JP2021511054A - Hpv免疫療法 - Google Patents
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- JP2021511054A JP2021511054A JP2020540347A JP2020540347A JP2021511054A JP 2021511054 A JP2021511054 A JP 2021511054A JP 2020540347 A JP2020540347 A JP 2020540347A JP 2020540347 A JP2020540347 A JP 2020540347A JP 2021511054 A JP2021511054 A JP 2021511054A
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Abstract
Description
本出願は、2018年1月24日に出願された米国特許仮出願第62/621,279号に対する優先権の利益を主張するものであり、これは、全体が参照により本明細書に組み込まれる。
自然免疫反応は、HPVの初感染の制御に重要な役割を果たすが、長期的防御は、液性及び細胞媒介性免疫を含む適応免疫反応に依存している。免疫応答性の個体では、HPV感染の大多数が、初感染から2年以内に除去される。CD4+及びCD8+T細胞の浸潤は、自然消退する病変において高い頻度で観察される。
便宜上、本明細書、実施例及び添付の特許請求の範囲で使用される特定の用語をここに集める。
細胞傷害性Tリンパ球(CTL)によって認識され、HPV感染及び/又はがん(例えば、本明細書中に提供されるHPVエピトープを発現するがん)及び/又は前癌性病変の予防及び/又は処置に有用なHPVエピトープを含むペプチドが本明細書において提供される。特定の実施形態では、HPVエピトープは、表1に列挙されたエピトープである。
本明細書に記載されているペプチドをコードする核酸分子が本明細書において提供される。一部の態様において、本明細書に開示される核酸を対象に投与することにより、がん、前癌性病変又はHPVを処置する方法が本明細書において提供される。核酸は、例えば細胞全体において、細胞溶解物中に、又は部分的に精製された若しくは実質的に純粋な形態で存在し得る。
一部の態様において、本明細書において提供される組成物及び方法は、HPV感染細胞又はがん細胞又は前癌性病変の原形質膜上に発現されるタンパク質(例えば、表1に列挙されるエピトープを含むタンパク質)に特異的に結合する抗体及びその抗原結合フラグメントに関する。いくつかの実施形態において、抗体は、本明細書に提供されるペプチドのうちの1つの特定のエピトープに結合する。いくつかの実施形態において、抗体は、表1におけるアミノ酸配列を有するエピトープを含むHPVタンパク質に結合する(ここでHPVタンパク質は全長HPVタンパク質ではない)。一部の実施形態では、エピトープは細胞外エピトープである。一部の実施形態では、エピトープは表1に列挙されるエピトープである。一部の実施形態では、HPVエピトープは、HPV E1、E2、E4、E5、E6、及び/又はE7に由来する。一部の実施形態では、HPVは、16又は18型である。いくつかの実施形態において、抗体はポリクローナル又はモノクローナルであり得、そして例えば、マウス、キメラ、ヒト化又は完全ヒトであり得る。一部の実施形態では、抗体は、全長免疫グロブリン分子、scFv、Fabフラグメント、Fab'フラグメント、F(ab')2フラグメント、Fv、ラクダ抗体、ジスルフィド結合Fv又は設計アンキリンリピートタンパク質(DARPin)である。
一部の態様では、本明細書に記載されるHPVエピトープを含む1以上のペプチドを提示するMHCをその表面上に発現する抗原提示細胞(APC)(例えば、表1に列挙された1以上のHPVエピトープを提示するAPC)が本明細書において提供される。一部の実施形態では、MHCはクラスI MHCである。一部の実施形態では、MHCはクラスII MHCである。一部の実施形態では、クラスI MHCは、HLA-A、HLA-B、HLA-C、HLA-E、HLA-F、HLA-g、HLA-K又はHLA-Lであるα鎖ポリペプチドを有する。一部の実施形態では、クラスII MHCは、HLA-DMA、HLA-DOA、HLA-DPA、HLA-DQA又はHLA-DRAであるα鎖ポリペプチドを有する。一部の実施形態では、クラスII MHCは、HLA-DMB、HLA-DOB、HLA-DPB、HLA-DQB又はHLA-DRBであるβ鎖ポリペプチドを有する。
