JP2004000216A - Tumor antigen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out a tumor antigen recognized by cytotoxic T-lymphocyte (CTL). <P>SOLUTION: The peptide or polypeptide recognized by CTL as restrained with at least one of HLA-A such as HLA-A31, HLA-A11 and HLA-A33, the polynucleotide containing a base sequence encoding one of them or its complementary sequence, the recombinant vector, the transformed body, the antibody against the peptide or polypeptide, the compound enhancing the recognition of the peptide or polypeptide by the CTL, the compounds accelerating the expression of the peptide or polypeptide, the CTL-inducing agent consisting of the peptide and/or polypeptide, the medicinal composition containing ≥1 kind of them, the method for inducing the CTL by using the peptide and/or polypeptide, the method for producing the peptide or polypeptide, the method for screening the compounds, the method for measuring them and the reagent kit used for the method for screening or the method for measuring them are provided. <P>COPYRIGHT: (C)2004,JPO

Description

【００２５】
これら遺伝子はＨＬＡ−Ａ３１拘束性に腫瘍特異的細胞傷害性Ｔ細胞により認識される腫瘍抗原をコードする遺伝子であり、上記のようにＨＬＡ−Ａ３１^＋細胞で発現させると、ＨＬＡ−Ａ３１拘束性に細胞傷害性Ｔ細胞を活性化してＩＦＮ−γ産生を促進した。これら遺伝子がコードするアミノ酸配列を、配列番号９から１４に記載した（表１参照）。
【００２６】
ヒト大腸癌細胞株ＳＷ６２０から得た上記遺伝子がコードするポリペプチドは、細胞傷害性Ｔ細胞を誘導するおよび／または細胞傷害性Ｔ細胞により認識される腫瘍抗原として使用できる。該ポリペプチドとして好ましくは、配列番号９から１４のいずれか１つに記載のアミノ酸配列からなるポリペプチドである。かかるポリペプチを認識する細胞傷害性Ｔ細胞としては、少なくともＨＬＡ−Ａ３１拘束性またはＨＬＡ−Ａ２拘束性の細胞傷害性Ｔ細胞が挙げられる。また、本ポリペプチドは、腫瘍抗原エピトープを特定して腫瘍抗原ペプチドを得るための材料として用いることもできる。
【００２７】
腫瘍抗原ペプチドは、上記ポリペプチドのアミノ酸配列に基づいて設計し合成したペプチドから細胞傷害性Ｔ細胞を誘導および／または活性化するものを選択することにより取得できる。細胞傷害性Ｔ細胞の誘導および／または活性化の判定方法としては、例えば、ペプチドをパルスした細胞とＯＫＡ３１−ＣＴＬ等の細胞傷害性Ｔ細胞とを共に培養して、活性化された細胞傷害性Ｔ細胞から産生されるＩＦＮ−γを測定し、これを指標として判定する方法等を例示できる。当該腫瘍抗原ペプチドは、ＨＬＡ−Ａ３１と結合して細胞表面上に提示され、かつ細胞傷害性Ｔ細胞により認識される腫瘍抗原エピトープとしての性質を有するものであればよく、少なくとも５個以上、好ましくは７個以上、さらに好ましくは９個乃至１１個のアミノ酸残基からなるペプチドである。さらに好ましくは、配列番号１から８のいずれか１つに記載のアミノ酸配列からなるペプチドである。これらペプチドは、ＨＬＡ−Ａ３１拘束性の腫瘍特異的な細胞傷害性Ｔ細胞を誘導するおよび／または細胞傷害性Ｔ細胞により認識される腫瘍抗原ペプチドとして使用できる。
【００２８】
配列番号１から８のいずれか１つに記載のアミノ酸配列からなるペプチドは、具体的には以下の方法により取得した。まず、上記ポリペプチドのアミノ酸配列に基づき、ＨＬＡ−Ａ３１結合モチーフに適合する９ｍｅｒから１１ｍｅｒのペプチドを設計し、常法により合成した。ペプチドの設計は、米国国立予防衛生研究所（ＮＩＨ）のバイオインフォーマティクス　アンド　モレキュラー　アナリシス　セクション（Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ　＆　Ｍｏｌｅｃｕｌａｒ　Ａｎａｌｙｓｉｓ　Ｓｅｃｔｉｏｎ；ＢＩＭＡＳ）が開示しているデータベース「ＨＬＡペプチド結合予測（ＨＬＡ　Ｐｅｐｔｉｄｅ　Ｂｉｎｄｉｎｇ　Ｐｒｅｄｉｃｔｉｏｎｓ」を用い（ｈｔｔｐ：／／ｂｉｍａｓ．ｄｃｒｔ．ｎｉｈ．ｇｏｖ／ｍｏｌｂｉｏ／ｈｌａ＿ｂｉｎｄ）、さらに既報（非特許文献１６−１８）を参照して行った。合成したペプチドからの、ＨＬＡ−Ａ３１拘束性に細胞傷害性Ｔ細胞により認識されるペプチドの選択は以下のように行った。各ペプチドを細胞（ＨＬＡ−Ａ３１０１を発現させたＣＯＳ−７細胞またはＯＫ−ＥＢＶ−Ｂ細胞株）にそれぞれパルスした後、ＯＫＡ３１−ＣＴＬと共に培養し、培養上清中に産生されるＩＦＮ−γを測定した。ネガティブコントロール（ＯＫＡ３１−ＣＴＬにより認識されないペプチド）と比較してＯＫＡ３１−ＣＴＬからのＩＦＮ−γ産生を促進したペプチドを、ＯＫＡ３１−ＣＴＬにより認識されるペプチドであると判定した。
【００２９】
合成したペプチドのうち８種類のペプチド（配列番号１から８）が、ＯＫＡ３１−ＣＴＬからのＩＦＮ−γ産生を用量依存的に促進した（実施例２参照。）。また、これらのペプチドは、癌患者から得た末梢血単核細胞（Ｐｅｒｉｐｈｅｒａｌ　Ｂｌｏｏｄ　Ｍｏｎｏｎｕｃｌｅａｒ　Ｃｅｌｌ；以下、ＰＢＭＣと略称することもある。）から、細胞傷害性Ｔ細胞を誘導した。健常人由来のＰＢＭＣにおいては癌患者由来のＰＢＭＣと比べて細胞傷害性Ｔ細胞が誘導される例が少なかった。誘導された細胞傷害性Ｔ細胞は、ＨＬＡ−Ａ３１^＋標的細胞、例えばヒト肺腺癌細胞株ＬＣ−１または誘導に用いたペプチドをパルスしたＨＬＡ−Ａ３１^＋細胞を認識してＩＦＮ−γを産生を促進し且つ該標的細胞を溶解したが、ＨＬＡ−Ａ３１⁻標的細胞は溶解しなかった（実施例３および４参照。）。細胞傷害性Ｔ細胞の誘導はペプチド特異的であり、誘導に用いたペプチドと、標的細胞により提示されたペプチドとが同一の場合に、誘導された細胞傷害性Ｔ細胞は標的細胞を認識してこれを傷害した（実施例４参照。）。これらから、誘導された細胞傷害性Ｔ細胞はＨＬＡ−Ａ３１拘束性かつペプチド特異的な細胞傷害性Ｔ細胞であることが判明した。かくして、ＨＬＡ−Ａ３１拘束性に腫瘍特異的細胞傷害性Ｔ細胞を活性化するおよび／または誘導する８種類の腫瘍抗原ペプチドを得た。これらペプチドは上記遺伝子において、ＨＬＡ−Ａ２拘束性腫瘍抗原ペプチドとは全く異なる領域にコードされるペプチドであった。
【００３０】
配列番号１から８に記載のペプチドは、ＨＬＡ−Ａ３１^＋癌患者由来のＰＢＭＣのみならず、ＨＬＡ表現型がＨＬＡ−Ａ１１またはＨＬＡ−Ａ３３である癌患者由来のＰＢＭＣからも、同様に各ＨＬＡ−Ａ拘束性に腫瘍細胞を溶解する細胞傷害性Ｔ細胞を誘導した（実施例５および６参照。）。ＨＬＡ−Ａ３１は、ＨＬＡ−Ａ３、ＨＬＡ−Ａ１１、ＨＬＡ−Ａ３３およびＨＬＡ−Ａ６８などの対立遺伝子が属するＨＬＡ−Ａ３スーパータイプ（ｓｕｐｅｒｔｙｐｅ）の１つである。今までに、ウイルス蛋白質あるいはメラノーマ抗原由来の同一のエピトープペプチドが、異なったＨＬＡ−Ａ３スーパータイプにより認識されることが報告されている（非特許文献１９および２０）。これらから、本ペプチドは、ＨＬＡ−Ａ３１分子のみならず、ＨＬＡ−Ａ３スーパータイプ、例えばＨＬＡ−Ａ１１分子またはＨＬＡ−Ａ３３分子などと結合し、各ＨＬＡ拘束性に細胞傷害性Ｔ細胞を誘導するおよび／または活性化することが明らかになった。
【００３１】
本明細書において、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａとは、ＨＬＡ−Ａ３、ＨＬＡ−Ａ１１、ＨＬＡ−Ａ３１、ＨＬＡ−Ａ３３およびＨＬＡ−Ａ６８など、ＨＬＡ−Ａ３スーパータイプであると報告されているＨＬＡ−Ａを意味する。好ましくはＨＬＡ−Ａ３１、ＨＬＡ−Ａ１１およびＨＬＡ−Ａ３３からなる群から選ばれる少なくとも１つのＨＬＡ−Ａであり、より好ましくはＨＬＡ−Ａ３１である。
【００３２】
本ペプチドは好ましくはＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性の腫瘍特異的な細胞傷害性Ｔ細胞を誘導するおよび／または該細胞傷害性Ｔ細胞により認識される腫瘍抗原ペプチドとして使用できる。また上記のとおり、１つのペプチドがＨＬＡ−Ａ３スーパータイプに属する複数のＨＬＡ−Ａ拘束性に腫瘍特異的な細胞傷害性Ｔ細胞を誘導および／または該細胞傷害性Ｔ細胞により認識される腫瘍抗原ペプチドとして使用できる。さらに、本ポリヌクレオチドは本ペプチドをその一部として有するため、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ表現型を示す細胞内で発現させると本ペプチド同様に該ＨＬＡ−Ａ拘束性の腫瘍特異的な細胞傷害性Ｔ細胞を誘導するおよび／または該細胞傷害性Ｔ細胞により認識されると考えられる。
【００３３】
このように特定されたポリペプチドまたはペプチドにおいて１個乃至数個のアミノ酸の欠失、置換、付加または挿入等の変異を有するものも本発明の範囲に包含される。変異を有するペプチドは天然に存在するものであってよく、また変異を導入したものであってもよい。好ましくは、かかる変異を有するポリペプチドまたはペプチドであって、細胞傷害性Ｔ細胞により、好ましくは少なくともＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性細胞傷害性Ｔ細胞により認識されるポリペプチドまたはペプチドが望ましい。変異を導入する手段は自体公知であり、例えば、部位特異的変異導入法、遺伝子相同組換え法、プライマー伸長法またはポリメラーゼ連鎖反応法（ＰＣＲ）等を単独でまたは適宜組合わせて用いることができる。例えば成書に記載の方法（非特許文献２１−２３）に準じて、あるいはそれらの方法を改変して実施することができ、ウルマーの技術（非特許文献２４）を利用することもできる。変異の導入において、当該ポリペプチドの基本的な性質（物性、機能、生理活性または免疫学的活性等）を変化させないという観点からは、例えば、同族アミノ酸（極性アミノ酸、非極性アミノ酸、疎水性アミノ酸、親水性アミノ酸、陽性荷電アミノ酸、陰性荷電アミノ酸および芳香族アミノ酸等）の間での相互置換は容易に想定される。さらに、これら利用できるペプチドは、その構成アミノ基またはカルボキシル基等を、例えばアミド化修飾する等、機能の著しい変更を伴わない程度に改変が可能である。
【００３４】
本発明に係るポリヌクレオチドは、上記ポリペプチドまたはペプチドをコードする塩基配列またはその相補的塩基配列からなるポリヌクレオチドである。好ましくは、配列番号１から８のいずれか１に記載のアミノ酸配列からなるペプチドまたは配列番号９から１４のいずれか１に記載のアミノ酸配列からなるポリペプチドをコードする塩基配列またはその相補的塩基配列からなるポリヌクレオチドである。より好ましくは、配列番号１５から１９のいずれか１に記載の塩基配列またはその相補的塩基配列からなるポリヌクレオチドである。該ポリヌクレオチドを含むポリヌクレオチドも本発明の範囲に包含される。さらに、本発明の範囲には、上記ポリペプチドのアミノ酸配列中でＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性の腫瘍抗原エピトープをコードする領域に対応する１５個以上、好ましくは２１個以上、より好ましくは３３個以上のヌクレオチドからなるポリヌクレオチドも包含される。本ポリヌクレオチドは、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ表現型を示す細胞で発現させたときに、該ＨＬＡ−Ａ拘束性の細胞傷害性Ｔ細胞を誘導および／または活性化できると考えられる。本ポリヌクレオチドにはその３′末端にポリ（Ａ）〔ｐｏｌｙ（Ａ）〕構造が存在するが、ポリ（Ａ）の数は腫瘍抗原として作用するアミノ酸のコード部位に影響するものではないため、その数は特に限定されない。かかる有用なポリヌクレオチドの選択は、例えば公知の蛋白質発現系を利用してポリペプチドまたはペプチドを発現させ、該ポリペプチドまたはペプチドの細胞傷害性Ｔ細胞誘導能および／または活性化能を測定することにより可能である。
【００３５】
さらに、上記ポリヌクレオチドにストリンジェントな条件下でハイブリダイゼーションするポリヌクレオチドも本発明の範囲に包含される。ハイブリダイゼーションの条件は、例えば成書に記載の方法（非特許文献２１）等に従うことができる。これらポリヌクレオチドは目的のポリヌクレオチドにハイブリダイゼーションするものであれば目的のポリヌクレオチドの相補的配列でなくてもよい。
【００３６】
本ポリヌクレオチドは、いずれも本ポリペプチドまたはペプチドの製造に有用な遺伝子情報を提供するものであり、あるいは核酸としての試薬や標準品としても利用できる。
【００３７】
ポリヌクレオチドを適当なベクターＤＮＡに組込むことにより、組換えベクターを取得できる。用いるベクターＤＮＡは、宿主の種類および使用目的により適宜選択される。ベクターＤＮＡは、天然に存在するものを抽出したもののほか、増殖に必要な部分以外のＤＮＡの部分が一部欠落しているものでもよい。例えば、染色体、エピソームおよびウイルス由来のベクター、例えば細菌プラスミド由来、バクテリオファージ由来、トランスポゾン由来、酵母エピソーム由来、挿入エレメント由来、酵母染色体エレメント由来、例えばバキュロウイルス、パポバウイルス、ＳＶ４０、ワクシニアウイルス、アデノウイルス、鶏痘ウイルス、仮性狂犬病ウイルスおよびレトロウイルス等のウイルス由来のベクター、並びにそれらを組合わせたベクター、例えばプラスミドおよびバクテリオファージの遺伝学的エレメント由来のベクター、例えばコスミドおよびファージミド等が挙げられる。また、目的により発現ベクターやクローニングベクター等を用いることができる。
【００３８】
組換えベクターは、目的の遺伝子配列と複製そして制御に関する情報を担持した遺伝子配列、例えばプロモーター、リボソーム結合部位、ターミネーター、シグナル配列、エンハンサー等とを構成要素とし、これらを自体公知の方法により組合わせて作製する。ベクターＤＮＡにポリヌクレオチドを組込む方法は、自体公知の方法を適用できる。例えば、適当な制限酵素を選択、処理してＤＮＡを特定部位で切断し、次いで同様に処理したベクターとして用いるＤＮＡと混合し、リガーゼによって再結合する方法が用いられる。あるいは、目的のポリヌクレオチドに適当なリンカーをライゲーションし、これを目的に適したベクターのマルチクローニングサイトへ挿入することによっても、所望の組換えベクターが得られる。
【００３９】
ポリヌクレオチドが組込まれたベクターＤＮＡを、自体公知の宿主に導入することにより形質転換体が得られる。宿主に導入するベクターＤＮＡは、１種のベクターＤＮＡであってもよく、２種以上のベクターＤＮＡを導入してもよい。宿主としては、例えば大腸菌、酵母、枯草菌、昆虫細胞または動物細胞等が例示できる。ベクターＤＮＡの宿主細胞への導入は、自体公知の手段が応用できる。例えば成書に記載されている標準的な方法（非特許文献２１）により実施できる。より好ましい方法としては、遺伝子の安定性を考慮するならば染色体内へのインテグレート法が挙げられるが、簡便には核外遺伝子を利用した自律複製系を使用できる。具体的には、リン酸カルシウムトランスフェクション、ＤＥＡＥ−デキストラン媒介トランスフェクション、マイクロインジェクション、陽イオン脂質媒介トランスフェクション、エレクトロポレーション、形質導入、スクレープ負荷（ｓｃｒａｐｅ　ｌｏａｄｉｎｇ）、バリスティック導入（ｂａｌｌｉｓｔｉｃ　ｉｎｔｒｏｄｕｃｔｉｏｎ）および感染等が挙げられる。
【００４０】
宿主に導入するベクターＤＮＡとして発現ベクターを使用すれば、該ベクターに組込んだ遺伝子を発現させることが可能である。所望の遺伝子を組込んだ発現ベクターＤＮＡを導入した形質転換体は、各宿主の培養条件に最適な条件を選択して培養される。培養は、形質転換体により発現される遺伝子産物の機能を指標にして実施できる。あるいは、宿主中または宿主外に産生された遺伝子産物量を指標にして行ってもよく、培地中の形質転換体量を指標にして行ってもよい。
【００４１】
本ポリペプチドまたはペプチドは、該ポリペプチドまたはペプチドをコードするポリヌクレオチドを組込んだ発現ベクター、または該ベクターを導入した形質転換体を用いて、遺伝子工学的技術により製造可能である。また、通常のペプチド化学において知られる方法でも製造できる。例えば成書に記載の方法（非特許文献２５および２６）が例示できるが、無論既知の方法が広く利用可能である。
【００４２】
本ポリペプチドまたはペプチドの精製および／または回収は、該ポリペプチドまたは該ペプチドの機能、例えば細胞傷害性Ｔ細胞誘導能および／または活性化能を指標にして実施できる。精製および／または回収の方法としては、硫安やアルコール等を用いた溶解度差に基づく分画手段、ゲルろ過、イオンカラムクロマトグラフィー、アフィニティクロマトグラフィー等が挙げられ、これらを単独でまたは組合わせて使用する。好ましくは、精製および／または回収しようとするポリペプチドまたはペプチドのアミノ酸配列の情報に基づき、これらに特異的なポリクローナル抗体またはモノクロ−ナル抗体を作成し、該抗体を用いて特異的に吸着回収する方法を使用する。
【００４３】
抗体は、本発明に係るポリペプチドまたはペプチドを抗原として用いて作製する。抗原は該ポリペプチドまたはペプチド、またはその断片でもよく、少なくとも８個、好ましくは少なくとも１０個、より好ましくは少なくとも１２個、さらに好ましくは１５個以上のアミノ酸で構成される。該ポリペプチドおよび／またはペプチドに特異的な抗体を作成するためには、該ポリペプチドまたはペプチドに固有なアミノ酸配列からなる領域を用いることが好ましい。この領域のアミノ酸配列は、必ずしも該ポリペプチドまたはペプチドのものと相同または同一である必要はなく、その立体構造上の外部への露出部位が好ましく、露出部位のアミノ酸配列が一次構造上で不連続であっても、該露出部位について連続的なアミノ酸配列であればよい。抗体は免疫学的に該ポリペプチドおよび／またはペプチドを結合または認識する限り特に限定されない。この結合または認識の有無は、抗原抗体結合反応を測定する公知方法によって決定できる。
【００４４】
抗体の産生には、自体公知の抗体作製法を利用できる。例えば、抗原をアジュバントの存在下または非存在下で、単独でまたは担体に結合して動物に投与し、体液性応答および／または細胞性応答等の免疫誘導を行うことにより抗体が得られる。担体はそれ自体が宿主に対して有害作用を示さずかつ抗原性を増強せしめるものであれば特に限定されず、例えばセルロース、重合アミノ酸、アルブミンおよびキーホールリンペットヘモシアニン等が例示できる。アジュバントとしては、フロイント完全アジュバント（ＦＣＡ）、フロイント不完全アジュバント（ＦＩＡ）、Ｒｉｂｉ（ＭＰＬ）、Ｒｉｂｉ（ＴＤＭ）、Ｒｉｂｉ（ＭＰＬ＋ＴＤＭ）、百日咳ワクチン（Ｂｏｒｄｅｔｅｌｌａ　ｐｅｒｔｕｓｓｉｓ　ｖａｃｃｉｎｅ）、ムラミルジペプチド（ＭＤＰ）、アルミニウムアジュバント（ＡＬＵＭ）、およびこれらの組合わせを例示できる。免疫される動物は、マウス、ラット、ウサギ、ヤギ、ウマ等が好適に用いられる。
【００４５】
ポリクローナル抗体は、免疫手段を施された動物の血清から自体公知の抗体回収法によって取得できる。好ましい抗体回収手段として免疫アフィニティクロマトグラフィー法が挙げられる。
【００４６】
モノクロ−ナル抗体は、免疫手段が施された動物から抗体産生細胞（例えば、脾臓またはリンパ節由来のリンパ球）を回収し、自体公知の永久増殖性細胞（例えば、Ｐ３−Ｘ６３−Ａｇ８細胞等のミエローマ株）への形質転換手段を導入することにより生産できる。例えば、抗体産生細胞と永久増殖性細胞とを自体公知の方法で融合させてハイブリドーマを作成してこれをクローン化し、本発明に係るポリペプチドおよび／またはペプチドを特異的に認識する抗体を産生するハイブリドーマを選別し、該ハイブリドーマの培養液から抗体を回収する。
【００４７】
かくして得られた、上記ポリペプチドまたはペプチドを認識し結合するポリクローナル抗体またはモノクローナル抗体は、該ポリペプチドまたは該ペプチドの精製用抗体、試薬、または標識マーカー等として利用できる。
【００４８】
本発明に係るポリペプチド、ペプチド、ポリヌクレオチド、組換えベクター、形質転換体または抗体は、単独でまたは複数を組合わせて用いることにより、細胞傷害性Ｔ細胞による該ポリペプチドまたは該ペプチドの認識を増強する物質のスクリーニングに有効な手段を提供する。スクリーニング方法は、自体公知の医薬品スクリーニングシステムを利用して構築可能である。
【００４９】
例えば、腫瘍抗原ペプチドをパルスした細胞を用いて細胞傷害性Ｔ細胞を刺激し、該細胞傷害性Ｔ細胞による腫瘍抗原ペプチドの認識および／または該細胞傷害性Ｔ細胞の活性化を測定する実験系を構築し、該実験系を用いて被検物質を試験することにより、本発明に係るペプチドの細胞傷害性Ｔ細胞による認識を増強する物質を選別できる。腫瘍抗原ペプチドをパルスする細胞としては、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ表現型を示す細胞、例えばＨＬＡ−Ａ３１^＋、ＨＬＡ−Ａ１１^＋および／またはＨＬＡ−Ａ３３^＋である細胞株が例示できる。あるいは、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ以外の表現型を示す細胞に、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−ＡのいずれかのｃＤＮＡを常法により遺伝子導入して細胞表面上に該ＨＬＡ−Ａ分子を発現させた細胞を用いることもできる。細胞への腫瘍抗原のパルスは、細胞と腫瘍抗原とを常法にしたがって共に培養することにより実施可能である（実施例２参照。）。該細胞傷害性Ｔ細胞としては、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性細胞傷害性Ｔ細胞株、例えばＯＫＡ３１−ＣＴＬ等を用いる。細胞傷害性Ｔ細胞と標的細胞の組合わせは、好ましくは両細胞のＨＬＡ表現型の少なくとも一方が一致することが望ましい。細胞傷害性Ｔ細胞による腫瘍抗原ペプチドの認識および／または細胞傷害性Ｔ細胞の活性化は、簡便には細胞傷害性Ｔ細胞からのＩＦＮ−γ産生量を指標にして測定できる。この実験系は同定方法の１つを説明するものであり、本発明に係る同定方法はこれに限定されない。
【００５０】
本発明は、上記スクリーニング方法によって得られた化合物も包含する。該化合物は、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性細胞傷害性Ｔ細胞による本発明に係るポリペプチドまたはペプチド、例えば配列番号１から８のいずれか１に記載のアミノ酸配列からなるペプチドまたは配列番号９から１４のいずれか１に記載のアミノ酸配列からなるポリペプチドの認識を増強する化合物であり得る。あるいは本発明に係るポリヌクレオチドの発現を増強する化合物であり得る。かくして選別された化合物は、生物学的有用性と毒性のバランスを考慮してさらに選別することにより、医薬組成物として調製可能である。
【００５１】
本発明に係るポリペプチドまたはペプチドは、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性の腫瘍特異的な細胞傷害性Ｔ細胞を誘導するおよび／または該細胞傷害性Ｔ細胞により認識されるため、腫瘍抗原または腫瘍抗原ペプチドとして使用できる。したがって、本ペプチドおよび／または本ポリペプチドを含有する細胞傷害性Ｔ細胞の誘導剤、並びに本ペプチドおよび／または本ポリペプチドを用いることを特徴とする細胞傷害性Ｔ細胞の誘導方法も本発明の範囲に包含される。本誘導剤および誘導方法により誘導される細胞傷害性Ｔ細胞としては、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性の細胞傷害性Ｔ細胞が例示できる。本ポリペプチドまたはペプチドは、細胞傷害性Ｔ細胞を誘導および／または活性化するために、単独で使用してもよいし、２つ以上を組合わせて使用してもよい。細胞傷害性Ｔ細胞は種々の抗原を認識する複数の細胞集団であることから、好ましくはこれらを２つ以上組合わせて用いることが推奨される。
【００５２】
