞のいずれかに対する非特異的抗体応答性を、ＥＬＩＳＡまたはフローサイトメトリを用いて検出し得た（データは示さず）。抗イディオタイプ抗体がワクチン接種された患者に存在するか否かをさらに確証するために、本発明者らは、抗体産生Ｂ細胞株のパネルを、ワクチン接種後の血液検体から、ＥＢＶ形質転換と限外希釈とを合わせた細胞培養に基づく技術を用いて、作製した（材料および方法を参照のこと）。
【００９９】
プレワクチン接種のＰＢＭＣは、実験に使用できなかったので、２つのランダムに選択された健常者由来の細胞をコントロール対象として用いて、同じ実験条件下で分析した。得られたＢ細胞株（各患者／個体由来の９２細胞株）の上清を、モチーフポジティブＴＣＲペプチドに対する抗体およびコントロールＴＣＲペプチドに対する抗体の存在について、それぞれＥＬＩＳＡで試験した。抗体がモチーフポジティブＴＣＲペプチドに対して特異的な応答を呈するが、コントロールＴＣＲペプチドに対しては応答しない場合、抗体を抗イディオタイプとして規定した。図９において示されるように、特異的抗イディオタイプ抗体を産生するＢ細胞は、２人の非免疫コントロール対象と比較して、ＭＳ７およびＭＳ２７患者において、それぞれ１．７５×１０^−６の頻度で前駆細胞に生じた。対照的に、コントロールペプチドに対する非特異的抗体の反応性が、同じ上清において検出された。この結果は、抗イディオタイプ抗体を産生するＢ細胞の高頻度が、Ｔ細胞ワクチン接種に関することを示唆する。
【０１００】
本明細書において開示および権利を主張する全ての組成物および／または方法は、本発明の開示を考慮することで過度の実験を伴わずに、作製および実行され得る。本発明の組成物および方法は、好ましい実施形態について記載されるが、この組成物ならびに／または方法および本明細書に記載される方法の工程もしくは工程の順序に、本発明の概念、趣旨および範囲から逸脱することなく改変が適用され得ることは当業者にとって明らかである。より詳細には、化学的および生理学的の両方に関連する特定の因子が、本明細書に記載される因子に置換され得、同じかまたは同様の結果が達成されることは明らかである。当業者に明らかである全てのこのような同様の置換および改変は、添付される特許請求の範囲に規定されるように、本発明の趣旨、範囲および概念に含まれるとみなされる。
【０１０１】
【表４】

【図面の簡単な説明】
以下の図面は、本明細書の一部をなし、そして本発明の特定の局面をさらに実証するために含まれる。本発明は、本明細書中に示される特定の実施形態の詳細な記載と組み合わせてこれらの図面の１以上を参照して、より理解され得る。
【図１】図１は、ＰＢＭＣ由来のＰＣＲ産物のクローニングおよび配列決定のための実験手順を示す。ＰＢＭＣ試料由来のｃＤＮＡを、５’Ｖβ１３．１プライマーおよび３’Ｊβプライマーによって増幅し、そしてＬＧＲＡＧＬＴＹモチーフの発現について陽性の４つのＰＢＭＣ試料由来のｃＤＮＡ、ＴＡクローニングベクターｐＣＲ２．１にライゲーションし、そしてＥ．ｃｏｌｉに形質転換した。プラスミドＤＮＡを、Ｍ１３プライマーおよびＬＧＲＡＧＬＴＹ特異的プライマーを用いてＰＣＲによってスクリーニングした。ＰＣＲによる明白な増幅を示した陽性のプラスミドを、Ｖβ１３．１プライマーを用いて、ＶβＤβＪβ配列について、Ｖβ１３．１プライマーを用いて配列決定した。
【図２】図２は、単一アラニン置換を有するアナログペプチドに対する、２つのＭＢＰ８３−９９　Ｔ細胞クローンの反応性パターンを示す。同一のＶβ１３．１再編成（ＭＳ７−Ｅ２．６およびＭＳ２７−Ｃ３．１について）および類似のＶα−Ｊα接合部配列（ＭＳ７−Ｅ２．６およびＭＳ７−Ｅ３．１について）を示す２対のＭＢＰ８３−９９　Ｔ細胞クローンを、［^３Ｈ］−チミジン取り込みアッセイにおいて、アラニン置換されたペプチドのパネルに対する反応性について試験した。ＤＲＢ１^＊１５０１を発現するマウス繊維芽細胞株を、抗原提示細胞の供給源として使用した。各アナログペプチドに対するこれらのクローンの増殖応答を、７２時間後に測定し、そして結果を取り込まれたＣＰＭとして示す。四角の囲み（ｓｈａｄｅｄ　ｂｏｘ）は、アナログペプチドに対する応答における、Ｔ細胞クローンの増殖の５０％より大きい減少を示す。
【図３】図３は、元のＴ細胞クローンおよび無関係のＴ細胞クローンを用いた、ＣＤＲ３オリゴヌクレオチドの特異性の交差試験を示す。ＴＣＲ　ＶＤＪ領域に特異的なオリゴヌクレオチドのセットを、元のＭＢＰ８３−９９　Ｔ細胞クローンならびに同じ個体および異なる個体由来の無関係のＭＢＰ８３−９９　Ｔ細胞クローンに存在する既知の標的ＤＮＡ配列の検出における、その特異性について試験した。ＣＤＲ３特異的オリゴヌクレオチドを順方向プライマーとして、そして３’−Ｃβプライマーを逆方向プライマーとして使用して、ＰＣＲ反応を実行した。塗りつぶした四角は、元のＴ細胞クローンまたは同じＣＤＲ３配列を共有するＴ細胞クローンに存在するＤＮＡ配列の陽性の検出を示す。全てのプライマーもまた、無関係のＣＤＲ３配列を有するランダムに選択されたＴ細胞クローンのＤＮＡ産物に対する結合について試験した（斜線の四角）。
【図４】図４は、ＭＳを有する患者に由来する、ランダムに選択されたＰＢＭＣ試料における、モチーフＶβ１３．１−ＬＧＲＡＧＬＴＹに相補的な標的ＤＮＡ配列の検出を示す。ランダムに選択されたＭＳ患者（ｎ＝４８）に由来するＰＢＭＣ試料から調製されたｃＤＮＡを、５’−Ｖβ１３．１特異的プライマーおよび３’−Ｃβプライマーを使用して、ＲＴ−ＰＣＲで最初に増幅した。次いで、増幅したＰＣＲ産物を、ＬＧＲＡＧＬＴＹモチーフに特異的なジゴキシゲニン標識したオリゴヌクレオチドプローブと、引き続いてハイブリダイズさせた。元のＭＢＰ８３−９９クローン（ＭＳ７−Ｅ２．６）および無関係のＴ細胞クローン（ＭＳ３２−Ｂ９．８）を、それぞれ、陽性コントロールおよび陰性コントロールとして使用した。ＭＳ−７およびＭＳ−２７は、クローンＭＳ７−Ｅ２．６（表１のＭＳ−７）およびクローンＭＳ２７−Ｃ３．１（表１のＭＳ−２７）が由来した元のＰＢＭＣ試料であった。星印は、ＤＲＢ１^＊１５０１の陽性の発現を示す。
【図５】図５は、正常な被験体に由来する、ランダムに選択されたＰＢＭＣ試料におけるＶβ１３．１−ＬＧＲＡＧＬＴＹモチーフの検出を示す。２０人の正常な被験体（ＮＳ）から得られたＰＢＭＣ試料を、図４の説明に記載されるのと同じ条件下で分析した。元のクローン（ＭＳ７−Ｅ２．６）および無関係のクローン（ＭＳ３２−Ｂ９．８）を、それぞれ、陽性コントロールおよび陰性コントロールとして使用した。星印は、ＤＲＢ１^＊１５０１の陽性の発現を示す。
【図６】図６は、ＭＳ患者および正常な被験体に由来するＰＢＭＣ試料におけるＬＧＲＡＧＬＴＹモチーフの発現の半定量的比較を示す。モチーフＶβ１３．１−ＬＧＲＡＧＬＴＹの発現を、ＭＳの個体および正常な個体のＰＢＭＣに由来する各ｃＤＮＡにおけるＣβ発現と比較して、半定量的ＰＣＲによって分析した。相対的発現レベルを、（ＬＧＲＡＧＬＴＹモチーフの発現／Ｃβの発現）×１００％として計算した。
【図７】図７は、ＭＳを有する患者に由来する短期ＭＢＰ８３−９９　Ｔ細胞株における、Ｖβ１３．１−ＬＧＲＡＧＬＴＹモチーフの検出を示す。独立した短期ＭＢＰ８３−９９　Ｔ細胞株のパネルを、ＭＢＰの合成８３−９９ペプチドを使用して、ＭＳを有する５人の患者から作製した。これら全てのＴ細胞株を、ＭＢＰ８３−９９ペプチドに対するその特異的反応性について確認した（ＭＢＰ８３−９９に応答したＣＰＭ／コントロールＣＰＭ＞５）。ｃＤＮＡ産物を、５’−Ｖβ１３．１特異的プライマーおよび３’−Ｃβプライマーを使用して、ＰＣＲで増幅した。増幅されたＰＣＲ産物を、サザンブロット分析において、Ｖβ１３．１−ＬＧＲＡＧＬＴＹモチーフに対応するジゴキシゲニン標識したオリゴヌクレオチドプローブと、引き続いてハイブリダイズさせた。元のＭＢＰ８３−９９クローン（ＭＳ７−Ｅ６．２）および無関係のＴ細胞クローン（ＭＳ３２−Ｂ９．８）由来のｃＤＮＡ産物を、それぞれ、陽性コントロールおよび陰性コントロールとして使用した。
【図８】図８は、免疫されたＭＳ患者におけるＭＢＰ反応性Ｔ細胞の頻度と関連付けた、免疫ＭＢＰ反応性Ｔ細胞クローンおよびＴＣＲペプチドに対する、ＰＢＭＣ（末梢血単核球）の増殖応答を示す。８Ａ：２人の免疫された患者から得られたＰＢＭＣの増殖応答を、以下のように定義される刺激指数として示す。共通ＣＤＲ３配列を発現する、照射された免疫ＭＢＰ反応性Ｔ細胞クローン（モチーフ陽性Ｔ細胞クローンの、ＭＳ７−Ｄ２．２およびＭＳ２７−Ｄ４．４）と共に培養したＰＢＭＣの数／分（ＣＰＭ）／単独培養されたＰＢＭＣのＣＰＭと単独培養された照射されたＴ細胞のＣＰＭとの合計。モチーフ陽性ペプチド（配列番号３２のアミノ酸２〜２１）およびコントロールＴＣＲペプチド（配列番号４８のアミノ酸１〜２０）に対する増殖応答を、増殖アッセイにおいて決定し、ここで、ＰＢＭＣを、それぞれ、ＴＣＲペプチド（２０μｇ／ｍｌ）と共に、１００，０００細胞／ウェルで５日間培養した。全ての実験を、基準（前）および４回目のワクチン接種後２ヶ月（後）に対応する２つの時点で実施した。８Ｂ：ＭＢＰに特異的なＴ細胞の前駆体頻度を、同じ時点で推定した。ＮＳ＝正常な被験体。
【図９】図９は、免疫された患者のＰＢＭＣにおいてＴＣＲペプチドに対する抗イディオタイプ抗体を産生するＢ細胞の推定前駆体頻度を示す。ＰＢＭＣを、２人の免疫されたＭＳ患者および、免疫されていない２人のランダムに選択された健康個体から得た。細胞を、ＥＢＶ産生細胞株およびシクロスポリンＡ（Ｓａｎｄｏｚ、Ｂａｓｅｌ、Ｓｗｉｔｚｅｒｌａｎｄ）に由来する上清の存在下で培養した。増殖陽性の全てのウェルを、ＥＬＩＳＡにおいて、モチーフ陽性ＴＣＲペプチドおよびコントロールのＴＣＲペプチドに対して反応性の抗体の存在について、評価した。ＴＣＲペプチドに対する抗イディオタイプ抗体を産生するＢ細胞の前駆体頻度を、陽性ウェルの数を、最初にプレートされたＰＢＭＣの総数で除算することによって、概算した。[0001]
(Background of the Invention)
This invention is a continuation-in-part of co-pending application Ser. No. 09 / 507,819, filed Feb. 22, 2000.
[0002]
The United States Government may have rights in the invention according to grant number NS36140 from the National Institute of Health.
[0003]
(1. Field of the Invention)
The present invention relates generally to the field of treating autoimmune diseases (eg, multiple sclerosis (MS)). More particularly, the present invention relates to T cell receptor sequences found in some MS patients and methods for their detection.
[0004]
(2. Description of Related Technology)
In humans and other mammals, T cell receptors are found on T cells. The T cell receptor contains an α-chain and a β-chain, the β-chain contains the following region from N-terminus to C-terminus: Vβ-Dβ-Jβ-Cβ. T cell receptors differ naturally in the Vβ-Dβ-Jβ region.
[0005]
When an antigen is presented to a T cell by an antigen presenting cell (APC), a T cell receptor having a variable region (Vβ-Dβ-Jβ) generated to recognize the antigen binds to the antigen on the APC I do. Then, T cells bearing the T cell receptor are activated (clonal expansion).
[0006]
The pathogenesis of many autoimmune diseases is believed to depend on an autoimmune T cell response to antigens normally displayed by this organism. An example of such a disease is multiple sclerosis (MS), which commonly occurs in T cell responses to myelin antigens, especially myelin basic protein (MBP). MBP-reactive T cells have been found to undergo in vivo activation and to occur at higher precursor frequencies in the blood and cerebrospinal fluid of patients with MS, as opposed to control individuals. These MBP-reactive T cells produce Th1 cytokines (eg, IL-2, TNF, and γ-interferon). These Th1 cytokines promote the migration of inflammatory cells into the central nervous system and exacerbate the myelin-destructive inflammatory response in MS.