いくつかの態様では、薬学的に許容される担体と共に製剤化された、本明細書に記載のペプチド(例えば表1からのエピトープを含む)、核酸、抗体、CTL、又はAPCを含有する組成物(例えば、ワクチン組成物などの医薬組成物)、並びにかかる医薬組成物を使用してがん、前癌性病変又はHPV感染を処置する方法が本明細書で提供される。いくつかの実施形態において、組成物は、本明細書に提供されている複数の(例えば、2つ以上の)薬剤の組み合わせを含む。
新規処置方法では、細胞傷害性T細胞(「キラー」T細胞としても知られる)により主に行われる、免疫系がウイルス感染細胞を除去し得る方法についての本発明者らの知識を利用する。これらの新規の治療法は、T細胞を活性化し、次いで、HPV感染細胞を位置づけ、殺傷することができるという考えによる、抗がんワクチン及び病変内免疫系に基づく治療の使用を含む。
アセグラトン;アルドホスファミドグリコシド;アミノレブリン酸;エニルウラシル(eniluracil);アムサクリン(amsacrine);ベストラブシル(bestrabucil);ビサントレン(bisantrene);エダトラキセート(edatraxate);デフォファミン(defofamine);デメコルシン(demecolcine);ジアジコン(diaziquone);エルフォミチン(elfomithine);酢酸エリプチニウム(elliptinium acetate);エポチロン(epothilone);エトグルシド(etoglucid);硝酸ガリウム;ヒドロキシウレア;レンチナン;ロニダイニン(lonidainine);メイタンシノイド(maytansinoid)、例えばメイタンシン(maytansine)及びアンサマイトシン(ansamitocin)等;ミトグアゾン(mitoguazone);ミトキサントロン;モピダンモール(mopidanmol);ニトラエリン(nitraerine);ペントスタチン;フェナメット(phenamet);ピラルビシン;ロソキサントロン(losoxantrone);ポドフィリン酸(podophyllinic acid);2-エチルヒドラジド;プロカルバジン;PSK多糖複合体);ラゾキサン(razoxane);リゾキシン(rhizoxin);シゾフラン(sizofuran);スピロゲルマニウム(spirogermanium);テニュアゾン酸(tenuazonic acid);トリアジコン(triaziquone);2,2',2''-トリクロロトリエチルアミン;トリコテセン(trichothecenes)(特にT-2トキシン、ベラキュリンA(verracurin A)、ロリジンA(roridin A)及びアングイジン(anguidine));ウレタン;ビンデシン;ダカルバジン;マンノムスチン(mannomustine);ミトブロニトール;ミトラクトール(mitolactol);ピポブロマン(pipobroman);ガシトシン(gacytosine);アラビノシド(「Ara-C」);シクロホスファミド;チオテパ;タキソイド、例えばパクリタキセル及びドセタキセル;クロラムブシル;ゲムシタビン;6-チオグアニン;メルカプトプリン;メトトレキセート;白金配位錯体、例えばシスプラチン、オキサリプラチン及びカルボプラチン等;ビンブラスチン;白金;エトポシド(VP-16);イホスファミド;ミトキサントロン;ビンクリスチン;ビノレルビン;ノバントロン(novantrone);テニポシド;エダトレキセート;ダウノマイシン;アミノプテリン;ゼローダ;イバンドロナート(ibandronate);イリノテカン(例えばCPT-11);トポイソメラーゼインヒビターRFS 2000;ジフルオロメチルオルニチン(DMFO);レチノイド、例えばレチノイン酸等;カペシタビン;並びに上述したものの薬学的に許容される塩、酸又は誘導体。
