JP4976853B2 - ボルデテラ(Bordetella)の組換えアデニル酸シクラーゼ毒素は腫瘍抗原に対するT細胞応答を誘発する - Google Patents
ボルデテラ(Bordetella)の組換えアデニル酸シクラーゼ毒素は腫瘍抗原に対するT細胞応答を誘発する Download PDFInfo
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Description
本発明は、がんを処置するための組成物及び方法に関する。
本発明は、免疫応答を誘発する組換えCyaAタンパク質を提供することにより当該技術分野における要求を達成するのを助ける。これらの応答は腫瘍抗原に向けて指向されうる。
形質細胞膜を横切って細胞質ゾル(そこで適当なプロセッシング及びMHCクラスI分子との相互作用が起こりうる)中に抗原性エピトープを送達することができるバクテリア毒素に基づいて、CTL活性化のための新規なアプローチが最近開発された。ボルデテラ ペルトゥッシスのアデニル酸シクラーゼ毒素(CyaA)(Glasser, P., et al. 1988 ボルデテラ ペルトゥッシス adenylate cyclase: the gene and the protein, Tokai J.Exp.Clin.Med., 13 Supp.: 239)は、その触媒ドメインを真核細胞の細胞質ゾルに送達する能力を有する(12)。CyaAの触媒ドメインに挿入されたCD8+T細胞エピトープの送達は、細胞内プロセッシング及び抗原提示細胞(13)の表面におけるMHCクラスI分子によるエピトープの提示をもたらす。更に、CyaAは、αMβ2インテグリン(CD11b/CD18)に特異的に結合し(14)、かくして、CD11b+DC亜集団を標的化し、これは一次免疫応答(15)を非常に有効に誘発する(15)。故に、ウイルスエピトープを有する組換えCyaA毒素によるマウスの免疫感作は、強いCTL応答の誘発及び致死的ウイルスチャレンジ(lethal viral challenge)に対する完全な保護をもたらす(16)。
実施例1
物質及び方法
マウス。HHDマウスは、H−2Db(D)のα3膜貫通及び細胞質ドメインに連結されたHLA−A*0201のα1(H)及びα2(H)ドメインを含むHHD導入遺伝子(該α1ドメインはβ2ミクログロブリンに連結されている)を発現するH−2D-/-β2m-/-ダブルノックアウトマウスである。かくして、HHDマウスにより発現されたMHCクラスI分子のみが、修飾されたHLA−A*0201分子である(19)。HHDマウスを繁殖させそしてInstitute Pasteurの動物施設に収容した。
25μl中でU底部マイクロプレート中に播種した。種々の濃度でX−Vivo10培地中に希釈されたCyaA調製物25μlをウエルに加えた。30分のインキュベーションの後に、対応するCTLクローンを、これらの細胞(104抗チロシナーゼCTLクローンIVS−B又は104抗GnT−V CTLクローンCMU5796/3を含有するX−vivo培地75μl)及びIL−2(25U/mlの最終濃度で)と共にインキュベーションした。上清を20時間後に集めそしてそれらのIFN−γ含有率をELISA(Biosource International, Carmarillo, CA)により決定した。種々の脱毒素化された組換えトキソイドとインキュベーションされたDCのCTLクローンを刺激する能力を制御するために、それらは、関連した抗原ペプチドを外因的にロードされ、関連したCTLクローンと共にインキュベーションされそしてIFN−γの産生を同様に評価された(データは示されていない)。
HLA−A*0201拘束性メラノーマエピトープを有する組換えCyaAによるHHDトランスジェニックマウスの免疫感作によるメラノーマ特異的CTL応答の誘発
CyaA毒素がヒト腫瘍抗原に対する特異的CTL応答を誘発することができるかどうかを決定するために、HLA−A*0201拘束性ヒトメラノーマエピトープを有する2つの組換えCyaAを構築した。第1の組換えCyaAは、チロシナーゼ抗原からのエピトープ369〜377を発現し(CyaA−Tyr)、そして第2の組換えCyaAは、N−アセチルグルコサミニルトランスフェラーゼVのイントロンに由来するエピトープNA17−Aを発現する(CyaA−GnT−V)。インビボでこれらの2つのエピトープに対するCTLを誘発する組換えCyaAの能力をHHDマウスで評価したが、このHHDマウスは、ヒトMHCクラスI分子HLA−A*0201に対してトランスジェニックでありそして腫瘍ペプチドに対してHLA−A*0201拘束性CTL応答を発生することが示された(26)。