[0007]
Numerous regulatory mechanisms can be utilized in the treatment of MS. One of these is vaccination with one or more of a limited number of T cell membrane associated peptides having an extracellular domain. Vandenbank, U.S. Pat. No. 5,614,192 discloses a self-administration by using an immunogenic T-cell receptor peptide of 15-30 amino acids comprising at least a portion of the second complementarity determining region (CDR2) of the T-cell receptor. Disclosed are treatments for immune disorders. A copending U.S. patent application by Zhang (60 / 099,102) discloses the treatment of autoimmune diseases by using an immunogenic T cell receptor peptide in combination with an immunogenic T cell activation marker peptide.
[0008]
One area of vaccination with T cell receptor peptides is to determine what common motifs (if any) are found in T cell receptors in patients with autoimmune diseases such as MS. Be improved. If such a motif is found, the patient can be vaccinated with a peptide identical to the motif to facilitate treatment.
[0009]
Thus, it may be desirable to have a common motif amino acid sequence found in T cell receptors of patients with autoimmune diseases. It would also be desirable to be able to easily detect such motifs in patient samples by convenient methods such as PCR. Furthermore, it may be desirable to use the same peptide for the detected motif for treatment of a patient with an autoimmune disease.
[0010]
The present invention discloses such a common motif found in the T cell receptor of a subset of Vβ13.1 T cells, the “LGRAGLTY motif”, and methods for its easy detection by PCR. This motif has the amino acid sequence Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3). This motif is found in some T cell receptors of some T cells that recognize amino acids 83-99 of MBP (hereinafter "MBP 83-99"). This motif in the case of this subset of Vβ13.1 T cells may be referred to hereafter as “Vβ13.1-LGRAGLTY”. A peptide identical to this motif can be vaccinated into a patient and used to treat or prevent an autoimmune disease associated with Vβ13.1-LGRAGLTY. One such autoimmune disease is MS.
[0011]
(Summary of the Invention)
In one embodiment, the present invention relates to an oligonucleotide of about 15-30 nucleotides in length comprising at least 10 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto or derived therefrom. Even more preferred is a sequence comprising about 15, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO: or a sequence complementary thereto. It is an oligonucleotide 30 nucleotides in length. Most preferred is an oligonucleotide about 15-30 nucleotides in length, comprising the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto.
[0012]
In a further series of embodiments, the oligonucleotide may be required in the amplification or detection of a nucleic acid sequence found in Vβ13.1-LGRAGLTY T cells. In one subseries of such embodiments, the oligonucleotide is used in a primer pair. This primer pair includes or is derived from the following, wherein the sequences (a) and (b) are not found on the same strand of the T cell receptor gene:
(A) a first primer, wherein the first primer is an oligonucleotide of about 15-30 nucleotides in length, comprising at least 10 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto; A primer; and
(B) a second primer, wherein the second primer is an oligonucleotide about 15-30 nucleotides in length, does not include sequence (a), and the second primer is a T cell T cell receptor The second primer, which can be found in the region from Vβ to Jβ of the Vβ13.1 gene (SEQ ID NO: 2).
[0013]
Preferably, the first primer is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO: 1, or complementary thereto. An oligonucleotide of about 15-30 nucleotides in length, including the sequence. Most preferred are oligonucleotides about 15-30 nucleotides in length, including the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto.
[0014]
In another subseries of such embodiments, the oligonucleotide is used as an oligonucleotide probe, which includes:
(A) an oligonucleotide that is about 15-30 nucleotides in length and comprises at least 10 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto;
(B) labeled moiety.
[0015]
Preferably, the oligonucleotide is about 15-30 nucleotides in length and is contiguous with 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of SEQ ID NO: 1. Includes nucleotides or sequences complementary thereto. Most preferred are oligonucleotides about 15-30 nucleotides in length, including the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto. The labeling moiety is preferably ³² It is selected from P or digoxigenin.
[0016]
In another embodiment, the present invention relates to a method of detecting MBP83-99 Vβ13.1 T cells that express the LGRAGLTY motif, comprising the following:
(I) obtaining a nucleic acid sample from MBP83-99 Vβ13.1 T cells;
(Ii) contacting the nucleic acid sample with a primer pair selected or derived from:
(A) a first primer comprising an oligonucleotide of about 15-30 nucleotides in length, wherein the first primer comprises at least 10 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto or a sequence derived therefrom; and
(B) a second primer comprising an oligonucleotide of about 15-30 nucleotides in length, not comprising the sequence of (a), and Vβ-Jβ of the Vβ13.1 gene (SEQ ID NO: 2) in Vβ13.1 T cells A second primer found in the region of
Wherein the sequences of (a) and (b) are not found on the same strand of the Vβ13.1 gene;
(Iii) detecting the presence of the nucleic acid encoding the LGRAGLTY motif.
[0017]
Preferably, the first primer comprises about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 23 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto. It is an oligonucleotide having a length of 15 to 30 nucleotides. Most preferred is an oligonucleotide about 15-30 nucleotides in length, comprising the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto.
