JP2004315433A - PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING PAPILLOMA VIRUS-INDUCED DISEASE CONTAINING ANTIGENIC PROTEIN OF PAPILLOMA VIRUS AND CpG-OLIGODEOXYNUCLEOTIDE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for preventing or treating diseases induced by papilloma virus which contains antigenic protein of papilloma virus and CpG-oligodeoxynucleotide. <P>SOLUTION: The composition for preventing or treating cellular proliferative diseases induced by papilloma virus contains immunologically effective amounts of E7 antigenic protein of papilloma virus and CpG-oligodeoxynucleotide. Here, the oligodeoxynucleotide contains 8-40 nucleotides and at least one CpG motif wherein at least one nucleotide separates contiguous CpG motifs in the oligodeoxynucleotide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００４０】
前記結果から明らかなように、ＣｐＧ−ＯＤＮがＥ７抗原特異反応、Ｔｈ増殖反応、ＩＦＮ−γを分泌するＣＤ４＋Ｔリンパ球（Ｔｈ１）及びＩＦＮ−γを分泌するＣＤ８＋Ｔリンパ球（ＣＴＬ）の活性を増加させる補助剤の役割をし、これによりＥ７蛋白質とＣｐＧ−ＯＤＮの同時利用によってのみ抗原特異Ｔｈ１型ＣＤ４＋及び特にＣＤ８＋Ｔ細胞免役反応から優れた乳頭腫ウィルス誘発疾患抑制免役効果、特に子宮頸部癌抑制免役効果を誘導するのに有用であることが立証される。
【００４１】
本発明の薬剤学的組成物は、乳頭腫ウィルスに感染されるか或いは感染されたと疑われる対象に対し予防的又は治療学的に投与することができる。このような投与対象は乳頭腫ウィルスにより誘発される疾患にかかったか或いは発病する可能性のある対象を含む。乳頭腫ウィルスにより誘発される疾患は例えばボーエン様丘疹症、肛門異形成、呼吸又は結膜乳頭腫、子宮頸部異形成、子宮頸部癌、陰部癌、前立腺癌などを含む。
【００４２】
特に、本発明の薬剤学的組成物は、乳頭腫ウィルスにより誘発される子宮頸部癌の予防及び治療に有用である。
【００４３】
本発明の薬剤学的組成物は、単独で投与するか、或いは当分野の他の公知処理、例えば化学療法治療、放射線治療、手術などと共に投与することができる。また、当分野によく知られている他の補助剤又はサイトカインと共同投与することができる。共同投与可能な、免役反応を刺激し得るサイトカインは、例えば顆粒球マクロファージコロニー刺激因子（ＧＭ−ＣＳＦ）、顆粒球コロニー刺激因子（ＧＣＳＦ）、ＩＬ−１、ＩＬ−２、ＩＬ−３、ＩＬ−４、ＩＬ−５、ＩＬ−６、ＩＬ−７、ＩＬ−１０、ＩＬ−１２、ＩＬ−１５、ＴＮＦ−α、ＩＮＦ−γ、Ｆｌｔ３リガンドなどを含む。
【００４４】
本発明の薬剤学的組成物は、当分野で広く知られている方法［参照：Ｄｏｎｎｅｌｌｙ ｅｔ ａｌ．， Ｊ． Ｉｍｍ． Ｍｅｔｈｏｄｓ， １７６： １４５， １９９４； Ｖｉｔｉｅｌｌｏ ｅｔ ａｌ．， Ｊ． Ｃｌｉｎ． Ｉｎｖｅｓｔ， ９５：３４１， １９９５］によって投与することができる。投与形態は錠剤、トローチ剤、分散剤、懸濁剤、液剤、カプセル剤、クリーム剤、軟膏剤、座剤、エアロゾル剤などを含む。前記投与形態は移殖される（ｉｍｐｌａｎｔｅｄ）徐放出考案物などをさらに含む。また、当分野の公知方法、例えば経口又は非経口、例えば筋肉内、静脈内、動脈内、皮内、腹腔内、鼻腔内、膣内、直腸内、舌下または皮下投与するか、胃腸管、粘膜又は呼吸器を介して投与することができる。
【００４５】
本発明の薬剤学的組成物は、局所投与或いは全身系投与を行うことができる。局所投与の場合には全身的吸収を最小化しながら投与部位に薬物を集中させることができて有利である。このような局所投与の際、他の経路による投与の場合に比べて一層少ない量を投与することができる。局所投与時の剤形は経皮考案物、エアロゾル、クリーム剤及び軟膏剤、ローション剤、散剤などを含む。
【００４６】
本発明の薬剤学的組成物は賦形剤を含んだ一つ以上の薬剤学的に許容される担体または製剤化を容易にする任意の補助剤を使用して剤形化することができる。剤形は投与経路によって異なる。注射するための場合、活性成分は水性溶液、好ましくは生理的食塩水中に剤形化することができる。粘膜伝達のための場合、透過される障害物に適した透過剤を剤形化に使用することができる。このような透過剤は当分野で一般的に広く知られている。経口投与の場合には、活性成分を錠剤、丸剤、糖衣錠剤、カプセル、液剤、ゲル剤、シロップ剤、スラリー剤、懸濁剤などに含むのに適した担体と配合することができる。吸入投与のためには、活性成分を適切な推進剤を用いて、加圧されたパックからエアロゾル噴霧形態で伝達されるか、カートリッジに剤形化することが可能な粉末の形態で伝達されることができる。また、注射により投与する場合、懸濁剤、液剤、乳化剤などで形成することができる。
【００４７】
投与用量は１回に約０．１μｇ／ｋｇ／日〜約３μｇ／ｋｇ／日、好ましくは０．５μｇ／ｋｇ／日〜乃至１μｇ／ｋｇ／日で最小限１週日間隔にて投与することができ、体重、年齢、性別、投与経路、剤形及び時間、一般的健康状態などいろいろの要因によって異なる。
【００４８】
以下、本発明を具体的な実施例を挙げて説明する。記述された実施例は説明のためのものに過ぎず、本発明を制限するものではない。
【００４９】
【実施例】
略語説明
ＯＤＮ：オリゴデオキシヌクレオチド、ＨＰＶ：ヒト乳頭腫ウィルス、ＰＢＳ：リン酸緩衝性生理食塩水、ＨＲＰ：ワサビ（ｈｏｒｓｅ ｒａｄｉｓｈ）ペルオキシダーゼ、ＨＳＶ：単純ヘルペスウィルス、ＯＤ：吸光度、ＩＰＴＧ：イソプロピル−Ｄ−チオガラクトピラノシド、ＲＴ−ＰＣＲ：逆転写酵素連鎖反応、ｉ．ｐ．：腹腔内、ｓ．ｃ．：皮下、ＳＩ：刺激指数。
統計分析は対応のあるＳｔｕｄｅｎｔ’ｓＴ検定（Ｔｈｅ ｐａｉｒｅｄ Ｓｔｕｄｅｎｔ’ｓ Ｔ Ｔｅｓｔ）を利用し、ｐ数値＜０．０５の際に統計的有意性を置いた。
【００５０】
実施例１：組換えＥ７蛋白質の製造
乳頭腫ウィルス１６型のＥ７組換え蛋白質を公知の方法［参照：Ｎｏｖａｇｅｎ会社のプロトコル及び Ｓｉｎ． Ｊ． Ｉ． Ｅｔ ａｌ．， Ｖａｃｃｉｎｅ １５： １８２７−１８３３， １９９７］を用いて製造分離した。
子宮頸部癌細胞株（Ｃａｓｋｉ細胞株）から２つのプライマー、すなわち５’−Ｔ ＴＧＧＧＡＴＣＣＡＣＣＡＴＧＣＡＴＧＧＡＧＡＴＡＣＡＣＣＴＡＣ−３’を （配列番号２）（ＢａｍＨＩ切断部位を含む）５’−ＣＧＧＡＡＴＴＣＡＴＴＣＴＴＡＴＧＧＴＴＴＣＴＧ −３’ （配列番号３）（ＥｃｏＲＩ切断部位を含む）を用いて逆転写酵素連鎖反応により乳頭腫ウィルス１６型のＥ７遺伝子を得た。得られたＥ７遺伝子を制限酵素（ＢａｍＨＩ及びＥｃｏＲＩ）を用いて切断した後、ＤＮＡゲルからＥ７遺伝子を分離した。その後、分離されたＥ７遺伝子を、ＢａｍＨＩとＥｃｏＲＩで酵素処理されたｐＥＴベクター（Ｎｏｖａｇｅｎ， Ｍａｄｉｓｏｎ， ＷＴ）にクローニングさせた。クローニングされたＤＮＡを用いてＥ．ｃｏｌｉ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＤＨ５を形質転換させ、カナマイシンの下で成長するＥ．ｃｏｌｉを選別してｐＥＴ−Ｅ７ ＤＮＡを分離した後、これを用いてＥ．ｃｏｌｉ ＢＬ２１（ＤＥ３）を形質転換させた。収得されたＥ．ｃｏｌｉ（ｐＥＴ−Ｅ７）をカナマイシン（３０μｇ／ｍｌ）含有のＬＢ溶液で培養して６００ｎｍ波長でＯＤ値が約０．６〜０．８に到達したとき、１ｍＭのＩＰＴＧを添加した後、３時間培養した。Ｅ．ｃｏｌｉを４ｋｒｐｍで２０分間遠心分離した後、−２０℃で冷凍−解凍過程を１回行った。細胞沈澱物を５ｍｌの８Ｍ尿素緩衝液（ｐＨ８．０）に溶かした後、常温で６０分間撹拌しながら細胞を破壊した。その後、１．５ｋｒｐｍで３０分間遠心分離し、上澄み液を得た後、８Ｍ尿素緩衝液（ｐＨ８．０）で洗浄した後、Ｎｉ−ＮＴＡ樹脂カラム（Ｑｉａｇｅｎ， Ｖａｌｅｎｃｉａ， ＣＡ）に通過させた。次に、樹脂カラムに５倍容積の緩衝液Ｂ（８Ｍ尿素緩衝液、ｐＨ８．０）を通過させた後、５〜１０倍容積の緩衝液Ｃ（８Ｍ尿素緩衝液、ｐＨ６．３）を通過させた。最後に、Ｈｉｓ−ｔａｇＥ７蛋白質を１０ｍｌの緩衝液Ｃ（２００ｍＭのイミダゾールが添加された緩衝液Ｂ）を用いて溶出させた。２時間間隔で蛋白質溶液を６Ｍ尿素緩衝液で透析した後、４Ｍ尿素緩衝液で再び透析した。最後に、生理食塩水で透析した。その後、Ｄｅｔｏｘｉ−Ｇｅｌ内毒素除去カラム（Ｐｉｅｒｃｅ， Ｒｏｃｋｆｏｒｄ， ＩＬ）に透過させて内毒素を除去した。蛋白質濃度は公知のＢｒａｄｆｏｒｄ方法で定量した［参照：Ｂｒａｄｆｏｒｄ， Ｍ． Ｍ．， Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ７２： ２４８−２５４， １９７６］。最終的にＥ７蛋白質を−７０℃で保管した。
【００５１】
前記製造された蛋白質サンプルを１２％のＳＤＳ−ＰＡＧＥから分離した後、ニトロセルロース膜（Ａｍｅｒｓｈａｍ， Ｐｉｓｃａｔａｗａｙ， ＮＪ）に付着させた。次いで、ニトロセルロース膜を２％ＢＳＡ含有のＴＢＳＴ溶液［１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、１５０ｍＭ ＮａＣｌ、０．１％ Ｔｗｅｅｎ２０］に１時間浸漬させた後、Ｅ７特異抗体（Ｏｎｃｏｇｅｎｅ， Ｂｏｓｔｏｎ， ＭＡ）と１時間反応させた。それぞれの反応後、ＴＢＳＴ溶液で数回洗浄し、最終的に抗−マウスＩｇＧ−ＨＲＰ（Ｓｉｇｍａ）と１時間反応させた後、ＥＣＬ溶液（Ａｍｅｒｓｈａｍ）で発色させた。
【００５２】
９８個のアミノ酸を含む組換えＥ７蛋白質がＥ．ｃｏｌｉで発現された。組換え蛋白質はＳＤＳ−ＰＡＧＥで２３ｋＤの蛋白質と確認され、免役ブロット検定（ｉｍｍｕｎｏｂｌｏｔ ａｓｓａｙ）でＥ７特異抗体と反応した。蛋白質の実際の大きさは予測より大きかった（１１ｋＤのＥ７蛋白質及びｐＥＴベクターのＨｉｓ−ｔａｇ４ｋＤ蛋白質）。ゲルにおける非正常的なＥ７蛋白質の移動特性は以前にも発表されたことがある［参照：Ａｒｍｓｔｒｏｎｇ， Ｄ． Ｊ． ｅｔ ａｌ．， Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍｍｕｎ．， １９２： １３８０−１３８７， １９９３； Ｆｅｒｎａｎｄｏ， Ｇ． Ｊ． Ｐ． ｅｔ ａｌ．， Ｃｌｉｎ． Ｅｘｐ． Ｉｍｍｕｎｏｌ．， １１５： ３９７−４０３， １９９９］。
【００５３】
Ｅ７蛋白質内の内毒素水準は内毒素検出キット（Ｓｉｇｍａ， Ｓａｉｎｔ Ｌｏｕｉｓ， ＭＯ）を用いて定量した。蛋白質内の内毒素水準は１００ＥＵ／ｍｇ以下と測定された。
【００５４】
実施例２：ＣｐＧ−ＯＤＮの製造
本実施例ではＣｐＧ−ＯＤＮ１８２６（５’−ＴＣＣＡＴＧＡＣＧＴＴＣＣＴＧＡＣＧＴＴ−３’）（配列番号１）を利用し、各塩基当たりホスホロチオエートで変化させたＯＤＮをＢｉｏｂａｓｉｃ Ｉｎｃ．（Ｃａｎａｄａ）に製造依頼して購入した。ＣｐＧ−ＯＤＮを内毒素の除去された水に溶解させて使用した。
【００５５】
実施例３：抗腫瘍活性