HHDマウスは、明礬を伴う組換えCyaA50μgの3回の腹腔内注射により免疫感作された。相当するペプチドによる脾臓細胞のインビトロ刺激の後に、HHD―マウスと同じ導入遺伝子を発現する、ペプチド−パルスされたRMA−S−HHD細胞を標的として使用する、クロム放出アッセイにおいてCTL応答を試験した。図1に示されたとおり、Tyr又はGnT−Vエピトープを有する両組換え毒素は、関連したペプチドをロードされた標的細胞に対する強いCTL応答を誘発する。これらのCTL応答は、抗原特異的であった。何故ならば、ペプチドで感作された標的細胞のみが殺され、そして無関係のペプチドをロードされた標的細胞に対するCTL活性は検出されなかったからである(データは示されていない)。予想されるとおり、野生型CyaAで免疫感作されたマウスで有意なCTL活性は観察されなかったが、これは、特異的CTL応答の誘発は、組換えCyaAに挿入されたエピトープによるインビボプライミング(in vivo priming)を必要とすることを示す。
組換えCyaA−Tyrは長く続く記憶CTL応答を誘発する
メラノーマエピトープを有する組換えCyaAにより誘発されたCTL応答の持続を分析するために、HHDマウスは、明礬の存在下にCyaA−Tyr50μgの2回の腹腔内注射を受け取った。最後の注射の3ヶ月及び5ヶ月後に、免疫感作されたマウスからの脾臓細胞をペプチドpTyrで5日間にわたりインビトロで刺激し、次いでペプチドパルスされたRMA−S−HHD標的細胞に対するそれらの細胞傷害性活性を試験した。図3に示されたとおり、CyaA−Tyrは、長く続く特異的CTL応答を誘発した。何故ならば、特異的細胞傷害性活性は、すべてのマウスにおいて、最後の注射の3ヶ月後に検出されることができ、そして1つの動物においては5ヶ月後ですら検出されることができたからである。
CyaAに挿入されたHLA−A*0201拘束性ペプチドは、HLA−A*0201ヒトDCによりプロセッシングされそして提示される
組換えCyaAによる特異的CTL応答のインビボ誘発は、挿入されたエピトープがマウスAPCにより有効にプロセッシングされそして提示されることを示す。しかしながら、ヒトAPCもまたCyaAに挿入されたこれらのHLA−A*0201拘束性エピトープをプロセッシングしそして提示することができることを証明することは重要である。DCは一次T細胞応答を誘発させるのに最も重要なAPCであるので、組換えCyaAとインキュベーションされたHLA−A*0201+DCの、組換えCyaAに挿入されたエピトープに対して特異的なヒトCTLクローンを刺激する能力を決定した。これらの実験では、ヒトDCを、GM−CSF及びIL−4の存在下にHLA−A*0201+接着性PBMCからインビトロで発生させた。次いで増加する用量のCyaA−E5−Tyr、CyaA−E5−GnT−V又はコントロールCyaA−E5を加えそして抗原性ペプチドの提示を、処理されたDCの関連したCTLを刺激する能力をIFN−γ産生アッセイにおいて測定することにより、評価した。
応答を誘発しないCyaA毒素構築
エピトープgp100〜280を含みそしてGP100メラノーマ関連腫瘍抗原を形成した挿入された配列YLEPGTVTAを含むCyaA−Mel21は、CTL応答を含まない。同様に、エピトープCEA571〜579を含みそしてがん胎児性抗原からの挿入されたYLSGANLNLを有するCyaA−CEA13は、CTL応答を誘発しない。これらの毒素のいずれもHHDマウスにおける挿入されたエピトープに特異的なCTL応答を誘発しない。更に、いくらかのヒト樹状細胞は、挿入されたエピトープCyaA−Mel21(エピトープgp100〜280)をヒトCTLクローンに提示することができない。ゆえに、この毒素は、多分ヒトにおいても有効ではない。これらの2つの毒素、CyaA−Mel21及びCyaA−CEA13は、CyaA−Tyr及びCyaA−GnTVと同一であるが、挿入された配列においてのみ異なる。故に、エピトープのみが異なりそしてそれ故CyaA−Tyr及びCyaA−GNTVに対する応答はエピトープ特異的である。
Claims (27)
- 組換えタンパク質を含む免疫原性組成物であって、該組換えタンパク質は、
(a)ボルデテラ種のCyaAタンパク質、又はCD11b/CD18レセプターに特異的に結合し、触媒ドメインのトランスロケーションのプロセスに影響されない、その断片、及び
(b)配列がYMDGTMSQVであるチロシナーゼに由来するHLA−A*0201エピトープを含むか又はそれからなるか、又は配列がVLPDVFIRCであるN−アセチルグルコサミニルトランスフェラーゼVに由来するHLA−A*0201エピトープを含むか又はそれからなるペプチド