[0018]
Another embodiment of the invention is directed to a peptide that comprises the LGRAGLTY motif (ie, comprises the sequence of SEQ ID NO: 3) and is between 8 and about 45 amino acid residues in length. In various aspects of the embodiments of the present invention, the peptide is SEQ ID NO: 32, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. , 24, 25, 26, 27, 28, 29, 30, 31, or 32 contiguous amino acids. Preferably, the peptide sequence is SEQ ID NO: 3, which is residues 2-21 of SEQ ID NO: 32. Most preferably, the peptide sequence is residues 2-21 of SEQ ID NO: 32.
[0019]
In yet another embodiment, the invention relates to a method for treating an autoimmune disease, the method comprising:
(A) obtaining MBP83-99Vβ13.1 T cells from a human;
(B) detecting the presence of a nucleic acid encoding a LGRAGLTY motif by the method described above;
And if this nucleic acid is detected,
(C) administering to the human one or more peptides, each having an LGRAGLTY motif, from 9 to about 45 amino acids in length. In various aspects of this embodiment of the invention, the peptide administered to a human is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, SEQ ID NO: 32, respectively. It comprises 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 contiguous amino acids. Preferably, the peptide sequence is SEQ ID NO: 3, or the peptide sequence comprises residues 2-21 of SEQ ID NO: 32. Most preferably, the peptide sequence is residues 2-21 of SEQ ID NO: 32.
[0020]
In yet a further embodiment, the invention relates to a method of monitoring an autoimmune disease, the method comprising:
(A) obtaining MBP83-99Vβ13.1 T cells from a human;
(B) detecting the presence of a nucleic acid encoding a LGRAGLTY motif by the method described above;
And if this nucleic acid is detected,
(C) a step of quantifying the nucleic acid.
[0021]
(Description of exemplary embodiments)
To aid in understanding the present invention, some terms are defined below.
[0022]
"PCR" refers to the polymerase chain reaction, for example, generally described in U.S. Patent No. 4,683,202, issued to Mullins on July 28, 1987, which is herein incorporated by reference. Which is incorporated herein by reference). PCR is an amplification technique in which selected oligonucleotides and primers are hybridized to a nucleic acid template in the presence of a polymerization agent (eg, a polymerase) and four nucleotide triphosphates, and the extension product is Formed from primer. These products are then denatured and used as templates in a cycling reaction to amplify the number and amount of nucleic acids present to facilitate subsequent detection. Various PCR techniques are available and can be used with the method according to the invention.
[0023]
“Primer” refers to an oligonucleotide, either natural or synthetic, that can serve as a starting point for DNA synthesis complementary to a particular DNA sequence on a template molecule.
[0024]
With respect to the term “primer or probe derived from”, “derived from” is not limited to the listed oligonucleotide sequences, but includes variations in the listed nucleotide sequences (variations to the listed sequences are Vβ13. 1-LGRAGLTY sequence, including nucleotide additions, deletions or substitutions, to the extent that it retains its ability to act as a primer in the detection of T cell receptor DNA encoding the Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3). Means a primer or a probe, also including
[0025]
"Immunogenic", when used to describe a peptide, means a peptide that is capable of eliciting an immune response, either T-cell mediated, antibody, or both. By "antigenic" is meant peptides that can be recognized in free form by antibodies and, in the case of antigen-specific T cells, MHC molecules.
[0026]
"Immune-related disease" refers to a disease in which the immune system is involved in the pathogenesis of this disease. A subset of immune-related diseases are autoimmune diseases. Autoimmune diseases contemplated by the present invention include, but are not limited to, rheumatoid arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, autoimmune thyroiditis (Hashimoto's thyroiditis). , Graves' disease, inflammatory bowel disease, autoimmune uveo-retinitis, polymyositis, and certain types of diabetes. In light of the present disclosure, one of skill in the art can readily recognize other autoimmune diseases that can be treated by the compositions and methods of the present invention. "T cell mediated disease" means a disease that is caused in an organism as a result of a T cell recognition peptide commonly found in the organism.
[0027]
"Treatment" or "treat", when referring to protecting an animal from a disease, means preventing, suppressing or repressing the disease. Preventing a disease involves administering a composition of the present invention to an animal prior to induction of the disease. Suppressing the disease involves administering a composition of the invention to an animal after the induction of the disease but before the clinical appearance. Suppressing the disease involves administering a composition of the invention to an animal after the clinical appearance of the disease. It is not always clear when during the course of disease induction, but it is understood that in human medicine the compositions of the present invention are administered.
[0028]
In one aspect, the invention relates to a primer pair comprising the following sequence or a sequence derived from:
(A) a first primer, which is an oligonucleotide about 15-30 nucleotides in length, comprising at least 10 contiguous nucleotides of SEQ ID NO: 1 or a nucleic acid sequence complementary thereto; and
(B) a second primer, which is an oligonucleotide about 15-30 nucleotides in length, that does not contain the sequence of (a) and is found in the Vβ-Jβ region of the T cell receptor gene in Vβ13.1 T cells. Be the primer. Here, the sequences (a) and (b) are not found on the same strand as the T cell receptor gene.
[0029]
Preferably, the first primer is an oligonucleotide about 15 to 30 nucleotides in length, and has a SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. Consecutive nucleotides or sequences complementary thereto are included. Most preferred are oligonucleotides about 15-30 nucleotides in length, including the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto.
[0030]
Primers according to the present invention are designed to amplify a fragment of the gene encoding the T cell receptor of human Vβ13.1 T cells, which fragment is an amino acid motif, Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3). including. A gene derived from Vβ13.1 T cells that encodes a T cell receptor, including the LGRAGLTY motif, has been submitted to GenBank and has accession number AF117132. The sequence of the Vβ13.1 T cell-derived gene encoding the T cell receptor, including the LGRAGLTY motif, is provided herein as SEQ ID NO: 2. In the method according to the invention, a fragment of about 400 bp of the T cell receptor gene from Vβ13.1 T cells is amplified using two primers, wherein the first primer is in the CDR3 region and The second primer is in the Cβ region. The Vβ-Dβ-Jβ region of the T cell receptor gene includes between the CDR3 region and the Cβ region. In a preferred embodiment, these primers are the primer pairs described above.
[0031]
Primers according to the present invention also include oligonucleotides derived from primers (a)-(b). The sequence has or comprises substantially the same sequence as one of the primers, and is substantially similar to the CDR3 or Cβ region of the Vβ-Dβ-Jβ region of the T cell receptor gene from Vβ13.1 T cells as described above. Derived from primer (a) or (b) when retaining the ability to selectively anneal. More specifically, this primer may have one or more of its length or sequence as long as it retains selectivity for the identified region of the Vβ-Dβ-Jβ region of the T cell receptor gene from the Vβ13.1 T cell. The type of nucleic acid at the position may be different from the primer (a) or (b). For example, the primer can be an oligonucleotide having at least 15 nucleotides, wherein the 15 nucleotides are selected from or derived from the sequences of primers (a)-(b). Identical to nucleic acids. The primer can also be any oligonucleotide of about 30 nucleotides or less, including a segment having a sequence selected from or derived from any of primers (a)-(b). The number of nucleotides in the primer should be high enough to retain selectivity, but low enough to retain efficacy and operability in primer synthesis and PCR procedures. This primer has variations including nucleotide deletions, additions or substitutions to the extent that the variations with respect to the sequences of primers (a) and (b) retain the ability to act as primers in the detection of Vβ13.1-LGRAGLTY. I can do it.
[0032]
The Vβ13.1-LGRAGLTY detection method according to the present invention uses the above-mentioned pair of primers in a procedure for detecting the presence of any Vβ13.1-LGRAGLTY in a sample. The sample tested for the presence of Vβ13.1-LGRAGLTY is a nucleic acid, preferably DNA. The DNA can be genomic DNA, cDNA, DNA previously amplified by PCR or any other form of DNA. This sample can be isolated directly or indirectly from any animal tissue or human body tissue that expresses the T cell receptor β chain gene. A preferred body tissue is a peripheral blood mononuclear cell (PBMC). If the sample is genomic DNA, it can be isolated directly from body tissue. If the sample is cCNA, it can be isolated indirectly by reverse transcription of mRNA directly isolated from body tissue. If the sample is DNA that has been pre-amplified by PCR, it may be indirectly isolated by amplification of genomic DNA, cDNA, or any other form of DNA.
[0033]
In a preferred embodiment, the portion of the T cell receptor gene derived from the Vβ13.1 T cell (the portion containing the sequence encoding the LGRAGLTY motif) is Vβ13.1-LGRAGLTY (5′-CTAGGGCGGGCGGGACTCACCTAC-3 ′ (SEQ ID NO: 1)). Amplified to enhance its ability to detect the presence of This amplification can be performed by a PCR reaction using any particular PCR technique or equipment that provides for sensitivity, selectivity and rapid amplification of the moiety in the sample.
[0034]
For example, this PCR amplification can be performed in the following procedure, where the reaction mixture is prepared containing the following components: 5 μL 10 × PCR Buffer II (100 mM Tris-HCl, pH 8.3, 500 mM KCl) , 3 μL 25 mM MgCl ₂ 1 μL 10 mM dNTP mixture, 0.3 μL Taq polymerase (5 U / μL) (AmpliTaq Gold, Perkin Elmer, Norwalk, CT), 30 pmol of primer A, and 30 pmol of primer B. In view of the disclosure of the present invention, one skilled in the art can select appropriate primers A and B for the purpose of PCR amplification of the T cell receptor gene portion from Vβ13.1 T cells. The above mixture is suitable for amplifying 1 μL of sample DNA. Hereinafter, the DNA to be amplified is referred to as a “template”.