インターロイキン-28 A(「IL-28A」)、インターロイキン29(「IL-29」)、インターロイキン31(「IL-31」)、C-X-Cモチーフケモカイン10(「IP-10」)、ケモカイン受容体CXCR3(「I-TAC」)、白血病抑制因子(「LIF」)、Light、ケモカイン(Cモチーフ)リガンド(「リンホタクチン」)、単球走化性タンパク質2(「MCP-2」)、単球走化性タンパク質3(「MCP-3」)、単球走化性タンパク質4(「MCP-4」)、マクロファージ由来ケモカイン(「MDC」)、マクロファージ遊走阻止因子(「MIF」)、ケモカイン(C-Cモチーフ)リガンド20(「MIP-3α」)、C-Cモチーフケモカイン19(「MIP-3β」)、ケモカイン(C-Cモチーフ)リガンド23(「MPIF-1」)、マクロファージ刺激タンパク質アルファ鎖(「MSPalpha」)、ヌクレオソーム集合タンパク質1様4(「NAP-2」)、分泌型ホスホタンパク質1(「オステオポンチン」)、肺及び活性化調節サイトカイン(「PARC」)、血小板因子4(「PF4」)、間質細胞由来因子-1アルファ(「SDF-1α」)、ケモカイン(C-Cモチーフ)リガンド17(「TARC」)、胸腺発現ケモカイン(「TECK」)、胸腺間質リンホポエチン(「TSLP 4-IBB」)、CD166抗原(「ALCAM」)、分化クラスター80(「B7-1」)、腫瘍壊死因子受容体スーパーファミリーメンバー17(「BCMA」)、分化クラスター14(「CD14」)、分化クラスター30(「CD30」)、分化クラスター40(「CD40リガンド」)、癌胎児性抗原関連細胞接着分子1(「胆汁糖タンパク質」)(「CEACAM-1」)、デスレセプター6(「DR6」)、デオキシチミジンキナーゼ(「Dtk」)、1型膜糖タンパク質(「エンドグリン」)、受容体型チロシンプロテインキナーゼerbB-3(「ErbB3」)、内皮白血球接着分子1(「E-セレクチン」)、アポトーシス抗原1(「Fas」)、Fms様チロシンキナーゼ3(「Flt-3L」)、腫瘍壊死因子受容体スーパーファミリーメンバー1(「GITR」)、腫瘍壊死因子受容体スーパーファミリーメンバー14(「HVEM」)、細胞間接着分子3(「ICAM-3」)、IL-1 R4、IL-1 RI、IL-10 Rベータ、IL-17R、IL-2Rガンマ、IL-21R、リソソーム膜タンパク質2(「LIMPII」)、好中球ゼラチナーゼ関連リポカリン(「リポカリン-2」)、CD62L(「L-セレクチン」)、リンパ内皮細胞(「LYVE-1」)、MHCクラスIポリペプチド関連配列A(「MICA」)、MHCクラスIポリペプチド関連配列B(「MICB」)、NRGl-ベータ1、ベータ型血小板由来成長因子受容体(「PDGF Rベータ」)、血小板内皮細胞接着分子(「PECAM-1」)、RAGE、A型肝炎ウイルス細胞受容体1(「TIM-1」)、腫瘍壊死因子受容体スーパーファミリーメンバーIOC(「TRAIL R3」)、トラピンタンパク質トランスグルタミナーゼ結合ドメイン(「トラピン-2」)、ウロキナーゼ受容体(「uPAR」)、血管細胞接着タンパク質1(「VCAM-1」)、XEDAR、アクチビンA、アグーチ(Agouti)関連タンパク質(「AgRP」)、リボヌクレアーゼ5(「アンギオゲニン」)、アンジオポエチン1、アンジオスタチン、カテプシンS、CD40、潜在性(Cryptic)ファミリータンパク質IB(「Cripto-1」)、DAN、Dickkopf関連タンパク質1(「DKK-1」)、E-カドヘリン、上皮細胞接着分子(「EpCAM」)、Fasリガンド(FasL又はCD95L)、Fcg RIIB/C、フルイスタチン(FoUistatin)、ガレクチン-7、細胞間接着分子2(「ICAM-2」)、IL-13 Rl、IL-13R2、IL-17B、IL-2 Ra、IL-2 Rb、IL-23、LAP、神経細胞接着分子(「NrCAM」)、プラスミノーゲンアクチベーターインヒビター-1(「PAI-1」)、血小板由来成長因子受容体(「PDGF-AB」)、レジスチン、間質細胞由来因子1(「SDF-1β」)、sgpl30、分泌型frizzled関連タンパク質2(「ShhN」)、シアル酸結合免疫グロブリン型レクチン(「Siglec-5」)、ST2、トランスフォーミング増殖因子ベータ2(「TGFβ2」)、Tie-2、トロンボポエチン(「TPO」)、腫瘍壊死因子受容体スーパーファミリーメンバー10D(「TRAIL