生後４〜６週齢のＣ５６ＢＬ／６マウス（Ｄａｅｈａｎ Ｂｉｏｌｉｎｋ ｃｏ．， Ｌｔｄ）を動物モデルとし、２０μｇの組換えＥ７蛋白質、２０μｇのＣｐＧ−ＯＤＮ又はこれらの全てを最終生理食塩水１００μｌに希釈して２８−ゲージ注射針（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ， Ｆｒａｎｋｌｉｎ Ｌａｋｅｓ， ＮＪ）で皮下注射した。
【００５６】
腫瘍誘導に使用された細胞株はＴＣ−１細胞であるが、ジョンホプキンス大学のＷｕ博士から提供を受け、ｃＲＰＭＩ（４００μｇ／ｍｌのＧ４１８）で培養された。ＴＣ−１はＥ７発現細胞株であって、Ｃ５７ＢＬ／６マウスの肺上皮細胞（ｐｒｉｍａｒｙ ｌｕｎｇ ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌｓ）に乳頭腫ウィルス１６型のＥ６とＥ７、及びｒａｓ遺伝子を用いて作った細胞株である［参照：Ｗｕ， Ｔ． Ｃ． ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ９２： １１６７１−１１６７５， １９９５］。それぞれの予防及び治療効果を検定するために２×１０^５及び５×１０^４個のＴＣ−１細胞株をＣ５７ＢＬ／６マウスの皮下に注射した。様々な注射容量が既に発表されたことがある［参照：Ｌｉｎ， Ｋ． Ｙ． ｅｔ ａｌ．， Ｃａｎｃｅｒ Ｒｅｓ．， ５６： ２１−２６， １９９６］。ＴＣ−１細胞株は生理食塩水で２回洗浄した後、注射に使用した。
【００５７】
実施例３ ． １：腫瘍予防活性
前記マウス動物モデルから、Ｅ７、ＣｐＧ−ＯＤＮ又はこれらの両者ともを使用した予防注射により誘導される抗腫瘍効果を観測した。各群の動物（ｎ＝１１〜１６）に対し、２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ又はこれら両者ともを０週及び２週に皮下注射した。最終注射後の３週目に各動物当たり２×１０^５個のＴＣ−１腫瘍細胞株を皮下注射した。
【００５８】
図１及び表２はＥ７（動物当たり２０μｇ）、ＣｐＧ−ＯＤＮ（動物当たり２０μｇ）又はこれら両者ともを２回予防注射し、２×１０^５個のＴＣ−１腫瘍細胞株を注射した後の抗腫瘍活性を示す。
【００５９】
【表２】

【００６０】
前記表２より分るように、Ｅ７又はＣｐＧ−ＯＤＮをそれぞれ単独で注射した場合には、非処理対照群でのように、ＴＣ−１腫瘍細胞株でチャレンジされた後腫瘍が現れたが、Ｅ７とＣｐＧ−ＯＤＮを同時に注射した群では時間が経過しても腫瘍が全く現われなかった。すなわち、Ｅ７又はＣｐＧ−ＯＤＮをそれぞれ単独で処理した場合には、いずれの腫瘍予防効果も示さなかったが、Ｅ７とＣｐＧ−ＯＤＮを同時に処理した場合には全てのマウス動物モデルで完全な腫瘍予防効果を示した。
【００６１】
また、腫瘍チャレンジ後の腫瘍の大きさを観測して示した図１の結果は、Ｅ７又はＣｐＧ−ＯＤＮをそれぞれ単独で注射する場合には時間経過に伴って腫瘍が段々大きくなるが、Ｅ７とＣｐＧ−ＯＤＮを同時に注射した場合には全く腫瘍が成長しないことを示す。この際、腫瘍が形成された動物はいずれも３０〜４０日の経過後に死んでしまった。Ｅ７とＣｐＧ−ＯＤＮを同時注射した動物は４ヶ月以上生存した。死んだ動物を剖検した結果、死因が悪液質ショック（ｃａｃｈｅｃｔｉｃ ｓｈｏｃｋ）であり、他の器官には全く転移されなかった。
【００６２】
前記表２及び図１から分るように、Ｅ７とＣｐＧ−ＯＤＮを同時に予防注射した場合にのみＥ７発現腫瘍細胞株の成長を抑制することが可能な予防効果がある。
【００６３】
実施例３ ． ２：腫瘍治療活性
前記マウス動物モデルから、既に形成された腫瘍に対するＥ７、ＣｐＧ−ＯＤＮ又はこれらの両者の注射により誘導される抗腫瘍効果を観測した。それぞれの群（グループ）の動物に５×１０^４個のＴＣ−１腫瘍細胞株を皮下注射し、腫瘍の大きさが１〜２ｍｍとなったとき、腫瘍注射部位から離れた箇所に２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ又は２０μｇのＥ７と２０μｇのＣｐＧ−ＯＤＮを皮下注射し、さらに１週後に皮下注射した。注射後１ヶ月間１週に２回ずつ測定器を用いて腫瘍の大きさを測定した。腫瘍の大きさは腫瘍の平均値［（ａ＋ｂ）／２、ａ：腫瘍の長軸長、ｂ：腫瘍の短縮長］で計算された。
【００６４】
腫瘍チャレンジ後、腫瘍を有するマウスの減少率を観測した結果を示す図２は、Ｅ７とＣｐＧ−ＯＤＮが同時に注射された動物群では２０％の動物でのみ腫瘍が探知され、残りの動物では腫瘍が無くなった反面、Ｅ７が単独注射された動物群及びＣｐＧ−ＯＤＮが単独注射された動物群では非処理対照群でのように全ての動物で腫瘍が抑制されなかったことを示す。一方、Ｅ７とＣｐＧ−ＯＤＮが同時注射された動物のうち１匹から観測された腫瘍の大きさは他の処理群に比べて相対的に小さかった。また、Ｅ７又はＣｐＧ−ＯＤＮがそれぞれ単独注射された動物群では腫瘍注射後４０日が経過してから全て死んだが、Ｅ７とＣｐＧ−ＯＤＮが同時に注射された処理群では２ヶ月以上長く生存した。
【００６５】
これはＥ７とＣｐＧ−ＯＤＮを同時注射する場合にのみ、既に形成されたＥ７発現腫瘍細胞株の成長を抑制することができて抗腫瘍治療効果があることを示す。
【００６６】
実施例３ ． ３：選択的免役細胞除去活性
ＣＤ４＋Ｔリンパ球及びＣＤ８＋リンパ球の数をＦＡＣＳ分析で測定した。Ｅ７とＣｐＧ−ＯＤＮが同時注射された前記のマウス動物モデルから採取した脾臓細胞（１×１０^５個）をＦＡＣＳ緩衝液（ＰＢＳ＋１％のＢＳＡ＋０．１％のアジ化ナトリウム）を用いて３回洗浄し、フィコエリトリン（ｐｈｙｃｏｅｒｙｔｈｒｉｎ）に結合された抗マウスＣＤ４及びＣＤ８抗体（Ｐｈａｒｍｉｎｇｅｎ， Ｓａｎ Ｄｉｅｇｏ， ＣＡ）と３０分間４℃で反応させた。その後、ＦＡＣＳ緩衝液で３回洗浄し、フローサイトメーター（ｆｌｏｗ ｃｙｔｏｍｅｔｅｒ： Ｃｏｕｌｔｅｒ−Ｅｐｉｃｓ ＸＬ， Ｍｉａｍｉ， ＦＬ）を用いてＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球の数を測定した。
【００６７】
ＣＤ４＋Ｔ細胞及びＣＤ８＋Ｔ細胞を試験管内及び生体内で除去するために公知の方法を利用した［参照：Ｓｉｎ， Ｊ． Ｉ． Ｅｔ ａｌ．， Ｈｕｍａｎ Ｇｅｎｅ Ｔｈｅｒａｐｙ， １２： １０９１−１１０２， ２００１； Ｓｉｎ， Ｊ． Ｉ． ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １６２： ２９１２−２９２１， １９９９］。
具体的に、試験管内リンパ球の除去は次のように処理した。Ｅ７とＣｐＧ−ＯＤＮが共同注射された動物からの脾臓細胞を抗−ＣＤ４抗体（Ｐｈａｒｍｉｎｇｅｎ）又は抗−ＣＤ８抗体（Ａｃｃｕｒａｔｅ Ｃｈｅｍｉｃａｌ ＆ Ｓｃｉｅｎｔｉｆｉｃ Ｃｏｒｐ．， Ｗｅｓｔｂｕｒｙ， ＮＹ）と４℃で１時間反応させた。その後、ウサギの補体（Ｓｉｇｍａ）と共に３７℃で１時間培養させた。細胞の生存率をトリパンブルー染料除去方法によって測定し、２回の分離除去過程後９８％以上のＴリンパ球が除去されたことをＦＡＣＳで確認した。
【００６８】
生体内リンパ球の除去は次のように処理した。予めプリスタンで処理されたヌードマウスにそれぞれのハイブリドーマ細胞株（Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ， Ｍａｎａｓｓａｓ， ＶＡ）を注射して抗−ＣＤ４抗体（クローンＧＫ１．５）及び抗−ＣＤ８抗体（クローン２．４３）腹水液を採取した。得られた１００μｌの腹水液（抗−ＣＤ４抗体及び抗−ＣＤ８抗体）を腫瘍注射する前後３、０及び３日に腹腔内注射した。ＦＡＣＳを用いて、９８％以上のＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球が除去されることを確認し、０日に腫瘍を注射した。
【００６９】
抗腫瘍活性に関与する細胞群を動物モデルから確認した結果が図６に示されている。図６はＥ７とＣｐＧ−ＯＤＮを同時注射した後、ＣＤ４＋Ｔ細胞、ＣＤ８＋Ｔ細胞それぞれ又はＣＤ４＋Ｔ細胞とＣＤ８＋Ｔ細胞を全て除去した後、これらの細胞群の抗腫瘍効果に対する重要性を確認することができる結果である。具体的に、各群（グループ）の動物（ｎ＝５）に対し２０μｇのＥ７と２０μｇのＣｐＧ−ＯＤＮを０週及び２週に皮下注射し、最終注射後の３週目に動物からＣＤ４＋Ｔ、ＣＤ８＋Ｔまたは２つの細胞群を全て除去した後、２×１０^５個のＴＣ−１腫瘍細胞株を皮下注射した後、腫瘍を有しないマウスの数を測定した。
【００７０】
これによれば、前記表１及び図１から立証されたように、Ｅ７とＣｐＧ−ＯＤＮを同時注射し、ＣＤ４＋Ｔ細胞及びＣＤ８＋細胞を除去していない場合には完全な腫瘍抑制効果が観測された。一方、ＣＤ４＋及びＣＤ８＋Ｔリンパ球の全てを除去した場合には非処理対照群と類似に腫瘍が形成された。特に、ＣＤ８＋Ｔリンパ球が除去された動物では、非処理対照群に比べてはやや遅いが、結局いずれも腫瘍が形成された。一方、ＣＤ４＋Ｔリンパ球が除去された動物では、５匹のうち４匹から腫瘍が観測されなかった。また、１匹に形成された腫瘍の大きさも相対的に小さかった。
【００７１】
これらの免役細胞群除去実験より、ＣｐＧ−ＯＤＮの存在下でＥ７がＣＤ４＋Ｔリンパ球及び特にＣＤ８＋Ｔリンパ球を活性化させて抗腫瘍効果を示すことが分る。従って、動物モデルにおける抗腫瘍効果がＣＤ４＋Ｔリンパ球及び特にＣＤ８＋Ｔリンパ球群により媒介されるという事実を確認することができる。
【００７２】
実施例４：免役反応誘導
実施例４ ． １：抗体反応誘導
公知の方法を用いてＥＬＩＳＡを行った［参照：Ｓｉｎ， Ｊ． Ｉ． ｅｔ ａｌ．， Ｖａｃｃｉｎｅ， １５： １８２７−１８３３， １９９７； Ｓｉｎ， Ｊ． Ｉ． ｅｔ ａｌ．， Ｊ． Ｖｉｒｏｌ．， ７４： １１１７３−１１１８０， ２０００］。
【００７３】
プレートをコートするために組換えＥ７蛋白質を生理食塩水に１μｇ／ｍｌの濃度で希釈させ、５０μｌずつ各ウェルに添加した後、次の日に利用した。ＥＬＩＳＡ緩衝液（２％のＢＳＡ）で遮断した後、抗−ネズミ（ｍｕｒｉｎｅ）ＩｇＧ−ＨＲＰを１：１０００の濃度で希釈して５０μｌずつ各ウェルに加えて反応させた。また、Ｅ７特異ＩｇＧサブクラスを検出するために、ＨＲＰの結合された抗−ネズミＩｇＧ１、ＩｇＧ２ａ、ＩｇＧ２ｂ又はＩｇＧ３（Ｚｙｍｅｄ， Ｓａｎ Ｆｒａｎｃｉｓｃｏ， ＣＡ）を抗−ネズミＩｇＧ−ＨＲＰに代えて使用した。
【００７４】
各群の動物に２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ、或いは２０μｇのＥ７及び２０μｇのＣｐＧ−ＯＤＮを０週及び２週に皮下注射した。初注射後２、４及び８週に各動物群（ｎ＝１０）から血清を採取して同一の容量で混合した各群別血清を２倍率ずつ希釈してＥＬＩＳＡ力価を測定した。また、４週及び８週の血清は１：１００で希釈してＥＬＩＳＡを行った。ＥＬＩＳＡ力価は対照群のＯＤ数値の２倍となる最も高い血清希釈倍率の逆数値とした。ＯＤ数値は４０５ｎｍで測定した。Ｅ７注射に比べてｐ＜０．０５で統計的有意性をもつ。
【００７５】
Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、或いはＥ７とＣｐＧ−ＯＤＮの共同注射によるＥ７特異抗体生成程度を測定した結果を図３に示した。図３ＡはＥ７が単独注射された動物の４週後の血清ＥＬＩＳＡ力価は１，６００であったが、Ｅ７とＣｐＧ−ＯＤＮが同時注射された動物の４週後の血清ＥＬＩＳＡ力価は６，４００であったことを示す。これはＥ７とＣｐＧ−ＯＤＮの同時使用により抗体生成が抗体力価において２倍以上増加したものである。また、注射後８週のＥＬＩＳＡ力価は、Ｅ７の場合には８００、Ｅ７とＣｐＧ−ＯＤＮを同時使用した場合には３，２００であって、類似の増加を示す。ところが、ＣｐＧ−ＯＤＮが単独注射された動物では非処理対照群でのように抗体反応が観測されなかった。
【００７６】
また、図３Ｂ、図３Ｃ、図３Ｄ及び図３ＥはＥ７特異抗体のＩｇＧイソ型に対する生成を示す。一番目の注射後４週及び８週の血清において、Ｅ７とＣｐＧ−ＯＤＮ同時注射の場合、Ｅ７単独注射に比べてＩｇＧイソ型の生成がさらに多く誘導された。