[0035]
Once sample DNA has been added to the above reaction mixture, the PCR reaction is performed at 95 ° C. for 1 minute (denaturation); 56 ° C. for 20 seconds (annealing) and 72 ° C. for 40 seconds (extension) for a total of 35 cycles. Using the amplification profile of
[0036]
In a PCR reaction, the template can be heat denatured and annealed for the two oligonucleotide primers. Oligonucleotides bracket the region of the nucleic acid sequence to be amplified. A thermostable DNA polymerase is included in the reaction mixture. The polymerase extends the primer annealed to the complementary DNA by adding the appropriate complementary nucleic acid. Preferred polymerases have the characteristics of being stable at a temperature of at least 95 ° C, have an advancement of 50-60, and have an extension rate of more than 50 nucleotides per minute.
[0037]
Approximately 40 PCR cycles are used in a typical PCR amplification reaction. However, certain PCR reactions are performed in as few as 15-20 or as few as 50 cycles. Each cycle consists of a melting step, where the template is warmed to a temperature above about 95 ° C.
[0038]
The temperature of the PCR reaction is then cooled to allow the primer to anneal to the template. In the annealing step, the reaction temperature is adjusted between about 55C to 72C for about 20 seconds. Longer or shorter times may be performed depending on the particular reaction.
[0039]
The temperature of the PCR reaction is then warmed to allow for maximum extension of the primers affected by the polymerase. In the extension step, the reaction temperature is adjusted between about 70 ° C to 75 ° C for approximately 40 seconds. Higher or lower temperatures and / or longer or shorter times are performed depending on the particular reaction.
[0040]
In addition, before the first cycle begins, the reaction mixture undergoes an initial denaturation in a period of about 5-15 minutes. Similarly, after the last cycle is completed, the reaction mixture may undergo a final extension in a period of about 5-10 minutes.
[0041]
Amplification can be performed using a two-step PCR. In this technique, a first PCR amplification reaction is performed by amplifying a first region longer than a target region and including the target region. Next, a second PCR amplification reaction is performed using the first region as a template for amplifying the region of interest. The second PCR reaction is "semi-nested" if any primers from the first PCR reaction can be used in the second PCR reaction. If neither primer from the first PCR reaction can be used in the second PCR reaction, the second PCR reaction is "nested".
[0042]
In a preferred manner of practicing the method of the invention, the Vβ13.1-LGRAGLTY motif is amplified by a two-step PCR. In a first PCR reaction, a first primer (annealing to the Vβ region of the T cell receptor gene) and a second primer (annealing to the Cβ region of the T cell receptor gene) are used to form the reaction mixture disclosed above. This sample is amplified using and the reaction profile. The first PCR reaction amplifies a first region (approximately 600 bp and extending from Vβ through the Vβ-Dβ-Jβ junction to Cβ). The second PCR reaction is nested or semi-nested; the first region portion is partially amplified using primer pairs (a)-(b). The second PCR reaction amplifies the region of interest.
[0043]
After amplification of any DNA encoding Vβ13.1-LGRAGLTY in the sample, the amplification product is detected. This detection can be performed by a number of procedures. For example, an aliquot of the amplification product can be loaded onto an electrophoresis gel (an electric field is applied to separate DNA molecules by size). In another method, aliquots of the amplification product are loaded onto a gel that has been stained with SYBR green, ethidium bromide or other molecules that bind to DNA and generate a detection signal. For example, ethidium bromide binds to DNA and emits visible light when irradiated with ultraviolet light. The dried gel alternatively contains radiolabeled or chemically labeled oligonucleotides (complementary to the sequence portion of the amplified template) (hereinafter termed "oligonucleotide probes") and the film An autoradiograph is taken by exposing the gel.
[0044]
In another embodiment, the present invention relates to oligonucleotide probes, comprising:
(A) an oligonucleotide about 15-30 nucleotides in length, comprising at least 10 contiguous nucleotides of SEQ ID NO: 1, or a nucleic acid sequence complementary thereto;
(B) Label part
Is included.
[0045]
Preferred "(a)" are oligonucleotides of about 15-30 nucleotides in length, which are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22 of SEQ ID NO: 1. Or 23 contiguous nucleotides, or sequences complementary thereto. Preferably, the label moiety is ³² It is selected from P or digoxingenin.
[0046]
Representative radiolabeled oligonucleotides useful for the detection of amplification products produced using the primers of the invention are derived from the Vβ-Dβ-Jβ region. The Vβ13.1-LGRAGLTY region is amplified by the two-step semi-nested PCR disclosed above, where primers corresponding to the sequence encoding the LGRAGLTY motif are used, and the amplified Vβ13.1-LGRAGLTY is used. Any oligonucleotide of about 10 or more nucleotides, and preferably about 18 or more nucleotides, complementary to a portion of either strand of the region can be used. More preferably, the oligonucleotide 5'-CTAGGGCGGGGCGGACTCACCTAC-3 '(SEQ ID NO: 1) or a nucleic acid sequence complementary thereto is used as the probe.
[0047]
The present invention also encompasses a test kit, comprising a first primer (a) about 15 to 30 nucleotides in length, comprising at least 10 contiguous nucleotides of SEQ ID NO: 1 or a nucleic acid sequence complementary thereto.
[0048]
In one preferred embodiment, the test kit further comprises a second primer (b), wherein the second primer is a nucleic acid sequence that is about 15-30 nucleotides in length without the sequence of (a). And Vβ13.1 in T cells is found in the region from Vβ to Jβ of the T cell receptor gene. Here, the sequences (a) and (b) are not found on the same strand of the T cell receptor gene.
[0049]
More preferably, the first primer is a contiguous nucleotide of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of SEQ ID NO: 1, or a sequence complementary thereto. And an oligonucleotide having a length of about 15 to 30 nucleotides. Most preferably, it is an oligonucleotide about 15-30 nucleotides in length, comprising the nucleotides of SEQ ID NO: 1 or their complementary sequences. Most preferred oligonucleotides are about 15-30 nucleotides in length, including the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary thereto.
[0050]
In this embodiment, the test kit further comprises at least one reagent useful in amplifying Vβ13.1-LGRAGLTY DNA by PCR techniques as described above. Exemplary reagents that can be included in the kit include buffers, deoxynucleoside triphosphates, a thermostable DNA polymerase (eg, Taq polymerase), Vβ13.1-LGRAGLTY DNA for a positive control and a negative control for a negative control. Non-Vβ13.1-LGRAGLTY DNA, including but not limited to. Other reagents that can be included in the test kit are known to those skilled in the art.
[0051]
In another preferred embodiment, the test kit further comprises a label moiety. Preferred label moieties are ³² P or digoxigenin.
[0052]
Another embodiment of the invention is directed to a peptide that comprises the LGRAGLTY motif (ie, comprises the sequence of SEQ ID NO: 3) and is 8 to about 45 amino acid residues in length. In various aspects of this embodiment of the invention, the peptide is SEQ ID NO: 32, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 contiguous amino acids. Preferably, the peptide sequence is residues 2 to 21 of SEQ ID NO: 3 or SEQ ID NO: 32. Most preferably, the peptide sequence is residues 2-21 of SEQ ID NO: 32. In a preferred aspect of this embodiment of the invention, the peptide is present as a purified peptide. "Purified peptide" refers to the majority (greater than 50%) of the polypeptides in a sample being the desired peptide. Preferably, the desired peptides make up more than 70% of the peptides in the purified peptide sample. More preferably, the peptide makes up more than 90% of the peptides in the sample. Even more preferably, the peptide makes up more than 95% of the peptides in the sample. Furthermore, the term "purified peptide" indicates that the sample is free of substances that interfere with the action of the present invention.
[0053]
Peptides according to this aspect of the invention may be derived from any source compatible with the invention, either natural or synthetic (available from commercial sources known to those skilled in the art).
[0054]
Another embodiment of the present invention provides a pharmaceutical composition comprising a peptide as described above. Methods for making pharmaceutical peptide compositions are known in the art (see, for example, US Pat. Nos. 6,066,619 and 6,068,850, which are incorporated herein by reference. Incorporated in the book))). Various aspects of this embodiment of the invention can include a pharmaceutically acceptable excipient, carrier or diluent, and do not include any biologically detrimental substances. The pharmaceutical compositions of the invention can be formulated by one skilled in the art. Exemplary pharmaceutical formulations are also described in Remington's Pharmaceutical Sciences (eds. Alfonso R. Gennaro, 16th ed., 1980), which is a standard reference in the field of pharmacy and is incorporated herein by reference. Incorporated.
[0055]
Pharmaceutical compositions may further include coloring or stabilizing agents, penetrants, antibacterial agents or any other substance that does not interfere with the function of the composition. The pharmaceutical compositions of the invention may be formulated, for example, as a solution, suspension, or emulsion in a pharmaceutically acceptable parenteral vehicle. The vehicle may include additives that maintain isotonicity, such as sodium chloride or mannitol, and additives that maintain chemical stability, such as buffers and preservatives. It should be understood that endotoxin contamination should be maintained at safe levels (eg, less than 0.5 ng / mg protein). In addition, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by United States Food and Drug Administration biological standards. This formulation can be sterilized by using common techniques, such as filtration.
[0056]
The phrase "pharmaceutically acceptable" refers to substances and compositions that do not produce an adverse, allergic, or another unintended reaction when administered to an animal or human as needed. Substances that cause any of these adverse effects are classified as "biologically harmful" within the scope of the present invention. Pharmaceutically acceptable substances and compositions include, but are not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. The use of any conventional ingredients is contemplated except for those incompatible with the present invention. In addition, auxiliary active ingredients that are utilized for some other pharmaceutically convenient purposes can also be incorporated into the instant compositions.