R4」)、骨髄細胞上に発現される誘発(Triggering)受容体1(「TREM-1」)、血管内皮増殖因子C(「VEGF-C」)、VEGFRl、アディポネクチン、アジプシン(「AND」)、アルファフェトプロテイン(「AFP」)、アンジオポエチン様4(「ANGPTL4」)、ベータ-2-ミクログロブリン(「B2M」)、基底細胞接着分子(「BCAM」)、炭水化物抗原125(「CA125」)、癌抗原15-3(「CA15-3」)、癌胎児性抗原(「CEA」)、cAMP受容体タンパク質(「CRP」)、ヒト上皮成長因子受容体2(「ErbB2」)、フォリスタチン、卵胞刺激ホルモン(「FSH」)、ケモカイン(C-X-Cモチーフ)リガンド1(「GROアルファ」)、ヒト絨毛性ゴナドトロピン(「βHCG」)、インスリン様成長因子1受容体(「IGF-1 sR」)、IL-1 sRII、IL-3、IL-18 Rb、
IL-21、レプチン(Leptin)、マトリックスメタロプロテイナーゼ-1(「MMP-1」)、マトリックスメタロプロテイナーゼ-2(「MMP-2」)、マトリックスメタロプロテイナーゼ-3(「MMP-3」)、マトリックスメタロプロテイナーゼ-8(「MMP-8」)、マトリックスメタロプロテイナーゼ-9(「MMP-9」)、マトリックスメタロプロテイナーゼ-10(「MMP-10」)、マトリックスメタロプロテイナーゼ-13(「MMP-13」)、神経細胞接着分子(「NCAM-1」)、エンタクチン(「ニドジェン-1」)、ニューロン特異的エノラーゼ(「NSE」)、オンコスタチンM(「OSM」)、プロカルシトニン、プロラクチン、前立腺特異抗原(「PSA」)、シアル酸結合Ig様レクチン9(「Siglec-9」)、ADAM 17エンドペプチダーゼ(「TACE」)、チログロブリン、メタロプロテイナーゼインヒビター4(「TIMP-4」)、TSH2B4、ディスインテグリン及びメタロプロテイナーゼドメイン含有タンパク質9(「ADAM-9」)、アンジオポエチン2、腫瘍壊死因子リガンドスーパーファミリーメンバー13/酸性ロイシンリッチ核ホスホタンパク質32ファミリーメンバーB(「APRIL」)、骨形成タンパク質2(「BMP-2」)、骨形成タンパク質9(「BMP-9」)、補体成分5a(「C5a」)、カテプシンL、CD200、CD97、ケメリン(Chemerin)、腫瘍壊死因子受容体スーパーファミリーメンバー6B(「DcR3」)、脂肪酸結合タンパク質2(「FABP2」)、線維芽細胞活性化タンパク質、アルファ(「FAP」)、線維芽細胞増殖因子19(「FGF-19」)、ガレクチン-3、肝細胞増殖因子受容体(「HGF R」)、IFN-アルファ/ベータR2、インスリン様増殖因子2(「IGF-2」)、インスリン様増殖因子2受容体(「IGF-2 R」)、インターロイキン-1受容体6(「IL-1R6」)、インターロイキン24(「IL-24」)、インターロイキン33(「IL-33」)、カリクレイン14、アスパラギンエンドペプチダーゼ(「レグマイン」)、酸化低密度リポタンパク質受容体1(「LOX-1」)、マンノース結合レクチン(「MBL」)、ネプリライシン(「NEP」)、Notchホモログ1、転座関連(Drosophila)(「Notch-1」)、腎芽細胞腫過剰発現(「NOV」)、オステオアクチビン、プログラム細胞死タンパク質1(「PD-1」)、N-アセチルムラモイル-L-アラニンアミダーゼ(「PGRP-5」)、セルピンA4、分泌型frizzled関連タンパク質3(「sFRP-3」)、トロンボモジュリン、Toll様受容体2(「TLR2」)、腫瘍壊死因子受容体スーパーファミリーメンバー10A(「TRAIL Rl」)、トランスフェリン(「TRF」)、WIF-lACE-2、アルブミン、AMICA、アンジオポエチン4、B細胞活性化因子(「BAFF」)、炭水化物抗原19-9(「CA19-9」)、CD163、クラステリン、CRT AM、ケモカイン(C-X-Cモチーフ)リガンド14(「CXCL14」)、シスタチンC、 