細胞傷害性Ｔ細胞の誘導方法は、その一態様として、本発明に係るペプチドを細胞にパルスする工程、および該工程で得られた細胞を用いて細胞傷害性Ｔ細胞の前駆細胞を含む細胞集団を刺激する工程を含む。ペプチドをパルスする細胞としては、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ表現型を示す細胞、例えば該表現系を示す公知の細胞株が例示できる。あるいは、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ以外の表現型を示す細胞に、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａのいずれか１つのｃＤＮＡを常法により遺伝子導入して細胞表面上に該ＨＬＡ−Ａ分子を発現させた細胞を用いることもできる。細胞へのペプチドのパルスは、細胞と腫瘍抗原ペプチドとを常法にしたがって共に培養することにより実施可能である（実施例２参照。）。細胞傷害性Ｔ細胞の前駆細胞を含む細胞集団は、例えばヒト末梢血より調製した末梢血細胞、より好ましくは末梢血単核細胞、さらに好ましくはＨＬＡ−Ａ３スーパータイプに属するＨＬＡ表現型を示す末梢血単核細胞が挙げられる。当該細胞集団の刺激は、当該細胞集団とペプチドをパルスしたＨＬＡ−Ａ３１^＋細胞とを常法にしたがって共に培養することにより実施可能である（実施例３参照。）。細胞集団と標的細胞との組合わせは、好ましくは両細胞のＨＬＡ表現型の少なくとも一方が一致することが望ましい。
【００５３】
本発明に係るポリペプチド、ペプチド、ポリヌクレオチド、組換えベクター、形質転換体、抗体または化合物を、単独でまたは複数を組合わせて利用することによって、これらからなる群から選ばれる少なくとも１つを含有する医薬組成物を提供できる。
【００５４】
本発明においては、本遺伝子のうちクローン４０、クローン５６、クロー７６およびクローン８５について、腫瘍細胞および組織における発現を検討し、いずれも上皮癌細胞株や腺癌細胞株（食道癌、肺腺癌、大腸腺癌および胃腺癌）で正常組織と比較して高レベルで発現していることを明らかにした（実施例７）。
【００５５】
本医薬組成物は癌の治療、好ましくは本発明に係る５種類の遺伝子の１つ以上を発現している癌の治療、より好ましくは該遺伝子の１つ以上を発現しておりＨＬＡ表現型の一方または両方がＨＬＡ−Ａ３スーパータイプに属するＨＬＡである癌の治療に使用できる。より具体的には、胃癌、食道癌、大腸癌、小腸癌、十二指腸癌、肺癌、肝臓癌、胆嚢癌、膵臓癌、腎臓癌、膀胱癌、口腔癌、骨癌、皮膚癌、乳癌、子宮癌、前立腺癌、脳腫瘍、神経芽腫、メラノーマ等の固形腫瘍、あるいは血液癌（白血病または悪性リンパ腫等）等の非固形腫瘍等のいずれにも適応可能である。好ましくは前立腺癌、大腸癌、胃癌、子宮癌、乳癌、肺癌、食道癌、膀胱癌またはメラノーマなどの治療に有用であると考えられる。癌のＨＬＡ表現型は、例えば癌患者のＨＬＡクラスＩ表現型を慣用の血清学的方法（非特許文献１５）で測定することにより判定できる。癌のＨＬＡ表現型の判定はこの方法に限らず、慣用の方法のいずれも使用することができる。
【００５６】
ＨＬＡ−Ａ３スーパータイプは、世界人口の多くで発現が認められるＨＬＡ−Ａ対立遺伝子である。例えば、白人、北アメリカ黒人、日本人、中国人およびラテンアメリカ人の、それぞれ３７．５％、４２．１％、４５．８％、５２．７％および４３．１％で認められている。したがって、本発明に係る医薬組成物は、大多数の癌患者の特異的免疫療法に適応できる可能性があり、非常に有用性が高い。
【００５７】
また、本ポリペプチドは、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性細胞傷害性Ｔ細胞により認識される腫瘍抗原エピトープと、ＨＬＡ−Ａ２拘束性細胞傷害性Ｔ細胞により認識される腫瘍抗原エピトープの両方を有する。そのため、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ^＋およびＨＬＡ−Ａ２^＋の細胞に本ポリペプチドを発現させることにより、ＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性およびＨＬＡ−２拘束性の細胞傷害性Ｔ細胞を共に誘導し得ると考えられる。これらから、本ポリペプチド、該ポリペプチドコードするポリヌクレオチド、該ポリヌクレオチドを含有する組換えベクター、および該組換えベクターを含む形質転換体を含む医薬組成物は、ＨＬＡ表現型としてＨＬＡ−Ａ３スーパータイプに属するＨＬＡとＨＬＡ−２とを有する癌の治療、例えばＨＬＡ表現型がＡ３１／Ａ２、Ａ１１／Ａ２またはＡ３３／Ａ２である癌に特に有効である場合があると考えている。
【００５８】
具体的には、例えば本発明に係るポリペプチドおよびペプチドから選ばれる１つ以上からなる医薬、さらに本発明に係るポリペプチドおよびペプチドから選ばれる１つ以上を含有する医薬組成物は、いわゆる癌ワクチンとして使用できる。ここでいう癌ワクチンとは、腫瘍細胞に対する特異的免疫応答の誘導および／または増強により、腫瘍細胞を選択的に傷害する薬物を意味する。その投与量は、細胞傷害性Ｔ細胞による該ポリペプチドまたは該ペプチドの認識の程度により適宜変更を加えて決定可能であるが、一般的には活性本体として０．０１ｍｇ乃至１００ｍｇ／日／成人ヒト、好ましくは０．１ｍｇ乃至１０ｍｇ／日／成人ヒトである。これを数日乃至数月に１回投与する。投与方法は、公知の医療用ペプチドの投与方法に準じて行えばよく、好適には皮下、静脈内、あるいは筋肉内投与にて行われる。投与に際して、免疫応答の誘導および／または増強のために、本発明に係るポリペプチドおよび／またはペプチドは適当なアジュバントの存在下または非存在下に、単独でまたは担体に結合して用いることができる。担体は、それ自体が人体に対して有害な作用を及ぼさなければ特に限定されず、例えばセルロース、重合アミノ酸またはアルブミン等が例示される。アジュバントは、通常のペプチドワクチン接種に用いられるものであればよく、フロイント不完全アジュバント（ＦＩＡ）、アルミニウムアジュバント（ＡＬＵＭ）、百日咳ワクチン（Ｂｏｒｄｅｔｅｌｌａ　ｐｅｒｔｕｓｓｉｓ　ｖａｃｃｉｎｅ）および鉱物油等が例示される。剤形は自体公知のペプチドを製剤化する手段を応用して適宜選択できる。
【００５９】
または例えば、患者の末梢血より単核細胞画分を採取して、本発明に係るポリペプチドまたはペプチドを用いて刺激した後に、細胞傷害性Ｔ細胞の誘導および／または細胞傷害性Ｔ細胞の活性化が認められた該単核細胞画分、あるいは該単核細胞画分から精製した細胞傷害性Ｔ細胞を、当該患者の血液中に戻すことによっても、有効な癌ワクチン効果が得られる。ポリペプチドまたはペプチドによる刺激は、単核細胞画分をポリペプチドまたはペプチド、ポリペプチドまたはペプチド発現させた細胞、またはペプチドをパルスした細胞と共に培養することにより行うことができる。培養の条件、例えば単核細胞濃度、ポリペプチドまたはペプチドの濃度、培養時間等は、簡単な実験により決定できる。培養時に、インターロイキン−２等のリンパ球増殖能を有する物質を添加してもよい。細胞傷害性Ｔ細胞の精製は常法により行うことができるが、例えばＣＤ８^＋細胞を回収することにより実施可能である。
【００６０】
癌ワクチンとして本発明に係るポリペプチドまたはペプチドを使用する場合、１種類のポリペプチドまたはペプチドのみでも癌ワクチンとして有効であるが、複数の種類の上記ポリペプチドまたはペプチドを組合わせて使用することもできる。複数ペプチドに基づく免疫療法が有効であると報告されていること（非特許文献１２−１４）、および癌患者の細胞傷害性Ｔ細胞は複数の腫瘍抗原を認識する細胞の集団であることから、１種類のペプチドを癌ワクチンとして使用するより複数種類を組合わせて使用する方が、より高い効果が得られるときがある。またこのとき、本発明のポリペプチドおよびペプチドからなる群から選ばれるものを組合わせて使用してもよいが、これらから選ばれる少なくとも１種類のポリペプチドまたはペプチドに、公知の腫瘍抗原または腫瘍抗原ペプチドから選ばれる少なくとも１種類を組合わせて使用してもよい。組合わせて使用する公知の腫瘍抗原または腫瘍抗原ペプチドはＨＬＡ−Ａ３スーパータイプに属するＨＬＡ−Ａ拘束性のものに限定されず、他の型のＨＬＡ、例えばＨＬＡ−Ａ２やＨＬＡ−Ａ２４等と共に細胞傷害性Ｔ細胞に認識される腫瘍抗原または腫瘍抗原ペプチドであってもよい。公知の腫瘍抗原または腫瘍抗原ペプチドとしては、ｌｃｋ遺伝子およびｓｒｃ遺伝子（特許文献１）、ＳＡＲＴ−１遺伝子（非特許文献２７）、ＳＡＲＴ−３遺伝子（非特許文献２８）、並びにサイクロフィリンＢ遺伝子（非特許文献７）等に由来する腫瘍抗原または腫瘍抗原ペプチドが例示されるが、これらに限定されない。
【００６１】
本発明に係るポリヌクレオチドは、癌の遺伝子治療に利用することができる。これらポリヌクレオチドをベクターに担持させ、直接体内に導入して発現させる方法またはヒトから細胞を採取した後に体外で細胞内に導入して発現させる方法のいずれも利用可能である。ベクターとしては、レトロウイルス、アデノウイルス、ワクシニアウイルス等が知られているが、レトロウイルス系が推奨される。無論これらウイルスは複製欠陥性のものを用いる。その投与量は、細胞傷害性Ｔ細胞による該ポリペプチドまたはペプチドの認識の程度により変化するが、一般的には本発明のポリペプチドまたはペプチドをコードするＤＮＡ含量として０．１μｇ乃至１００ｍｇ／日／成人ヒト、好ましくは１μｇ乃至５０ｍｇ／日／成人ヒトである。これを数日乃至数月に１回投与する。
【００６２】
（測定方法および試薬）
本発明に係るポリペプチド、ペプチド、ポリヌクレオチド、ベクターおよび抗体は、それ自体を単独で、診断マーカーや試薬等として使用可能である。これらは試薬であるとき、緩衝液、塩、安定化剤、および／または防腐剤等の物質を含んでいてもよい。また本発明は、これらのうちの１つまたはそれ以上を充填した、１個またはそれ以上の容器を含んでなる試薬キットも提供する。なお、製剤化にあたっては、自体公知のポリペプチド、ペプチド、ポリヌクレオチド、ベクターまたは抗体それぞれに応じた製剤化手段を導入すればよい。該試薬および試薬キットは、本発明に係る同定方法、細胞傷害性Ｔ細胞の誘導方法、または該ポリペプチド、ペプチドおよびポリヌクレオチドの定量的または定性的測定に使用できる。当該測定をするための方法は、当業者に周知の方法を利用して構築できる。このような測定法には、ラジオイムノアッセイ、競合結合アッセイ、ウェスタンブロット分析および酵素免疫固相法（ＥＬＩＳＡ）等がある。また、核酸は、例えば増幅、ＰＣＲ、逆転写ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）、ＲＮアーゼ保護、ノーザンブロッティングおよびその他のハイブリダイゼーション法を用いてＲＮＡレベルでの検出および定量が可能である。
【００６３】
測定される試料としては、個体由来の細胞、血液、尿、唾液、髄液、組織生検または剖検材料等が挙げられる。また、測定される核酸は、上記各試料から自体公知の核酸調製法で得られる。核酸は、ゲノムＤＮＡを検出に直接使用してもよく、あるいは分析前にＰＣＲまたはその他の増幅法を用いて酵素的に増幅してもよい。ＲＮＡまたはｃＤＮＡを同様に用いてもよい。正常遺伝子型との比較において、増幅生成物のサイズ変化により欠失および挿入を検出できる。増幅ＤＮＡを標識した上記ポリペプチドをコードするＤＮＡにハイブリダイゼーションさせることにより点突然変異を同定できる。
【００６４】
かかる測定により本発明に係るポリペプチド、ペプチドまたはポリヌクレオチドの変異、減少または増加を検出することにより、当該ポリペプチドまたはペプチドが関連する疾患、例えば上皮性癌または腺癌等の診断が可能になる。
【００６５】
【実施例】
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
また、実施例においてヒト由来の試料を用いた実験は、試料提供者である全ての患者のインフォームドコンセントを得て行なったものである。
【００６６】
【実施例１】
（腫瘍抗原をコードするｃＤＮＡクローンの同定）
腫瘍特異的細胞傷害性Ｔ細胞として、大腸癌患者（ＨＬＡ−Ａ０２０７／３１０１、ＨＬＡ−Ｂ４６／５１、ＨＬＡ−Ｃｗ１）の腫瘍浸潤リンパ球から文献（非特許文献７および１９）に記載の方法に準拠して樹立したＯＫ−ＣＴＬのサブラインであるＯＫＡ３１−ＣＴＬを用いた。ＯＫＡ３１−ＣＴＬは、他のサブラインと比較して相対的に強くＨＬＡ−Ａ３１に反応し、ＨＬＡ−Ａ３１拘束性に腫瘍抗原を認識して活性化する。例えば、ヒト胃腺癌細胞株ＭＫＮ２８（遺伝子型はＨＬＡ−Ａ３１であるが、表現型としてはＨＬＡ−Ａ３１が検出されない細胞株）にＨＬＡ−Ａ３１を遺伝子導入により発現させたもの（以下、ＭＫＮ２８−Ａ３１と称する。）、またはヒト肺腺癌細胞株ＬＣ−１（Ａ３１０１／３３０２）などのＨＬＡ−Ａ３１^＋標的細胞を認識してＩＦＮ−γ産生を促進した。さらに、^５１Ｃｒで標識したこれら標的細胞を用いた慣用の細胞傷害性試験において、標的細胞からの^５１Ｃｒ遊離が認められたことから、ＯＫＡ３１−ＣＴＬがこれら標的細胞を傷害して溶解することが明らかになった。しかし、ＨＬＡ−Ａ３１⁻腫瘍細胞（例えばＭＫＮ２８、大腸腺癌細胞株ＳＷ６２０（Ａ０２０１／２４０２）および肺腺癌細胞株１−８７（Ａ０２０７／１１０１））、およびＰＨＡで刺激したＨＬＡ−Ａ３１^＋正常Ｔ細胞に対して細胞傷害活性を示さなかった。ＯＫＡ３１−ＣＴＬによるＭＫＮ２８−Ａ３１またはＨＬＡ−Ａ３１^＋ＬＣ−１の認識の結果認められたＩＦＮ−γ産生の促進は、抗ＨＬＡ−クラスＩ　モノクローナル抗体または抗ＣＤ８モノクローナル抗体により阻害された。これらから、ＯＫＡ３１−ＣＴＬは、ＨＬＡ−Ａ３１拘束性に腫瘍抗原を認識するＣＴＬであると考えられる。
【００６７】
本発明者らは既に、ヒト大腸癌細胞株ＳＷ６２０のｃＤＮＡライブラリーから遺伝子発現クローニング法を用いて、ＨＬＡ−Ａ２に強く反応するＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブラインを活性化する腫瘍抗原をコードする遺伝子を単離・同定した。これらの遺伝子を対象にして、ＯＫＡ３１−ＣＴＬをＨＬＡ−Ａ３１拘束性に活性化する腫瘍抗原をコードする遺伝子を同定した。
【００６８】
ＳＷ６２０細胞のｃＤＮＡライブラリーからの、ＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブラインを活性化する腫瘍抗原をコードする遺伝子の単離・同定は以下のように行った。まず、ＳＷ６２０細胞のｐｏｌｙ（Ａ）^＋ＲＮＡをｃＤＮＡに転換してＳａｌＩアダプターにライゲーションし、発現ベクターｐＣＭＶ−ＳＰＯＲＴ−２（Ｉｎｖｉｔｒｏｇｅｎ）に挿入した。また、ＨＬＡ−Ａ０２０７のｃＤＮＡを、ＲＴ−ＰＣＲによって得、真核細胞発現ベクターｐＣＲ３（Ｉｎｖｉｔｒｏｇｅｎ）　にクローン化した。
【００６９】
ＳＷ６２０細胞ｃＤＮＡライブラリーは１００クローンずつプールし、各ウエル毎にプールしたｃＤＮＡ２００ｎｇとＨＬＡ−Ａ０２０７ｃＤＮＡ２００ｎｇとを、１００μｌのｌｉｐｏｆｅｃｔｏａｍｉｎｅ（Ｉｎｖｉｔｒｏｇｅｎ）／Ｏｐｔｉ−ＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ）１：２００混液中で３０分間混合した。この混合物の５０μｌをＣＯＳ−７細胞（細胞数１×１０^４）に加え、６時間インキュベーションして共遺伝子導入した。次いで１０％ＦＣＳを含むＲＰＭＩ−１６４０培地を加えて２日間培養した後、ＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブライン（細胞数２×１０^５）を各ウエルに添加した。さらに１８時間インキュベーション後、上清を１００μｌ採り、産生されたＩＦＮ−γをＥＬＩＳＡ法で測定した。このとき、ネガティブコントロールとして遺伝子を導入していないＣＯＳ−７細胞を用い、ＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブラインによるＩＦＮ−γ産生を検討し、産生されたＩＦＮ−γの値をバックグランドとして各測定値から減算した。ＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブラインからのＩＦＮ−γ産生の促進が認められたｃＤＮＡをＣＴＬ活性化能が陽性であると判定し、この判定基準によりｃＤＮＡライブラリーのプールをスクリーニングした。
【００７０】
ＣＴＬ活性化能が認められた上記ＳＷ６２０ｃＤＮＡライブラリーのプールから個別にクローンを取り出してさらにスクリーニングを行うことにより、ＣＴＬ活性化能陽性クローンを選別した。得られたクローンのＨＬＡ−Ａ２拘束性ＯＫ−ＣＴＬサブライン活性化における用量依存性を検討し、最終的に６５個のクローンを得た。得られたｃＤＮＡクローンの塩基配列の決定は、ＤＮＡシーケンシングキット（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ）を用い、ＡＢＩ　ＰＲＩＳＭ^ＴＭ３７７ＤＮＡ　Ｓｅｑｕｅｎｃｅｒ（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ）を使用して、ダイデオキシヌクレオチドシーケンシング法で行った。さらに、ｃＤＮＡクローンがコードするアミノ酸配列を塩基配列から推定した。
【００７１】
次に、上記６５個の各ｃＤＮＡクローンを、ｐＣＲ３にクローン化したＨＬＡ−Ａ３１０１　ｃＤＮＡと共に、上記同様の方法でＣＯＳ−７細胞に共遺伝子導入した。このＣＯＳ−７細胞によるＯＫＡ３１−ＣＴＬの活性化を上記同様の方法で検討した。
【００７２】
その結果、クローン４０、クローン４５、クローン５６、クローン７６およびクローン８５の５個のｃＤＮＡクローンが、それぞれＨＬＡ−Ａ３１拘束性かつ用量依存的にＯＫＡ３１−ＣＴＬを活性化し、ＩＦＮ−γ産生を促進した。一方、発現ベクターｐＣＭＶ−ＳＰＯＲＴ−２のみをＨＬＡ−Ａ３１０１と共に共遺伝子導入したＣＯＳ−７細胞では、ＯＫＡ３１−ＣＴＬからのＩＦＮ−γ産生は促進されなかった。
【００７３】
得られた５個の各遺伝子についてＧｅｎＢａｎｋに対して相同性検索を行った。その結果、上記表１に示した遺伝子と高い相同性を有することが明らかになった。しかし、これら遺伝子がＨＬＡ−Ａ３１拘束性にＣＴＬを誘導および／または活性化する腫瘍抗原をコードするというデータは、今まで報告されていない。
【００７４】
【実施例２】
（腫瘍抗原ペプチドの同定）
腫瘍抗原をコードする上記遺伝子から腫瘍抗原ペプチドを得るために、クローン４０、クローン４５、クローン５６、クローン７６およびクローン８５がそれぞれコードするアミノ酸配列に基づいて、ＢＩＭＡＳが開示しているデータベース「ＨＬＡペプチド結合予測（ＨＬＡ　Ｐｅｐｔｉｄｅ　Ｂｉｎｄｉｎｇ　Ｐｒｅｄｉｃｔｉｏｎｓ）」（ｈｔｔｐ：／／ｂｉｍａｓ．ｄｃｒｔ．ｎｉｈ．ｇｏｖ／ｍｏｌｂｉｏ／ｈｌａ＿ｂｉｎｄ）を用い、さらに既報（非特許文献１６−１８）を参照して、それぞれ異なる複数個の９ｍｅｒから１１ｍｅｒのペプチドを設計し合成した。
【００７５】
ＨＬＡ−Ａ３１０１を常法により遺伝子導入して発現させたＣＯＳ−７細胞またはＯＫ−ＥＢＶ−Ｂ細胞に、合成したそれぞれのペプチドをパルスした後、ＯＫＡ３１−ＣＴＬと共に培養し、ＯＫＡ３１−ＣＴＬから産生されるＩＦＮ−γを定量することにより、ＨＬＡ−Ａ３１拘束性にＣＴＬを活性化するペプチドを選択した。ＯＫ−ＥＢＶ−Ｂ細胞は、大腸癌患者ＯＫの末梢血Ｂ細胞にエプスタイン・バーウイルス（Ｅｐｓｔｅｉｎ−Ｂａｒｒ　Ｖｉｒｕｓ）（ＥＢＶ）を感染させて得た無限増殖性を有する培養細胞株である。すなわち、ＯＫＡ３１−ＣＴＬとＯＫ−ＥＢＶ−Ｂ細胞は同一個体に由来する細胞株である。
【００７６】
詳しくは、ＨＬＡ−Ａ３１０１遺伝子を導入したＣＯＳ−７細胞またはＯＫ−ＥＢＶ−Ｂ細胞を上記合成された各ペプチド（１０μＭ）と共に、５％ＣＯ_２−９５％エア（Ａｉｒ）の下で３７℃にて３時間インキュベーションすることにより、細胞表面上に発現されたＨＬＡ−Ａ３１にペプチドを結合させた。このようにペプチドをパルスしたＣＯＳ−７細胞またはＯＫ−ＥＢＶ−Ｂ細胞を標的細胞（Ｔ）として用いた。また、ＯＫＡ３１−ＣＴＬをエフェクター細胞（Ｅ）として用いた。細胞数１×１０^４の標的細胞と標的細胞の５倍乃至２０倍量のエフェクター細胞とを混合し（Ｅ／Ｔ比＝５〜２０）、１８時間インキュべーションした。このとき、エフェクター細胞の誘導に用いたペプチドと標的細胞にパルスしたペプチドとが同じものになるように、エフェクター細胞と標的細胞とを組合わせた。インキュベーション後の上清１００μｌを回収してＥＬＩＳＡでＩＦＮ−γを測定した。ＯＫＡ３１−ＣＴＬに認識されないペプチドをパルスしたＣＯＳ−７細胞をネガティブコントロールとして用いて同様に測定を行った。その結果、配列番号１から８のいずれか１に記載したアミノ酸配列からなる８種類のペプチドがＯＫＡ３１−ＣＴＬにより認識され、該ＯＫＡ３１−ＣＴＬからのＩＦＮ−γ産生を促進した。得られた８種類のペプチドは、クローン４０由来の４０−Ｐ４７（配列番号１）、クローン４５由来の４５−Ｐ５４（配列番号２）、クローン５６由来の５６−Ｐ２８、５６−Ｐ３１、および５６−Ｐ３２（配列番号３から５）、クローン７６由来の７６−Ｐ２０（配列番号６）、並びにクローン８５由来の８５−Ｐ１０（配列番号７）および８５−Ｐ１１（配列番号８）である。結果は図１から図７に示した。
【００７７】
【実施例３】
（ペプチドによる癌患者末梢血単核細胞からのＣＴＬの誘導）
実施例２で得た８種類のペプチドのうち、４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）および８５−Ｐ１１（配列番号８）について、ヒトＰＢＭＣからのＣＴＬ誘導能を検討した。ＰＢＭＣは、ＨＬＡ−Ａ３１^＋癌患者１０例（前立腺癌患者４例、大腸癌患者２例、胃癌患者１例、子宮頚癌患者２例および乳癌患者１例）およびＨＬＡ−Ａ３１^＋健常人６例の各血液試料から常法に従って調製した。ＰＢＭＣのＨＬＡクラスＩ表現型の決定は慣用の血清学的方法により行った（非特許文献１５）。
【００７８】
各ＰＢＭＣ（細胞数１×１０^５）を、培養培地〔４５％ＲＰＭＩ−１６４０培地、４５％ＡＩＭ−Ｖ培地（Ｉｎｖｉｔｒｏｇｅｎ社）、１００Ｕ／ｍｌのＩＬ−２、０．１ｍＭのＭＥＭノンエッセンシャルアミノ酸溶液（Ｉｎｖｉｔｒｏｇｅｎ社）および１０％牛胎児血清（ＦＣＳ）からなる。〕２００μｌを加えた９６ウエルＵ底型マイクロカルチャープレート（Ｎｕｎｃ）の各ウエル中で、ペプチド各１０μＭとインキュベーションした。培養３日目および６日目に半量の培地を除き、対応する各ペプチドを含む上記組成の培地と交換した。培養９日目に細胞を回収し、ＩＬ−２のみを加えてペプチド非存在下でさらに培養し、培養１２日目に細胞を回収して洗浄しエフェクター細胞（Ｅ）として用いた。エフェクター細胞は、種々の標的細胞（Ｔ）とＥ／Ｔ比１０で混合し、５％ＣＯ_２−９５％Ａｉｒの条件下で３７℃にて１８時間インキュベーションした。インキュベーション後の上清１００μｌを回収して、産生されたＩＦＮ−γ量をＥＬＩＳＡにより測定した。標的細胞として、ＨＬＡ−Ａ３１^＋ヒト肺腺癌細胞株ＬＣ−１（Ａ３１０１／３３０２）、ＨＬＡ−Ａ３１⁻腫瘍細胞（例えば大腸腺癌細胞株ＳＷ６２０（Ａ０２０１／２４０２）、肺腺癌細胞株１−８７（Ａ０２０７／１１０１）、およびＭＫＮ２８細胞）、ＭＫＮ２８−Ａ３１細胞、または培養に用いたペプチドと同じペプチドをパルスした細胞を用いた。ペプチドをパルスする細胞としては、ＨＬＡ−Ａ３１　ｃＤＮＡを導入したＣ１Ｒ細胞（以下、Ｃ１Ｒ−Ａ３１と略称する。）を用いた。また、標的細胞としてペプチドをパルスしたＣ１Ｒ−Ａ３１を用いる場合は、ネガティブコントロールとしてＨＬＡ−Ａ３１拘束性ＣＴＬを誘導しないヒト免疫不全ウイルス由来ペプチド（ＨＩＶペプチド：ＲＬＲＦＬＬＬＩＶＴＲ、配列番号２０）を用い、ＨＩＶペプチドをパルスした細胞を認識したＣＴＬのＩＦＮ−γ産生量をバックグランドとして各測定値から減算した。Ｃ１Ｒ−Ａ３１以外の標的細胞を用いる場合は、エフェクター細胞のみをインキュベーションした時のＩＦＮ−γ産生量をバックグランドとして各測定値から減算した。算出された各測定値（ＮＥＴ）はスチューデントｔ検定で統計処理し、Ｐ値が０．０５以下であるものを有意差があると見なした。
【００７９】
ペプチドでパルスしたＣ１Ｒ−Ａ３１細胞を標的細胞として用いた結果の代表的な例を表２に示した。４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）または８５−Ｐ１１（配列番号８）と共にそれぞれ培養した１０例の癌患者由来のＰＢＭＣのうち、それぞれ５例（５０％）、３例（３０％）、４例（４０％）、４例（４０％）、５例（５０％）、５例（５０％）、および７例（７０％）のＰＢＭＣがＩＦＮ−γ産生を促進した。すなわち、これらペプチドが標的細胞に対する反応における癌患者由来ＰＢＭＣからのＩＦＮ−γ産生を促進したと考えられる。一方、６例の健常人由来のＰＢＭＣからのＩＦＮ−γ産生については、５６−Ｐ３１（配列番号４）が２例、４０−Ｐ４７（配列番号１）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）および８５−Ｐ１０（配列番号７）がそれぞれ１例で促進したが、５６−Ｐ２８（配列番号３）および８５−Ｐ１１（配列番号８）は促進しなかった。
【００８０】
【表２】