を含む、免疫原性組成物。 - ボルデテラ CyaA又はその断片と前記ペプチドが、化学的に互いに結合している、請求項1に記載の免疫原性組成物。
- ボルデテラ CyaA又はその断片と前記ペプチドが、遺伝子的に融合されている、請求項1に記載の免疫原性組成物。
- ボルデテラ CyaAが脱毒素化されている、請求項1又は2に記載の免疫原性組成物。
- 上記ペプチドが、YMDGTMSQVからなる、請求項1〜4のいずれか1項に記載の免疫原性組成物。
- 上記ペプチドが、SSMHNALHIYMDGTMSQVQGSANDPIからなる、請求項1〜4のいずれか1項に記載の免疫原性組成物。
- 上記ペプチドが、VLPDVFIRCからなる、請求項1〜4のいずれか1項に記載の免疫原性組成物。
- 上記ペプチドが、MVLPDVFIRCVVFCLからなる、請求項1〜4のいずれか1項に記載の免疫原性組成物。
- ボルデテラ CyaAが、B.ペルトゥッシス、B.パラペルトゥッシス又はB.ブロンキセプチカに由来する、請求項1〜8のいずれか1項に記載の免疫原性組成物。
- 組換えタンパク質が、1つより多くのペプチドを含む、請求項1〜9のいずれか1項に記載の免疫原性組成物。
- ペプチドが同じであるか、又は少なくとも1つが他とは異なる、請求項10に記載の免疫原性組成物。
- 該ペプチドが、CyaAのいかなる許容される部位に挿入される、請求項1及び3〜13のいずれか1項に記載の免疫原性組成物。
- 該許容される部位が、B.ペルトゥッシス CyaAの残基107−108(Gly−His)、残基132−133(Met−Ala)、残基137−138(Val−Ala)、残基224−225(Arg−Ala)、残基228−229(Glu−Ala)、残基232−233(Gly−Leu)、残基235−236(Arg−Glu)、残基317−318(Ser−Ala)、残基335−336(Gly−Gln)及び残基336−337からなる群より選択される、請求項12に記載の免疫原性組成物。
- (1)残基188と189との間のジペプチドLeu−Glnの挿入によって及びコドン224と240の間のペプチドPASVLPDVFIRCGTの挿入によって改変された、ボルデテラ ペルトゥッシスCysA、及び
(2)残基188と189との間のジペプチドLeu−Glnの挿入及びコドン224と240の間のペプチドPASYMDGTMSQVGTRARLKの挿入によって改変された、ボルデテラ ペルトゥッシスCysA
からなる群より選択される組換えタンパク質を含む免疫原性組成物。 - アジュバント及び/又は賦形剤を含む、請求項1〜14のいずれか1項に記載の免疫原性組成物。
- 該組成物が、免疫療法において用いられる、請求項1〜15のいずれか1項に記載の免疫原性組成物。
- 請求項1及び3〜14のいずれか1項に記載の組換えタンパク質をコードする核酸。
- 請求項1及び3〜14のいずれか1項に記載の組換えタンパク質を発現するベクター。
- 受託番号I−3111の下に、2003年10月13日にC.N.C.Mに寄託された宿主細胞に含まれるプラスミド。
- 受託番号I−2679の下に、2001年5月31日にC.N.C.Mに寄託された宿主細胞に含まれるプラスミド。
- 請求項18〜20のいずれか1項に記載のベクター又はプラスミドを含む免疫原性組成物。
- 癌を有する患者の処置において用い、該患者にT細胞応答を誘導するための、請求項1〜15のいずれか1項に記載の免疫原性組成物。
- 癌を有する患者の処置において用い、該患者にT細胞応答を誘導するための、請求項21に記載の免疫原性組成物。
- T細胞応答がCTL応答である、請求項22又は23に記載の免疫原性組成物。
- がんの処置用の薬物の製造のための、請求項1〜15のいずれか1項に記載の免疫原性組成物の使用。
- がんの処置のための、請求項1〜15のいずれか1項に記載の免疫原性組成物。
- メラノーマの処置のための、請求項26に記載の免疫原性組成物。
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ATE438409T1 (de) | 2000-09-15 | 2009-08-15 | Pasteur Institut | Proteinhaltigen vektoren zur einführung von moleküle in cd11b exprimirende zellen |