[0057]
The invention also includes a method of treating an autoimmune disease. The disease is a disease in which, for at least some patients, T cell receptors, including LGRAGLTY, are found on Vβ13.1 T cells. Other types of T cells and / or Vβ13.1 T cells (lacking the T cell receptor containing the LGRAGLTY motif) can be represented by this patient. The method is as follows:
(A) obtaining MBP83-99 Vβ13.1 T cells from a human;
(B) detecting the presence of a nucleic acid encoding a LGRAGLTY motif by the method described above;
(C) administering to a human a pharmaceutical composition comprising one or more peptides from 9 to about 45 amino acid residues in length, wherein each peptide comprises an LGRAGLTY motif.
[0058]
In various aspects of this method, the pharmaceutical composition is administered to a human, which comprises the sequence of SEQ ID NO: 32, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 consecutive amino acids. Preferably, the peptide sequence is a peptide sequence comprising residues 2 to 21 of SEQ ID NO: 3 or SEQ ID NO: 32. Most preferably, the peptide sequence is residues 2-21 of SEQ ID NO: 32.
[0059]
The autoimmune disease can be any autoimmune disease in which a T cell receptor containing the LGRAGLTY motif is found on Vβ13.1 T cells. Autoimmune diseases contemplated by the present invention include, but are not limited to, rheumatoid arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus, autoimmune thyroiditis (Hashimoto thyroiditis) ), Graves' disease, inflammatory bowel disease, autoimmune uveo-retinitis, polymyositis and certain types of diabetes. A preferred autoimmune disease is multiple sclerosis (MS).
[0060]
If a nucleic acid encoding a LGRAGLTY motif is detected by the methods disclosed above, the autoimmune disease can be treated by administering a pharmaceutical composition comprising one or more peptides as described above. The pharmaceutical compositions of the present invention can be administered alone or in combination with a T cell activity marker peptide. Preferably, the pharmaceutical composition is combined with a T cell activity marker peptide according to the disclosure of Zhang, US Patent Application No. 60 / 099,102, which is incorporated herein by reference. Is administered. Administration of the peptide can elicit an immunogenic response, wherein the patient recognizes the LGRAGLTY motif of the T cell receptor found on Vβ13.1 T cells and recognizes antibodies and T cell receptors that bind to it. To develop.
[0061]
Since Vβ13.1-LGRAGLTY can be present in both patients with MS and normal individuals without the disease, the pharmaceutical compositions of the present invention can be used in both patients with MS and normal individuals. It is contemplated that it can be administered.
[0062]
In an alternative embodiment, if a nucleic acid encoding a LGRAGLTY motif is detected by the methods disclosed above, an autoimmune disease can be monitored by quantifying the nucleic acid. The more abundant nucleic acid is present in a sample (eg, PBMC), the greater the number of Vβ13.1 T cells and probably the more severe the symptoms of autoimmunity. Also, depending on the time between presentation of elevated Vβ13.1 T cell levels and appearance of symptoms, clinicians have the opportunity to apply treatments to minimize the severity of symptoms, and The disease may be treated before / or before the symptoms appear.
[0063]
The following examples are included to demonstrate preferred embodiments of the present invention. The techniques disclosed in these examples, which are described below, have been discovered by the present inventors and function well in the practice of the present invention, and thus are configured in a manner preferred for that practice. It should be understood by those skilled in the art that it may be considered However, in view of the present disclosure, many modifications may be made in the specific embodiments disclosed, and further, the same or similar results may be obtained without departing from the spirit and scope of the invention. It should be understood that
[0064]
(Example)
Example 1: T cell receptor Vβ-Dβ-Jβ DNA sequence and sequence motif shared between MBP83-99 specific T cell clones from different patients with MS
Twenty panels of CD4 + independent T cell clones were generated from seven patients with MS. All T cell clones were DRB1 as antigen-presenting cells. ^* Recognized the 83-99 peptide of myelin basic protein (MBP83-99) in the context of HLA-DR2 as determined by using mouse fibroblasts (L cells) transfected at 1510. T cell clones were characterized for TCR V gene rearrangement in reverse transcription PCR (RT-PCR) using Vα- and Vβ-specific oligonucleotide primers, followed by the Vα-Jα junction region and The Vβ-Dβ-Jβ junction region was sequenced. The sequences of these junction regions are shown in Tables 1 and 2.
[0065]
Table 1 uses reverse transcription PCR, which uses a panel of oligonucleotide primers specific to the Vα gene family (the sequence of the specific primers used is underlined in the DNA sequence corresponding to each clone). The results of an analysis using 20 panels of independent MBP83-99-specific T cell clones, characterized by Vα gene usage according to (shown)), are summarized. The amino acid sequences of the “Vα”, “n”, “Jα” and “Cα” portions of each clone are shown in Table 1 as follows: “n” portions are underlined and “Vα” and “Jα” The sequences are shown in bold on each side of the "n" sequence, and the "Cα" sequence is not underlined and is shown in normal font. The amplified PCR product was hybridized with a digoxingenin labeled Cα cDNA probe and subsequently analyzed for DNA sequence.
[0066]
Table 2 summarizes the analysis of 20 panels of independent MBP83-99 specific T cell clones. These clones were subjected to reverse transcription PCR, which uses a set of oligonucleotide primers specific to the 26 Vβ gene family (the sequence of the specific primers for each clone is indicated by underlining in the corresponding DNA sequence). As indicated))) was analyzed for Vβ gene usage. The “Vβ”, “D”, “Jβ” and “Cβ” portions of each clone are shown in Table 2 as follows: The “D” portion is underlined and the “Vβ” and “Jβ” sequences are The “D” sequence is shown in bold on each side, and the remaining sequence, the “Cβ” sequence, is in normal font (not underlined or bold). The amplified PCR product was hybridized with a digoxigenin-labeled Cβ cDNA probe and subsequently analyzed for DNA sequence.
[0067]
(Table 1: TCR Vα gene sequence specific for MBP83-99 peptide)
[0068]
[Table 1]

(Table 2: TCR Vβ gene sequence specific for MBP83-99 peptide)
[0069]
[Table 2]

Although the rearrangement of Vα and Vβ varied between individual MBP83-99 T cell clones, many of these independent T cell clones from a given individual showed that the same Vα-Jα and Vβ-Dβ- The same Vα and Vβ chains were shared with the Jβ junction region sequence. This finding is consistent with the in vivo clonal expansion of MBP83-99-specific T cells in certain patients with MS, as previously reported (Vandevyver 1995, Wucherpfenning 1994).
[0070]
Interestingly, as shown in Tables 1 and 2, an independent T cell clone (clone E2.6) from one patient (MS-1) was obtained from another patient (MS-2). Three of the four T cell clones obtained (clones C3.1, D7.16 and F3.4) shared the same Vβ13.1 and Vα17. Vβ13.1 of these T cell clones shared the same DNA sequence within the Vβ-Dβ-Jβ junction region.
[0071]
(Example 2: Vβ-Dβ-Jβ specific oligonucleotide primers bind the corresponding DNA sequences present in the original MBP83-99 T cell clone and the PBMCs containing the original MBP83-99 T cells. Highly specific and sensitive when detected)
A set of 14 oligonucleotide primers was synthesized according to the DNA sequence within the Vβ-Dβ-Jβ junction region of the independent MBP83-99 T cell clone and subsequently tested for their specificity in RT-PCR. . Table 3 shows the DNA sequences of these oligonucleotide primers.
[0072]
(Table 3: DNA sequence of Vβ-Dβ-Jβ-specific oligonucleotide primer)
[0073]
[Table 3]

These Vβ-Dβ-Jβ-specific primers bound exclusively to the DNA sequence present in the original MBP83-99 T cell clone, but contained sequences from unrelated MBP83-99 T cell clones. Did not bind (FIG. 2). This suggests their high specificity for the original Vβ-Dβ-Jβ DNA sequence. Only the exceptions for clones MS1-E2.6 and MS2-C3.1 were noted (in which the same primers bound to the DNA sequence of the Vβ-Dβ-Jβ junction shared by the T cell clones).
[0074]
Given the specificity of Vβ-Dβ-Jβ oligonucleotide primers and the high sensitivity of the PCR detection system, we use 5 ′ Vβ primers and Vβ-Dβ-Jβ specific oligonucleotide primers, This two-step PCR detection system was used to determine whether to detect the corresponding Vβ-Dβ-Jβ DNA sequence present in the peripheral blood mononuclear cell (PBMC) specimen from which the MBP83-99 T cell clone was derived. I asked. The results of two separate experiments showed positive detection of the Vβ-Dβ-Jβ sequence in the original PBMC specimen. Therefore, the PCR detection system in which the Vβ-Dβ-Jβ sequence acts as a fingerprint, is capable of specifically tracking MBP83-99 T cells present in peripheral blood mononuclear cells by searching for the same DNA sequence. And that they are sensitive.
[0075]
Example 3: Detection of a common Vβ-Dβ-Jβ DNA sequence in PBMC specimens from different patients with MS and healthy individuals
We then found that the DNA sequence corresponding to the Vβ-Dβ-Jβ junction region of the MBP83-99 T cell clone was found in PBMC specimens randomly selected from a group of patients with MS and a group of healthy individuals. It was examined whether it could be detected. The same PCR amplification system using specific primers for the corresponding Vβ family (in the first PCR) and Vβ-Dβ-Jβ sequences (in the second half-nested PCR) was used. Hybridization was performed using this in combination with Southern blot analysis using the corresponding Vβ-Dβ-Jβ probe. Given the specific requirements of this two-step PCR detection system and the specificity of the Vβ-Dβ-Jβ primers and probes, the identified DNA sequence is from a particular TCR Vβ chain and the DNA sequence is Represents either identity or similarity to the Vβ-Dβ-Jβ sequence.