【００７７】
実際に、ＩｇＧ１及びＩｇＥはＴｈ２に連関した抗体であり、ＩｇＧ２ａはＴｈ１に連関した抗体である［参照：Ｆｉｎｋｅｌｍａｎ， Ｆ． Ｄ． ｅｔ ａｌ．， Ａｎｎ． Ｒｅｖ． Ｉｍｍｕｎｏｌ．， ８： ３０３−３３３， １９９０］。特に、Ｅ７とＣｐＧ−ＯＤＮを同時注射する場合には、Ｅ７単独注射に比べてＩｇＧ２ａの生成がさらに増加した。また、ＩｇＧ２ａ／ＩｇＧ１の数値は０．２（Ｅ７）及び０．２６（Ｅ７＋ＣｐＧ−ＯＤＮ）で計算された。
【００７８】
このような結果から確認されるように、ＣｐＧ−ＯＤＮが全般的な抗体免役反応を増加させるため、Ｅ７とＣｐＧ−ＯＤＮの同時注射の場合、Ｅ７単独注射に比べてＩｇＧ及びＩｇＧサブクラスの生成が増加する。
【００７９】
実施例４ ． ２：Ｔヘルパー（Ｔｈ）細胞増殖反応の誘導
Ｔｈ細胞増殖反応は細胞免役反応に重要な役割を果たす。Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、或いはＥ７及びＣｐＧ−ＯＤＮの共同注射が行われた各動物群から脾臓免役細胞を採取してＴｈ細胞増殖反応を測定した。
【００８０】
Ｔｈ細胞増殖反応を公知の方法で行った［参照：Ｓｉｎ， Ｊ． Ｉ． ｅｔ ａｌ．， Ｈｕｍａｎ Ｇｅｎｅ Ｔｈｅｒａｐｙ， １２： １０９１−１１０２， ２００１； Ｓｉｎ， Ｊ． Ｉ． ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １６２： ２９１２−２９２１， １９９９］。
【００８１】
各群の動物に対し２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ、或いは２０μｇのＥ７及び２０μｇのＣｐＧ−ＯＤＮを０週及び２週に皮下注射し、最終注射後の３週目に動物群から脾臓免役細胞を採取した。採取した脾臓細胞を様々な濃度（０．５、１及び５μｇ／ｍｌ）のＥ７で３日間刺激した後、標識^３［Ｈ］の付いたチミジン（各ウェル当たり１μＣｉ）を添加した後、次の日に細胞を回収してβ計数器（ＰｅｒｋｉｎＥｌｍｅｒ， Ｂｏｓｔｏｎ， ＭＡ）によって各免役群のｃｐｍを測定した。ＳＩは［実験群ｃｐｍ−対照群ｃｐｍ］／［対照群ｃｐｍ］で計算した。
【００８２】
図４ＡはＥ７とＣｐＧ−ＯＤＮの同時注射がＥ７単独注射に比べてＴｈ細胞増殖反応を増加させることを示す。その結果は同じ結果を示す重複実験の一つである。＊は対照群に比較してｐ＜０．０５で統計的有意性を有し、＊＊はＥ７注射グループに比較してｐ＜０．０５で統計的有意性を有する。Ｅ７単独注射の際、Ｅ７特異Ｔｈ細胞増殖反応が現われたが、Ｅ７とＣｐＧ−ＯＤＮを同時注射した場合にはさらに増加したＴｈ細胞増殖反応を示した。反面、ＣｐＧ−ＯＤＮ単独注射の際には非処理対照群でのようにＴｈ細胞増殖反応が観測されなかった。
【００８３】
実施例４ ． ３：細胞毒性Ｔリンパ球（ＣＴＬ）反応の誘導
５時間^５１Ｃｒ探知方法を用いてＣＴＬ検定を行った。
各群の動物に対し２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ、或いは２０μｇのＥ７及び２０μｇのＣｐＧ−ＯＤＮを０週及び２週に皮下注射し、最終注射後の３週目に動物群から脾臓免役細胞を採取した。採取した脾臓細胞を、２０Ｕ／ｍｌのＩＬ−２（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ， Ｍｉｎｎｅａｐｏｌｉｓ， ＭＮ）と予めマイトマイシンＣ（３０μｇ／ｍｌ）で３時間処理されたＴＣ−１細胞株と共に５日間培養して活性化させた。その後、１００μＣｉ／ｍｌＮａ_２ ^５１ＣｒＯ_４と共に２時間培養されたＴＣ−１細胞を前記活性化された免役細胞とは異なる比率で５時間培養して細胞溶解（ｃｙｔｏｌｙｔｉｃ）活性をＴＣ−１細胞主に対する^５１Ｃｒ分泌程度で測定したものである。その後、１００μｌの細胞培養液を採取してγ−計数器（ＰｅｒｋｉｎＥｌｍｅｒ）を用いてｃｐｍを測定した。ＣＴＬ活性（％）は１００×［（実験群ｃｐｍ−自然積分比ｃｐｍ）／（最大分泌ｃｐｍ−自然積分比ｃｐｍ）］である。最大分泌ｃｐｍは１％のＴｒｉｔｏｎＸ−１００を添加した標的ＴＣ−１細胞のｃｐｍとした。
【００８４】
図４ＢはＥ７単独注射の場合及びＣｐＧ−ＯＤＮ単独注射の場合にはＣＴＬ反応が観測されない反面、Ｅ７とＣｐＧ−ＯＤＮの同時注射の場合にはＣＴＬ反応が観測されることを示す。その結果は同じ結果を示す重複実験の一つである。
前記図４Ｂから立証されるように、Ｅ７とＣｐＧ−ＯＤＮを同時注射した場合にのみＣＴＬ反応が誘導された。
これはＥ７がＣｐＧ−ＯＤＮの存在下でＥ７特異ＣＴＬ反応を誘導することができることを示す。
【００８５】
実施例４ ． ４：ＩＦＮ−γの生成
ＩＦＮ−γはＴｈ１型免役反応及びＣＴＬ反応の誘導に重要な役割をする。ＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球におけるＩＦＮ−γの生成程度を測定した。
【００８６】
各群の動物に対し２０μｇのＥ７、２０μｇのＣｐＧ−ＯＤＮ、或いは２０μｇのＥ７及び２０μｇのＣｐＧ−ＯＤＮを０週及び２週に皮下注射し、最終注射後の３週目に動物群から脾臓免役細胞を採取した。採取した脾臓細胞を６×１０^６個／ｍｌで２４ウェルフラスコに加え、組換えＥ７蛋白質を１μｇ／ｍｌ添加し或いは予めマイトマイシンＣ（３０μｇ／ｍｌ）で３時間処理されたＴＣ−１細胞株（５×１０^５個の細胞／ｍｌ）を添加した後、３７℃の５％ＣＯ_２培養条件で３日間培養した。細胞培養液を取ってＩＦＮ−γ検出キット（Ｂｉｏｓｏｕｒｃｅ， Ｉｎｔｌ．， Ｃａｍａｒｉｌｌｏ， ＣＡ）を用いて、分泌されたＩＦＮ−γの量を測定した。
【００８７】
具体的に、ＣＤ４＋Ｔリンパ球におけるＩＦＮ−γの生成程度を測定するために、動物から採取した脾臓免役細胞をＥ７蛋白質で刺激した。
【００８８】
図５ＡはＥ７単独注射、ＣｐＧ−ＯＤＮ単独注射、或いはＥ７及びＣｐＧ−ＯＤＮの共同注射で免役誘導された脾臓免役細胞を１μｇのＥ７蛋白質／ｍｌに添加した後、３日間培養した場合のＩＦＮ−γの分泌を示す。ＩＦＮ−γの生成は、Ｅ７とＣｐＧ−ＯＤＮが同時注射された動物群のみから観測され、Ｅ７単独注射又はＣｐＧ−ＯＤＮ単独注射の行われた群からは観測されなかった。
【００８９】
図５ＢはＥ７とＣｐＧ−ＯＤＮの同時注射が行われた脾臓免役細胞からＣＤ４＋Ｔ又はＣＤ８＋Ｔリンパ球をそれぞれ除去した後、１μｇのＥ７蛋白質／ｍｌに添加した後、３日間培養した場合のＩＦＮ−γの分泌を示す。脾臓免役細胞からＣＤ４＋Ｔリンパ球を除去した場合にはＩＦＮ−γの生成が観測されなかったが、ＣＤ＋８Ｔリンパ球を除去した場合にはＩＦＮ−γの生成が観測された。数値及びバー（ｂａｒ）はＩＦＮ−γの平均値と誤差を意味する。前記の結果は２回繰り返した結果であり、類似の結果が測定された。
【００９０】
このような結果はＴｈ１型のＣＤ４＋Ｔリンパ球がＥ７とＣｐＧ−ＯＤＮの同時注射によってのみ活性化され、ＣｐＧ−ＯＤＮがＥ７特異Ｔｈ１型の免役反応を誘導することを意味する。
【００９１】
また、ＣＤ８＋Ｔリンパ球におけるＩＦＮ−γの生成程度を測定するために、Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、或いはＥ７とＣｐＧ−ＯＤＮの共同注射を行って処理した動物から採取した脾臓免役細胞を、不活性化されたＴＣ−１細胞株（ＭＨＣ ｃｌａｓｓ Ｉ^＋， ｃｌａｓｓ ＩＩ⁻）と共に３日間培養した。
図５ＣはＥ７単独注射、ＣｐＧ−ＯＤＮ単独注射、或いはＥ７とＣｐＧ−ＯＤＮの共同注射で免役誘導された脾臓免役細胞を予めマイトマイシンＣで処理されたＴＣ−１と３日間培養した場合のＩＦＮ−γの分泌を示す。ＩＦＮ−γの生成は、Ｅ７とＣｐＧ−ＯＤＮの同時注射が行われた動物群からのみ観測され、Ｅ７単独注射又はＣｐＧ−ＯＤＮ単独注射の行われた群からは観測されなかった。このような結果はＣＴＬ（ＩＦＮ−γ分泌ＣＤ８＋Ｔ細胞）結果とも同一である（図４Ｂ）。
【００９２】
また、図５ＤはＥ７とＣｐＧ−ＯＤＮが同時注射された脾臓免役細胞からＣＤ４＋Ｔ又はＣＤ８＋Ｔリンパ球をそれぞれ除去した後、予めマイトマイシンＣで処理されたＴＣ−１と３日間培養した場合のＩＦＮ−γの分泌を示す。脾臓免役細胞からＣＤ８＋Ｔリンパ球を除去した場合にはＩＦＮ−γの生成が観測されなかったが、ＣＤ＋４Ｔリンパ球を除去した場合にはＩＦＮ−γの生成が観測された。数値及びバーはＩＦＮ−γの平均値と誤差を意味する。前記の結果は２回繰り返した結果であり、類似の結果が測定された。
【００９３】
このような結果はＣＤ８＋Ｔリンパ球がＥ７とＣｐＧ−ＯＤＮの同時注射によってのみＭＨＣｃｌａｓｓＩに連繋されてＩＦＮ−γを生成することを意味する。以上、本発明を具体的様態によって説明したが、本発明の趣旨及び範囲内で様々な変形が可能であることを当業者なら理解するであろう。従って、本発明は本明細書に例示された具体的様態に制限されない。
【００９４】
【発明の効果】
乳頭腫ウィルス抗原蛋白質及びＣｐＧ−オリゴデオキシヌクレオチドを含む本発明の薬剤学的組成物は、これらを同時に投与する場合にのみ動物モデルで抗腫瘍活性を有し、ＣＴＬ反応を示し、ＣＤ４＋Ｔリンパ球及びＣＤ８＋Ｔリンパ球におけるＩＦＮ−γの生成を誘導する強力な免役反応を誘導することができるため、乳頭腫ウィルスにより誘発される疾患を効果的に予防又は治療することができる。

【図面の簡単な説明】
【図１】Ｅ７単独注射、ＣｐＧ−オリゴデオキシヌクレオチド（ＣｐＧ−ＯＤＮ）単独注射、及びＥ７とＣｐＧ−ＯＤＮの共同注射による抗腫瘍予防効果を示す。
【図２】Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、及びＥ７とＣｐＧ−ＯＤＮの共同注射による抗腫瘍治療効果を示す。
【図３】Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、及びＥ７とＣｐＧ−ＯＤＮの共同注射によるＥ７特異抗体反応（ＩｇＧ、ＩｇＧ１、ＩｇＧ２ａ、ＩｇＧ２ｂ及びＩｇＧ３）を示す。
【図４】Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、及びＥ７とＣｐＧ−ＯＤＮの共同注射により誘導されるＥ７特異Ｔｈ細胞増殖反応及びＣＴＬ反応を示す。
【図５】Ｅ７単独注射、ＣｐＧ−ＯＤＮ単独注射、及びＥ７とＣｐＧ−ＯＤＮの共同注射により誘導される免疫細胞におけるＩＦＮ−γの分泌程度、及びＥ７とＣｐＧ−ＯＤＮが同時注射された脾臓免疫細胞からＣＤ４＋Ｔ又はＣＤ８＋Ｔリンパ球をそれぞれ除去した後のＩＦＮ−γの分泌程度を示す。
【図６】抗腫瘍効果に対する免疫細胞群の役割を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition comprising a papilloma virus antigen protein and CpG-oligodeoxynucleotide (hereinafter, referred to as CpG-ODN) for preventing or treating a disease induced by papilloma virus.
[0002]
[Prior art]
Papillomavirus (PV) is an insidious and serious disease that is known to induce positive, dysplastic and malignant hyperproliferation of skin and mucosal epidermis [see: Mansur et al. , Biochim Biophys Acta, 1155: 323-345, 1993; Pfilter, Rev. Physiol. Biochem. Pharmacol. , 99: 111-181, 1984; Broker et al. , Cancer Cells, 4: 17-36, 1986].
[0003]