【００８１】
また、各ペプチドで培養したＰＢＭＣは、標的細胞としてＨＬＡ−Ａ３１^＋腫瘍細胞ＬＣ−１を用いたときにもＩＦＮ−γ産生を促進した。一方、ＨＬＡ−Ａ３１⁻腫瘍細胞（ＳＷ６２０、１−８７、ＭＫＮ−２８）を標的細胞として用いたときにはＩＦＮ−γ産生を促進しなかった。しかし、ＨＬＡ−Ａ３１　ｃＤＮＡを導入して発現させたＭＫＮ２８細胞に対しては反応し、ＩＦＮ−γ産生を促進した。代表的な結果を患者例４（前立腺癌）、患者例５（大腸癌）および患者例８（子宮頚癌）について図８に示した。標的細胞に対する反応における癌患者ＰＢＭＣからのＩＦＮ−γ産生は、抗ＨＬＡ−クラスＩ抗体または抗ＣＤ８抗体により阻害されたが、抗ＨＬＡ−クラスＩＩ抗体、抗ＣＤ４抗体、抗ＣＤ１４抗体または抗ＨＬＡ−Ａ２４抗体によっては阻害されなかった。すなわち、各ペプチドで培養したＰＢＭＣ中のＩＦＮ−γ産生細胞は、ＣＤ８^＋の表現型を有するＨＬＡ−クラスＩ拘束性のＣＴＬであると考えられる。
【００８２】
また、各ペプチドで培養したＰＢＭＣは、培養に用いたペプチドを同じペプチドをコードするｃＤＮＡとＨＬＡ−Ａ３１　ｃＤＮＡとを共遺伝子導入したＣＯＳ−７細胞と共に培養したときに、ＩＦＮ−γ産生を促進した。
【００８３】
これらから、４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）および８５−Ｐ１１（配列番号８）が、癌患者ＰＢＭＣから、ＨＬＡ−Ａ３１拘束性ＣＴＬを誘導することが判明した。
【００８４】
【実施例４】
（ペプチドにより誘導されたＣＴＬの細胞傷害活性）
実施例３においてペプチドで１２日間培養したＰＢＭＣをさらにＩＬ−２の存在下で１０日間以上培養後にエフェクター細胞として用い、各種標的細胞に対する細胞傷害活性を標準的な６時間の^５１Ｃｒ遊離試験（非特許文献１５）で測定した。すなわち、エフェクター細胞と^５１Ｃｒ標識した各標的細胞とを、Ｅ／Ｔ比３、１０または３０で混合して６時間インキュベーションした後に、上清中に遊離された^５１Ｃｒの放射活性を測定した。得られた結果は、標的細胞を完全に溶解したときの放射活性を１００％として算出した％溶解（％ｌｙｓｉｓ）で表した。標的細胞として、ＨＬＡ−Ａ３１^＋腫瘍細胞ＬＣ−１、ＨＬＡ−Ａ３１⁻腫瘍細胞ＳＷ６２０または１−８７、ＨＬＡ−Ａ３１^＋正常細胞であるＰＨＡ芽球化Ｔ細胞（ＰＨＡ−ｂｌａｓｔｏｉｄ　Ｔ　ｃｅｌｌ）、エプスタインバーウイルスで形質転換したＢ細胞株（ＥＢＶ−Ｂ）およびＣ１Ｒ−Ａ３１を用いた。
【００８５】
各ペプチドで培養したＰＢＭＣは、ＨＬＡ−Ａ３１^＋腫瘍細胞を特異的に溶解した。代表的な結果を図９に示した。このことから、４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）および８５−Ｐ１１（配列番号８）は癌患者のＰＢＭＣからＨＬＡ−Ａ３１拘束性の腫瘍特異的ＣＴＬを誘導することが明らかになった。
【００８６】
さらにペプチドによるＰＢＭＣからのＣＴＬ誘導がペプチド特異的であることを実証するために、コールドターゲットインヒビションアッセイを行った。すなわち、エフェクター細胞と^５１Ｃｒ標識したＭＫＮ２８−Ａ３１細胞（ｈｏｔということもある。）とを用いて行う^５１Ｃｒ遊離試験において、エフェクター細胞を得るために用いたペプチドと同じペプチドでパルスしたＣ１Ｒ−Ａ３１細胞（ｃｏｌｄということもある。）を、ｃｏｌｄ／ｈｏｔ比１０になるように添加し、６時間インキュベーション後に上清中に遊離した^５１Ｃｒの放射活性を測定した。ネガティブコントロールとして、ＨＩＶペプチド（配列番号２０）でパルスしたＣ１Ｒ−Ａ３１細胞を用いた。図１０に代表的な例として、５６−Ｐ２８（配列番号３）で培養した患者例５（大腸癌）由来のＰＢＭＣをエフェクター細胞として用いた結果を示す。エフェクター細胞は、ＭＫＮ２８−Ａ３１細胞に対して細胞傷害活性を示し、ＭＫＮ２８−Ａ３１細胞を溶解した。しかし、標的細胞としてＭＫＮ２８−Ａ３１細胞にｃｏｌｄを添加したものを用いたとき、エフェクター細胞のＭＫＮ２８−Ａ３１細胞に対する細胞傷害活性は有意に低下した。一方、ネガティブコントロールを加えても、エフェクター細胞の細胞傷害活性に影響は見られなかった。このことから、ペプチドによりＰＢＭＣから誘導されたＣＴＬは該ペプチドに特異的であることが判明した。
【００８７】
【実施例５】
（ペプチドによる癌患者末梢血単核細胞からのＣＴＬの誘導）
ペプチド４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）および８５−Ｐ１１（配列番号８）について、ヒトＰＢＭＣからのＣＴＬ誘導能を検討した。ＰＢＭＣは、ＨＬＡ−Ａ３３^＋癌患者６例（肺癌患者２例、前立腺癌患者３例および大腸癌患者１例）およびＨＬＡ−Ａ１１^＋癌患者９例（肺癌患者２例、前立腺癌患者５例、膀胱癌患者１例およびメラノーマ患者１例）の各血液試料から常法に従って調製した。
【００８８】
各ＰＢＭＣ（細胞数１×１０^５）はペプチド各１０μＭと共に実施例３に記載の方法で培養し、エフェクター細胞として用いた。標的細胞として、各ペプチドでパルスしたＣ１Ｒ−Ａ３３細胞またはＣ１Ｒ−Ａ１１細胞を用いた。Ｃ１Ｒ−Ａ３３細胞およびＣ１Ｒ−Ａ１１細胞はそれぞれ、Ｃ１Ｒ細胞にＨＬＡ−３３　ｃＤＮＡおよびＨＬＡ−Ａ１１　ｃＤＮＡを常法により遺伝子導入して作作成した。エフェクター細胞と標的細胞とは、培養またはパルスに用いたペプチドが一致するように組合わせて、Ｅ／Ｔ比１０で混合し、５％ＣＯ_２−９５％Ａｉｒの条件下で３７℃にて１８時間インキュベーションした。インキュベーション後の上清１００μｌを回収して、産生されたＩＦＮ−γ量をＥＬＩＳＡにより測定した。また、ネガティブコントロールとしてＨＬＡ−Ａ３１拘束性ＣＴＬを誘導しないＨＩＶペプチド（配列番号２０）を用い、ＨＩＶペプチドをパルスした細胞を認識したＣＴＬのＩＦＮ−γ産生量をバックグランドとして各測定値から減算した。ポジティブコントロールとしてはインフルエンザウイルス由来ペプチド（Ｆｌｕペプチド：ＮＶＫＮＬＹＥＫＶＫ、配列番号２１）およびエプスタインバーウイルス由来ペプチド（ＥＢＶペプチド：ＩＶＴＤＦＳＶＩＫ、配列番号２２）を用いた。算出された各測定値（ＮＥＴ）はスチューデントｔ検定で統計処理し、Ｐ値が０．０５以下であるものを有意差があると見なした。ＨＬＡ−Ａ３３^＋癌患者由来のＰＢＭＣおよびＨＬＡ−Ａ１１^＋癌患者由来のＰＢＭＣをエフェクター細胞として用いた結果をそれぞれ表３および４に示した。これらの表中で、ＩＦＮ−γ産生の促進が有意であると認められたものを下線で示した。
【００８９】
５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）または８５−Ｐ１１（配列番号８）と共にそれぞれ培養した６例のＨＬＡ−Ａ３３^＋癌患者由来のＰＢＭＣのうち、それぞれ２例（３３％）、１例（１７％）、１例（１７％）、１例（１７％）、４例（６７％）、および４例（６７％）のＰＢＭＣがＩＦＮ−γ産生を促進した（表３）。すなわち、これらペプチドが、ペプチドでパルスしたＣ１Ｒ−Ａ３３細胞に対する反応におけるＨＬＡ−Ａ３３^＋癌患者由来ＰＢＭＣからのＩＦＮ−γ産生を促進したと考えられる。
【００９０】
また、４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、または８５−Ｐ１０（配列番号７）と共にそれぞれ培養した９例のＨＬＡ−Ａ１１^＋癌患者由来のＰＢＭＣのうち、それぞれ２例（２２％）、４例（４４％）、２例（２２％）、１例（１１％）、２例（２２％）、および４例（４４％）のＰＢＭＣがＩＦＮ−γ産生を促進した（表４）。すなわち、これらペプチドが、ペプチドでパルスしたＣ１Ｒ−Ａ１１細胞に対する反応におけるＨＬＡ−Ａ１１^＋癌患者由来ＰＢＭＣからのＩＦＮ−γ産生を促進したと考えられる。
【００９１】
【表３】ＨＬＡ−Ａ３３^＋癌患者由来のＰＢＭＣからのＣＴＬ誘導