CA2546452C (en) | 2003-11-21 | 2015-01-20 | Institut Pasteur | Recombinant adenylate cyclase toxin of bordetella induces t cell responses against tumoral antigens |
ES2293178T3 (es) * | 2004-03-18 | 2008-03-16 | Institut Pasteur | Proteina recombinante que contiene epitopos del papilomavirus humano insertados en un proteina adenilato ciclasa o un fragmento de la misma y usos terapeuticos de la misma. |
RU2448729C2 (ru) * | 2006-09-01 | 2012-04-27 | Жантисель | Композиции, вызывающие специфический ответ цитотоксических т-лимфоцитов, включающие лимфо-аблативное соединение и молекулу, содержащую антигенные последовательности и нацеленную на специализированные антиген-презентирующие клетки |
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WO2008088771A2 (en) * | 2007-01-12 | 2008-07-24 | Cornell Research Foundation, Inc. | Adenylyl cyclases as novel targets for the treatment of infection by eukaryotic pathogens |
WO2008121171A1 (en) * | 2007-01-12 | 2008-10-09 | Cornell Research Foundation, Inc. | Adenylyl cyclases as novel targets for antibacterial interventions |
EP2478915A1 (en) | 2011-01-24 | 2012-07-25 | Genticel | CyaA-carried polypeptide(s) and use to induce both therapeutic and prophylactic immune responses |
US9345755B2 (en) * | 2012-02-20 | 2016-05-24 | University Of Virginia Patent Foundation | Composition and methods for treating melanoma |
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US20100310594A1 (en) | 2010-12-09 |
US20050271679A1 (en) | 2005-12-08 |
ATE450272T1 (de) | 2009-12-15 |
US20140227323A1 (en) | 2014-08-14 |
EP2193804B1 (en) | 2015-12-23 |
PT1684801E (pt) | 2010-03-08 |
EP2193804A3 (en) | 2010-06-30 |
CA2546452A1 (en) | 2005-06-16 |
ES2337694T3 (es) | 2010-04-28 |
WO2005053738A1 (en) | 2005-06-16 |
HK1093314A1 (en) | 2007-03-02 |
EP1684801B1 (en) | 2009-12-02 |
DE602004024440D1 (de) | 2010-01-14 |
EP2193804A2 (en) | 2010-06-09 |
PL1684801T3 (pl) | 2010-07-30 |
JP2007511233A (ja) | 2007-05-10 |
EP1684801A1 (en) | 2006-08-02 |
US20090117143A1 (en) | 2009-05-07 |
US9410139B2 (en) | 2016-08-09 |
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