Human papillomavirus (HPV) is a DNA tumor virus that is associated with human squamous epithelium hyperproliferation, malignancy (carcinoma) and the like. Although the reproductive system of many women is infected with human papillomavirus, papillomavirus infection progresses to cervical cancer in a significant number of women. Thus, 20% of women's cancer mortality is associated with HPV.
HPVs are categorized as high-risk HPV (eg, HPV16, HPV18) and low-risk HPV (eg, HPV6, HPV11) based on their relative propensity to lesions that progress to cancer and associated clinical lesions. HPV type 16 infection is a major cause of cervical cancer development [see: zur Hausen, H. et al. , Virol. , 184: 9-13, 1991], and among the HPV proteins, the E6 and E7 proteins are known to play an important role in the formation and maintenance of cervical cancer [see: Scheffner, M., et al. et al. , Proc. Natl. Acc. Sci. USA, 88; 5523-5527, 1991; A. et al. Dyson, N., Science, 248: 76-79, 1990; et al. , Science, 243: 934-937, 1989]. The amino acid sequence of the HPV16 type E7 protein was deduced from this nucleic acid sequence and was described in the literature [see: N. et al. Salzman and P.S. Hawley, "The Papoviridae", Vol. 2, p. 379, Plenum Press, N.M. Y. (1987)].
[0004]
An E7-specific immune response is detected in cervical cancer patients [see de Gruijl, T. et al. D. et al. , J. et al. Gen. Virol. , 77: 2183-2191, 1996], which means that the E7 protein can be a target for cervical cancer immunotherapy. Therefore, the possibility of using prophylactic and therapeutic vaccines using the E7 protein in various animal models has been studied. Examples include the E7 recombinant protein [see: Fernando, G .; J. P. et al. , Clin. Exp. Immunol. , 115, 1999], an E7 expressing gene vaccine [see Hung, C. et al. F. et al. , Cancer Res. 61: 3698-3703, 2001], microorganisms and viral vectors expressing E7 or cytotoxic T lymphocyte (CTL) epitopes of E7 [see: Lamikanra, A. et al. et al. , J. et al. Virol. Cheng, W., 75: 9654-9664, 2001; F. et al. , Hum. Gene Ther. Liu, D., 13: 553-568, 2002; W. et al. , J. et al. Virol. 74: 2888-2894, 2000; Londono, L .; P. et al. , Vaccine, 14: 545-552, 1996] and the E7 CTL epitope [see Feltkamp, M. et al. C. et al. , Eur. J. Immunol. , 23: 2242-2249, 1993]. In these studies, CD4 + T lymphocytes, and especially CTLs, emerged as a population of immune cells involved in suppressing tumorigenesis.
[0005]
On the other hand, the immune system recognizes the DNA of lower organisms, including bacteria, due to structural and hierarchical differences between pathogens and host DNA. In particular, short stretch DNA derived from invertebrates or short oligodeoxynucleotide-type short stretch DNA containing cytosine-guanine dinucleotide (CpG) that is unmethylated at specific base contexts was targeted.
Recently, bacterial DNA itself has been shown to stimulate the immune response of mammals. The major difference between bacterial DNA and mammalian DNA is that bacterial DNA contains a large number of unmethylated CpG (cytosine-phosphorothioate-guanine) dinucleotides that are not included in mammalian DNA. Based on this, synthetic CpG-ODNs containing unmethylated CpG motifs have been used as immunostimulants.
[0006]
ODNs having an unmethylated form of the CpG motif are known to activate B lymphocytes, macrophages and NK cells, and to induce Th1-type immune responses [see Bohl, B]. . et al. , Eur. J. Immunol. Klinman, D., 29: 2344-2353, 1999; M. et al. , Proc. Natl. Acad. Sci. ScL USA, 93: 2879-2883, 1996; M. et al. Ballas, Z., Nature, 374: 546-549, 1995; K. et al. , J. et al. Immunol. , 157: 1840-1845, 1996; Sparwasser, T .; et al. , Eur. J. Immunol. Sparwasser, T., 30: 3591-3597, 2000; et al. , Eur. J. Immunol. Chu, R., 28: 2045-2054, 1998; S. et al. , J. et al. Exp. Med. , 186; 1623-1631, 1997; Leclecr, C. et al. et al. , Cell. Immunol. Klinman, D., 179: 97-106, 1997; M. et al. , J. et al. Immunol. , 158: 3635-3639, 1997; Jakob, T .; et al. , J. et al. Immunol. , 161: 3042-3049, 1998. ]. CpG-ODN is known to induce the production of IL-12 from macrophages, induces the production of IFN-γ from NK cells, and selectively induces a Th1-type immune response [see: Chu, R .; S. et al. , J. et al. Exp. Med. , 186: 1623-1631, 1997; Roman, M .; et al. , Nature Med. , 3: 849-854, 1997]. In addition, CpG-ODN selectively induces the production of an IgG2a type antibody (Th1 type antibody) among antibodies [see: Chu, R .; S. et al. , J. et al. Exp. Med. Davis, H., 186: 1623-1631, 1997; L. et al. , J. et al. Immunol. , 160: 870-876, 1998], and are known to also increase the CTL response [see: Krieg, A. et al. M. et al. , Nature, 374: 546-549, 1995; Warren, T .; L. et al. , J. et al. Immunol. , 165: 6244-6251, 2000]. These CpG-ODNs are currently being extensively studied as immunoactive agents [see: Davis, H .; L. et al. , J. et al. Immunol. , 160: 870-876, 1998; Weiner, G. et al. J. et al. , Proc. Natl. Acad. Sci. ScL USA, 94: 10833-10837, 1997; L. et al. Pro. Natl. Acad. Sci. USA, 93; 7213-7218, 1996; N. et al. , J. et al. Immunol. , 160: 2555-2559, 1998; Scott Gallican, W.W. et al. , J. et al. Immunol. , 166: 3451-3457, 2001]. However, there is no description of the anti-immune effect of CpG-ODN on papillomavirus-induced diseases including cervical cancer.