【００９２】
【表４】ＨＬＡ−Ａ１１^＋癌患者由来のＰＢＭＣからのＣＴＬ誘導

表３および４において、ＮＴとは試験しなかったこと意味する。
【００９３】
８５−Ｐ１１（配列番号８）で培養したＨＬＡ−Ａ３３^＋癌患者由来ＰＢＭＣの、該ペプチドをパルスしたＣ１Ｒ−Ａ３３細胞に対する反応におけるＩＦＮ−γ産生の促進は、抗クラスＩ抗体または抗クラスＣＤ８抗体により阻害された（図１１Ａ）。また、８５−Ｐ１０（配列番号７）で培養したＨＬＡ−Ａ１１^＋癌患者由来ＰＢＭＣの、該ペプチドをパルスしたＣ１Ｒ−Ａ１１細胞に対する反応におけるＩＦＮ−γ産生の促進も、抗クラスＩ抗体または抗クラスＣＤ８抗体により阻害された（図１１Ｂ）。これらから、上記ペプチドによりＩＦＮ−γ産生が促進された細胞は、クラスＩ抗原およびＣＤ８の表現型を示す細胞、すなわちＣＴＬであると考えられる。
【００９４】
このように、本ペプチドは、ＨＬＡ−Ａ３１^＋癌患者のみならず、ＨＬＡ−Ａ１１^＋癌患者またはＨＬＡ−Ａ３３^＋癌患者のＰＢＭＣから各ＨＬＡ−Ａ拘束性のＣＴＬを誘導することが明らかになった。
【００９５】
【実施例６】
（ペプチドにより誘導されたＣＴＬの細胞傷害活性）
実施例５においてペプチドで１２日間培養したＰＢＭＣをさらにＩＬ−２の存在下で１０日間以上培養後にエフェクター細胞として用い、各種標的細胞に対する細胞傷害活性を標準的な６時間の^５１Ｃｒ遊離試験（非特許文献１５）で測定した。標的細胞として、肺腺癌細胞株ＬＣ−１（Ａ３１０１／３３０２）、子宮頚癌細胞株ＲＫＮ細胞（Ａ３３０２／）、肺腺癌細胞株１１−１８（Ａ０２０１／Ａ２４０２）、肺腺癌細胞株１−８７（Ａ０２０７／１１０１）、肺扁平上皮癌細胞株ＱＧ５６（Ａ２６０１／）、食道癌細胞株ＫＥ５（Ａ１１０１／）、大腸癌細胞株ＳＷ６２０（Ａ０２０１／２４０２）、ＨＬＡ−Ａ３３^＋またはＨＬＡ−Ａ１１^＋正常細胞であるＰＨＡ芽球化Ｔ細胞を用いた。
【００９６】
各ペプチドで培養したＨＬＡ−Ａ３３^＋癌患者由来ＰＢＭＣは、ＨＬＡ−Ａ３３^＋腫瘍細胞を特異的に溶解した。代表的な結果を図１２に示した。このことから、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）または８５−Ｐ１１（配列番号８）はＨＬＡ−Ａ３３^＋癌患者のＰＢＭＣから腫瘍特異的ＣＴＬを誘導することが明らかになった。
【００９７】
また、各ペプチドで培養したＨＬＡ−Ａ１１^＋癌患者由来ＰＢＭＣは、ＨＬＡ−Ａ１１^＋腫瘍細胞を特異的に溶解した。代表的な結果を図１３に示した。このことから、４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、または８５−Ｐ１０（配列番号７）はＨＬＡ−Ａ１１^＋癌患者のＰＢＭＣから腫瘍特異的ＣＴＬを誘導することが明らかになった。
【００９８】
さらに実施例４と同様の方法により、コールドターゲットインヒビションアッセイを行った。エフェクター細胞として、５６−Ｐ３１（配列番号４）と共に培養した肺癌患者由来ＰＢＭＣ（ＨＬＡ−Ａ１１／）または８５−Ｐ１０（配列番号７）と共に培養した肺癌患者由来ＰＢＭＣ（ＨＬＡ−Ａ１１／）を用いた。ポジティブコントロールとして、Ｆｌｕペプチド（配列番号２１）と共に培養したこれらＰＢＭＣを用いた。ｈｏｔとして、^５１Ｃｒ標識した肺扁平上皮癌細胞株ＱＧ５６（Ａ２６０１／）または食道癌細胞株ＫＥ５（Ａ１１０１／）を用いた。ｃｏｌｄとして、５６−Ｐ３１（配列番号４）、８５−Ｐ１０（配列番号７）またはＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ１１細胞を用いた。
【００９９】
図１４Ａに示したように、５６−Ｐ３１（配列番号４）と共に培養した肺癌患者由来ＰＢＭＣ（ＨＬＡ−Ａ１１／）は、ＫＥ５細胞に対してＱＧ５６細胞と比較して強い細胞傷害作用を示した。このことから、該ＰＢＭＣはＨＬＡ−Ａ１１拘束性に細胞傷害活性を示すことが明らかになった。標的細胞としてＫＥ５細胞にｃｏｌｄ（５６−Ｐ３１（配列番号４）をパルスしたＣ１Ｒ−Ａ１１細胞）を添加したものを用いたとき、ＫＥ５細胞に対する該ＰＢＭＣの細胞傷害活性は有意に低下した。一方、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ１１細胞を加えても、ＫＥ５細胞に対する該ＰＢＭＣの細胞傷害活性に影響は見られなかった。また、図１４Ｂに示したように、８５−Ｐ１０（配列番号７）と共に培養した肺癌患者由来ＰＢＭＣ（ＨＬＡ−Ａ１１／）のＫＥ５細胞に対する細胞傷害活性も同様に、ｃｏｌｄ（８５−Ｐ１０（配列番号７）をパルスしたＣ１Ｒ−Ａ１１細胞）の添加により有意に低下した。
【０１００】
これらから、これらペプチドによりＰＢＭＣから誘導されたＨＬＡ−Ａ１１拘束性ＣＴＬはペプチド特異的であることが判明した。また、該ＣＴＬは、ＨＬＡ−Ａ１１と該ペプチドとの複合体を認識することが明らかになった。
【実施例７】
（組織および各種癌細胞における各クローンの発現）
実施例１で得られたクローン４０、クローン４５、クローン５６、クローン７６およびクローン８５のうち、クローン４０、クローン５６、クローン７６およびクローン８５について、組織および各種癌細胞におけるｍＲＮＡの発現をノザンブロッティング分析により検討した。ノザンブロッティング分析は、各種腫瘍細胞（肺腺癌細胞株１−８７、胃腺癌細胞株ＭＫＮ−２８、肺腺癌細胞株ＬＣ−１、大腸癌細胞株ＳＷ６２０および食道癌細胞株ＫＥ４）または健常人由来のＰＢＭＣから抽出した総ＲＮＡ、あるいは各組織由来の総ＲＮＡを用いて、既報（非特許文献２９）に記載の方法に準じて行った。プローブｃＤＮＡは、^３２Ｐ標識した以下のものを用いた；ＥｃｏＲＩで消化したクローン４０の第４２０〜１，３１４番目の０．９ｋｂ断片ｃＤＮＡ；ＸｂａＩで消化したクローン５６の第３３４〜１，５９８番目の１．３ｋｂｐ断片ｃＤＮＡ；ＢａｒＩおよびＮｃｏＩで消化したクローン７８の第４９４〜１，１６９番目の０．７ｋｂｐ断片ｃＤＮＡ；ＨｉｎｄＩＩＩで消化したクローン８５の第８５８〜１，７９８番目の０．９ｋｂｐ断片ｃＤＮＡ。また、β−アクチンｍＲＮＡを発現程度のコントロールとして同様に検出した。β−アクチンｍＲＮＡ検出用プローブとして、ヒトβ−アクチンｃＤＮＡ（Ｃｌｏｎｅｔｅｃｈ）を用いた。得られた結果を基に、下記式１により相対的発現レベルを算出し、表５に示した。
【０１０１】
【式１】
インデックス＝（試料の各クローン濃度／試料のβ−アクチン濃度）×（ＳＷ６２０細胞のβ−アクチン濃度／ＳＷ６２０細胞の各クローン濃度）
【０１０２】
【表５】

【０１０３】
クローン４０、クローン５６、クローン７６およびクローン８５はいずれも、検討した全ての腫瘍細胞で高いレベルで発現していた。クローン４０、クローン５６、およびクローン７６は精巣においても発現していたが、そのレベルは腫瘍細胞と同等か、より低かった。一方、精巣以外の正常組織における発現レベルは低かった。クローン８５は、正常組織においても発現レベルが高く、精巣において強く発現していたが、それ以外の正常組織にける発現レベルは腫瘍細胞における発現レベルより低かった。
【０１０４】
【発明の効果】
ＨＬＡ−Ａ３１拘束性腫瘍特異的細胞傷害性Ｔ細胞に認識される腫瘍抗原をコードする遺伝子を、ヒト大腸癌細胞株ＳＷ６２０から単離・同定した。さらに得られた遺伝子にコードされるポリペプチドに基づいて、該腫瘍抗原のエピトープを有するペプチドを見出した。
本ペプチドは、ＨＬＡ−Ａ３１^＋癌患者、ＨＬＡ−Ａ１１^＋癌患者またはＨＬＡ−Ａ３３^＋癌患者の血液より調製した末梢血単核細胞から、該ペプチドおよびポリペプチドを認識して腫瘍細胞を傷害する腫瘍特異的細胞傷害性Ｔ細胞を誘導した。ＨＬＡ−Ａ３１対立遺伝子、ＨＬＡ−Ａ１１対立遺伝子またはＨＬＡ−Ａ３３対立遺伝子はＨＬＡ−Ａ３スーパータイプに属するＨＬＡであり、世界人口の多くで発現が認められるＨＬＡ−Ａ対立遺伝子である。このように本ペプチドは、１つのペプチドであってもＨＬＡ−Ａ３スーパータイプに属する１つまたは複数のＨＬＡ−Ａ拘束性に腫瘍特異的な細胞傷害性Ｔ細胞を誘導し得るという特徴を有する。したがって、本発明は、大多数の癌患者の特異的免疫療法に適応できる可能性があり、非常に有用性が高い。
また、本ポリペプチドまたはペプチドは、既に報告されている腫瘍抗原ペプチドと組合わせて使用することにより、複数種類のＨＬＡ拘束性細胞傷害性Ｔ細胞の誘導および／または活性化が可能であるため、本発明により癌の多様性に対応することが可能になる。
このように本発明により、癌等の、例えば前立腺癌、大腸癌、胃癌、子宮頚癌、乳癌、肺腺癌、食道癌、膀胱癌またはメラノーマ等の特異的免疫療法が可能になり、癌治療において多大な貢献を期待できる。特に、ＨＬＡ表現型の一方または両方がＨＬＡ−Ａ３スーパータイプに属するＨＬＡである癌患者の特異的免疫療法の開発に有用である。また、本発明は、細胞傷害性Ｔ細胞による癌の認識に関する分子の基礎的研究にも多大に寄与するものである。
【０１０５】
【配列表フリーテキスト】
配列番号１：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号２：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号３：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号４：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号５：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号６：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号７：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号８：ＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞に認識される設計されたペプチド。
配列番号２０：ＨＩＶ蛋白質から設計したペプチド
配列番号２１：インフルエンザウイルス蛋白質から設計したペプチド
配列番号２２：ＥＢＶ蛋白質から設計したペプチド
【０１０６】
【配列表】