[0076]
These results indicate that only one Vβ-Dβ-Jβ oligonucleotide primer (MSI-E2.6, Vβ13.1-LGRAGLTY) was used in 15 of 48 PBMC specimens from different patients with MS. It was shown that the complementary TCR Vβ13.1 DNA sequence was detected in PBMC specimens (31%). Thus, this finding indicates the presence of MBP83-99 T cells expressing Vβ13.1-LGRAGLTY in these patients with MS. Under similar experimental conditions, the same primers also detected the corresponding DNA sequence in 5 out of 20 PBMC specimens (25%) obtained from healthy individuals. The remaining 13 Vβ-Dβ-Jβ primers failed to identify any sequence signal in the same panel of PBMC specimens. These results were reproducible in three separate experiments. Since 13.1 specific primers were used in the first PCR for amplification, the identified DNA product amplified by the E2.6 primer was derived from T cells expressing Vβ13.1.
[0077]
In addition, the identified Vβ13.1-LGRAGLTY sequence also generated 24 short-term MBP83-99 T cell lines generated from 5 patients with MS (MS-35, MS36 and MS39). Out of 13 cell lines (54%), and PBMC specimens from these patients were shown to contain the Vβ13.1-LGRAGLTY sequence. Therefore, these results confirmed that the Vβ13.1-LGRAGLTY DNA sequence detected in these PBMC specimens was derived from several T cells recognizing MBP83-99. This finding also indicates that MBP83-99 T cells expressing the Vβ13.1-LGRAGLTY sequence may be all or most of the MBP83-99 T cell lines found in patients with MS. Implies that
[0078]
The combined PCR-DNA hybridization detection system (where the Vβ-Dβ-Jβ sequence was used as a fingerprint) has the potential to track antigen-specific T cells by detecting the same Vβ-Dβ-Jβ junction sequence. Tools provided. The high specificity and sensitivity of this detection system allowed the identification of specific Vβ-Dβ-Jβ sequences in peripheral blood T cells. This study shows that a common subset of Vβ13.1 T cells, which recognize the immunodominant 83-99 peptide of MBP and uniformly express the same Vβ-Dβ-Jβ sequence, are among the patients with MS It was demonstrated for the first time that it was present in about 30%. This result was obtained based on the step-by-step experiments described herein. First, the same DNA sequence (Vβ13.1-LGRAGLTY) was found in independent MBP83-99 T cell clones from different patients with MS. Second, the sequence was identified in a cDNA product amplified from TCR Vβ13.1 in PBMC specimens from different patients with MS. Third, the DNA sequence was detected in a short-term, independent MBP83-99 T cell line generated from a PBMC specimen that was shown to contain the Vβ13.1-LGRAGLTY sequence. MBP83-99 T cells expressing the Vβ13.1-LGRAGLTY sequence appear to represent all or most of the MBP83-99 T cell line that originated from several patients with MS. Finally, the presence of the Vβ13.1-LGRAGLTY sequence in the PBMC specimen was confirmed by recombinant DNA cloning and direct DNA sequencing.
[0079]
Furthermore, it is not surprising that MBP83-99 T cells expressing the common Vβ13.1-LGRAGLTY sequence are also present in some healthy individuals. Previously reported studies indicate that MBP-reactive T cells, including those that recognize the immunodominant 83-99 peptide, are also present in some healthy individuals (Zhang). 1994, Ota 1990). However, there is a functional difference that these T cells undergo in vivo activation and clonal expansion in patients with MS, as opposed to healthy individuals (Zhang 1994).
[0080]
These Vβ13.1 MBP83-99 T cells, which share a common Vβ-Dβ-Jβ sequence, may represent a significant portion of the MBP83-99 T cells found in some patients with MS. This possibility is supported by the observation that the Vβ13.1-LGRAGLTY sequence was present in 40% of the short-term MBP83-99 T cell lines arising from patients with MS after two stimulation cycles.
[0081]
The identified common Vβ-Dβ-Jβ sequence is used as a specific marker in a quantitative PCR detection system for the purpose of monitoring in vivo clonal growth and in vivo activity potentially associated with this disease. Can detect a common subset of MBP83-99 T cells in blood and cerebrospinal fluid in a large group of MS patients. This method is superior to conventional cell culture-based assays because the in vitro selection and expansion of MBP-reactive T cells is often hampered by various inhibitory factors specific to cell culture. is there. This is because a recent study found that the frequency of MBP-reactive T cells was surprisingly high in patients with MS when quantifying MBP-reactive T cells using direct ex vivo analysis (Hafler). (Last listed author) JEM 1997).
[0082]
In addition, synthetic peptides corresponding to this TCR have been shown to induce anti-idiotype T cell responses to MBP-reactive T cells in patients with MS (Chou et al., JI). Thus, a TCR peptide containing a common CDR sequence may be used to inhibit anti-idiotype T cells to suppress a specific subset of MBP-reactive T cells in a group of patients whose MBP83-99 T cells possess a common CDR3 sequence motif. It can be of great potential to attract cells. Immunization with such a common CDR3 peptide is more beneficial than a CDR2 peptide or an individual dependent CDR3 peptide as a potential treatment procedure in patients with MS (Vandenbark 1996).
[0083]
Example 4: Induction of an immune response to immune MBP-reactive T cell clones and a 20-mer TCR peptide by T cell vaccination in patients with MS and specificity for TCR peptides incorporating a common CDR3 sequence from immunized patients Generation of B cell lines that produce specific antibodies)
Subcutaneous inoculation with irradiated autologous MBP-reactive T cell clones demonstrates that a substantial anti-idiotype T cell response is induced in patients with MS, which is Correlated with progressive depletion of MBP-reactive T cells (Medaer et al., 1995).
[0084]
(Materials and methods)
(Reagents and peptides)
The medium used for cell culture was Aim-V serum-free medium (Life Technologies, Grand Island, NY). The immunodominant peptide of MBP (residues 83-99) and two TCR peptides of 20 amino acids were synthesized by Chiron Mimotope (San Diego, CA). The purity of this peptide was over 95%.
[0085]
(Evaluation of precursor frequency of MBP-reactive T cells)
PBMC were plated at 200,000 cells / well (for a total of 96 wells) in the presence of MBP (40 μg / mL). After 7 days, all cultures were restimulated with MBP in the presence of irradiated autologous PBMC. After an additional week, each well was divided into four aliquots (approximately 10 ⁴ Cells) and, in the presence and absence of MBP, 10 ⁵ Were cultured in duplicate using the irradiated self-PMBC. The culture is maintained for 3 days and at 1 μCi per well during the last 16 hours of culture [ ³ [H] thymidine (Nycomed Amersham, Arlington Heights, IL). The cells are then harvested using an automatic cell harvester and [ ³ [H] thymidine incorporation was measured.
[0086]
If the counts per minute (CPM) is greater than 1000 and exceeds the reference CPM (in the absence of MBP) by at least three times, the wells / cultures are treated with MBP or MBP. Was determined to be specific. The precursor frequency of MBP-reactive T cells was then determined by the total number of PBMC seeded in the initial culture (19.2 × 10 ⁶ Cells) divided by the number of specific wells.
[0087]
(Myelin-reactive T cell clone)
Positively identified T cell lines were cloned using a limiting dilution assay in the presence of 2 μg / ml phytohemagglutinin-protein (PHA-P). Cultures were fed with fresh medium every 3-4 days. Growth positive wells were tested for specific reactivity to MBP83-99 peptide in a proliferation assay. The resulting MBP83-99 specific T cell clone was further characterized and used for T cell vaccination.
[0088]
(TCR V gene analysis and DNA sequencing)
The T cell receptor V gene rearrangement of immune MBP-reactive T cell clones was analyzed using reverse transcription PCR. The genes for the TCRa and TCRβ chains were amplified and sequenced directly as described elsewhere (Vandevyer et al., 1995; Zhang, YCQ. Et al., 1998). Briefly, total RNA was analyzed using RNeasy mini kit (QIAGEN, Santa Clarita, CA). ⁶ Cells were extracted from each MBP83-99 reactive T cell clone. First-strand cDNA reverse transcribed from total RNA was specific for the TCR Vα and Vβ gene families, whose sequences were published (Vandevyer et al., 1995; Zhang, YCQ. Et al., 1998). PCR amplification using one set of primers. The amplified PCR products were separated by electrophoresis in a 1% agarose gel and stained with ethidium bromide. The visualized PCR product was excised and then purified using a QIAquick® gel extraction kit (QIAGEN, Santa Clarita, CA) prior to sequence analysis. Purified PCR products were sequenced directly with a T7 sequencing kit (Pharmacia, Uppsala, Sweden). 1.5 μg of template was sequenced using 2 pmol of the corresponding V gene primer using the dideoxy chain termination method.