[0007]
[Problems to be solved by the invention]
To date, there are a number of available methods for treating papillomavirus-induced disease, but none of the treatments has been consistently effective in eliminating persistent HPV-induced disease .
[0008]
The present invention increases the E7-specific antibody response and the Th cell proliferative response when papillomavirus antigen protein is used together with CpG-ODN as compared to their use alone. It has antitumor activity for prevention and treatment, shows cytotoxic T lymphocyte reaction, and has an excellent effect of producing IFN-γ from CD4 + T cells and CD8 + T cells.
[0009]
Based on this, the present invention provides a powerful immunity induction method capable of selectively increasing Th1-type CD4 + T lymphocytes and CTL responses and effectively treating diseases induced by papillomavirus. Can be.
[0010]
[Means for Solving the Problems]
The present invention relates to a pharmaceutical composition for preventing or treating a disease induced by papillomavirus, comprising an immunologically effective amount of papillomavirus E7 antigen protein and CpG-oligodeoxynucleotide. The composition of the present invention is a CpG-oligodeoxynucleotide, comprising one or more unmethylated CpG dinucleotides, wherein one or more nucleotides separate a contiguous CpG, from about 8 to 40 nucleotides. And oligodeoxynucleotides containing
Preferably, the composition of the invention comprises a papillomavirus antigen protein, the human papillomavirus type 16 E7 protein.
Particularly preferably, the composition of the present invention is a papillomavirus antigen protein, which comprises a human papillomavirus type 16 E7 recombinant protein.
[0011]
Particularly preferably, the composition according to the invention is a CpG-oligodeoxynucleotide and comprises 5'-TCCATGACGTTCCTGACGTT-3 '.
Preferably, the composition of the present invention relates to a pharmaceutical composition for preventing or treating cervical cancer.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides a pharmaceutical composition for preventing or treating a disease induced by papilloma virus, which comprises an immunologically effective amount of papilloma virus E7 antigen protein and CpG-oligodeoxynucleotide.
In the above, the term “immunologically effective amount” means an amount of an active ingredient capable of inducing an immune response effective in preventing or treating papillomavirus-induced disease in an animal to be administered.
[0013]
The term "papilloma virus-induced disease" refers to a cell-proliferative disease of a group of malignant and non-malignant cells that is induced by papilloma virus and is morphologically different from surrounding tissues.
In the term "prevention or treatment", prevention means that the drug is administered before exposure to papillomavirus, and treatment means that the drug is administered after infection or after the onset of the disease.
[0014]
The term "antigen" refers to a molecule capable of producing an immune response. For the purpose of the present invention, "papilloma virus antigen" used is one isolated from nature or one produced by a recombinant method.
In a preferred embodiment, the present invention uses the human papillomavirus type 16 E7 protein as the papillomavirus E7 antigen protein.
[0015]
A papillomavirus antigen protein, which can be included as a papillomavirus antigen protein in the pharmaceutical composition of the present invention and is isolated from nature or produced by a recombinant method, is a protein having a natural protein sequence. Of course, it means a protein which has the same sequence as the natural protein sequence in 85% or more, preferably 90% or more, and can cause substantially the same immune reaction as the natural papillomavirus antigen protein. . The protein isolated from nature can be isolated and purified from a large-scale culture of papillomavirus using a conventional method in the art.
[0016]
Particularly preferred as the papillomavirus antigen protein of the present invention is a human papillomavirus type 16 E7 recombinant protein produced by a gene recombination method. The E7 recombinant protein used in the present invention can be produced using various known recombinant expression vectors.
[0017]
The term "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. For the purpose of the present invention, any recombinant expression vector can be used without limitation as long as it can express papillomavirus antigen protein. For example, plasmids, phages, other viruses and the like can be used. Generally, when a compatible host is transformed with a recombinant vector, the vector can replicate and function independently of the host genome, and in some cases can also be integrated into the genome itself.
[0018]
Expression vectors that can be used in eukaryotic hosts suitable for expressing the papillomavirus antigen protein of the present invention include, for example, SV40, retrovirus, Adenovirus, herpes simplex virus, and poxvirus. (Poxvirus), lentivirus (Lentivirus), adeno-associated virus (Adeno-associated virus), cytomegalovirus (Cytomegalovirus) and the like. Examples of expression vectors that can be used in bacterial hosts include pBluescript, pMAL-c2X, pGELXC E. coli such as pUC vector, pCR1, pBR322, pMB9 and derivatives thereof. bacterial plasmids from E. coli, plasmids with a wider host range such as RP4, phage DNA exemplified by a wide variety of phage λ derivatives such as λgt10, λgt11, M13 and filamentous single DNA phage Other DNA phages and the like are included. Expression vectors useful for yeast cells include the 2μ plasmid and derivatives thereof, and vectors useful for insect cells include pVL941.
[0019]
Generally, recombinant expression vectors contain expression control sequences, which are essential for the expression of the operably linked coding sequence from the particular host organism. A wide variety of expression regulatory sequences can be used to express a DNA sequence according to the present invention. It includes, for example, a promoter for performing transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating termination of transcription and decoding. For example, regulatory sequences suitable for prokaryotes include promoters, operators, ribosome binding sites, and the like. Regulatory sequences suitable for eukaryotic cell expression include promoters, polyadenylation signals, enhancers, and the like. The factor that most affects the expression level of the plasmid gene is the promoter, and the SRα promoter or cytomegalovirus-derived promoter is preferably used as the promoter for high expression.
[0020]
Also, when one nucleic acid sequence is placed in a functional relationship with another nucleic acid sequence, it can be operably linked. For example, a sequence encoding a transcriptional activator protein may be linked to a regulatory sequence (or the like) to allow gene expression, or a sequence encoding a pre-sequence or secretory leader may be a protein or peptide of interest. A ribosome binding site that is linked to an encoding nucleic acid sequence, or that affects transcription, is linked to a target protein or peptide coding sequence, or a ribosome binding site that facilitates decoding is a target protein or peptide coding sequence. Linked to
[0021]
The recombinant vector according to the present invention can be introduced into cells using a conventional transformation method, for example, a method such as DEAE-dextran, calcium phosphate, and electroporation. Techniques for transforming host cells and methods for expressing foreign DNA sequences cloned within them are already well known in the art [see: Maniatis et al. , Molecular Cloning:; (. Eds) A Laboratory Manual, Cold Spring Harbor Laboratory (1982) Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink, Academic Press, San Diego, Calif. Hitman et al., 1991; , J. et al. Biol. Chem. , 255, 12073-12080, 1980].
[0022]
The expressed protein can be expressed as a fusion protein in which the target protein is fused with another protein. For example, the E7 protein and glutathione-S-transferase (GST) fusion protein can be expressed as E. coli. E. coli [see: Fernando G .; J. , Clin. Exp. Immunol, 115 (3): 397-403, 1999 Mar]. Alternatively, host cells can be transfected with an adeno-associated virus (AAV) having a gene encoding the E7 protein fused to the heat shock protein gene, and the fusion protein produced therefrom [see Liu D. et al. W. et al. , J Virol. , 74 (6) 2888-94, 2000, Mar].
[0023]
Also, the papillomavirus antigen protein can be glycosylated or lipidated and derivatized to include molecules that enhance antigen presentation and enhance the targeting of antigen to antigen presenting cells. Can be.
The papillomavirus antigen protein is preferably separated and purified before use. Therefore, ordinary techniques for separating and purifying proteins can be used. In addition, in order to facilitate separation and purification of the papillomavirus recombinant protein, it can be produced into a fusion protein with, for example, GST, His-tag and the like.
[0024]
The E7 recombinant protein produced according to the present invention is a protein containing 98 amino acids of the E7 protein. Escherichia coli and expressed as a fusion protein of the E7 protein and the His-tag peptide of the pET vector, and the His-tag peptide residue facilitates protein purification. The E7 recombinant protein thus separated and purified is preferably used after removing endotoxin.
[0025]
In addition, the natural or recombinant papillomavirus antigen protein used in the composition of the present invention can be modified by an ordinary technique capable of enhancing an immune response.
The term "CpG-oligodeoxynucleotide (CpG-ODN)" means an oligonucleotide comprising one or more unmethylated CpG (cytosine-phosphorothioate-guanine) dinucleotides.
[0026]
Preferably, the CpG-oligodeoxynucleotide is an ODN comprising one or more nucleotides separating a continuous CpG and comprising about 8 to 40 nucleotides.
Particularly preferably, 5'-TCCATGACGTTCCTGACGTT-3 '(SEQ ID NO: 1).
CpG-ODN can be chemically synthesized, recombinantly produced, or derived from natural sources. Of course, other CpG-ODN mixtures can also be used.
[0027]
Chemically synthesized CpG-ODN can be de novo synthesized using a number of known methods in the art. For example, the β-cyanoethyl phosphoramidate method [see: S. L. See Beaucage et al. , Tet. Let. , 22: 1859, 1981], the nucleoside H-phosphonate method [see Garegg et al. , Tet. Let. , 27: 4051-4054, 1986; Froehler et al. , Nucl. Acid. Res. , 14: 5399-5407; Garegg et al. , Tet. Let. 29: 2619-2622, 1988]. Such chemical synthesis methods can be performed using various commercially available automated oligonucleotide synthesizers. Alternatively, oligonucleotides can be produced from existing nucleic acid sequences (eg, genomic or cDNA) using known methods, for example, restriction enzymes, exonucleases or endonucleases.
[0028]
In addition, CpG-ODN can be suitably modified to have resistance to degradation in vivo. Preferably, it comprises a phosphorothioate variant. Phosphorothioate deformation can occur from the end. For example, the last two or three 5 'or 3' nucleotides can be linked by a phosphorothioate linkage. Also, CpG-ODN can be modified to include a secondary structure (eg, a stem-loop structure) that makes it resistant to degradation. Preferred stabilized nucleic acids are those having one or more partially phosphorothioate modified backbones. Phosphorothioates can be synthesized using automated techniques by phosphoramidate or H-phosphonate chemistry. Aryl- and alkyl-phosphonates can be prepared, for example, as described in U.S. Patent No. 4,469,863; (Alkylated as described in Patent No. 092,574) can be produced by automated solid-phase synthesis using commercially available reagents. Methods for making and replacing other DNA backbone variants are described in the literature [see: Uhlmann, E .; et al. Chem., Chem. Rev .. 90: 544, 1990; Goodchild, J. et al. , Bioconjugate Chem. , 1: 165, 1990]. Another variant that makes the degradation a little slower is that which involves atypical bases such as inosine and quesin, as well as acetyl-, thio- and similar variants of adenine, cytosine, guanine, thymine and uridine.
[0029]
CpG-ODNs containing diols such as tetraethylene glycol or hexaethylene glycol at the ends also proved to be more resistant to degradation.
For in vivo administration, CpG-ODNs can form "nucleic acid transduction complexes" by binding with high affinity binding to the surface of target cells or with molecules for increased cell uptake. Nucleic acids can be ionically or covalently linked to suitable molecules using techniques well known in the art. As a suitable coupling agent or crosslinking agent, for example, protein A, carbodiimide, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP; N-Succinimidyl-3- (2-pyridyldithio) propionate) and the like can be used. it can. In addition, CpG-ODN can be encapsulated in liposomes or virosome using known techniques.