【図面の簡単な説明】
【図１】ペプチド４０−Ｐ４７（配列番号１）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図２】ペプチド４５−Ｐ５４（配列番号２）および４０−Ｐ４７（配列番号１）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図３】ペプチド５６−Ｐ２８（配列番号３）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図４】ペプチド５６−Ｐ２８（配列番号３）および５６−Ｐ３１（配列番号４）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図５】ペプチド５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）および７６−Ｐ２０（配列番号６）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図６】ペプチド８５−Ｐ１０（配列番号７）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図７】ペプチド８５−Ｐ１１（配列番号８）がＨＬＡ−Ａ３１拘束性細胞傷害性Ｔ細胞（ＯＫＡ３１−ＣＴＬ）を活性化してＩＦＮ−γ産生を促進したことを示す図である。図中、ＮＣはネガティブコントロールを意味する。
【図８】図８Ａは、ペプチド８５−Ｐ１１（配列番号８）または５６−Ｐ２８（配列番号３）で培養したＨＬＡ−Ａ３１^＋癌患者由来の末梢血単核細胞（ＰＢＭＣ）が、ＨＬＡ−Ａ３１^＋肺腺癌細胞株に対する反応におけるＩＦＮ−γ産生を促進したが、ＨＬＡ−Ａ３１⁻腫瘍細胞（１−８７およびＳＷ６２０）に対する反応におけるＩＦＮ−γ産生は促進しなかったことを示す図である。図８Ｂは、ペプチド８５−Ｐ１１（配列番号８）で培養したＨＬＡ−Ａ３１^＋癌患者由来ＰＢＭＣが、ＨＬＡ−Ａ３１⁻腫瘍細胞ＭＫＮ２８に対する反応におけるＩＦＮ−γ産生を促進しないが、ＨＬＡ−Ａ３１　ｃＤＮＡを導入したＭＫＮ２８（ＭＫＮ２８−Ａ３１）に対する反応におけるＩＦＮ−γ産生は促進したことを示す図である。図８Ｃは、ペプチド５６−Ｐ２８（配列番号３）で培養したＨＬＡ−Ａ３１^＋癌患者由来ＰＢＭＣが、当該ペプチドをパルスしたＣ１Ｒ−Ａ３１細胞に対する反応におけるＩＦＮ−γ産生を促進したが、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ３１細胞に対する反応におけるＩＦＮ−γ産生は促進しなかったことを示す図である。図８ＡからＣにおいて、ペプチドにより促進されたＩＦＮ−γ産生は、抗ＨＬＡ−クラスＩ抗体または抗ＣＤ８抗体により阻害された。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図９】ペプチド４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、５６−Ｐ３２（配列番号５）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）または８５−Ｐ１１（配列番号８）で培養したＨＬＡ−Ａ３１^＋癌患者由来の末梢血単核細胞が、ＨＬＡ−Ａ３１^＋肺腺癌細胞株ＬＣ−１を溶解したが、ＨＬＡ−Ａ３１⁻肺腺癌細胞株１−８７およびＨＬＡ−Ａ３１^＋ＰＨＡ芽球化正常Ｔ細胞は溶解しなかったことを示す図である。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図１０】コールドターゲットインヒビションアッセイにおいて、ペプチド５６−Ｐ２８（配列番号３）で培養したＨＬＡ−Ａ３１^＋癌患者由来の末梢血単核細胞のＭＫＮ２８−Ａ３１細胞に対する溶解作用が、当該ペプチドをパルスしたＣ１Ｒ−Ａ３１細胞の添加により阻害されたが、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ３１では阻害されなかったことを示す図である。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図１１】図１１Ａは、ペプチド８５−Ｐ１１（配列番号８）で培養したＨＬＡ−Ａ３３^＋癌患者由来の末梢血単核細胞（ＰＢＭＣ）が、該ペプチドをパルスしたＣ１Ｒ−Ａ３１に対する反応におけるＩＦＮ−γ産生を促進したことを示す図である。一方、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ３１は認識せずＩＦＮ−γ産生を促進を促進しなかった。また、ＩＦＮ−γ産生の促進は、抗ＨＬＡ−クラスＩ抗体または抗ＣＤ８抗体により阻害された。図１１Ｂは、ペプチド８５−Ｐ１０（配列番号７）で培養したＨＬＡ−Ａ１１^＋癌患者由来の末梢血単核細胞（ＰＢＭＣ）が、該ペプチドをパルスしたＣ１Ｒ−Ａ１１に対する反応におけるＩＦＮ−γ産生を促進したことを示す図である。一方、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ１１は認識せずＩＦＮ−γ産生を促進を促進しなかった。また、ＩＦＮ−γ産生の促進は、抗ＨＬＡ−クラスＩ抗体または抗ＣＤ８抗体により阻害された。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図１２】ペプチド５６−Ｐ２８（配列番号３）、５６−Ｐ３１（配列番号４）、７６−Ｐ２０（配列番号６）、８５−Ｐ１０（配列番号７）または８５−Ｐ１１（配列番号８）で培養したＨＬＡ−Ａ３３^＋癌患者由来の末梢血単核細胞が、ＨＬＡ−Ａ３３^＋腫瘍細胞株（ＬＣ−１およびＲＫＮ）を溶解したが、ＨＬＡ−Ａ３１⁻腫瘍細胞株（１１−８７および１−８７）およびＨＬＡ−Ａ３３^＋ＰＨＡ芽球化正常Ｔ細胞（ＰＨＡ−ｂｌａｓｔ）は溶解しなかったことを示す図である。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図１３】ペプチド４０−Ｐ４７（配列番号１）、５６−Ｐ２８（配列番号３）、７６−Ｐ２０（配列番号６）または８５−Ｐ１０（配列番号７）で培養したＨＬＡ−Ａ１１^＋癌患者由来の末梢血単核細胞が、ＨＬＡ−Ａ１１^＋腫瘍細胞株（１−８７およびＫＥ５）を溶解したが、ＨＬＡ−Ａ１１⁻腫瘍細胞株（ＭＫＮ２８、ＲＫＮおよびＳＷ６２０）およびＨＬＡ−Ａ１１^＋ＰＨＡ芽球化正常Ｔ細胞（ＰＨＡ−ｂｌａｓｔ）は溶解しなかったことを示す図である。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。
【図１４】図１４Ａは、コールドターゲットインヒビションアッセイにおいて、ペプチド５６−Ｐ３１（配列番号４）で培養したＨＬＡ−Ａ１１^＋肺癌患者由来の末梢血単核細胞のＫＥ５細胞に対する溶解作用が、当該ペプチドをパルスしたＣ１Ｒ−Ａ１１細胞の添加により阻害されたが、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ１１では阻害されなかったことを示す図である。図１４Ｂは、同アッセイにおいてペプチド８５−Ｐ１０（配列番号７）で培養したＨＬＡ−Ａ１１^＋膀胱癌癌患者由来の末梢血単核細胞のＫＥ５細胞に対する溶解作用が、当該ペプチドをパルスしたＣ１Ｒ−Ａ１１細胞の添加により阻害されたが、ＨＩＶペプチド（配列番号２０）をパルスしたＣ１Ｒ−Ａ１１では阻害されなかったことを示す図である。図中、アスタリスク（＊）はスチューデントｔ検定により有意差が認められたことを示す。[0001]
[Industrial applications]
The present invention relates to tumor antigens and more particularly to HLA-A belonging to the HLA-A3 supertype, such as HLA-A31, HLA-A11 or at least one of HLA-A33, HLA-A restricted tumor-specific cytotoxic T. The present invention relates to a peptide recognized by a cell or a polypeptide containing the peptide. In addition, the present invention relates to a medicine, a cancer vaccine and an inducer of cytotoxic T cells containing one or more of the peptide or the polypeptide. Furthermore, the present invention relates to a method for inducing cytotoxic T cells using one or more of the peptide or the polypeptide. Furthermore, the present invention relates to a polynucleotide containing the nucleotide sequence encoding the peptide or the polypeptide or a nucleotide sequence complementary thereto, a recombinant vector containing the polynucleotide, and a transformant containing the recombinant vector. The present invention also relates to an antibody against the peptide and / or the polypeptide, a compound that enhances recognition of the peptide or the polypeptide by cytotoxic T cells, and a compound that promotes expression of the polynucleotide. Furthermore, the present invention relates to a pharmaceutical composition or a reagent kit containing at least one selected from the peptide, the polypeptide, the polynucleotide, the recombinant vector, the transformant, the antibody, and the compound. Furthermore, the present invention relates to a method for producing the peptide or the polypeptide, a method for screening the compound, and a method for measuring the peptide or the polypeptide or the polynucleotide.
[0002]
[Prior art]
The immune system, in particular, cytotoxic T lymphocytes (Cytotoxic T Lymphocytotes; hereinafter, sometimes abbreviated as CTLs) plays an important role in eliminating cancer in a living body. Infiltration of cytotoxic T cells exhibiting cytotoxic activity against tumor cells has been observed in tumor sites of cancer patients (Non-Patent Document 1). This tumor-specific cytotoxic T cell target molecule (tumor antigen) was first discovered in melanoma. Tumor antigens generated in tumor cells are degraded in the cells into peptides consisting of 8 to 11 amino acids (tumor antigen peptides), and human leukocyte antigen (HLA) which is a major histocompatibility antigen (MHC) And b) binding to molecules and presented on the surface of tumor cells. Cytotoxic T cells recognize a complex consisting of HLA and a tumor antigen peptide and damage tumor cells. That is, cytotoxic T cells recognize tumor cells in an HLA-restricted manner.
[0003]
HLA is a cell membrane antigen and is expressed on almost all nucleated cells. HLA is roughly classified into a class I antigen and a class II antigen. HLA recognized together with an antigenic peptide by cytotoxic T cells is a class I antigen. HLA class I antigens are further classified into HLA-A, HLA-B, HLA-C, etc., and in humans, nucleated cells have different amounts of HLA-A, HLA-B, and HLA-C, respectively. It is also reported that the gene is rich in polymorphism. For example, HLA-A has polymorphisms such as A1, A2, A24, A26, and A31, HLA-B has polymorphisms such as B8, B27, and B46, and HLA-C has polymorphisms such as Cw3 and Cw6. Therefore, the type of HLA possessed by each individual is not necessarily the same. One of the HLA-A subregion polymorphisms, the HLA-A31 allele, is about 65% of Brazilian Indians, about 60% of American Indians, about 20% of Mongolians, about 20% of Japanese, , About 10% of Chinese, Koreans and Brazilians, and about 5% of Caucasians and Hispanics (Non-Patent Documents 2-4).
[0004]
When cytotoxic T cells recognize the complex of HLA class I antigen and tumor antigen peptide, they also recognize the type of HLA. It is known that the amino acid sequence of a tumor antigen peptide that binds to an HLA molecule has a motif (regular sequence) that differs depending on the type of HLA.
[0005]
In recent years, molecules involved in specific immunity, such as tumor rejection antigen gene and T cell antigen receptor, have been identified in melanoma, esophageal cancer and other cancers. It has been studied (Non-Patent Documents 5-10). For example, in the United States and Europe, a cancer vaccine therapy for activating cytotoxic T cells in the body of a cancer patient by administering a tumor antigen is being developed, and the results of clinical trials on melanoma-specific tumor antigens have been reported. For example, by subcutaneously administering a melanoma antigen gp100 peptide to a melanoma patient and intravenously administering interleukin-2, tumor reduction has been observed in 42% of patients (Non-Patent Document 11). Thus, an effective cancer therapeutic effect can be expected by using a tumor antigen as a cancer vaccine.
[0006]
However, given the diversity of cancers, it is not possible to treat all cancers with a cancer vaccine consisting of one type of tumor antigen. In fact, it has been reported that immunotherapy using multiple peptides (multi-peptide based immunotherapy) is effective in treating cancer (Non-patent Documents 12 to 14). In addition, it is considered that the type and expression amount of the expressed tumor antigen vary depending on the type and tissue of the cancer cell. Many of the identified tumor antigens are derived from melanoma, and there are few reports of tumor antigens derived from epithelial cancers, adenocarcinomas, and the like, which frequently occur.
[0007]
Furthermore, since the types of tumor antigen peptides that function in each individual differ depending on the polymorphism of the HLA gene, identifying a tumor antigen peptide corresponding to each HLA is important for obtaining a high effect in cancer treatment. .
[0008]
Of course, a cancer vaccine therapy that activates cytotoxic T cells using a single tumor antigen can also provide a therapeutic effect on cancer having the tumor antigen. However, in order to induce and / or activate antigen-specific cytotoxic T cells in the treatment of cancer and to obtain a high therapeutic effect corresponding to the diversity of cancer, HLA restriction and cancer diversity are required. It is important to discover and utilize a number of new tumor antigens in response.
[0009]
The documents cited in the present specification are listed below.
[Patent Document 1] International Publication WO01 / 11044
[Non-Patent Document 1] "Archives of Surgery", 1990, Vol. 126, p. 200-205.
[Non-Patent Document 2] Aizawa M., "The Proceedings of the Third Asia-Oceania Histocompatibility, Ford Press Office, Ford Oxford University Press", "The Proceedings of the Third Asia-Oceania Histo-Compatibility Workshop Conference" Press), 1986.
[Non-Patent Document 3] Berich MP et al., "Nature", 1992, Vol. 357, p. 326-329.
[Non-Patent Document 4] Sette A., "Immunogenetics", 1999, Vol. 50, p. 201-212.
[Non-Patent Document 5] "Science", 1991, vol. 254, p. 1643-1647.
[Non-Patent Document 6] "Journal of Experimental Medicine", 1996, Vol. 183, p. 1185-1192.
[Non-Patent Document 7] Gomi, S. et al., "Journal of Immunology", 1999, vol. 163, p. 4994-5004.
[Non-Patent Document 8] "Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, America, Vol. 95, America," Proceedings of the National Academy of Sciences of the United States of America. " 432-436.
[Non-Patent Document 9] "Science", 1995, Vol. 269, p. 1281-1284.
[Non-Patent Document 10] "Journal of Experimental Medicine", 1997, Vol. 186, p. 785-793.
[Non-Patent Document 11] "Nature Medicine", 1998, vol. 4, p. 321-327.
[Non-Patent Document 12] "Clinical Cancer Research", 2001, Vol. 7, p. 3950-3962.
[Non-Patent Document 13] "Journal of Clinical Oncology", 2001, Vol. 19, p. 3836-3847.
[Non-Patent Document 14] "Nature Medicine", 1998, vol. 4, p. 328-332.
[Non-Patent Document 15] Ito et al., "Cancer Research", 2001, Vol. 61, p. 2038-2046.
[Non-Patent Document 16] Parker, KC. Et. Al., "Journal of Immunology", 1994, Vol. 152, p. 163-.
[Non-Patent Document 17] Wang et al. (Wang, RF. Et. Al.), "Journal of Experimental Medicine", 1995, Vol. 181, p. 799-804.
[Non-Patent Document 18] Wang, RF. Et. Al., "Journal of Experimental Medicine", 1996, Vol. 183, p. 1131-1140.
Non-Patent Document 19: Wang, RF. Et. Al., "Journal of Immunology", 1998, Vol. 160, p. 890-897.
[Non-Patent Document 20] Missale, G. et. Al., "Journal of Experimental Medicine", 1993, Vol. 177, p. 751-762.
[Non-Patent Document 21] Ed. Sambrook [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989.
[Non-Patent Document 22] Masami Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988.
[Non-Patent Document 23] Ehrlich, H .; E. FIG. Ed. [PCR Technology, Principles and Applications of DNA Amplification] Stockton Press, 1989.
[Non-Patent Document 24] Ulmer, "Science", 1983, Vol. 219, p. 666-.
[Non-Patent Document 25] "Peptide Synthesis", Maruzen Co., Ltd., 1975.
[Non-Patent Document 26] "Peptide Synthesis", Interscience, New York, 1996.
[Non-Patent Document 27] "International Journal of Cancer", 1999, Vol. 81, p. 459-466.
[Non-Patent Document 28] "Cancer Research", 1999, Vol. 59, p. 4056-4063.
[Non-Patent Document 29] Nishizaka, S. et al., "Cancer Research", 2000, Vol. 60, p. 4830-4837.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a polypeptide or peptide having an action as a tumor antigen or a tumor antigen peptide, for example, a polypeptide recognized by cytotoxic T cells useful for specific immunotherapy of colorectal cancer patients Or finding and providing the peptide. At present, many HLA-A2-restricted or HLA-A24-restricted tumor antigens or tumor antigen peptides have been found, but no HLA-A31-restricted one has been reported.
[0011]
One of the objects of the present invention is to provide a polypeptide or peptide that is recognized by and / or induces cytotoxic T cells at least in an HLA-A31 restricted manner. Another object of the present invention is to provide a polynucleotide comprising the polypeptide or a nucleotide sequence encoding the peptide or a complementary nucleotide sequence thereof. Yet another issue is an antibody against the polypeptide and / or peptide, a compound that enhances recognition of at least one of the polypeptide or the peptide by cytotoxic T cells, and promotes the expression of the polynucleotide. An object of the present invention is to provide a compound, the polypeptide and / or a cytotoxic T cell inducer comprising the peptide. Yet another problem is to provide a pharmaceutical composition or a reagent kit comprising one or more of these. Still another object is to provide a method for inducing cytotoxic T cells using the polypeptide and / or the peptide, a method for screening the compound, and a method for measuring the polypeptide or the peptide or the polynucleotide. It is to provide.
[0012]
[Means for solving the problem]
In order to solve the above-mentioned problems, the present inventors have made intensive efforts to use tumor-specific cytotoxic T cells established from tumor-infiltrating lymphocytes derived from colorectal cancer patients. A gene encoding a tumor antigen that activates cells in an HLA-A31-restricted manner was identified from a cDNA clone derived from human colon cancer cell line SW620. Then, a polypeptide or peptide that activates cytotoxic T cells in an HLA-A31-restricted manner is obtained from the gene product of the gene, and the peptide has an HLA phenotype of HLA-A31, HLA-A11 or HLA-A33. The present invention was completed by finding that HLA-A-restricted tumor-specific cytotoxic T cells are induced from peripheral blood mononuclear cells of a cancer patient.
[0013]
That is, the present invention
1. A peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing,
2. Induces HLA-A-restricted cytotoxic T cells consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing and belonging to one or more HLA-A3 supertypes and / or A polypeptide recognized by cytotoxic T cells,
3. HLA-A belonging to the HLA-A3 supertype is HLA-A selected from the group consisting of HLA-A31, HLA-A11 and HLA-A33. A polypeptide,
4. A peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8 and one or more HLA-A3 superscripts consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing A medicament comprising one or more peptides and / or polypeptides selected from the group consisting of polypeptides which induce HLA-A-restricted cytotoxic T cells belonging to the type and / or are recognized by cytotoxic T cells ,
5. 3. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is selected from the group consisting of HLA-A31, HLA-A11 and HLA-A33. Medicine
6. A peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8 and one or more HLA-A3 superscripts consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing It contains one or more peptides and / or polypeptides selected from the group consisting of polypeptides that induce HLA-A-restricted cytotoxic T cells belonging to the type and / or are recognized by cytotoxic T cells. Cancer vaccines,
7. 5. Use for a cancer whose HLA phenotype is HLA-A belonging to the HLA-A3 supertype. Cancer vaccine,
8. 6. The cancer wherein the cancer is prostate cancer, colon cancer, gastric cancer, cervical cancer, breast cancer, lung cancer, esophageal cancer, bladder cancer or melanoma. Cancer vaccine,
9. 5. The HLA-A belonging to the HLA-A3 supertype is one or more HLA-A selected from HLA-A31, HLA-A11 and HLA-A33. From 8. Any of the cancer vaccines,
10. One or more peptides selected from the peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and the polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14 in the sequence listing An inducer of HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes, comprising a peptide and / or polypeptide;
11. 9. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. A cytotoxic T cell inducer,
12. 1 selected from the group consisting of a peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14 in the sequence listing; A method for inducing HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes, comprising using one or more peptides and / or polypeptides.
13. 11. HLA-A belonging to the HLA-A3 supertype, at least one HLA-A selected from HLA-A31, HLA-A11 and HLA-A33; A method for inducing a cytotoxic T cell,
14． A polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8, or a complementary nucleotide sequence thereof,
15. Induces HLA-A-restricted cytotoxic T cells consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing and belonging to one or more HLA-A3 supertypes and / or A polynucleotide comprising a nucleotide sequence encoding a polypeptide recognized by cytotoxic T cells or a complementary nucleotide sequence thereof;
16. The nucleotide sequence according to any one of SEQ ID NO: 15 to SEQ ID NO: 19, wherein the polypeptide comprising the amino acid sequence encoded by the nucleotide sequence belongs to one or more HLA-A3 supertypes. A polynucleotide comprising a nucleotide sequence that induces A-restricted cytotoxic T cells and / or is recognized by cytotoxic T cells, or a complementary nucleotide sequence thereof;
17. 14. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. Or 16. A polynucleotide,
18. 14. From 17. A polynucleotide that hybridizes under stringent conditions with any of the polynucleotides of
19. 14. From 18. A recombinant vector containing any of the polynucleotides,
20. 19. The above-mentioned 19. wherein the recombinant vector is an expression recombinant vector. A recombinant vector,
21. 19. Or 20. A transformant into which the recombinant vector has been introduced,
22. 20. Culturing a transformant into which the recombinant vector has been introduced. Or the peptide of 2. Or 3. A method for producing a polypeptide,
23. 1. Antibodies that immunologically recognize the peptide of
24. 1. And / or 2. Or 3. 13. a compound that enhances the recognition of at least one of the polypeptides by HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes, and / or 14. From 17. 2. A method for screening a compound that enhances the expression of any one of the polynucleotides described in 1. The peptide of 2. Or 3. 14. the polypeptide of the above, From 18. 19. the polynucleotide according to any of the above, Or 20. The recombinant vector of 21. Or a transformant of the above 23. A screening method, characterized by using at least one of the above antibodies,
25. 25. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. Screening method,
26. 24. Or 25. A compound obtained by the screening method of
27. 1. Or the peptide of 2. Or 3. 13. a compound that enhances the recognition of at least one of the polypeptides by HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes; From 17. A compound that enhances the expression of any one of the polynucleotides,
28. 27. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. A compound of
29. 1. The peptide of 2. Or 3. 14. the polypeptide of the above, From 17. 19. the polynucleotide according to any of the above, Or 20. The recombinant vector of 21. Or a transformant of the above 23. The antibody of 26. From 28. A pharmaceutical composition for use in treating cancer, comprising at least one compound of any of the following:
30. 29. The cancer treatment according to the above 29, wherein the cancer treatment is a cancer for which the HLA phenotype is HLA-A belonging to the HLA-A3 supertype. A pharmaceutical composition of
31. 30. The HLA-A belonging to the HLA-A3 supertype, wherein the HLA-A is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. A pharmaceutical composition of
32. 29. The cancer wherein the cancer is prostate cancer, colon cancer, gastric cancer, cervical cancer, breast cancer, lung cancer, esophageal cancer, bladder cancer or melanoma. To 31. Any of the pharmaceutical compositions of
33. 1. The peptide of 2. Or 3. Or the polypeptide of 14. From 17. Any of the polynucleotide quantitative or qualitative measurement method,
34. 1. The peptide of 2. Or 3. 14. the polypeptide of the above, From 17. 19. the polynucleotide according to any of the above, Or 20. Or the recombinant vector of 23. A reagent kit comprising at least one or more of the antibodies of
35. 24. Or 25. Screening method or 27. A reagent kit used for the measurement method of 1. The peptide of 2. Or 3. 14. the polypeptide of the above, From 17. 19. the polynucleotide according to any of the above, Or 20. Or the recombinant vector of 23. A reagent kit comprising at least one or more of the antibodies of
Consists of
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
For understanding the present invention, terms used in the present specification will be described first. A tumor antigen is one that is recognized by tumor-specific cytotoxic T cells and / or can induce cytotoxic T cells, and refers to a protein or peptide possessed by tumor cells. The tumor antigen peptide is a peptide generated by decomposing the tumor antigen in tumor cells, and is recognized by tumor-specific cytotoxic T cells by binding to HLA molecules and presenting on the cell surface. And / or capable of inducing cytotoxic T cells. Further, a site of an amino acid sequence which can induce and / or activate a tumor-specific cytotoxic T cell possessed by a tumor antigen is referred to as a tumor antigen epitope (tumor antigen determinant).
[0015]
Hereinafter, a tumor antigen or a tumor antigen peptide that binds to an HLA-A molecule and is presented on the cell surface and is capable of inducing and / or activating cytotoxic T cells is used as an HLA-A-restricted tumor antigen or HLA-A-restricted tumor. Sometimes called an antigen peptide. For example, a tumor antigen or a tumor antigen peptide which binds to an HLA-A31 molecule and is presented on the cell surface and can induce and / or activate cytotoxic T cells is used as an HLA-A31-restricted tumor antigen or HLA-A31-restricted tumor antigen A cytotoxic T cell that recognizes such a tumor antigen or a tumor antigen peptide as a peptide may be referred to as an HLA-A31-restricted cytotoxic T cell.
[0016]
Here, “recognize” means that the recognizing object recognizes and recognizes the recognized object from other objects, for example, combines with the recognized object. In particular, in the present specification, the expression that cytotoxic T cells recognize tumor cells or tumor antigen peptides means that cytotoxic T cells bind to tumor antigen peptides presented by HLA molecules via T cell antigen receptors. Means that. “Activate” means to further enhance or activate an entity or condition that has a certain activity or effect. In particular, in the present specification, activation of a cytotoxic T cell means that the cytotoxic T cell recognizes an antigen presented by an HLA molecule, for example, interferon-γ (hereinafter abbreviated as IFN-γ). ) Or the cytotoxic T cells exhibit cytotoxic activity against the recognized target cells. "Induce" means to produce an activity or action from a substance or state that has little activity or action. In particular, as used herein, to induce antigen-specific cytotoxic T cells means to differentiate and / or expand cytotoxic T cells that specifically recognize a certain antigen in vitro or in vivo. . As used herein, the term "cytotoxic T cell inducer" refers to a cell that recognizes an antigen from a state in which CD8 positive T cells that specifically recognize the antigen do not exist or exist at a very low rate. An agent that acts to alter a state in which a very high percentage of injurious T cells are present is meant.
[0017]
In the present specification, a long-chain peptide of any peptide containing two or more amino acids linked to each other by a peptide bond or a modified peptide bond is referred to as a polypeptide. For example, a protein is also included in the polypeptide in this specification. In addition, short-chain peptides, which are also referred to as oligopeptides and oligomers, are simply referred to as peptides. Hereinafter, the amino acid sequence may be represented by one letter or three letters.
[0018]
Hereinafter, embodiments of the present invention will be described. The following detailed description is illustrative and is illustrative only and does not limit the invention in any way.
[0019]
The polypeptide and peptide according to the present invention are a polypeptide encoded by a cDNA clone derived from human colon cancer cell line SW620 and a partial peptide of the polypeptide. When the cDNA is introduced into a cell and expressed, the cell is recognized by a tumor-specific cytotoxic T cell and can induce and / or activate the cytotoxic T cell. More specifically, when expressed in cells having HLA-A31, it is recognized by tumor-specific cytotoxic T cells in an HLA-A31-restricted manner, and induces and / or activates the cytotoxic T cells. be able to.
[0020]
Among the partial peptides of the polypeptide encoded by the cDNA, a peptide having a tumor antigen epitope is recognized as one of its characteristics by tumor-specific cytotoxic T cells, and induces the cytotoxic T cells. Or can be activated. More specifically, it is recognized by HLA-A31-restricted tumor-specific cytotoxic T cells, and can induce and / or activate the cytotoxic T cells.
[0021]
The above cDNA was identified as follows. First, a tumor-infiltrating lymphocyte of a colorectal cancer patient (HLA-A0207 / 3101, HLA-B46 / 51, HLA-Cw1) (this colorectal cancer patient may be hereinafter referred to as OK) has already been reported (Non-Patent Document 15). Using the subline OKA31-CTL of the tumor-specific cytotoxic T cell line OK-CTL established according to the method described in the above section, a tumor antigen that activates this was identified. OKA31-CTL recognizes HLA-A31 relatively strongly compared to other sublines, and its antigen recognition is HLA-A locus-restricted. That is, HLA-A31 positive (hereinafter, HLA-A31 ⁺ Notation. ) Recognize the antigen presented by the cells, but negative for HLA-A31 (hereinafter HLA-A31) ⁻ Notation. 2.) It does not recognize antigens presented by cells via other types of HLA molecules. HLA-A31-positive means that the HLA phenotype is HLA-A31. HLA-A31 negative means that the HLA phenotype is other than A31.
[0022]
The present inventors have already isolated and identified a gene encoding a tumor antigen that activates the HLA-A2-restricted OK-CTL subline from a cDNA library of human colon cancer cell line SW620 by using gene expression cloning. are doing. In the present invention, five types of genes encoding tumor antigens that activate OKA31-CTL in an HLA-A31-restricted manner have been found among the genes.
[0023]
Identification of the five genes was carried out by co-transfecting a cDNA encoding a tumor antigen that activates the HLA-A2-restricted OK-CTL subline and HLA-A3101 cDNA into monkey kidney cell line COS-7, Was carried out by selecting cells that promote IFN-γ production from OKA31-CTL from cells in which was expressed (see Example 1). The nucleotide sequences of the obtained five types of cDNA clones are shown in SEQ ID NOs: 15 to 19 in the sequence listing. These nucleotide sequences were found to have homology to those of known genes shown in Table 1 by homology search using an existing database such as GenBank. However, no data has been reported to date that these known genes encode tumor antigens that induce and / or activate cytotoxic T cells in an HLA-A31 restricted manner.
[0024]
[Table 1]