[0089]
(Immunization of MS patients with irradiated autologous MBP-reactive T cell clones)
Two patients with clinically defined MS confirmed by magnetic resonance imaging were included in this study. They were diagnosed as having relapsing-remitting MS for over two years. These patients had not taken any immunomodulatory drug for at least three months prior to the study. Immunization with irradiated autologous MBP83-99 reactive T cell clones was performed as previously described (J. Zhang et al., 1992, 1993). Briefly, MBP83-99 reactive T cell clones were activated and expanded in the presence of PHA for 7 days before injection. The T cells then ⁶⁰ Irradiated at 10,000 rad (using a Co source) and washed thoroughly with sterile saline. 4 × 10 in total from two autologous T cell clones ⁷ Cells were resuspended in 2 ml of sterile saline, which was injected subcutaneously in the arm. Each patient received a total of four injections at two-month intervals and achieved a sufficient immune response as defined by PBMC expansion against immune T cell clones. This protocol was approved by the Institute Human Subjects Committee at Baylor College of Medicine. Consent was obtained from the patient after explaining the experimental procedure. These patients were evaluated before and after each immunization for adverse event and disability score (Expanded Disability Scale Score). Gadolinium enhanced MRI scans were performed before immunization and at different time points after immunization. This clinical and radiographic evaluation is part of a separate clinical study.
[0090]
(Generation of B cell line producing antibody by EBV transformation)
This method has been described elsewhere (J. Zhang, 1989, 1991). Briefly, PBMCs were cultured in microtiter plates in the presence of cell-free supernatant of EBV producing strain B95.8 (ATCC, Bethesda, MD) and 0.5 μg / ml Cyclosporin A (Sandoz, Basel, Switzerland). (Costar, Cambridge, MA) at 20,000 cells / well to selectively inhibit T cell proliferation. Cells were cultured for 14 days, changing media every 3-4 days. On day 14, the growth positive wells were visualized and the culture supernatant was collected for testing. The precursor frequency of B cells producing a particular antibody was assessed by dividing the number of positive wells by the total number of PBMCs plated. Positive B cell lines were then transferred to 24-well plates (Costar, Cambridge, MA) for growth. This B cell line typically produced relatively pure antibodies at 2-10 μg / ml.
[0091]
(Detection of anti-TCR antibody by ELISA)
Culture supernatants were collected from individual B cell lines and they were tested for the presence of anti-idiotype antibodies using ELISA. Briefly, microtiter plates were coated overnight at 4 ° C. with this motif-positive TCR peptide or control TCR peptide at a concentration of 1 μg / ml each. The wells were then blocked with PBS containing 2% bovine serum albumin (BSA) (Sigma, St. Louis, Mo.) for 2 hours at 37 ° C. and these were added to 0.02% in 0.9% NaCl solution. Washed 4 times with% Tween® 20 (Sigma, St. Louis, MO). Each sample and its controls were added to adjacent wells and incubated for 2 hours. Plates were washed four times and incubated with goat anti-human IgG / IgM antibody conjugated to horseradish peroxidase (Sigma, St. Louis, MO) at a 1: 1500 dilution for 30 minutes. 0.0125% tetramethylbenzidine / 0.008% H in citrate buffer (pH = 5.0) ₂ O ₂ Is used as a substrate and the color is ₂ SO ₄ Stopped using. Optical density was measured using an ELISA reader (Biorad, Hercules, CA). Wells containing medium alone served as background controls.
[0092]
(Immunoblot analysis)
Lysates were prepared from a representative MBP-reactive T cell clone (MS7.E2.6) expressing the consensus CDR3 sequence using standard methods described elsewhere (Hjelmeland et al., 1984). did. Briefly, 5 × 10 ⁶ T cells were lysed in 100 μl lysis buffer containing: 150 M NaCl, 50 mM Tris (pH 7.6), 0.5% Triton X-100, 1 mM PMSF, 10 μg / ml aprotinin, 10 μg / ml leupeptin. . Cell debris was centrifuged at 13,000 g for 20 minutes at 4 ° C. The resulting lysate was electrophoresed using 10% SDS-PAGE. After blotting, the nitrocellulose membrane was cut into strips and then blocked with 5% low fat milk powder (Milk-TBST) in Tris buffered saline containing 0.1% Tween®20. did. The strip was then incubated with the undiluted supernatant in a mini-incubation tray for 1 hour at room temperature. Goat anti-human IgG and IgM (heavy and light chains) conjugated to horseradish peroxidase were used as secondary antibodies (100 ng / ml in 2% milk-TBST) and were used for 45 min with the washed strips. Incubation followed by enhanced chemiluminescence visualization of the protein on the membrane (Amersham, Piscataway, NJ). Supernatant from an EBV transformed B cell line producing non-reactive antibodies was used as a negative control. Rabbit polyclonal anti-human TCR β chain antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was included as a positive control.
[0093]
(Flow cytometry)
A representative MBP-reactive T cell clone expressing the consensus CDR3 sequence (MS7-E2.6) was incubated with anti-idiotype antibodies from individual B cell lines at 4 ° C for 30 minutes. Supernatant from an EBV-transformed B cell line producing non-reactive antibodies was used as a control. After washing by centrifugation with FACS buffer (PBS containing 5% fetal calf serum and 0.01% sodium azide) at 2300 rpm for 2 minutes at 4 ° C., the cells are resuspended and fluorescein Was stained with a goat anti-human IgG / IgM antibody (FITC) conjugated to. After two washes, the cells were resuspended in 300 μl FACS buffer and analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.). FITC-conjugated anti-IgG ₁ Was used to detect background staining (Becton Dickinson, San Jose, CA).
[0094]
(Inhibition assay)
20,000 cells of immune MBP-reactive T cell clones (motif positive and motif negative T cell clones) were irradiated in the presence and absence of 83-99 peptides of MBP (20 μg / ml). With autologous PBMC (100,000 cells / well). 50 μl of undiluted supernatant was added to each well. Cell proliferation ³ H] -thymidine incorporation assay was measured after 72 hours. Supernatant from an EBV-transformed B cell line producing non-reactive antibodies was used as a control.
[0095]
(result)
(Functional and structural characteristics of immune MBP-reactive T cell clones)
A panel of four MBP-reactive T cell clones was generated from two patients with recurrent flaccid MS. These T cell clones show a CD4 phenotype and have a DR4 molecule or DR2 (DRB1 ^* 1501) Recognized 83-99 immunodominant peptides of MBP for the molecule. These were analyzed for TCR V gene rearrangement by RT-PCR using primers specific for Vα and Vβ, and then sequenced for Vα-Jα and Vβ-Dβ-Jβ junction regions. An independent T cell clone from patient MS7 (E2.6) shared the same TCR Vα17 and Vβ13.1 genes with another T cell clone (C3.1) from a different patient (MS27). The two T cell clones had the same sequence in the Vβ13.1-Dβ-Jβ junction region (SEQ ID NO: 3), while their Vα17 chains had two distinct Vα-Jα junction region sequences. It had. As mentioned above, the identified LueGlyArgAlaGlyLeuThrTyr (SEQ ID NO: 3) sequence corresponded to a common CDR3 motif in Vβ13.1T cells recognizing the 83-99 immunodominant region of MBP in different patients with MS (23).
[0096]
(Induction of an immune response to immunization with MBP-reactive T cell clones and 20-mer TCR peptide by T cell vaccination in patients with MS)
Each patient received a total of four subcutaneous inoculations of two irradiated autologous MBP-reactive T cell clones (2 x 10 ⁷ Cells / clone) for two months. The proliferative response of PBMC to the immunized autologous T cell clone was tested at two time points, corresponding to baseline and two months from the last immunization. As shown in FIG. 8A, the proliferative response to both irradiated immunized T cell clones increased in these patients after T cell vaccination and substantially exceeded baseline values. In addition, the response to the TCR peptide incorporating the CDR3 consensus sequence (motif positive peptide, consisting of amino acids 2-21 of SEQ ID NO: 32) was determined by the response of a control CDR3 peptide (motif negative peptide, SEQ ID NO: 48) derived from a non-immunized T cell clone. (Consisting of amino acids 1 to 20). However, the intensity of specific proliferation in response to the motif positive peptide was significantly lower than induction by irradiated immunized T cells (FIG. 8A). The proliferative response to immunized T cells was inversely correlated with a decrease in the frequency of circulating MBP-responsive T cells in the immunized patient (FIG. 8B).
[0097]
(Generation of B cell lines producing specific antibodies to TCR peptides incorporating the CER3 consensus sequence from immunized patients)
We then tested whether immunization with irradiated T cells elicited a specific anti-idiotypic antibody response in patients. Since whole T cells expressed an array of surface molecules that could interfere with the detection of serum anti-idiotype antibodies, a TCR peptide (motif positive peptide) incorporating a common CDR3 sequence was used as an antigen for screening. A 20-mer TCR peptide from a non-immunized MBP-responsive T cell clone (motif negative peptide) was included in all experiments as a control. The CDR3 sequence of the control peptide was not detected in the immunized T cell clone.
[0098]
When tested with sera from two patients, non-specific antibody responsiveness to either the TCR peptide or the original immunized T cells could be detected using ELISA or flow cytometry ( Data not shown). To further confirm whether anti-idiotypic antibodies are present in vaccinated patients, we used a panel of antibody-producing B cell lines from post-vaccination Made using a technique based on cell culture combined with ultra dilution (see Materials and Methods).
[0099]
Since pre-vaccinated PBMCs could not be used in the experiments, they were analyzed under the same experimental conditions using cells from two randomly selected healthy individuals as controls. Supernatants of the resulting B cell lines (92 cell lines from each patient / individual) were tested by ELISA for the presence of antibodies to the motif positive TCR peptide and to the control TCR peptide, respectively. If the antibody exhibited a specific response to the motif positive TCR peptide but no response to the control TCR peptide, the antibody was defined as an anti-idiotype. As shown in FIG. 9, B cells producing specific anti-idiotypic antibodies were 1.75 × 10 5 each in MS7 and MS27 patients compared to two non-immune control subjects. ^-6 Occurred in progenitor cells at a frequency of. In contrast, the reactivity of the non-specific antibody to the control peptide was detected in the same supernatant. This result suggests that the high frequency of B cells producing anti-idiotype antibodies is related to T cell vaccination.