[0030]
In the present invention, E7 protein and CpG-ODN were co-injected to observe antitumor effects from animal models, and E7-specific antibody response, Th cell proliferation response, CTL response, IFN-γ by CD4 + T lymphocytes and CD8 + T lymphocytes. Was observed.
Specifically, the antitumor effect of papillomavirus type 16 E7-expressing tumor cell line upon injection was injected simultaneously with E7 protein and CpG-ODN (FIG. 1 and Table 2). As a result, when the E7 protein or CpG-ODN was injected respectively, as shown in FIG. 1 and Table 2, the antitumor preventive effect as described above was not observed at all, and the E7 protein and CpG-ODN were injected simultaneously. Only when this was done, an antitumor preventive effect was observed. This is similar in terms of the antitumor therapeutic effect, and the antitumor therapeutic effect was observed only when the E7 protein and CpG-ODN were simultaneously injected (FIG. 2). This indicates that papillomavirus antigen protein and CpG-ODN are essential for prevention and treatment of tumors induced by papillomavirus.
[0031]
As described above, in the case of the present invention, when the papilloma virus antigen protein and CpG-ODN were used, no antitumor effect was observed, and the antitumor effect was observed only when both of them were used. The effect was observed. In view of this, the antitumor effect as in the present invention cannot be predicted at all from the conventional technology using CpG-ODN by itself or as a known adjuvant for enhancing the immune effect.
[0032]
In addition, as a result of observing an E7-specific antibody reaction by co-injection of E7 protein and Cpg-ODN, when the E7 protein and CpG-ODN were injected simultaneously, a higher ELISA titer was shown, and the E7 protein was more intense than the single injection of E7 protein. It showed an enhanced antibody response (FIG. 3A) and further enhanced the production of IgG isoforms, IgG1, IgG2a, IgG2b and IgG3 antibodies (FIGS. 3B, C, D and E).
[0033]
When the Th cell proliferative response due to the simultaneous injection of the E7 protein and CpG-ODN was observed, the Th cell proliferative response was considerably increased as compared with the case where the E7 protein and the CpG-ODN were respectively injected (FIG. 4A). On the other hand, when CD4 + T lymphocytes were removed, no Th cell proliferation reaction was detected. This means that there is a direct correlation between the antibody response and the Th cell proliferative response associated with CD4 + T lymphocytes.
[0034]
Moreover, as a result of observing the CTL reaction by simultaneous injection of the E7 protein and CpG-ODN, a CTL reaction was observed only when the E7 protein and CpG-ODN were simultaneously injected, and when the E7 or CpG-ODN was injected, respectively. No CTL reaction was observed (FIG. 4B). These results indicate that CpG-ODN is essential for the induction of an antigen-specific CTL reaction by the E7 protein.
[0035]
In addition, generation of IFN-γ by simultaneous injection of the E7 protein and ODN was observed. There have been reports of the effect of CpG-ODN on the production of IFN-γ [see Chu, R. et al. S. et al. , J. et al. Exp. Med. In the present invention, when stimulated with an antigen, IFN-γ production was induced from CD4 + T lymphocytes only in a group of animals to which E7 protein and CpG-ODN were simultaneously injected (FIG. 5B). ). This means that CD4 + T lymphocytes that are not CD8 + T lymphocytes produce IFN-γ upon antigen stimulation. Similarly, when stimulated with the TC-1 cell line, IFN-γ is produced from CD8 + T lymphocytes only in the group of animals into which the E7 protein and CpG-ODN have been simultaneously injected, while E7 or CpG-ODN is No IFN-γ production could be observed in the injected animals (FIG. 5D). This means that upon stimulation of the TC-1 cell line, CD8 + T lymphocytes, but not CD4 + T lymphocytes, secrete IFN-γ.
[0036]
These results are consistent with the results of CTL reaction induction induced by simultaneous injection of E7 protein and CpG-ODN. This ultimately means that simultaneous use of the E7 protein and CpG-ODN is necessary to induce the production of IFN-γ from CD4 + and CD8 + T lymphocytes in response to MHC I and II. In this connection, the protective immunity of IFN-γ against viral infections or antitumor effects has been demonstrated [see Samuel, C. et al. E. FIG. , Virol. , 183: 1-11, 1991; Smith, P .; M. et al. , Virol. , 202: 76-88, 1994; Boehm, U.S.A. et al. , Annu. Rev .. Immunol. , 15: 749-795, 1997; Yang, Y .; et al. , Proc. Natl. Acad. Sci. ScL USA, 89: 4928-4932, 1992].
[0037]
In addition, the role of CD4 + and CD8 + T lymphocytes involved in the anti-tumor immunizing effect on the TC-1 cell line was investigated (FIG. 6). When all the CD4 + and CD8 + T lymphocyte groups were removed from the animals co-injected with the E7 protein and CpG-ODN, the tumor formation was similar to that of the untreated control group. On the other hand, when only CD8 + T lymphocytes were removed, tumors were generated in all animals, although slightly slower than the control group. This demonstrates that CD8 + T lymphocytes are the most important population of immune cells. When the CD4 + T lymphocyte group was removed, one out of five tumors formed. This means that the CD4 + T lymphocyte group is also a group of immune cells involved in the formation of protective immune characteristics. This means that the CD4 + and especially the CD8 + T lymphocyte population has an antitumor effect. Antibodies did not contribute much in terms of the anti-tumor immunizing effect on TC-1.
[0038]
These results are consistent with other findings that all CD4 + or CD8 + effector T lymphocyte populations have antitumor function against the TC-1 tumor cell line [see Hung, C. et al. F. et al. , Cancer Res. , 61: 3698-3703, 2001; Lamikanra, A .; et al. , J. et al. Virol. Cheng, W., 75: 9654-9664, 2001; F. et al. , Hum. Gene Ther. Liu, D., 13: 553-568, 2002; W. et al. , J. et al. Virol. Lin, K., 74: 2888-2894, 2000; Y. et al. , Cancer Res. , 56: 21-26, 1996].
[0039]
[Table 1]

[0040]
As is clear from the above results, CpG-ODN increases the activity of E7 antigen-specific reaction, Th proliferation reaction, CD4 + T lymphocytes secreting IFN-γ (Th1) and CD8 + T lymphocytes secreting IFN-γ (CTL). It has a role of an adjuvant to thereby suppress the effect of papillomavirus-induced disease, especially the suppression of cervical cancer, from the antigen-specific Th1-type CD4 + and especially the CD8 + T cell immune response only by simultaneous use of the E7 protein and CpG-ODN. It proves to be useful in inducing an immunity effect.
[0041]
The pharmaceutical compositions of the present invention can be administered prophylactically or therapeutically to a subject infected or suspected of being infected with papillomavirus. Such administration subjects include those who have or are at risk of developing a disease induced by papillomavirus. Diseases induced by papillomavirus include, for example, Bowen's papulosis, anal dysplasia, respiratory or conjunctival papilloma, cervical dysplasia, cervical cancer, genital cancer, prostate cancer, and the like.
[0042]
In particular, the pharmaceutical compositions of the present invention are useful for preventing and treating cervical cancer induced by papillomavirus.
[0043]
The pharmaceutical compositions of the present invention can be administered alone or with other known treatments in the art, such as chemotherapy treatment, radiation treatment, surgery and the like. It can also be co-administered with other adjuvants or cytokines well known in the art. Cytokines capable of stimulating an immune response that can be co-administered include, for example, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF), IL-1, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, TNF-α, INF-γ, Flt3 ligand and the like.
[0044]
The pharmaceutical compositions of the present invention can be prepared by any of the methods well known in the art [see: Donnelly et al. , J. et al. Imm. Methods, 176: 145, 1994; Vitiello et al. , J. et al. Clin. Invest, 95: 341, 1995]. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, suppositories, aerosols, and the like. The dosage form may further include an implanted controlled release device or the like. Also, methods known in the art, for example, oral or parenteral, such as intramuscular, intravenous, intraarterial, intradermal, intraperitoneal, intranasal, vaginal, rectal, sublingual or subcutaneous administration, or gastrointestinal tract, It can be administered via the mucosa or the respiratory tract.
[0045]
The pharmaceutical composition of the present invention can be administered locally or systemically. Local administration is advantageous because it allows the drug to be concentrated at the site of administration while minimizing systemic absorption. In such local administration, smaller amounts can be administered than in the case of administration by other routes. Dosage forms for topical administration include transdermal devices, aerosols, creams and ointments, lotions, powders and the like.
[0046]
The pharmaceutical compositions of the present invention can be formulated using one or more pharmaceutically acceptable carriers, including excipients, or any adjuvant that facilitates formulation. The dosage form depends on the route of administration. For injection, the active ingredients can be formulated in aqueous solutions, preferably in physiological saline. For mucosal delivery, penetrants appropriate to the obstacle to be permeated can be used in formulating. Such penetrants are generally and widely known in the art. For oral administration, the active ingredients can be combined with carriers suitable for inclusion in tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. For administration by inhalation, the active ingredient is delivered from a pressurized pack using an appropriate propellant in the form of an aerosol spray or in the form of a powder that can be formulated into cartridges. be able to. When administered by injection, it can be formed as a suspension, liquid, emulsifier, or the like.
[0047]
The dose may be about 0.1 μg / kg / day to about 3 μg / kg / day, preferably 0.5 μg / kg / day to 1 μg / kg / day at a time, with a minimum of one week interval. It depends on various factors such as body weight, age, gender, route of administration, dosage form and time, and general health.
[0048]
Hereinafter, the present invention will be described with reference to specific examples. The described embodiments are merely illustrative and do not limit the invention.
[0049]
【Example】
Abbreviation explanation
ODN: oligodeoxynucleotide, HPV: human papilloma virus, PBS: phosphate buffered saline, HRP: horse radish peroxidase, HSV: herpes simplex virus, OD: absorbance, IPTG: isopropyl-D-thiogalacto Pyranoside, RT-PCR: reverse transcriptase chain reaction, i. p. : Intraperitoneal, s. c. : Subcutaneous, SI: stimulation index.
Statistical analysis utilized the paired Student's T test (The paired Student's T Test) and placed statistical significance when p-value <0.05.
[0050]
Example 1: Production of recombinant E7 protein
Papillomavirus type 16 E7 recombinant protein was prepared by known methods [see: Novagen protocol and Sin. J. I. Et al. , Vaccine 15: 1827-1833, 1997].
Two primers from a cervical cancer cell line (Caski cell line), namely 5′-TTGGATCCACCATGCCATGGAGATACACCTAC-3 ′ (SEQ ID NO: 2) (including BamHI cleavage site) 5′-CGGAATTCATTCTTTATGGTTCTCG-3 ′ (SEQ ID NO: 3) The papillomavirus type 16 E7 gene was obtained by reverse transcriptase chain reaction (including the EcoRI cleavage site). After the obtained E7 gene was cut with restriction enzymes (BamHI and EcoRI), the E7 gene was separated from the DNA gel. Thereafter, the isolated E7 gene was cloned into a pET vector (Novagen, Madison, WT) which had been treated with BamHI and EcoRI. Using the cloned DNA, E. coli was used. E. coli (Escherichia coli) DH5 is transformed and E. coli growing under kanamycin. E. coli was selected to separate pET-E7 DNA. coli BL21 (DE3) was transformed. The obtained E. E. coli (pET-E7) was cultured in an LB solution containing kanamycin (30 μg / ml), and when the OD value reached about 0.6 to 0.8 at a wavelength of 600 nm, 3 mM was added after adding 1 mM IPTG. Cultured. E. FIG. After centrifugation of E. coli at 4 krpm for 20 minutes, a freeze-thaw process was performed once at -20 ° C. After dissolving the cell precipitate in 5 ml of 8M urea buffer (pH 8.0), the cells were disrupted with stirring at room temperature for 60 minutes. Thereafter, the mixture was centrifuged at 1.5 krpm for 30 minutes to obtain a supernatant, washed with an 8 M urea buffer (pH 8.0), and passed through a Ni-NTA resin column (Qiagen, Valencia, CA). Next, 5 times volume of buffer B (8M urea buffer, pH 8.0) was passed through the resin column, and then 5 to 10 times volume of buffer C (8M urea buffer, pH 6.3) was passed. I let it. Finally, the His-tagE7 protein was eluted using 10 ml of buffer C (buffer B to which 200 mM imidazole was added). At 2-hour intervals, the protein solution was dialyzed against a 6 M urea buffer and then again against a 4 M urea buffer. Finally, it was dialyzed against saline. Thereafter, endotoxin was removed by permeation through a Detoxi-Gel endotoxin removal column (Pierce, Rockford, IL). Protein concentration was quantified by the known Bradford method [see: Bradford, M. et al. M. , Anal. Biochem. , 72: 248-254, 1976]. Finally, the E7 protein was stored at -70 ° C.