[0025]
These genes encode tumor antigens that are recognized by HLA-A31-restricted tumor-specific cytotoxic T cells, and as described above, HLA-A31 ⁺ When expressed in cells, it activated HLA-A31-restricted cytotoxic T cells and promoted IFN-γ production. The amino acid sequences encoded by these genes are shown in SEQ ID NOs: 9 to 14 (see Table 1).
[0026]
The polypeptide encoded by the above gene obtained from the human colon cancer cell line SW620 can be used as a tumor antigen to induce cytotoxic T cells and / or recognized by cytotoxic T cells. The polypeptide is preferably a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 9 to 14. Examples of cytotoxic T cells that recognize such polypeptides include at least HLA-A31-restricted or HLA-A2-restricted cytotoxic T cells. Further, the present polypeptide can be used as a material for obtaining a tumor antigen peptide by specifying a tumor antigen epitope.
[0027]
The tumor antigen peptide can be obtained by selecting a peptide that induces and / or activates cytotoxic T cells from a peptide designed and synthesized based on the amino acid sequence of the above polypeptide. Methods for determining the induction and / or activation of cytotoxic T cells include, for example, culturing both peptide-pulsed cells and cytotoxic T cells such as OKA31-CTL to obtain activated cytotoxic T cells. An example is a method of measuring IFN-γ produced from T cells and using this as an index to determine. The tumor antigen peptide may be one which binds to HLA-A31, is presented on the cell surface, and has a property as a tumor antigen epitope recognized by cytotoxic T cells. Is a peptide consisting of 7 or more, more preferably 9 to 11 amino acid residues. More preferably, it is a peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8. These peptides can be used as tumor antigen peptides that induce HLA-A31 restricted tumor-specific cytotoxic T cells and / or are recognized by cytotoxic T cells.
[0028]
The peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 was specifically obtained by the following method. First, a 9-mer to 11-mer peptide conforming to the HLA-A31 binding motif was designed based on the amino acid sequence of the above polypeptide, and synthesized by a conventional method. The peptide design was performed using the database "HLA PeptideBinding Prediction (HLA PeptideBinding)" disclosed in the Bioinformatics and Molecular Analysis Section (BIMAS) of the National Institutes of Health (NIH). (Http://bimas.dcrt.nih.gov/molbio/hla_bind), and further referred to in the previous report (Non-Patent Documents 16 to 18.) HLA-A31-restricted cytotoxicity from a synthesized peptide Selection of peptides recognized by sex T cells was performed as follows: Each peptide was converted into cells (COS-7 cells expressing HLA-A3101 or OK-EB). -B cell line), and cultured with OKA31-CTL to measure IFN-γ produced in the culture supernatant.OKA31- was compared with a negative control (a peptide not recognized by OKA31-CTL). A peptide that promoted IFN-γ production from CTL was determined to be a peptide recognized by OKA31-CTL.
[0029]
Eight of the synthesized peptides (SEQ ID NOS: 1 to 8) promoted IFN-γ production from OKA31-CTL in a dose-dependent manner (see Example 2). In addition, these peptides induced cytotoxic T cells from peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell; hereinafter sometimes abbreviated as PBMC) obtained from cancer patients. In PBMCs derived from healthy individuals, there were fewer cases where cytotoxic T cells were induced as compared to PBMCs derived from cancer patients. The induced cytotoxic T cells were HLA-A31 ⁺ HLA-A31 pulsed with target cells such as the human lung adenocarcinoma cell line LC-1 or the peptide used for induction ⁺ Recognized cells to promote IFN-γ production and lysed the target cells, but HLA-A31 ⁻ Target cells did not lyse (see Examples 3 and 4). The induction of cytotoxic T cells is peptide-specific, and if the peptide used for induction is the same as the peptide presented by the target cells, the induced cytotoxic T cells will recognize the target cells and This was injured (see Example 4). From these, it was found that the induced cytotoxic T cells were HLA-A31-restricted and peptide-specific cytotoxic T cells. Thus, eight types of tumor antigen peptides that activate and / or induce tumor-specific cytotoxic T cells in an HLA-A31-restricted manner were obtained. These peptides were peptides encoded in a region completely different from the HLA-A2-restricted tumor antigen peptide in the above gene.
[0030]
The peptides described in SEQ ID NOs: 1 to 8 are HLA-A31 ⁺ In addition to PBMCs derived from cancer patients as well as PBMCs derived from cancer patients whose HLA phenotype is HLA-A11 or HLA-A33, cytotoxic T cells that lyse tumor cells in an HLA-A-restricted manner are also used. Induced (see Examples 5 and 6). HLA-A31 is one of the HLA-A3 supertypes to which alleles such as HLA-A3, HLA-A11, HLA-A33 and HLA-A68 belong. So far, it has been reported that the same epitope peptide derived from a viral protein or melanoma antigen is recognized by different HLA-A3 supertypes (Non-Patent Documents 19 and 20). From these, the present peptide binds not only to the HLA-A31 molecule but also to the HLA-A3 supertype, for example, the HLA-A11 molecule or the HLA-A33 molecule, and induces each HLA-restricted cytotoxic T cell. And / or activated.
[0031]
In the present specification, HLA-A belonging to the HLA-A3 supertype is reported to be an HLA-A3 supertype such as HLA-A3, HLA-A11, HLA-A31, HLA-A33 and HLA-A68. HLA-A. Preferably it is at least one HLA-A selected from the group consisting of HLA-A31, HLA-A11 and HLA-A33, more preferably HLA-A31.
[0032]
The present peptide can be used as a tumor antigen peptide that induces and / or is recognized by HLA-A-restricted tumor-specific cytotoxic T cells, preferably belonging to the HLA-A3 supertype. As described above, one peptide induces a plurality of HLA-A-restricted tumor-specific cytotoxic T cells belonging to the HLA-A3 supertype and / or a tumor antigen recognized by the cytotoxic T cells Can be used as a peptide. Furthermore, since the present polynucleotide has the present peptide as a part thereof, when expressed in a cell showing an HLA phenotype belonging to the HLA-A3 supertype, the HLA-A-restricted tumor-specific cell It is thought to induce and / or be recognized by cytotoxic T cells.
[0033]
Polypeptides or peptides specified in this way that have mutations such as deletion, substitution, addition or insertion of one or several amino acids are also included in the scope of the present invention. The peptide having a mutation may be a naturally occurring peptide or a peptide having a mutation introduced therein. Preferably, a polypeptide or peptide having such a mutation, which is recognized by cytotoxic T cells, preferably by at least HLA-A restricted cytotoxic T cells belonging to the HLA-A3 supertype. Is desirable. Means for introducing a mutation are known per se, and for example, site-directed mutagenesis, homologous recombination, primer extension or polymerase chain reaction (PCR) can be used alone or in appropriate combination. . For example, the method can be carried out in accordance with the method described in a compendium (Non-Patent Documents 21 to 23) or by modifying those methods, and the technique of Ulmer (Non-Patent Document 24) can also be used. From the viewpoint of not changing the basic properties (physical properties, functions, physiological activities or immunological activities, etc.) of the polypeptide upon introduction of the mutation, for example, homologous amino acids (polar amino acids, non-polar amino acids, hydrophobic amino acids) , Hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids) are readily envisioned. Furthermore, these available peptides can be modified to such an extent that no significant change in the function is involved, such as by amidation modification of the constituent amino group or carboxyl group.
[0034]
The polynucleotide according to the present invention is a polynucleotide comprising a base sequence encoding the above polypeptide or peptide or a complementary base sequence thereof. Preferably, a nucleotide sequence encoding a peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 or a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14, or a complementary nucleotide sequence thereof Or a polynucleotide consisting of More preferably, it is a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOS: 15 to 19 or a complementary nucleotide sequence thereof. A polynucleotide including the polynucleotide is also included in the scope of the present invention. Furthermore, within the scope of the present invention, 15 or more, preferably 21 or more, corresponding to the region encoding the HLA-A-restricted tumor antigen epitope belonging to the HLA-A3 supertype in the amino acid sequence of the polypeptide, More preferably, a polynucleotide comprising 33 or more nucleotides is also included. This polynucleotide is considered to be capable of inducing and / or activating the HLA-A-restricted cytotoxic T cell when expressed in a cell exhibiting the HLA phenotype belonging to the HLA-A3 supertype. Although the present polynucleotide has a poly (A) [poly (A)] structure at its 3 'end, the number of poly (A) does not affect the coding site of the amino acid acting as a tumor antigen. The number is not particularly limited. Selection of such a useful polynucleotide includes, for example, expressing a polypeptide or peptide using a known protein expression system, and measuring the ability of the polypeptide or peptide to induce cytotoxic T cells and / or activate it. Is possible.
[0035]
Furthermore, polynucleotides that hybridize to the above polynucleotides under stringent conditions are also included in the scope of the present invention. Hybridization conditions can be according to, for example, the method described in a textbook (Non-Patent Document 21). These polynucleotides need not be complementary sequences to the target polynucleotide as long as they hybridize to the target polynucleotide.
[0036]
Each of the present polynucleotides provides useful genetic information for producing the present polypeptide or peptide, or can be used as a reagent or standard as a nucleic acid.
[0037]
By incorporating the polynucleotide into an appropriate vector DNA, a recombinant vector can be obtained. The vector DNA to be used is appropriately selected depending on the type of the host and the purpose of use. The vector DNA may be obtained by extracting a naturally occurring DNA, or may be a DNA in which a part of DNA other than a part necessary for growth is partially deleted. For example, chromosomal, episomal and viral derived vectors such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion element derived, yeast chromosomal element derived such as baculovirus, papovavirus, SV40, vaccinia virus, adenovirus, Examples include vectors derived from viruses such as fowlpox virus, pseudorabies virus and retrovirus, and vectors obtained by combining them, such as vectors derived from genetic elements of plasmids and bacteriophages, such as cosmids and phagemids. In addition, an expression vector, a cloning vector, or the like can be used depending on the purpose.
[0038]
A recombinant vector is composed of a gene sequence of interest and a gene sequence carrying information on replication and control, such as a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer, etc., and combining these by a method known per se. To make. As a method of incorporating a polynucleotide into a vector DNA, a method known per se can be applied. For example, a method is used in which a DNA is cleaved at a specific site by selecting and treating an appropriate restriction enzyme, then mixed with a DNA used as a vector similarly treated, and religated with ligase. Alternatively, a desired recombinant vector can also be obtained by ligating a suitable linker to a desired polynucleotide and inserting this into a multicloning site of a vector suitable for the purpose.
[0039]
A transformant can be obtained by introducing the vector DNA into which the polynucleotide has been incorporated into a host known per se. The vector DNA to be introduced into the host may be one kind of vector DNA, or two or more kinds of vector DNAs may be introduced. Examples of the host include Escherichia coli, yeast, Bacillus subtilis, insect cells and animal cells. Means known per se can be applied for introducing the vector DNA into the host cell. For example, it can be implemented by a standard method described in a compendium (Non-Patent Document 21). A more preferable method is an integration method into a chromosome if gene stability is taken into consideration, but an autonomous replication system using an extranuclear gene can be used simply. Specifically, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection, etc. Is mentioned.
[0040]
When an expression vector is used as a vector DNA to be introduced into a host, it is possible to express a gene incorporated in the vector. The transformant into which the expression vector DNA incorporating the desired gene has been introduced is cultured under conditions that are optimal for the culture conditions of each host. The culturing can be performed using the function of the gene product expressed by the transformant as an index. Alternatively, the determination may be performed using the amount of the gene product produced in or outside the host as an index, or may be performed using the amount of the transformant in the medium as an index.
[0041]
The present polypeptide or peptide can be produced by a genetic engineering technique using an expression vector incorporating a polynucleotide encoding the polypeptide or peptide, or a transformant into which the vector has been introduced. It can also be produced by a method known in ordinary peptide chemistry. For example, the method described in a compendium (Non-Patent Documents 25 and 26) can be exemplified, but, of course, known methods are widely available.
[0042]
Purification and / or recovery of the polypeptide or peptide can be carried out using the function of the polypeptide or peptide, for example, the ability to induce and / or activate cytotoxic T cells as an index. Examples of the method of purification and / or recovery include fractionation means based on a difference in solubility using ammonium sulfate, alcohol, or the like, gel filtration, ion column chromatography, affinity chromatography, and the like. These may be used alone or in combination. I do. Preferably, based on the amino acid sequence information of the polypeptide or peptide to be purified and / or recovered, a polyclonal antibody or a monoclonal antibody specific thereto is prepared, and the antibody is specifically adsorbed and recovered using the antibody. Use method.
[0043]
Antibodies are prepared using the polypeptide or peptide according to the present invention as an antigen. The antigen may be the polypeptide or peptide, or a fragment thereof, and is composed of at least 8, preferably at least 10, more preferably at least 12, even more preferably 15 or more amino acids. In order to prepare an antibody specific to the polypeptide and / or peptide, it is preferable to use a region consisting of an amino acid sequence unique to the polypeptide or peptide. The amino acid sequence of this region does not necessarily need to be homologous or identical to that of the polypeptide or peptide, and preferably has an exposed site on the three-dimensional structure, and the amino acid sequence of the exposed site is discontinuous on the primary structure. May be any amino acid sequence that is continuous for the exposed site. The antibody is not particularly limited as long as it binds or recognizes the polypeptide and / or peptide immunologically. The presence or absence of this binding or recognition can be determined by a known method for measuring an antigen-antibody binding reaction.
[0044]
For the production of the antibody, a known antibody production method can be used. For example, an antibody can be obtained by administering an antigen alone or in the presence of an adjuvant to an animal, alone or in combination with a carrier, to induce immunity such as a humoral response and / or a cellular response. The carrier is not particularly limited as long as it does not itself exhibit a harmful effect on the host and enhances antigenicity, and examples thereof include cellulose, polymerized amino acids, albumin, and keyhole limpet hemocyanin. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordeterra pertussis vaccine), muramyl dipeptide (MDP). Adjuvants (ALUM) and combinations thereof can be exemplified. As the animal to be immunized, mice, rats, rabbits, goats, horses and the like are suitably used.
[0045]
A polyclonal antibody can be obtained from the serum of an animal that has been subjected to immunization by a method known per se for recovering antibodies. Preferred means for collecting antibodies include immunoaffinity chromatography.
[0046]
Monoclonal antibodies are obtained by recovering antibody-producing cells (for example, lymphocytes derived from spleen or lymph node) from an animal to which immunization has been performed, and obtaining perpetually proliferating cells (for example, P3-X63-Ag8 cells and the like) known per se (Myeloma strain). For example, an antibody-producing cell and a permanently proliferating cell are fused by a method known per se to prepare a hybridoma, which is cloned to produce an antibody that specifically recognizes the polypeptide and / or peptide according to the present invention. The hybridoma is selected, and the antibody is recovered from the culture of the hybridoma.
[0047]
The thus obtained polyclonal antibody or monoclonal antibody that recognizes and binds to the polypeptide or peptide can be used as an antibody, reagent, or marker for purification of the polypeptide or peptide.
[0048]
The polypeptide, peptide, polynucleotide, recombinant vector, transformant, or antibody according to the present invention can be used alone or in combination of two or more to recognize the polypeptide or the peptide by cytotoxic T cells. It provides an effective means for screening for a potentiating substance. The screening method can be constructed using a drug screening system known per se.
[0049]
For example, an experimental system for stimulating cytotoxic T cells using cells pulsed with a tumor antigen peptide and measuring the recognition of the tumor antigen peptide by the cytotoxic T cells and / or the activation of the cytotoxic T cells Is constructed, and a test substance is tested using the experimental system, whereby a substance that enhances recognition of the peptide of the present invention by cytotoxic T cells can be selected. Cells that pulse with a tumor antigen peptide include cells that exhibit the HLA phenotype belonging to the HLA-A3 supertype, such as HLA-A31. ⁺ , HLA-A11 ⁺ And / or HLA-A33 ⁺ Can be exemplified. Alternatively, any cDNA of HLA-A belonging to the HLA-A3 supertype is transfected into cells exhibiting a phenotype other than HLA-A belonging to the HLA-A3 supertype by a conventional method, and the HLA is placed on the cell surface. Cells expressing the -A molecule can also be used. The cells can be pulsed with a tumor antigen by culturing the cells and the tumor antigen together in a conventional manner (see Example 2). As the cytotoxic T cell, an HLA-A-restricted cytotoxic T cell line belonging to the HLA-A3 supertype, such as OKA31-CTL, is used. The combination of a cytotoxic T cell and a target cell preferably has the same HLA phenotype of both cells. The recognition of the tumor antigen peptide by the cytotoxic T cells and / or the activation of the cytotoxic T cells can be simply measured using the amount of IFN-γ production from the cytotoxic T cells as an index. This experimental system describes one of the identification methods, and the identification method according to the present invention is not limited to this.
[0050]
The present invention also includes a compound obtained by the above screening method. The compound may be a polypeptide or a peptide according to the present invention by an HLA-A-restricted cytotoxic T cell belonging to the HLA-A3 supertype, for example, a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 8 or It may be a compound that enhances recognition of a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14. Alternatively, it may be a compound that enhances the expression of the polynucleotide according to the present invention. The compound thus selected can be prepared as a pharmaceutical composition by further selecting in consideration of the balance between biological utility and toxicity.
[0051]
Since the polypeptide or peptide according to the present invention induces and / or is recognized by HLA-A-restricted tumor-specific cytotoxic T cells belonging to the HLA-A3 supertype, It can be used as a tumor antigen or tumor antigen peptide. Therefore, the present invention also provides a cytotoxic T cell-inducing agent containing the present peptide and / or the present polypeptide, and a method for inducing cytotoxic T cells using the present peptide and / or the present polypeptide. Included in the scope. HLA-A-restricted cytotoxic T cells belonging to the HLA-A3 supertype can be exemplified as cytotoxic T cells induced by the present inducer and method. The polypeptide or peptide may be used alone or in combination of two or more to induce and / or activate cytotoxic T cells. Since cytotoxic T cells are a plurality of cell populations that recognize various antigens, it is preferable to use a combination of two or more of them.
[0052]
As one embodiment of the method for inducing cytotoxic T cells, a step of pulsing the cells with the peptide according to the present invention, and a cell population containing precursor cells of cytotoxic T cells using the cells obtained in the step Stimulating Examples of the cells to be pulsed with the peptide include cells exhibiting the HLA phenotype belonging to the HLA-A3 supertype, for example, known cell lines exhibiting the expression system. Alternatively, any one of the cDNAs of HLA-A belonging to the HLA-A3 supertype is transfected into cells having a phenotype other than HLA-A belonging to the HLA-A3 supertype by a conventional method, and the cells are transferred onto the cell surface. Cells expressing the HLA-A molecule can also be used. The cells can be pulsed with the peptide by culturing the cells and the tumor antigen peptide together in a conventional manner (see Example 2). Cell populations containing cytotoxic T cell precursor cells include, for example, peripheral blood cells prepared from human peripheral blood, more preferably peripheral blood mononuclear cells, and even more preferably peripheral blood exhibiting the HLA phenotype belonging to the HLA-A3 supertype. Mononuclear cells. The stimulation of the cell population was performed by pulsed HLA-A31 with the cell population and peptide. ⁺ It can be carried out by culturing the cells together with the cells according to a conventional method (see Example 3). The combination of the cell population and the target cell preferably has the same HLA phenotype of both cells.
[0053]
The polypeptide, the peptide, the polynucleotide, the recombinant vector, the transformant, the antibody or the compound according to the present invention contains at least one selected from the group consisting of these, by using the compound alone or in combination. Pharmaceutical compositions can be provided.
[0054]
In the present invention, expression of the clone 40, clone 56, claw 76 and clone 85 of the present gene in tumor cells and tissues was examined, and all of them were epithelial cancer cell lines and adenocarcinoma cell lines (esophageal cancer, lung adenocarcinoma , Colorectal adenocarcinoma and gastric adenocarcinoma) (Example 7).
[0055]
The pharmaceutical composition may be for the treatment of cancer, preferably for the treatment of cancer expressing one or more of the five genes according to the invention, more preferably for the expression of one or more of said genes and for the HLA phenotype. One or both can be used to treat cancers that are HLA belonging to the HLA-A3 supertype. More specifically, gastric cancer, esophageal cancer, colon cancer, small intestine cancer, duodenal cancer, lung cancer, liver cancer, gallbladder cancer, pancreatic cancer, kidney cancer, bladder cancer, oral cancer, bone cancer, skin cancer, breast cancer, uterine cancer The present invention is applicable to any of solid tumors such as prostate cancer, brain tumor, neuroblastoma and melanoma, and non-solid tumors such as blood cancer (leukemia or malignant lymphoma). Preferably, it is considered to be useful for treating prostate cancer, colorectal cancer, gastric cancer, uterine cancer, breast cancer, lung cancer, esophageal cancer, bladder cancer or melanoma. The HLA phenotype of a cancer can be determined, for example, by measuring the HLA class I phenotype of a cancer patient by a conventional serological method (Non-Patent Document 15). The determination of the HLA phenotype of the cancer is not limited to this method, and any conventional method can be used.
[0056]
The HLA-A3 supertype is an HLA-A allele that is expressed in many of the world's population. For example, it is found in 37.5%, 42.1%, 45.8%, 52.7% and 43.1% of whites, North American blacks, Japanese, Chinese and Latin Americans, respectively. Therefore, the pharmaceutical composition according to the present invention may be applicable to specific immunotherapy of the majority of cancer patients, and is extremely useful.
[0057]
Further, the present polypeptide comprises a tumor antigen epitope recognized by HLA-A-restricted cytotoxic T cells belonging to the HLA-A3 supertype, and a tumor antigen epitope recognized by HLA-A2-restricted cytotoxic T cells. Having both. Therefore, HLA-A belonging to the HLA-A3 super type ⁺ And HLA-A2 ⁺ It is thought that by expressing the present polypeptide in cells of the present invention, both HLA-A-restricted and HLA-2-restricted cytotoxic T cells belonging to the HLA-A3 supertype can be induced. From these, a pharmaceutical composition comprising the present polypeptide, a polynucleotide encoding the polypeptide, a recombinant vector containing the polynucleotide, and a transformant containing the recombinant vector has an HLA-A3 super It is believed that the treatment of cancers with HLA and HLA-2 belonging to the type, for example, cancers with an HLA phenotype of A31 / A2, A11 / A2 or A33 / A2 may be particularly effective.
[0058]
Specifically, for example, a drug comprising one or more selected from the polypeptides and peptides according to the present invention, and a pharmaceutical composition containing one or more selected from the polypeptides and peptides according to the present invention are so-called cancer vaccines Can be used as The term “cancer vaccine” as used herein means a drug that selectively damages tumor cells by inducing and / or enhancing a specific immune response against the tumor cells. The dose can be determined by appropriately changing the degree of recognition of the polypeptide or the peptide by cytotoxic T cells, but is generally 0.01 mg to 100 mg / day / adult human , Preferably 0.1 mg to 10 mg / day / adult human. It is administered once every few days to several months. The administration method may be in accordance with a known method for administering a medical peptide, and is preferably performed by subcutaneous, intravenous, or intramuscular administration. Upon administration, the polypeptide and / or peptide of the present invention can be used alone or in combination with a carrier in the presence or absence of a suitable adjuvant, for the induction and / or enhancement of an immune response. . The carrier is not particularly limited as long as it does not have a harmful effect on the human body, and examples thereof include cellulose, polymerized amino acids, and albumin. The adjuvant may be any adjuvant used for ordinary peptide vaccination, and examples thereof include incomplete Freund's adjuvant (FIA), aluminum adjuvant (ALUM), pertussis vaccine (Bordetella pertussis vaccine), and mineral oil. The dosage form can be appropriately selected by applying a known means for preparing a peptide.
[0059]
Alternatively, for example, after collecting a mononuclear cell fraction from a patient's peripheral blood and stimulating it with the polypeptide or peptide of the present invention, induction of cytotoxic T cells and / or activity of cytotoxic T cells An effective cancer vaccine effect can also be obtained by returning the mononuclear cell fraction that has been transformed or the cytotoxic T cells purified from the mononuclear cell fraction to the blood of the patient. Stimulation with a polypeptide or peptide can be performed by culturing the mononuclear cell fraction with the polypeptide or peptide, cells expressing the polypeptide or peptide, or cells pulsed with the peptide. Culture conditions, such as mononuclear cell concentration, polypeptide or peptide concentration, culture time, etc., can be determined by simple experiments. During the culture, a substance having lymphocyte proliferation ability, such as interleukin-2, may be added. Purification of cytotoxic T cells can be performed by a conventional method. ⁺ It can be performed by collecting cells.
[0060]
When the polypeptide or peptide according to the present invention is used as a cancer vaccine, only one type of polypeptide or peptide is effective as a cancer vaccine, but a plurality of types of the above polypeptides or peptides may be used in combination. it can. It has been reported that immunotherapy based on multiple peptides is effective (Non-patent Documents 12 to 14), and cytotoxic T cells of cancer patients are a population of cells that recognize multiple tumor antigens. In some cases, a higher effect can be obtained by using a combination of a plurality of types of peptides than using one type of peptide as a cancer vaccine. Further, at this time, those selected from the group consisting of the polypeptides and peptides of the present invention may be used in combination, but at least one type of polypeptide or peptide selected from these may be a known tumor antigen or tumor antigen. At least one kind selected from peptides may be used in combination. Known tumor antigens or tumor antigen peptides used in combination are not limited to HLA-A-restricted ones belonging to the HLA-A3 supertype, and may be used together with other types of HLA, such as HLA-A2 and HLA-A24. It may be a tumor antigen or tumor antigen peptide that is recognized by cytotoxic T cells. Known tumor antigens or tumor antigen peptides include lck gene and src gene (Patent Document 1), SART-1 gene (Non-patent Document 27), SART-3 gene (Non-patent Document 28), and cyclophilin B gene ( Examples include, but are not limited to, tumor antigens and tumor antigen peptides derived from Non-Patent Document 7) and the like.
[0061]
The polynucleotide according to the present invention can be used for gene therapy of cancer. Either a method in which these polynucleotides are carried on a vector and directly introduced into the body to express them, or a method in which cells are collected from a human and then introduced into cells outside the body and expressed, can be used. As a vector, a retrovirus, an adenovirus, a vaccinia virus and the like are known, but a retrovirus system is recommended. Of course, these viruses use replication-defective ones. The dose varies depending on the degree of recognition of the polypeptide or peptide by cytotoxic T cells, but is generally 0.1 μg to 100 mg / day / DNA content encoding the polypeptide or peptide of the present invention. Adult humans, preferably 1 μg to 50 mg / day / adult human. It is administered once every few days to several months.
[0062]
(Measurement method and reagent)
The polypeptide, peptide, polynucleotide, vector and antibody according to the present invention can be used alone as a diagnostic marker, a reagent or the like. When they are reagents, they may contain substances such as buffers, salts, stabilizers, and / or preservatives. The present invention also provides a reagent kit comprising one or more containers filled with one or more of these. In addition, in formulating, it is sufficient to introduce a formulating means corresponding to each of the known polypeptides, peptides, polynucleotides, vectors or antibodies. The reagents and reagent kits can be used for the identification method according to the present invention, the method for inducing cytotoxic T cells, or the quantitative or qualitative measurement of the polypeptide, peptide and polynucleotide. A method for performing the measurement can be constructed using a method well known to those skilled in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis and enzyme-linked immunosorbent assays (ELISA). Nucleic acids can also be detected and quantified at the RNA level using, for example, amplification, PCR, reverse transcription polymerase chain reaction (RT-PCR), RNase protection, Northern blotting, and other hybridization methods.
[0063]
Samples to be measured include cells, blood, urine, saliva, cerebrospinal fluid, tissue biopsy or autopsy material from an individual. The nucleic acid to be measured can be obtained from each of the above-mentioned samples by a nucleic acid preparation method known per se. Nucleic acids may be used directly for detection of genomic DNA or may be amplified enzymatically using PCR or other amplification methods prior to analysis. RNA or cDNA may be used as well. In comparison with the normal genotype, deletions and insertions can be detected by changes in the size of the amplification product. The point mutation can be identified by hybridizing the amplified DNA to the DNA encoding the labeled polypeptide.
[0064]
By detecting the mutation, decrease or increase of the polypeptide, peptide or polynucleotide according to the present invention by such measurement, it becomes possible to diagnose diseases associated with the polypeptide or peptide, such as epithelial cancer or adenocarcinoma. .
[0065]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
In the examples, experiments using human-derived samples were performed with informed consent of all patients who are sample donors.
[0066]
Embodiment 1
(Identification of cDNA clone encoding tumor antigen)
As a tumor-specific cytotoxic T cell, a method described in the literature (Non-Patent Documents 7 and 19) was used from a tumor-infiltrating lymphocyte of a colon cancer patient (HLA-A0207 / 3101, HLA-B46 / 51, HLA-Cw1). OKA31-CTL, a subline of OK-CTL established according to the standard, was used. OKA31-CTL responds to HLA-A31 relatively strongly as compared with other sublines, and recognizes and activates a tumor antigen in an HLA-A31-restricted manner. For example, a human gastric adenocarcinoma cell line MKN28 (a cell line whose genotype is HLA-A31 but whose phenotype does not detect HLA-A31) is obtained by expressing HLA-A31 by gene transfer (hereinafter referred to as MKN28-A31). ) Or HLA-A31 such as the human lung adenocarcinoma cell line LC-1 (A3101 / 3302). ⁺ Recognized target cells and promoted IFN-γ production. further, ⁵¹ In a conventional cytotoxicity test using these Cr-labeled target cells, ⁵¹ Cr release was observed, indicating that OKA31-CTL damages and lyses these target cells. However, HLA-A31 ⁻ Tumor cells (eg, MKN28, colon adenocarcinoma cell line SW620 (A0201 / 2402) and lung adenocarcinoma cell line 1-87 (A0207 / 1101)), and HLA-A31 stimulated with PHA ⁺ It did not show cytotoxic activity against normal T cells. MKN28-A31 or HLA-A31 by OKA31-CTL ⁺ The promotion of IFN-γ production observed as a result of recognition of LC-1 was inhibited by an anti-HLA-class I monoclonal antibody or an anti-CD8 monoclonal antibody. From these, OKA31-CTL is considered to be a CTL that recognizes a tumor antigen in an HLA-A31-restricted manner.
[0067]
The present inventors have already used a gene expression cloning method from a cDNA library of human colon cancer cell line SW620 to encode a tumor antigen that activates an HLA-A2-restricted OK-CTL subline that strongly reacts to HLA-A2. And identified genes. Using these genes as targets, genes encoding tumor antigens that activate OKA31-CTL in an HLA-A31-restricted manner were identified.
[0068]
The isolation and identification of the gene encoding the tumor antigen that activates the HLA-A2-restricted OK-CTL subline from the cDNA library of SW620 cells was performed as follows. First, poly (A) of SW620 cells ⁺ The RNA was converted to cDNA, ligated to a SalI adapter, and inserted into the expression vector pCMV-SPORT-2 (Invitrogen). In addition, HLA-A0207 cDNA was obtained by RT-PCR and cloned into the eukaryotic cell expression vector pCR3 (Invitrogen).
[0069]
The SW620 cell cDNA library was pooled by 100 clones each, and 200 ng of cDNA pooled for each well and 200 ng of HLA-A0207 cDNA were mixed for 30 minutes in 100 μl of lipofectamine (Invitrogen) / Opti-MEM (Invitrogen) 1: 200 mixture. 50 μl of this mixture was used for COS-7 cells (cell number 1 × 10 ⁴ ), And co-transfected by incubation for 6 hours. Then, after adding RPMI-1640 medium containing 10% FCS and culturing for 2 days, the HLA-A2-restricted OK-CTL subline (2 × 10 ⁵ ) Was added to each well. After further incubation for 18 hours, 100 μl of the supernatant was taken, and the produced IFN-γ was measured by ELISA. At this time, IFN-γ production by a HLA-A2-restricted OK-CTL subline was examined using COS-7 cells into which no gene was introduced as a negative control, and the value of IFN-γ produced was used as a background value. Subtracted from measurements. A cDNA in which the promotion of IFN-γ production from the HLA-A2-restricted OK-CTL subline was recognized was determined to have a positive CTL activating ability, and a pool of the cDNA library was screened by the criteria.
[0070]
Clones were individually taken out from the pool of the SW620 cDNA library in which the CTL activating ability was recognized, and further subjected to screening to select CTL activating ability-positive clones. The dose dependence of the obtained clones in activating the HLA-A2-restricted OK-CTL subline was examined, and finally 65 clones were obtained. The nucleotide sequence of the obtained cDNA clone was determined using a DNA sequencing kit (Perkin-Elmer) using ABI PRISM. ^TM Dideoxynucleotide sequencing was performed using a 377 DNA Sequencer (Perkin-Elmer). Further, the amino acid sequence encoded by the cDNA clone was deduced from the nucleotide sequence.
[0071]
Next, each of the 65 cDNA clones was co-transfected into COS-7 cells together with the HLA-A3101 cDNA cloned into pCR3 in the same manner as described above. Activation of OKA31-CTL by the COS-7 cells was examined in the same manner as described above.
[0072]
As a result, the five cDNA clones of clone 40, clone 45, clone 56, clone 76 and clone 85 activated OKA31-CTL in a HLA-A31-restricted and dose-dependent manner, respectively, and promoted IFN-γ production. . On the other hand, in COS-7 cells into which only the expression vector pCMV-SPORT-2 was co-transfected together with HLA-A3101, IFN-γ production from OKA31-CTL was not promoted.
[0073]
A homology search was performed on GenBank for each of the five genes obtained. As a result, it was revealed that the gene had high homology to the genes shown in Table 1 above. However, no data has been reported so far that these genes encode tumor antigens that induce and / or activate CTL in an HLA-A31 restricted manner.
[0074]
Embodiment 2
(Identification of tumor antigen peptide)
In order to obtain a tumor antigen peptide from the above gene encoding a tumor antigen, BIMAS discloses a database "HLA peptide" based on the amino acid sequences encoded by clones 40, 45, 56, 76 and 85, respectively. A plurality of different 9mers each using the “HLA Peptide Binding Predictions” ”(http://bimas.dcrt.nih.gov/molbio/hla_bind) and referring to a previously reported report (Non-Patent Documents 16-18). And designed and synthesized an 11-mer peptide.
[0075]
After synthesizing each peptide into COS-7 cells or OK-EBV-B cells in which HLA-A3101 was transfected and expressed by a conventional method, the cells were cultured with OKA31-CTL and produced from OKA31-CTL. By quantifying the IFN-γ, a peptide that activates CTL in an HLA-A31-restricted manner was selected. OK-EBV-B cells are infinitely proliferating cultured cell lines obtained by infecting peripheral blood B cells of colorectal cancer patient OK with Epstein-Barr Virus (EBV). That is, OKA31-CTL and OK-EBV-B cells are cell lines derived from the same individual.
[0076]
More specifically, COS-7 cells or OK-EBV-B cells into which the HLA-A3101 gene was introduced were combined with the above synthesized peptides (10 μM) in 5% CO 2. ₂ The peptide was bound to HLA-A31 expressed on the cell surface by incubation for 3 hours at 37 ° C under -95% air (Air). COS-7 cells or OK-EBV-B cells pulsed with the peptide in this manner were used as target cells (T). OKA31-CTL was used as an effector cell (E). Cell count 1 × 10 ⁴ Was mixed with an effector cell in an amount 5 to 20 times that of the target cell (E / T ratio = 5 to 20), and the mixture was incubated for 18 hours. At this time, the effector cell and the target cell were combined so that the peptide used for inducing the effector cell and the peptide pulsed to the target cell were the same. After the incubation, 100 µl of the supernatant was recovered, and IFN-γ was measured by ELISA. The same measurement was performed using COS-7 cells pulsed with a peptide not recognized by OKA31-CTL as a negative control. As a result, eight kinds of peptides consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8 were recognized by OKA31-CTL, and promoted the production of IFN-γ from the OKA31-CTL. The resulting eight peptides were 40-P47 from clone 40 (SEQ ID NO: 1), 45-P54 from clone 45 (SEQ ID NO: 2), 56-P28, 56-P31, and 56-P from clone 56. P32 (SEQ ID NOS: 3-5), 76-P20 from clone 76 (SEQ ID NO: 6), and 85-P10 (SEQ ID NO: 7) and 85-P11 (SEQ ID NO: 8) from clone 85. The results are shown in FIGS.
[0077]
Embodiment 3
(Induction of CTL from peripheral blood mononuclear cells of cancer patients by peptide)
Of the eight peptides obtained in Example 2, 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76 -P20 (SEQ ID NO: 6), 85-P10 (SEQ ID NO: 7) and 85-P11 (SEQ ID NO: 8) were examined for their ability to induce CTL from human PBMC. PBMC is HLA-A31 ⁺ 10 cancer patients (4 prostate cancer patients, 2 colorectal cancer patients, 1 gastric cancer patient, 2 cervical cancer patients and 1 breast cancer patient) and HLA-A31 ⁺ Each sample was prepared from blood samples of 6 healthy subjects according to a conventional method. The determination of the HLA class I phenotype of PBMC was performed by a conventional serological method (Non-Patent Document 15).
[0078]
Each PBMC (cell count 1 × 10 ⁵ ) With a culture medium [45% RPMI-1640 medium, 45% AIM-V medium (Invitrogen), 100 U / ml IL-2, 0.1 mM MEM non-essential amino acid solution (Invitrogen) and 10% fetal calf Consists of serum (FCS). In each well of a 96-well U-bottomed microculture plate (Nunc) to which 200 µl was added, each well was incubated with 10 µM of each peptide. On the third and sixth days of the culture, half of the medium was removed, and replaced with a medium having the above composition containing the corresponding peptide. The cells were collected on the 9th day of the culture, further cultured in the absence of peptide by adding only IL-2, and collected and washed on the 12th day of the culture to be used as effector cells (E). Effector cells were mixed with various target cells (T) at an E / T ratio of 10 and 5% CO2 ₂ Incubation was performed at 37 ° C. for 18 hours under the condition of −95% Air. 100 μl of the supernatant after the incubation was collected, and the amount of IFN-γ produced was measured by ELISA. As a target cell, HLA-A31 ⁺ Human lung adenocarcinoma cell line LC-1 (A3101 / 3302), HLA-A31 ⁻ Tumor cells (for example, colon adenocarcinoma cell line SW620 (A0201 / 2402), lung adenocarcinoma cell line 1-87 (A0207 / 1101), and MKN28 cells), MKN28-A31 cells, or the same peptide as the peptide used in the culture. Pulsed cells were used. C1R cells transfected with HLA-A31 cDNA (hereinafter abbreviated as C1R-A31) were used as cells to be pulsed with the peptide. When C1R-A31 pulsed with a peptide is used as a target cell, a human immunodeficiency virus-derived peptide that does not induce HLA-A31-restricted CTL (HIV peptide: RLRFLLLIVTR, SEQ ID NO: 20) is used as a negative control. The amount of IFN-γ production of the CTL that recognized the cells pulsed with, was subtracted from each measurement value as a background. When target cells other than C1R-A31 were used, the amount of IFN-γ production when only effector cells were incubated was subtracted from each measurement value as a background. Each of the calculated measurement values (NET) was statistically processed by the Student's t test, and those having a P value of 0.05 or less were regarded as having a significant difference.
[0079]
Table 2 shows representative examples of the results obtained when the C1R-A31 cells pulsed with the peptide were used as target cells. 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85-P10 (Sequence No. No. 7) or PBMCs derived from 10 cancer patients cultured with 85-P11 (SEQ ID NO: 8), respectively, 5 (50%), 3 (30%), 4 (40%), 4 Example (40%), 5 (50%), 5 (50%), and 7 (70%) PBMCs promoted IFN-γ production. That is, it is considered that these peptides promoted IFN-γ production from PBMCs derived from cancer patients in response to target cells. On the other hand, regarding IFN-γ production from PBMCs derived from 6 healthy individuals, 56-P31 (SEQ ID NO: 4) was 2 cases, 40-P47 (SEQ ID NO: 1), 56-P32 (SEQ ID NO: 5), 76 -P20 (SEQ ID NO: 6) and 85-P10 (SEQ ID NO: 7) promoted in one case each, while 56-P28 (SEQ ID NO: 3) and 85-P11 (SEQ ID NO: 8) did not.
[0080]
[Table 2]