[0100]
All compositions and / or methods claimed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention are described in terms of preferred embodiments, the concepts, spirit, and scope of the present invention may be embodied in the compositions and / or the steps or sequence of steps of the methods described herein. It will be apparent to those skilled in the art that modifications may be applied without departing from More specifically, it is clear that certain factors, both chemical and physiological, can be substituted for the factors described herein, with the same or similar results being achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
[0101]
[Table 4]

[Brief description of the drawings]
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more of these drawings, in conjunction with the detailed description of the specific embodiments set forth herein.
FIG. 1 shows the experimental procedure for cloning and sequencing of PCR products from PBMC. The cDNA from the PBMC sample was amplified with the 5'Vβ13.1 and 3'Jβ primers and ligated into the TA cloning vector pCR2.1, the cDNA from four PBMC samples positive for the expression of the LGRAGLTY motif, and . E. coli. Plasmid DNA was screened by PCR using M13 primers and LGRAGLTY specific primers. Positive plasmids that showed overt amplification by PCR were sequenced using the Vβ13.1 primer and the VβDβJβ sequence using the Vβ13.1 primer.
FIG. 2 shows the reactivity pattern of two MBP83-99 T cell clones to an analog peptide with a single alanine substitution. Two pairs of MBP83s showing identical Vβ13.1 rearrangements (for MS7-E2.6 and MS27-C3.1) and similar Vα-Jα junction sequences (for MS7-E2.6 and MS7-E3.1) The -99 T cell clone was identified as [ ³ The alanine-substituted peptides were tested for reactivity in a panel of [H] -thymidine incorporation assays. DRB1 ^* A mouse fibroblast cell line expressing 1501 was used as a source of antigen presenting cells. The proliferative response of these clones to each analog peptide was measured after 72 hours and the results are shown as incorporated CPM. The boxed boxes indicate a greater than 50% reduction in the proliferation of T cell clones in response to the analog peptide.
FIG. 3 shows a cross test of the specificity of CDR3 oligonucleotides using original and unrelated T cell clones. The set of oligonucleotides specific for the TCR VDJ region was used to detect the known target DNA sequences present in the original MBP83-99 T cell clone and unrelated MBP83-99 T cell clones from the same and different individuals. Tested for specificity. PCR reactions were performed using the CDR3 specific oligonucleotide as a forward primer and the 3'-Cβ primer as a reverse primer. Filled boxes indicate positive detection of DNA sequences present in the original T cell clones or T cell clones sharing the same CDR3 sequence. All primers were also tested for binding to DNA products of randomly selected T cell clones with irrelevant CDR3 sequences (shaded boxes).
FIG. 4 shows the detection of a target DNA sequence complementary to the motif Vβ13.1-LGRAGLTY in randomly selected PBMC samples from a patient with MS. CDNA prepared from PBMC samples from randomly selected MS patients (n = 48) was first subjected to RT-PCR using 5′-Vβ13.1 specific primers and 3′-Cβ primers. Amplified. The amplified PCR product was subsequently hybridized with a digoxigenin-labeled oligonucleotide probe specific for the LGRAGLTY motif. The original MBP83-99 clone (MS7-E2.6) and an unrelated T cell clone (MS32-B9.8) were used as positive and negative controls, respectively. MS-7 and MS-27 were the original PBMC samples from which clones MS7-E2.6 (MS-7 in Table 1) and clone MS27-C3.1 (MS-27 in Table 1) were derived. The star is DRB1 ^* Shows 1501 positive expression.
FIG. 5 shows the detection of the Vβ13.1-LGRAGLTY motif in randomly selected PBMC samples from normal subjects. PBMC samples obtained from 20 normal subjects (NS) were analyzed under the same conditions as described in the description of FIG. The original clone (MS7-E2.6) and an unrelated clone (MS32-B9.8) were used as positive and negative controls, respectively. The star is DRB1 ^* Shows 1501 positive expression.
FIG. 6 shows a semi-quantitative comparison of the expression of the LGRAGLTY motif in PBMC samples from MS patients and normal subjects. The expression of the motif Vβ13.1-LGRAGLTY was analyzed by semi-quantitative PCR in comparison to Cβ expression in each cDNA from PBMCs of MS and normal individuals. Relative expression levels were calculated as (expression of LGRAGLTY motif / expression of Cβ) x 100%.
FIG. 7 shows detection of the Vβ13.1-LGRAGLTY motif in a short-term MBP83-99 T cell line from a patient with MS. A panel of independent short-term MBP83-99 T cell lines was generated from five patients with MS using the synthetic 83-99 peptide of MBP. All these T cell lines were confirmed for their specific reactivity to the MBP83-99 peptide (CPM in response to MBP83-99 / control CPM> 5). The cDNA product was amplified by PCR using 5′-Vβ13.1 specific primers and 3′-Cβ primers. The amplified PCR product was subsequently hybridized in a Southern blot analysis with a digoxigenin-labeled oligonucleotide probe corresponding to the Vβ13.1-LGRAGLTY motif. CDNA products from the original MBP83-99 clone (MS7-E6.2) and an unrelated T cell clone (MS32-B9.8) were used as positive and negative controls, respectively.
FIG. 8 shows the proliferative response of PBMC (peripheral blood mononuclear cells) to immune MBP-reactive T cell clones and TCR peptides in relation to the frequency of MBP-reactive T cells in immunized MS patients. . 8A: Proliferative response of PBMCs obtained from two immunized patients is shown as stimulation index defined as follows. Number / min (CPM) / single of PBMCs cultured with irradiated immune MBP-reactive T cell clones (motif positive T cell clones, MS7-D2.2 and MS27-D4.4) expressing the consensus CDR3 sequence Sum of CPM of cultured PBMC and CPM of irradiated T cells cultured alone. The proliferative response to the motif positive peptide (amino acids 2-21 of SEQ ID NO: 32) and the control TCR peptide (amino acids 1-20 of SEQ ID NO: 48) was determined in a proliferation assay, where PBMCs were each isolated from the TCR peptide (20 μg / Ml) at 100,000 cells / well for 5 days. All experiments were performed at two time points, corresponding to baseline (pre) and two months (post) after the fourth vaccination. 8B: Precursor frequency of T cells specific for MBP was estimated at the same time point. NS = normal subject.
FIG. 9 shows the estimated precursor frequency of B cells producing anti-idiotypic antibodies to the TCR peptide in PBMC of immunized patients. PBMC were obtained from two immunized MS patients and two non-immunized randomly selected healthy individuals. Cells were cultured in the presence of EBV producing cell line and supernatant from cyclosporin A (Sandoz, Basel, Switzerland). All growth positive wells were evaluated in an ELISA for the presence of antibodies reactive against the motif positive TCR peptide and the control TCR peptide. The precursor frequency of B cells producing anti-idiotype antibodies to the TCR peptide was estimated by dividing the number of positive wells by the total number of PBMCs initially plated.

Claims (13)

A peptide 8 to about 45 amino acids in length, comprising the sequence of SEQ ID NO: 3. The peptide according to claim 1, which is SEQ ID NO: 3. 2. The peptide according to claim 1, comprising amino acids 2 to 21 of SEQ ID NO: 32. The peptide according to claim 1, consisting of amino acids 2 to 21 of SEQ ID NO: 32. A method of treating an autoimmune disease in a human, comprising:
(A) obtaining MBP83-99 Vβ13.1 T cells from a human;
(B) obtaining a nucleic acid sample from MBP83-99 Vβ13.1 T cells;
(C) contacting the nucleic acid sequence with a primer pair selected from or derived from:
(I) a first oligonucleotide about 15-30 nucleotides in length, comprising at least 10 contiguous nucleotides of SEQ ID NO: 1 or a nucleic acid complementary thereto; and (ii) about 15 and 30 And is found in the Vβ13.1 gene Vβ to Jβ region in the T cell receptor of the T cell, without the first oligonucleotide sequence. Oligonucleotides,
Wherein the sequence of said first and said second oligonucleotide is not found on the same strand of said T cell receptor gene; and (d) detecting the presence of a nucleic acid encoding a LGRAGLTY motif; and When the nucleic acid is detected,
(E) administering a peptide of 9 to about 45 amino acids in length to a human, wherein the polypeptide comprises the sequence of SEQ ID NO: 3;
A method comprising: The method according to claim 5, wherein the Vβ13.1 gene sequence is SEQ ID NO: 2. 6. The method of claim 5, wherein said administering further comprises administering a T cell activation marker peptide. 6. The method of claim 5, wherein said peptide comprises amino acids 2-21 of SEQ ID NO: 32. 6. The method of claim 5, wherein said peptide consists of amino acids 2-21 of SEQ ID NO: 32. A pharmaceutical composition for treating an autoimmune disease, comprising a peptide from 8 to about 45 amino acids in length, wherein said peptide comprises the sequence of SEQ ID NO: 3. The pharmaceutical composition according to claim 10, wherein the peptide is SEQ ID NO: 3. The pharmaceutical composition according to claim 10, wherein the peptide comprises amino acids 2-21 of SEQ ID NO: 32. The pharmaceutical composition according to claim 10, wherein the peptide consists of amino acids 2-21 of SEQ ID NO: 32.

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