[0051]
The prepared protein sample was separated from 12% SDS-PAGE and then attached to a nitrocellulose membrane (Amersham, Piscataway, NJ). Next, the nitrocellulose membrane was immersed in a TBST solution containing 2% BSA [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20] for 1 hour, and then an E7 specific antibody (Oncogene, Boston, Mass.). ) For 1 hour. After each reaction, the plate was washed several times with a TBST solution, and finally reacted with anti-mouse IgG-HRP (Sigma) for 1 hour, and then developed with an ECL solution (Amersham).
[0052]
A recombinant E7 protein containing 98 amino acids was obtained from E. coli. coli. The recombinant protein was identified as a 23 kD protein by SDS-PAGE, and reacted with an E7-specific antibody by immunoblotting assay. The actual size of the protein was larger than expected (11 kD E7 protein and His-tag 4 kD protein in pET vector). The migratory properties of abnormal E7 proteins in gels have been previously published [see Armstrong, D .; J. et al. , Biochem. Biophys. Res. Commun. , 192: 1380-1387, 1993; Fernando, G .; J. P. et al. , Clin. Exp. Immunol. , 115: 397-403, 1999].
[0053]
Endotoxin levels in the E7 protein were quantified using an endotoxin detection kit (Sigma, Saint Louis, MO). Endotoxin levels in the protein were determined to be less than 100 EU / mg.
[0054]
Example 2: Production of CpG-ODN
In this example, CpG-ODN1826 (5'-TCCATGACGTTCCTGACGTTT-3 ') (SEQ ID NO: 1) was used, and ODN changed with phosphorothioate for each base was used for Biobasic Inc. (Canada) and purchased. CpG-ODN was used by dissolving in endotoxin-free water.
[0055]
Example 3: Antitumor activity
A 4-6 week old C56BL / 6 mouse (Daehan Biolink co., Ltd) was used as an animal model, and 20 μg of the recombinant E7 protein, 20 μg of CpG-ODN, or all of them were diluted in 100 μl of final saline. It was injected subcutaneously with a 28-gauge needle (Becton Dickinson, Franklin Lakes, NJ).
[0056]
The cell line used for tumor induction was TC-1 cells, provided by Dr. Wu of John Hopkins University, and cultured in cRPMI (400 μg / ml G418). TC-1 is an E7-expressing cell line, which is a cell line produced by using papillomavirus type 16 E6 and E7 and ras genes in primary lung epithelial cells of C57BL / 6 mice [ See Wu, T .; C. et al. , Proc. Natl. Acad. Sci. ScL USA, 92: 11671-11675, 1995]. 2 × 10 to test each preventive and therapeutic effect⁵And 5 × 10⁴TC-1 cell lines were injected subcutaneously into C57BL / 6 mice. Various injection volumes have already been published [see Lin, K. et al. Y. et al. , Cancer Res. , 56: 21-26, 1996]. The TC-1 cell line was washed twice with physiological saline and then used for injection.
[0057]
Example 3 . 1: Tumor preventive activity
From the mouse animal model, anti-tumor effects induced by vaccination using E7, CpG-ODN or both were observed. Animals in each group (n = 11-16) were injected subcutaneously at week 0 and week 2 with 20 μg E7, 20 μg CpG-ODN or both. At 3 weeks after the last injection, 2 x 10 per animal⁵TC-1 tumor cell lines were injected subcutaneously.
[0058]
FIG. 1 and Table 2 show that two vaccinations with E7 (20 μg per animal), CpG-ODN (20 μg per animal) or both were performed at 2 × 10⁵2 shows antitumor activity after injection of one TC-1 tumor cell line.
[0059]
[Table 2]

[0060]
As can be seen from Table 2, when E7 or CpG-ODN was injected alone, tumors appeared after challenge with the TC-1 tumor cell line, as in the untreated control group. In the group injected with E7 and CpG-ODN at the same time, no tumor appeared at all over time. That is, when E7 or CpG-ODN was treated alone, no tumor preventive effect was shown, but when E7 and CpG-ODN were simultaneously treated, complete tumor prevention was observed in all mouse animal models. The effect was shown.
[0061]
In addition, the results of FIG. 1 showing the size of the tumor after the tumor challenge are shown, the results show that when E7 or CpG-ODN is injected alone, the tumor gradually grows with time, but E7 It shows that no tumor grows when CpG-ODN is injected simultaneously. At this time, all animals in which the tumor was formed died after 30 to 40 days. Animals co-injected with E7 and CpG-ODN survived for more than 4 months. Necropsy of the dead animal revealed that the cause of death was cachectic shock and was not metastasized to other organs.
[0062]
As can be seen from Table 2 and FIG. 1, there is a preventive effect capable of suppressing the growth of an E7-expressing tumor cell line only when E7 and CpG-ODN are simultaneously injected.
[0063]
Example 3 . 2: Tumor therapeutic activity
From the mouse animal model, antitumor effects induced by injection of E7, CpG-ODN, or both on already formed tumors were observed. 5 × 10 animals in each group⁴SC-1 tumor cell lines were injected subcutaneously and when the tumor size was 1-2 mm, 20 μg of E7, 20 μg of CpG-ODN or 20 μg of E7 and 20 μg of E7 were placed away from the tumor injection site. CpG-ODN was injected subcutaneously, and one week later, it was injected subcutaneously. Tumor size was measured twice a week for 1 month after injection using a measuring instrument. The tumor size was calculated by the average value of the tumor [(a + b) / 2, a: major axis length of the tumor, b: shortened length of the tumor].
[0064]
FIG. 2 shows the result of observing the decrease rate of the tumor-bearing mice after the tumor challenge. FIG. 2 shows that only 20% of the animals in the group of the animals injected with E7 and CpG-ODN had tumors detected, and the remaining animals had tumors. However, in the group of animals injected with E7 alone and the group of animals injected with CpG-ODN alone, tumors were not suppressed in all animals as in the untreated control group. On the other hand, the size of the tumor observed from one of the animals co-injected with E7 and CpG-ODN was relatively smaller than that of the other treatment groups. In the group of animals injected with E7 or CpG-ODN alone, all died 40 days after the tumor injection, but the group treated with both E7 and CpG-ODN survived for 2 months or longer.
[0065]
This indicates that only when E7 and CpG-ODN are co-injected, the growth of the already formed E7-expressing tumor cell line can be suppressed and the antitumor therapeutic effect can be obtained.
[0066]
Example 3 . 3: Selective immune cell removal activity
The numbers of CD4 + T lymphocytes and CD8 + lymphocytes were determined by FACS analysis. Spleen cells (1 × 10 5) collected from the mouse animal model co-injected with E7 and CpG-ODN⁵Were washed three times with FACS buffer (PBS + 1% BSA + 0.1% sodium azide), and with anti-mouse CD4 and CD8 antibodies conjugated to phycoerythrin (Pharmingen, San Diego, CA). The reaction was performed at 4 ° C. for 30 minutes. Thereafter, the cells were washed three times with a FACS buffer, and the numbers of CD4 + T lymphocytes and CD8 + T lymphocytes were measured using a flow cytometer (Coulter-Epics XL, Miami, FL).
[0067]
Known methods were used to remove CD4 + and CD8 + T cells in vitro and in vivo [see Sin, J. et al. I. Et al. Sin, J. et al., Human Gene Therapy, 12: 1091-1102, 2001; I. et al. , J. et al. Immunol. , 162: 2912-2921, 1999].
Specifically, in vitro lymphocyte removal was performed as follows. Spleen cells from animals co-injected with E7 and CpG-ODN were reacted with anti-CD4 antibody (Pharmingen) or anti-CD8 antibody (Accurate Chemical & Scientific Corp., Westbury, NY) for 1 hour at 4 ° C. Thereafter, the cells were cultured at 37 ° C. for 1 hour together with rabbit complement (Sigma). The viability of the cells was measured by the trypan blue dye removal method, and it was confirmed by FACS that 98% or more of T lymphocytes were removed after the two separation removal steps.
[0068]
Removal of lymphocytes in vivo was processed as follows. Nude mice previously treated with pristane were injected with each hybridoma cell line (American Type Culture Collection, Manassas, VA) to ascites anti-CD4 antibody (clone GK1.5) and anti-CD8 antibody (clone 2.43). The liquid was collected. 100 μl of the obtained ascites fluid (anti-CD4 antibody and anti-CD8 antibody) was injected intraperitoneally on days 3, 0 and 3 before and after tumor injection. Using FACS, it was confirmed that 98% or more of CD4 + T lymphocytes and CD8 + T lymphocytes were removed, and tumors were injected on day 0.
[0069]
The results of confirming the cell group involved in the antitumor activity from the animal model are shown in FIG. FIG. 6 shows that after the co-injection of E7 and CpG-ODN, CD4 + T cells, CD8 + T cells, or all CD4 + T cells and CD8 + T cells were removed, and the importance of these cell groups on the antitumor effect was confirmed. It is. Specifically, 20 μg of E7 and 20 μg of CpG-ODN were subcutaneously injected into the animals (n = 5) of each group (n = 5) at weeks 0 and 2 and at week 3 after the final injection, CD4 + T, After removing CD8 + T or all two cell groups, 2 × 10⁵After subcutaneous injection of the TC-1 tumor cell lines, the number of tumor-free mice was determined.
[0070]
According to this, as shown in Table 1 and FIG. 1, when E7 and CpG-ODN were co-injected and CD4 + T cells and CD8 + cells were not removed, a complete tumor suppression effect was observed. . On the other hand, when all CD4 + and CD8 + T lymphocytes were removed, tumors were formed similarly to the non-treated control group. In particular, in the animals from which CD8 + T lymphocytes had been removed, tumors were formed, although slightly slower than the untreated control group. On the other hand, in the animals from which CD4 + T lymphocytes had been removed, no tumor was observed in 4 out of 5 animals. Also, the size of the tumor formed in one animal was relatively small.
[0071]
These immunized cell group removal experiments show that E7 activates CD4 + T lymphocytes and especially CD8 + T lymphocytes in the presence of CpG-ODN to exhibit an antitumor effect. Thus, one can confirm the fact that the antitumor effect in animal models is mediated by CD4 + T lymphocytes and especially the CD8 + T lymphocyte population.
[0072]
Example 4: Immunization reaction induction
Example 4 . 1: Induction of antibody reaction
ELISA was performed using known methods [see: Sin, J. et al. I. et al. Sin, J., Vaccine, 15: 1827-1833, 1997; I. et al. , J. et al. Virol. , 74: 11173-11180, 2000].
[0073]
To coat the plate, the recombinant E7 protein was diluted in physiological saline at a concentration of 1 μg / ml, added to each well in an amount of 50 μl, and used the next day. After blocking with an ELISA buffer (2% BSA), anti-murine IgG-HRP was diluted at a concentration of 1: 1000, and added to each well by 50 μl for reaction. Also, to detect E7-specific IgG subclasses, anti-mouse IgG1, IgG2a, IgG2b or IgG3 (Zymed, San Francisco, Calif.) Conjugated with HRP was used instead of anti-mouse IgG-HRP.