[0081]
In addition, PBMC cultured with each peptide was used as a target cell for HLA-A31. ⁺ The use of tumor cell LC-1 also promoted IFN-γ production. On the other hand, HLA-A31 ⁻ When tumor cells (SW620, 1-87, MKN-28) were used as target cells, they did not promote IFN-γ production. However, it reacted with MKN28 cells into which HLA-A31 cDNA was introduced and expressed, and promoted IFN-γ production. Representative results are shown in FIG. 8 for patient example 4 (prostate cancer), patient example 5 (colorectal cancer) and patient example 8 (cervical cancer). IFN-γ production from cancer patient PBMC in response to target cells was inhibited by anti-HLA-class I or anti-CD8 antibodies, but not by anti-HLA-class II, anti-CD4, anti-CD14 or anti-HLA-antibodies. It was not inhibited by the A24 antibody. That is, IFN-γ producing cells in PBMC cultured with each peptide were CD8 ⁺ Is considered to be an HLA-class I restricted CTL with a phenotype of
[0082]
PBMC cultured with each peptide promoted IFN-γ production when the peptide used for culture was cultured with COS-7 cells into which cDNA encoding the same peptide and HLA-A31 cDNA had been co-transfected. .
[0083]
From these, 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85- P10 (SEQ ID NO: 7) and 85-P11 (SEQ ID NO: 8) were found to induce HLA-A31 restricted CTL from PBMCs with cancer.
[0084]
Embodiment 4
(Cytotoxic activity of CTL induced by peptide)
In Example 3, PBMC cultured for 12 days with the peptide was further cultured for 10 days or more in the presence of IL-2, and then used as effector cells. ⁵¹ It was measured by a Cr release test (Non-Patent Document 15). That is, with effector cells ⁵¹ After being mixed with each Cr-labeled target cell at an E / T ratio of 3, 10 or 30, and incubated for 6 hours, it was released into the supernatant. ⁵¹ The radioactivity of Cr was measured. The obtained results were expressed as% lysis (% lysis) calculated assuming radioactivity when the target cells were completely lysed to be 100%. As a target cell, HLA-A31 ⁺ Tumor cells LC-1, HLA-A31 ⁻ Tumor cells SW620 or 1-87, HLA-A31 ⁺ Normal cells, PHA-blastoid T cells, P-cell lines transformed with Epstein Barr virus (EBV-B) and C1R-A31 were used.
[0085]
PBMC cultured with each peptide were HLA-A31 ⁺ Tumor cells were specifically lysed. Representative results are shown in FIG. From this, 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85 -P10 (SEQ ID NO: 7) and 85-P11 (SEQ ID NO: 8) were found to induce HLA-A31-restricted tumor-specific CTL from PBMC of cancer patients.
[0086]
To further demonstrate that peptide induced CTL induction from PBMCs was peptide specific, a cold target inhibition assay was performed. That is, with effector cells ⁵¹ It is performed using Cr-labeled MKN28-A31 cells (sometimes called hot). ⁵¹ In the Cr release test, C1R-A31 cells (sometimes called cold) pulsed with the same peptide as that used to obtain effector cells were added at a cold / hot ratio of 10 and incubated for 6 hours. Released in the supernatant ⁵¹ The radioactivity of Cr was measured. As a negative control, C1R-A31 cells pulsed with the HIV peptide (SEQ ID NO: 20) were used. FIG. 10 shows, as a typical example, the results of using PBMC derived from patient example 5 (colorectal cancer) cultured on 56-P28 (SEQ ID NO: 3) as effector cells. The effector cells exhibited cytotoxic activity against MKN28-A31 cells, and lysed the MKN28-A31 cells. However, when a cell obtained by adding cold to MKN28-A31 cells was used as a target cell, the cytotoxic activity of the effector cells on MKN28-A31 cells was significantly reduced. On the other hand, addition of the negative control did not affect the cytotoxic activity of the effector cells. This proved that CTLs derived from PBMC by the peptide were specific to the peptide.
[0087]
Embodiment 5
(Induction of CTL from peripheral blood mononuclear cells of cancer patients by peptide)
Peptides 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85-P10 ( CTL inducibility from human PBMC was examined for SEQ ID NO: 7) and 85-P11 (SEQ ID NO: 8). PBMC is HLA-A33 ⁺ 6 cancer patients (2 lung cancer patients, 3 prostate cancer patients and 1 colon cancer patient) and HLA-A11 ⁺ It was prepared from each blood sample of 9 cancer patients (2 lung cancer patients, 5 prostate cancer patients, 1 bladder cancer patient, and 1 melanoma patient) according to a conventional method.
[0088]
Each PBMC (cell count 1 × 10 ⁵ ) Was cultured with 10 μM of each peptide by the method described in Example 3 and used as effector cells. C1R-A33 cells or C1R-A11 cells pulsed with each peptide were used as target cells. C1R-A33 cells and C1R-A11 cells were prepared by introducing HLA-33 cDNA and HLA-A11 cDNA into C1R cells by a conventional method, respectively. The effector cells and the target cells are combined so that the peptides used for culture or pulse coincide, mixed at an E / T ratio of 10 and mixed with 5% CO2. ₂ Incubation was performed at 37 ° C. for 18 hours under the condition of −95% Air. 100 μl of the supernatant after the incubation was collected, and the amount of IFN-γ produced was measured by ELISA. In addition, an HIV peptide that does not induce HLA-A31-restricted CTL (SEQ ID NO: 20) was used as a negative control, and the amount of IFN-γ production of CTL that recognized cells pulsed with the HIV peptide was subtracted from each measurement value as a background. . Influenza virus-derived peptides (Flu peptide: NVKNLYEKVK, SEQ ID NO: 21) and Epstein-Barr virus-derived peptides (EBV peptide: IVTDFSVIK, SEQ ID NO: 22) were used as positive controls. Each of the calculated measurement values (NET) was statistically processed by the Student's t test, and those having a P value of 0.05 or less were regarded as having a significant difference. HLA-A33 ⁺ PBMC and HLA-A11 from cancer patients ⁺ The results of using PBMCs derived from cancer patients as effector cells are shown in Tables 3 and 4, respectively. In these tables, those in which promotion of IFN-γ production was found to be significant are underlined.
[0089]
56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85-P10 (SEQ ID NO: 7) or 85-P11 (SEQ ID NO: 7) No. 8) and 6 cases of HLA-A33 cultured respectively. ⁺ Of PBMCs derived from cancer patients, 2 (33%), 1 (17%), 1 (17%), 1 (17%), 4 (67%), and 4 (67%) ) PBMC promoted IFN-γ production (Table 3). That is, these peptides were used in response to HLA-A33 in C1R-A33 cells pulsed with the peptide. ⁺ It is considered that IFN-γ production from PBMCs derived from cancer patients was promoted.
[0090]
Also, 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), or 85-P20 (SEQ ID NO: 6) 9 cases of HLA-A11 cultured together with P10 (SEQ ID NO: 7) ⁺ Of PBMCs from cancer patients, 2 (22%), 4 (44%), 2 (22%), 1 (11%), 2 (22%), and 4 (44%), respectively ) Promoted IFN-γ production (Table 4). That is, these peptides were used in response to HLA-A11 in C1R-A11 cells pulsed with the peptide. ⁺ It is considered that IFN-γ production from PBMCs derived from cancer patients was promoted.
[0091]
[Table 3] HLA-A33 ⁺ CTL induction from PBMCs derived from cancer patients

[0092]
[Table 4] HLA-A11 ⁺ CTL induction from PBMCs derived from cancer patients

In Tables 3 and 4, NT means not tested.
[0093]
HLA-A33 cultured on 85-P11 (SEQ ID NO: 8) ⁺ The promotion of IFN-γ production in the response of PBMCs derived from cancer patients to C1R-A33 cells pulsed with the peptide was inhibited by an anti-class I antibody or an anti-class CD8 antibody (FIG. 11A). In addition, HLA-A11 cultured on 85-P10 (SEQ ID NO: 7) ⁺ The promotion of IFN-γ production in the response of cancer patient-derived PBMC to the peptide-pulsed C1R-A11 cells was also inhibited by anti-class I or anti-class CD8 antibodies (FIG. 11B). From these results, it is considered that cells in which IFN-γ production was promoted by the above-mentioned peptide are cells showing the phenotype of the class I antigen and CD8, that is, CTLs.
[0094]
Thus, the present peptide is HLA-A31 ⁺ HLA-A11 as well as cancer patients ⁺ Cancer patient or HLA-A33 ⁺ It was revealed that each HLA-A-restricted CTL was induced from PBMC of a cancer patient.
[0095]
Embodiment 6
(Cytotoxic activity of CTL induced by peptide)
In Example 5, PBMC cultured for 12 days with the peptide was further cultured for at least 10 days in the presence of IL-2, and then used as effector cells. ⁵¹ It was measured by a Cr release test (Non-Patent Document 15). As target cells, lung adenocarcinoma cell line LC-1 (A3101 / 3302), cervical cancer cell line RKN cells (A3302 /), lung adenocarcinoma cell line 11-18 (A0201 / A2402), lung adenocarcinoma cell line 1 -87 (A0207 / 1101), lung squamous cell carcinoma cell line QG56 (A2601 /), esophageal cancer cell line KE5 (A1101 /), colon cancer cell line SW620 (A0201 / 2402), HLA-A33 ⁺ Or HLA-A11 ⁺ PHA blast-forming T cells, which are normal cells, were used.
[0096]
HLA-A33 cultured with each peptide ⁺ PBMCs derived from cancer patients are HLA-A33 ⁺ Tumor cells were specifically lysed. Representative results are shown in FIG. From this, 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), 85-P10 (SEQ ID NO: 7) or 85 -P11 (SEQ ID NO: 8) is HLA-A33 ⁺ It has been revealed that tumor-specific CTLs are induced from PBMC of cancer patients.
[0097]
In addition, HLA-A11 cultured with each peptide ⁺ PBMCs derived from cancer patients are HLA-A11 ⁺ Tumor cells were specifically lysed. Representative results are shown in FIG. From this, 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), or 85-P10 (SEQ ID NO: 7) is HLA-A11 ⁺ It has been revealed that tumor-specific CTLs are induced from PBMC of cancer patients.
[0098]
Further, a cold target inhibition assay was performed in the same manner as in Example 4. As effector cells, PBMC derived from a lung cancer patient cultured with 56-P31 (SEQ ID NO: 4) (HLA-A11 /) or PBMC derived from a lung cancer patient cultured with 85-P10 (SEQ ID NO: 7) (HLA-A11 /) were used. . As a positive control, these PBMCs cultured with the Flu peptide (SEQ ID NO: 21) were used. As a hot, ⁵¹ Cr-labeled lung squamous cell carcinoma cell line QG56 (A2601 /) or esophageal cancer cell line KE5 (A1101 /) were used. As the cold, C1R-A11 cells pulsed with 56-P31 (SEQ ID NO: 4), 85-P10 (SEQ ID NO: 7) or HIV peptide (SEQ ID NO: 20) were used.
[0099]
As shown in FIG. 14A, lung cancer patient-derived PBMC (HLA-A11 /) cultured with 56-P31 (SEQ ID NO: 4) showed stronger cytotoxicity against KE5 cells than QG56 cells. From this, it became clear that the PBMC exhibited cytotoxic activity in an HLA-A11-restricted manner. When a cell (C1R-A11 cell pulsed with 56-P31 (SEQ ID NO: 4)) was added to KE5 cell as a target cell, the cytotoxic activity of the PBMC on KE5 cell was significantly reduced. On the other hand, addition of C1R-A11 cells pulsed with the HIV peptide (SEQ ID NO: 20) did not affect the cytotoxic activity of the PBMC on KE5 cells. In addition, as shown in FIG. 14B, the cytotoxic activity of PBMC (HLA-A11 /) derived from a lung cancer patient cultured with 85-P10 (SEQ ID NO: 7) on KE5 cells was also similar to that of cold (85-P10 (SEQ ID NO: 7)). 7) was significantly reduced by the addition of (C1R-A11 cells pulsed).
[0100]
These revealed that HLA-A11-restricted CTLs derived from PBMC by these peptides were peptide-specific. Further, it was revealed that the CTL recognizes a complex of HLA-A11 and the peptide.
Embodiment 7
(Expression of each clone in tissues and various cancer cells)
Among clones 40, 45, 56, 76 and 85 obtained in Example 1, Northern blotting analysis of mRNA expression in tissues and various cancer cells was carried out for clones 40, 56, 76 and 85. It was examined by. Northern blotting analysis was performed using various tumor cells (lung adenocarcinoma cell line 1-87, gastric adenocarcinoma cell line MKN-28, lung adenocarcinoma cell line LC-1, colon cancer cell line SW620 and esophagus cancer cell line KE4) or healthy individuals. It was performed according to the method described in a previously reported report (Non Patent Literature 29) using total RNA extracted from PBMCs derived from the cells or total RNA derived from each tissue. The probe cDNA is ³² The following were used, which were P-labeled; the cDNA of the 0.9-kb fragment at positions 420 to 1,314 of clone 40 digested with EcoRI; the 1.3-kbp fragment of positions 334 to 1,598 of clone 56 digested with XbaI. cDNA; cDNA of the 0.749 kbp fragment at positions 494 to 1,169 of clone 78 digested with BarI and NcoI; cDNA of the 0.9 kbp fragment at positions 858 to 1,798 of clone 85 digested with HindIII. In addition, β-actin mRNA was similarly detected as a control of the expression level. As a probe for detecting β-actin mRNA, human β-actin cDNA (Clonetech) was used. Based on the obtained results, the relative expression levels were calculated by the following formula 1, and are shown in Table 5.
[0101]
(Equation 1)
Index = (concentration of each clone in sample / β-actin concentration of sample) × (β-actin concentration of SW620 cells / concentration of each clone of SW620 cells)
[0102]
[Table 5]

[0103]
Clone 40, clone 56, clone 76 and clone 85 were all expressed at high levels in all tumor cells examined. Clone 40, clone 56, and clone 76 were also expressed in testis, but at levels similar or lower than tumor cells. On the other hand, the expression level in normal tissues other than the testis was low. Clone 85 had a high expression level in normal tissues and was strongly expressed in testis, but the expression level in other normal tissues was lower than that in tumor cells.
[0104]
【The invention's effect】
A gene encoding a tumor antigen recognized by HLA-A31-restricted tumor-specific cytotoxic T cells was isolated and identified from human colon cancer cell line SW620. Furthermore, a peptide having an epitope of the tumor antigen was found based on the polypeptide encoded by the obtained gene.
The peptide is HLA-A31 ⁺ Cancer patient, HLA-A11 ⁺ Cancer patient or HLA-A33 ⁺ From peripheral blood mononuclear cells prepared from the blood of a cancer patient, tumor-specific cytotoxic T cells that recognize the peptide and polypeptide and damage tumor cells were induced. The HLA-A31, HLA-A11 or HLA-A33 allele is an HLA belonging to the HLA-A3 supertype and is an HLA-A allele that is expressed in many of the world's population. Thus, the present peptide is characterized in that even one peptide can induce one or more HLA-A-restricted tumor-specific cytotoxic T cells belonging to the HLA-A3 supertype. Therefore, the present invention may be applicable to the specific immunotherapy of the majority of cancer patients, and is very useful.
In addition, by using the present polypeptide or peptide in combination with a previously reported tumor antigen peptide, it is possible to induce and / or activate a plurality of types of HLA-restricted cytotoxic T cells. The present invention makes it possible to respond to the diversity of cancer.
Thus, the present invention allows for specific immunotherapy of cancer and the like, for example, prostate cancer, colon cancer, stomach cancer, cervical cancer, breast cancer, lung adenocarcinoma, esophageal cancer, bladder cancer, melanoma, etc. A great contribution can be expected. In particular, it is useful for the development of specific immunotherapy for cancer patients in which one or both of the HLA phenotypes are HLA belonging to the HLA-A3 supertype. The present invention also greatly contributes to basic research on molecules related to recognition of cancer by cytotoxic T cells.
[0105]
[Sequence List Free Text]
SEQ ID NO: 1: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells.
SEQ ID NO: 2: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells.
SEQ ID NO: 3: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells.
SEQ ID NO: 4: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells
SEQ ID NO: 5: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells.
SEQ ID NO: 6: Designed peptide recognized by HLA-A31-restricted cytotoxic T cells.
SEQ ID NO: 7: designed peptide recognized by HLA-A31-restricted cytotoxic T cell
SEQ ID NO: 8: designed peptide recognized by HLA-A31-restricted cytotoxic T cell
SEQ ID NO: 20: Peptide designed from HIV protein
SEQ ID NO: 21: Peptide designed from influenza virus protein
SEQ ID NO: 22: Peptide designed from EBV protein
[0106]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows that peptide 40-P47 (SEQ ID NO: 1) activated HLA-A31-restricted cytotoxic T cells (OKA31-CTL) and promoted IFN-γ production. In the figure, NC means a negative control.
FIG. 2. Peptides 45-P54 (SEQ ID NO: 2) and 40-P47 (SEQ ID NO: 1) activated HLA-A31-restricted cytotoxic T cells (OKA31-CTL) to promote IFN-γ production FIG. In the figure, NC means a negative control.
FIG. 3 shows that peptide 56-P28 (SEQ ID NO: 3) activated HLA-A31-restricted cytotoxic T cells (OKA31-CTL) and promoted IFN-γ production. In the figure, NC means a negative control.
FIG. 4. Peptides 56-P28 (SEQ ID NO: 3) and 56-P31 (SEQ ID NO: 4) activate HLA-A31-restricted cytotoxic T cells (OKA31-CTL) to promote IFN-γ production FIG. In the figure, NC means a negative control.
FIG. 5. Peptides 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5) and 76-P20 (SEQ ID NO: 6) activate HLA-A31 restricted cytotoxic T cells (OKA31-CTL) FIG. 2 shows that IFN-γ production was promoted by the method. In the figure, NC means a negative control.
FIG. 6 shows that peptide 85-P10 (SEQ ID NO: 7) activated HLA-A31-restricted cytotoxic T cells (OKA31-CTL) and promoted IFN-γ production. In the figure, NC means a negative control.
FIG. 7 shows that peptide 85-P11 (SEQ ID NO: 8) activated HLA-A31-restricted cytotoxic T cells (OKA31-CTL) and promoted IFN-γ production. In the figure, NC means a negative control.
FIG. 8A shows HLA-A31 cultured with peptide 85-P11 (SEQ ID NO: 8) or 56-P28 (SEQ ID NO: 3). ⁺ Peripheral blood mononuclear cells (PBMCs) derived from cancer patients are HLA-A31 ⁺ Promoted IFN-γ production in response to lung adenocarcinoma cell lines, but with HLA-A31 ⁻ FIG. 7 shows that IFN-γ production was not promoted in response to tumor cells (1-87 and SW620). FIG. 8B shows HLA-A31 cultured with peptide 85-P11 (SEQ ID NO: 8). ⁺ PBMC derived from a cancer patient is HLA-A31 ⁻ FIG. 7 shows that IFN-γ production was not promoted in response to tumor cell MKN28, but IFN-γ production was promoted in response to MKN28 into which HLA-A31 cDNA was introduced (MKN28-A31). FIG. 8C shows HLA-A31 cultured with peptide 56-P28 (SEQ ID NO: 3). ⁺ PBMCs derived from cancer patients promoted IFN-γ production in response to C1R-A31 cells pulsed with the peptide, but promoted IFN-γ production in response to C1R-A31 cells pulsed with HIV peptide (SEQ ID NO: 20). It is a figure showing that it did not do. 8A-C, peptide-stimulated IFN-γ production was inhibited by anti-HLA-class I or anti-CD8 antibodies. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 9 shows peptides 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 56-P32 (SEQ ID NO: 5), 76-P20 (SEQ ID NO: 6), HLA-A31 cultured on 85-P10 (SEQ ID NO: 7) or 85-P11 (SEQ ID NO: 8) ⁺ Peripheral blood mononuclear cells from a cancer patient are HLA-A31 ⁺ Lung adenocarcinoma cell line LC-1 was lysed, but HLA-A31 ⁻ Lung adenocarcinoma cell lines 1-87 and HLA-A31 ⁺ It is a figure which shows that PHA blasting normal T cells did not lyse. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 10: HLA-A31 cultured with peptide 56-P28 (SEQ ID NO: 3) in a cold target inhibition assay ⁺ The lytic effect of peripheral blood mononuclear cells from cancer patients on MKN28-A31 cells was inhibited by the addition of C1R-A31 cells pulsed with the peptide, whereas C1R-A31 pulsed with the HIV peptide (SEQ ID NO: 20) It is a figure which shows that it was not inhibited. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 11A shows HLA-A33 cultured with peptide 85-P11 (SEQ ID NO: 8). ⁺ FIG. 2 shows that peripheral blood mononuclear cells (PBMC) derived from a cancer patient promoted IFN-γ production in response to C1R-A31 pulsed with the peptide. On the other hand, C1R-A31 pulsed with the HIV peptide (SEQ ID NO: 20) did not recognize and did not promote IFN-γ production. Further, promotion of IFN-γ production was inhibited by the anti-HLA-class I antibody or the anti-CD8 antibody. FIG. 11B shows HLA-A11 cultured with peptide 85-P10 (SEQ ID NO: 7). ⁺ FIG. 4 shows that peripheral blood mononuclear cells (PBMC) derived from a cancer patient promoted IFN-γ production in response to C1R-A11 pulsed with the peptide. On the other hand, C1R-A11 pulsed with the HIV peptide (SEQ ID NO: 20) did not recognize and did not promote IFN-γ production. Further, promotion of IFN-γ production was inhibited by the anti-HLA-class I antibody or the anti-CD8 antibody. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 12. Peptide 56-P28 (SEQ ID NO: 3), 56-P31 (SEQ ID NO: 4), 76-P20 (SEQ ID NO: 6), 85-P10 (SEQ ID NO: 7) or 85-P11 (SEQ ID NO: 8) Cultured HLA-A33 ⁺ Peripheral blood mononuclear cells from a cancer patient are HLA-A33 ⁺ Tumor cell lines (LC-1 and RKN) were lysed, but HLA-A31 ⁻ Tumor cell lines (11-87 and 1-87) and HLA-A33 ⁺ It is a figure showing that PHA blasting normal T cells (PHA-blast) were not lysed. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 13: HLA-A11 cultured with peptides 40-P47 (SEQ ID NO: 1), 56-P28 (SEQ ID NO: 3), 76-P20 (SEQ ID NO: 6) or 85-P10 (SEQ ID NO: 7) ⁺ Peripheral blood mononuclear cells from a cancer patient are HLA-A11 ⁺ Tumor cell lines (1-87 and KE5) were lysed, but HLA-A11 ⁻ Tumor cell lines (MKN28, RKN and SW620) and HLA-A11 ⁺ It is a figure showing that PHA blasting normal T cells (PHA-blast) were not lysed. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.
FIG. 14A shows HLA-A11 cultured with peptide 56-P31 (SEQ ID NO: 4) in a cold target inhibition assay. ⁺ The lytic effect of peripheral blood mononuclear cells derived from lung cancer patients on KE5 cells was inhibited by the addition of C1R-A11 cells pulsed with the peptide, but not with C1R-A11 pulsed with the HIV peptide (SEQ ID NO: 20). It is a figure showing that there was no. FIG. 14B shows HLA-A11 cultured with peptide 85-P10 (SEQ ID NO: 7) in the same assay. ⁺ The lytic effect of peripheral blood mononuclear cells from bladder cancer cancer patients on KE5 cells was inhibited by the addition of C1R-A11 cells pulsed with the peptide, whereas C1R-A11 pulsed with the HIV peptide (SEQ ID NO: 20) It is a figure which shows that it was not inhibited. In the figure, an asterisk (*) indicates that a significant difference was recognized by the Student's t test.

Claims (35)

A peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 8 in the sequence listing. Induces HLA-A-restricted cytotoxic T cells consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing and belonging to one or more HLA-A3 supertypes and / or A polypeptide recognized by cytotoxic T cells. The polypeptide according to claim 2, wherein the HLA-A belonging to the HLA-A3 supertype is HLA-A selected from the group consisting of HLA-A31, HLA-A11 and HLA-A33. A peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8 and one or more HLA-A3 superscripts consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing A medicament comprising one or more peptides and / or polypeptides selected from the group consisting of polypeptides which induce HLA-A-restricted cytotoxic T cells belonging to the type and / or are recognized by cytotoxic T cells . The medicament according to claim 4, wherein the HLA-A belonging to the HLA-A3 supertype is HLA-A selected from the group consisting of HLA-A31, HLA-A11 and HLA-A33. A peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8 and one or more HLA-A3 superscripts consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing It contains one or more peptides and / or polypeptides selected from the group consisting of polypeptides that induce HLA-A-restricted cytotoxic T cells belonging to the type and / or are recognized by cytotoxic T cells. Cancer vaccine. The cancer vaccine according to claim 6, which is used for a cancer whose HLA phenotype is HLA-A belonging to the HLA-A3 supertype. The cancer vaccine according to claim 7, wherein the cancer is prostate cancer, colon cancer, gastric cancer, cervical cancer, breast cancer, lung cancer, esophageal cancer, bladder cancer or melanoma. The cancer vaccine according to any one of claims 6 to 8, wherein the HLA-A belonging to the HLA-A3 supertype is one or more HLA-A selected from HLA-A31, HLA-A11 and HLA-A33. One or more peptides selected from the peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and the polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14 in the sequence listing An agent for inducing HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes, comprising a peptide and / or polypeptide. The cytotoxic T cell inducer according to claim 10, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11 and HLA-A33. 1 selected from the group consisting of a peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8 and a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14 in the sequence listing; A method for inducing HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes, comprising using one or more peptides and / or polypeptides. The method for inducing cytotoxic T cells according to claim 12, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. A polynucleotide comprising a nucleotide sequence encoding a peptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8, or a complementary nucleotide sequence thereof. Induces HLA-A-restricted cytotoxic T cells consisting of the amino acid sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 14 in the sequence listing and belonging to one or more HLA-A3 supertypes and / or A polynucleotide comprising a nucleotide sequence encoding a polypeptide recognized by cytotoxic T cells or a complementary nucleotide sequence thereof. The nucleotide sequence according to any one of SEQ ID NO: 15 to SEQ ID NO: 19, wherein the polypeptide comprising the amino acid sequence encoded by the nucleotide sequence belongs to one or more HLA-A3 supertypes. A polynucleotide comprising a nucleotide sequence that induces A-restricted cytotoxic T cells and / or is recognized by cytotoxic T cells or a complementary nucleotide sequence thereof. The polynucleotide according to claim 15 or 16, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11 and HLA-A33. A polynucleotide that hybridizes under stringent conditions with the polynucleotide of any one of claims 14 to 17. A recombinant vector containing the polynucleotide according to any one of claims 14 to 18. The recombinant vector according to claim 19, wherein the recombinant vector is an expression recombinant vector. A transformant into which the recombinant vector according to claim 19 or 20 has been introduced. The method for producing the peptide according to claim 1 or the polypeptide according to claim 2 or 3, comprising a step of culturing a transformant into which the recombinant vector according to claim 20 has been introduced. An antibody that immunologically recognizes the peptide of claim 1. Recognition of at least one of the peptide of claim 1 and / or the polypeptide of claim 2 or 3 by HLA-A-restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes. A method for screening a compound that enhances the expression of a polynucleotide and / or a compound that enhances expression of the polynucleotide according to any one of claims 14 to 17, wherein the peptide according to claim 1 or the peptide according to claim 2 or 3. The polypeptide according to claim 14, the polynucleotide according to any one of claims 14 to 18, the recombinant vector according to claim 19 or 20, the transformant according to claim 21, or the transformant according to claim 23. A screening method using at least one of the antibodies. The screening method according to claim 24, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. A compound obtained by the screening method according to claim 24 or 25. Enhanced recognition of at least one of the peptide of claim 1 or the polypeptide of claim 2 or 3 by HLA-A restricted cytotoxic T cells belonging to one or more HLA-A3 supertypes. A compound that enhances expression of the polynucleotide according to any one of claims 14 to 17. 28. The compound according to claim 27, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11 and HLA-A33. 22. A peptide according to claim 1, a polypeptide according to claim 2 or 3, a polynucleotide according to any one of claims 14 to 17, a recombinant vector according to claim 19 or 20, 21. 29. A medicament for treating cancer, comprising the transformant of claim 23, the antibody of claim 23, or at least one of the compounds of any one of claims 26 to 28. Composition. 30. The pharmaceutical composition according to claim 29, wherein the cancer treatment is a cancer treatment for a cancer whose HLA phenotype is HLA-A belonging to the HLA-A3 supertype. 31. The pharmaceutical composition according to claim 30, wherein the HLA-A belonging to the HLA-A3 supertype is at least one HLA-A selected from HLA-A31, HLA-A11, and HLA-A33. The pharmaceutical composition according to any one of claims 29 to 31, wherein the cancer is prostate cancer, colorectal cancer, gastric cancer, cervical cancer, breast cancer, lung cancer, esophageal cancer, bladder cancer or melanoma. A method for quantitatively or qualitatively measuring the peptide according to claim 1, the polypeptide according to claim 2 or 3, or the polynucleotide according to any one of claims 14 to 17. The peptide according to claim 1, the polypeptide according to claim 2 or 3, the polynucleotide according to any one of claims 14 to 17, the recombinant vector according to claim 19 or 20, or the claim. A reagent kit comprising at least one of the antibodies according to 23. A reagent kit used in the screening method according to claim 24 or 25 or the measurement method according to claim 27, wherein the peptide according to claim 1, the polypeptide according to claim 2 or 3, and the reagent according to claim 2 or 3. A reagent kit comprising at least one of the polynucleotide according to any one of claims 1 to 17, the recombinant vector according to claim 19 or 20, or the antibody according to claim 23.

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