[0074]
Animals in each group were injected subcutaneously at week 0 and week 2 with 20 μg E7, 20 μg CpG-ODN, or 20 μg E7 and 20 μg CpG-ODN. At 2, 4 and 8 weeks after the first injection, serum was collected from each animal group (n = 10), and the serum of each group mixed in the same volume was diluted by a factor of 2 to measure the ELISA titer. In addition, the sera at 4 weeks and 8 weeks were diluted 1: 100 and subjected to ELISA. The ELISA titer was the reciprocal of the highest serum dilution that was twice the OD value of the control group. OD values were measured at 405 nm. Has statistical significance at p <0.05 compared to E7 injection.
[0075]
FIG. 3 shows the results of measuring the degree of E7-specific antibody production by E7 single injection, CpG-ODN single injection, or co-injection of E7 and CpG-ODN. FIG. 3A shows that the serum ELISA titer after 4 weeks of animals injected with E7 alone was 1,600, whereas the serum ELISA titer after 4 weeks of animals co-injected with E7 and CpG-ODN was 6 weeks. , 400. This is because antibody production increased more than 2-fold in antibody titer by simultaneous use of E7 and CpG-ODN. The ELISA titer at 8 weeks after injection was 800 for E7, and 3,200 when E7 and CpG-ODN were used simultaneously, showing a similar increase. However, no antibody reaction was observed in animals injected with CpG-ODN alone as in the non-treated control group.
[0076]
FIGS. 3B, 3C, 3D and 3E show the generation of E7-specific antibodies against IgG isoforms. In serum 4 weeks and 8 weeks after the first injection, co-injection of E7 and CpG-ODN induced more generation of IgG isoforms than injection of E7 alone.
[0077]
Indeed, IgG1 and IgE are Th2 linked antibodies, and IgG2a is a Th1 linked antibody [see Finkelman, F. et al. D. et al. , Ann. Rev .. Immunol. , 8: 303-333, 1990]. In particular, when E7 and CpG-ODN were co-injected, the production of IgG2a was further increased as compared with E7 alone injection. The values of IgG2a / IgG1 were calculated at 0.2 (E7) and 0.26 (E7 + CpG-ODN).
[0078]
As can be seen from these results, since CpG-ODN increases the overall antibody immune response, the co-injection of E7 and CpG-ODN results in the generation of IgG and IgG subclasses as compared to E7 alone injection. To increase.
[0079]
Example 4 . 2: Induction of T helper (Th) cell proliferation reaction
Th cell proliferative response plays an important role in cell immune response. Spleen immune cells were collected from each animal group that had been injected with E7 alone, CpG-ODN alone, or co-injected with E7 and CpG-ODN, and the Th cell proliferation response was measured.
[0080]
Th cell proliferative reactions were performed by known methods [see Sin, J. et al. I. et al. Sin, J. et al., Human Gene Therapy, 12: 1091-1102, 2001; I. et al. , J. et al. Immunol. , 162: 2912-2921, 1999].
[0081]
Animals in each group were injected subcutaneously with 20 μg of E7, 20 μg of CpG-ODN, or 20 μg of E7 and 20 μg of CpG-ODN at weeks 0 and 2 and spleen immunization from the animal group 3 weeks after the last injection. Cells were harvested. The collected spleen cells were stimulated with various concentrations (0.5, 1, and 5 μg / ml) of E7 for 3 days, and then labeled.³After addition of thymidine with [H] (1 μCi per well), cells were harvested the next day and cpm of each immune group was measured with a β counter (PerkinElmer, Boston, Mass.). SI was calculated as [experimental group cpm-control group cpm] / [control group cpm].
[0082]
FIG. 4A shows that co-injection of E7 and CpG-ODN increases the Th cell proliferative response compared to E7 alone injection. The result is one of the duplicate experiments showing the same result. * Has statistical significance at p <0.05 compared to control group, ** has statistical significance at p <0.05 compared to E7 injection group. When E7 was injected alone, an E7-specific Th cell proliferation reaction appeared, but when E7 and CpG-ODN were co-injected, the Th cell proliferation reaction was further increased. On the other hand, when CpG-ODN was injected alone, no Th cell proliferation reaction was observed as in the non-treated control group.
[0083]
Example 4 . 3: Induction of cytotoxic T lymphocyte (CTL) response
5 hours⁵¹A CTL test was performed using the Cr detection method.
Animals in each group were injected subcutaneously with 20 μg of E7, 20 μg of CpG-ODN, or 20 μg of E7 and 20 μg of CpG-ODN at weeks 0 and 2 and spleen immunization from the animal group 3 weeks after the last injection. Cells were harvested. The collected spleen cells were activated by culturing for 5 days together with 20 U / ml of IL-2 (R & D Systems, Minneapolis, MN) and a TC-1 cell line previously treated with mitomycin C (30 μg / ml) for 3 hours. Was. Then, 100 μCi / ml Na₂ ⁵¹CrO₄And TC-1 cells cultured for 2 hours at a different ratio from the activated immune cells for 5 hours to increase cytolytic activity against TC-1 cells.⁵¹It is measured by the degree of Cr secretion. Thereafter, 100 μl of the cell culture was collected and cpm was measured using a γ-counter (PerkinElmer). The CTL activity (%) is 100 × [(experimental group cpm−natural integration ratio cpm) / (maximum secreted cpm−natural integration ratio cpm)]. The maximum secreted cpm was the cpm of the target TC-1 cells to which 1% Triton X-100 was added.
[0084]
FIG. 4B shows that the CTL reaction was not observed in the case of the E7 single injection and the CpG-ODN alone injection, but the CTL reaction was observed in the case of the simultaneous injection of E7 and CpG-ODN. The result is one of the duplicate experiments showing the same result.
As shown in FIG. 4B, the CTL response was induced only when E7 and CpG-ODN were co-injected.
This indicates that E7 can induce an E7-specific CTL response in the presence of CpG-ODN.
[0085]
Example 4 . 4: Generation of IFN-γ
IFN-γ plays an important role in inducing Th1-type immune response and CTL response. The degree of IFN-γ production in CD4 + T lymphocytes and CD8 + T lymphocytes was measured.
[0086]
Animals in each group were injected subcutaneously with 20 μg of E7, 20 μg of CpG-ODN, or 20 μg of E7 and 20 μg of CpG-ODN at weeks 0 and 2 and spleen immunization from the animal group 3 weeks after the last injection. Cells were harvested. 6 × 10 6 spleen cells collected⁶Cells / ml in a 24-well flask, 1 μg / ml of recombinant E7 protein or TC-1 cell line (5 × 10 5 cells) previously treated with mitomycin C (30 μg / ml) for 3 hours.⁵Cells / ml) at 37 ° C. and 5% CO 2₂The cells were cultured under culture conditions for 3 days. The cell culture was taken and the amount of secreted IFN-γ was measured using an IFN-γ detection kit (Biosource, Intl., Camarillo, CA).
[0087]
Specifically, spleen immune cells collected from animals were stimulated with E7 protein in order to measure the degree of IFN-γ production in CD4 + T lymphocytes.
[0088]
FIG. 5A shows IFN- when the spleen immune cells induced by injection of E7 alone, CpG-ODN alone, or co-injection of E7 and CpG-ODN were added to 1 μg of E7 protein / ml and cultured for 3 days. Fig. 4 shows the secretion of γ. The production of IFN-γ was observed only from the group of animals co-injected with E7 and CpG-ODN, and not from the group injected with E7 alone or CpG-ODN alone.
[0089]
FIG. 5B shows IFN-γ when CD4 + T or CD8 + T lymphocytes were removed from spleen immune cells co-injected with E7 and CpG-ODN, added to 1 μg of E7 protein / ml, and cultured for 3 days. Indicates secretion of When CD4 + T lymphocytes were removed from the spleen immune cells, the production of IFN-γ was not observed, but when CD + 8 T lymphocytes were removed, the production of IFN-γ was observed. Numerical values and bars mean IFN-γ mean and error. The above results were repeated twice and similar results were measured.
[0090]
These results indicate that Th1-type CD4 + T lymphocytes are activated only by co-injection of E7 and CpG-ODN, and that CpG-ODN induces an E7-specific Th1-type immune response.
[0091]
To measure the degree of IFN-γ production in CD8 + T lymphocytes, spleen immune cells collected from animals treated with E7 alone injection, CpG-ODN alone injection, or co-injection of E7 and CpG-ODN were used. , An inactivated TC-1 cell line (MHC class I⁺, Class II⁻) For 3 days.
FIG. 5C shows IFN- when the spleen immune cells induced by E7 alone injection, CpG-ODN alone injection, or co-injection of E7 and CpG-ODN were cultured with TC-1 previously treated with mitomycin C for 3 days. Fig. 4 shows the secretion of γ. The production of IFN-γ was observed only from the group of animals co-injected with E7 and CpG-ODN, but not from the group injected with E7 alone or CpG-ODN alone. These results are the same as those of CTL (IFN-γ secreting CD8 + T cells) (FIG. 4B).
[0092]
FIG. 5D shows IFN-γ obtained by removing CD4 + T or CD8 + T lymphocytes from spleen immune cells co-injected with E7 and CpG-ODN, and then culturing with TC-1 previously treated with mitomycin C for 3 days. Indicates secretion of When CD8 + T lymphocytes were removed from spleen immune cells, IFN-γ production was not observed, but when CD + 4 T lymphocytes were removed, IFN-γ production was observed. Numerical values and bars indicate the average value and error of IFN-γ. The above results were repeated twice and similar results were measured.
[0093]
These results indicate that CD8 + T lymphocytes are linked to MHC class I to produce IFN-γ only by co-injection of E7 and CpG-ODN. Although the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various modifications can be made within the spirit and scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments illustrated herein.
[0094]
【The invention's effect】
The pharmaceutical composition of the present invention comprising papillomavirus antigen protein and CpG-oligodeoxynucleotide has an antitumor activity in an animal model only when they are simultaneously administered, shows a CTL response, shows CD4 + T lymphocytes and Since a potent immune response that induces the production of IFN-γ in CD8 + T lymphocytes can be induced, a disease induced by papillomavirus can be effectively prevented or treated.

[Brief description of the drawings]
FIG. 1 shows the antitumor preventive effects of E7 alone injection, CpG-oligodeoxynucleotide (CpG-ODN) alone injection, and co-injection of E7 and CpG-ODN.
FIG. 2 shows the antitumor therapeutic effect of E7 alone injection, CpG-ODN alone injection, and co-injection of E7 and CpG-ODN.
FIG. 3 shows E7-specific antibody reactions (IgG, IgG1, IgG2a, IgG2b, and IgG3) by injection of E7 alone, CpG-ODN alone, and co-injection of E7 and CpG-ODN.
FIG. 4 shows E7-specific Th cell proliferation and CTL responses induced by E7 alone injection, CpG-ODN alone injection, and co-injection of E7 and CpG-ODN.
FIG. 5 shows the degree of IFN-γ secretion in immune cells induced by E7 alone injection, CpG-ODN alone injection, and co-injection of E7 and CpG-ODN, and spleen immunity co-injected with E7 and CpG-ODN. FIG. 4 shows the degree of IFN-γ secretion after removing CD4 + T or CD8 + T lymphocytes from cells, respectively.
FIG. 6 shows the role of immune cell groups on antitumor effects.

Claims (5)

An immunologically effective amount of a papillomavirus E7 antigen protein and a CpG-oligodeoxynucleotide, wherein the oligodeoxynucleotide has one or more nucleotides separating a contiguous CpG motif within the oligodeoxynucleotide. A pharmaceutical composition for preventing or treating cell proliferative disease induced by papillomavirus, comprising 8 to 40 nucleotides having one or more CpG motifs. The pharmaceutical composition according to claim 1, wherein the papillomavirus antigen protein is human papillomavirus type 16 E7 protein. The pharmaceutical composition according to claim 2, wherein the human papillomavirus type 16 E7 protein is a recombinant protein. The pharmaceutical composition according to any one of claims 1 to 3, wherein the CpG-oligodeoxynucleotide is 5'-TCCATGACGTTCCTGACGTTT-3 '(SEQ ID NO: 1). The pharmaceutical composition according to any one of claims 1 to 4, wherein the disease induced by the papillomavirus is cervical cancer.

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