デコリン(Decorin)(「DCN」)、Dickkopf関連タンパク質3(「Dkk-3」)、デルタ様タンパク質1(「DLL1」)、フェツインA、ヘパリン結合成長因子1(「aFGF」)、葉酸受容体アルファ(「FOLR1」)、フーリン(Furin)、GPCR関連ソーティングタンパク質1(「GASP-1」)、GPCR関連ソーティングタンパク質2(「GASP-2」)、顆粒球コロニー刺激因子受容体(「GCSF R」)、セリンプロテアーゼヘプシン(「HAI-2」)、インターロイキン-17B受容体(「IL-17B R」)、インターロイキン27(「IL-27」)、リンパ球活性化遺伝子3(「LAG-3」)、アポリポタンパク質A-V(「LDL R」)、ペプシノーゲンI、レチノール結合タンパク質4(「RBP4」)、SOST、ヘパラン硫酸プロテオグリカン(「シンデカン-1」)、腫瘍壊死因子受容体スーパーファミリーメンバー13B(「TACI」)、組織因子経路阻害剤(「TFPI」)、TSP-1、腫瘍壊死因子受容体スーパーファミリー、メンバー10b(「TRAIL R2」)、TRANCE、トロポニンI、ウロキナーゼプラスミノーゲンアクチベーター(「uPA」)、カドヘリン5、タイプ2又はCD144としても知られるVE-カドヘリン(血管内皮)(「VE-カドヘリン」)、WNTl誘導性シグナル伝達経路タンパク質1(「WISP-1」)、及び核因子κBの受容体活性化因子(「RANK」)。
HPVのための免疫療法戦略の開発を促進するためのHPV特異的T細胞応答の特性評価
総計50人の志願者が、この試験に採用される。これには、HPV関連中咽頭がんについて、処置を未だ受けていないか、現在処置しているか、又は根治的放射線療法±全身治療を完了した患者を含む。患者コホートに加えて、最大21人の健康なドナーが、この試験に採用される。最大70mLの初期血液試料を各参加者から採取し、約20mLをHLA分類のため、及び約50mLを免疫学的アッセイのためにEDTA管内に採取する。さらなる3つの血液試料(各50mL)を参加者から採取して、HPV免疫についての追跡調査を行う(特に、HPV陽性がんと診断されている患者において)。参加者が同意期間の後に手術を受けることを必要とする場合、手術時に新鮮な癌性又は前癌性病変の検体を採取する。標準的病理解析のための検体の除去後に十分な組織が利用可能である場合は、試料を病理組織学及び免疫学的試験について調査する。
HPV16及びHPV18抗原に対するCD8+及びCD4+T細胞応答
HNC(頭頚部がん)から採取したPBMC由来のウイルス特異的T細胞をインビトロで増殖させた。簡単に説明すると、各図1及び2にそれぞれ説明されているように、HPV16又はHPV18のいずれかのHPV抗原ペプミックスペプチドプールとともにPBMC試料をインキュベートし、これらの細胞をIL-2の存在下で14日間培養した。
HPV T細胞エピトープマッピング
HNC患者由来の末梢血単核球細胞(PBMC)は、上記のように、単離し、別々の培養物中のHPV16又はHPV18のいずれかのHPV抗原ペプミックスペプチドプールで刺激し、細胞内サイトカイン染色(ICS)アッセイのために準備した(非刺激対照は平行して実行した)。ペプミックスペプチドプールに対するT細胞応答を解析して、個々のペプチドを同定した。これらの個々のペプチドをT細胞増殖についてさらに評価し、ICS解析で潜在的エピトープを同定した。15merのペプチドを同定した後、エピトープ配列のさらなる最小化を実行して、最適なT細胞エピトープ配列を同定した。15merのペプチド配列を、N及びCの両末端から最小の9〜14アミノ酸長のペプチドにトリミングした。最小ペプチド配列を同定した後、制限用量滴定ICSアッセイを使用して、さらなる確認を行った。最小エピトープ配列をマッピングした後、エピトープのHLA制限を、ペプチド負荷HLA適合及び不適合PHAブラスト(PHA)を使用してT細胞を刺激することにより同定した。エピトープマッピングの完全プロセスを図3に提供するフローチャートに示す。
HPV CD8及びCD4ポリエピトープ配列
一連のHPVポリエピトープペプチドを設計した。CD8+T細胞エピトープを選択して(表1を参照)HPV CD8ポリエピトープを生成した(図7Aを参照、個々のエピトープは、交互の太字及び下線イタリック文字により示す)。同様に、CD4+T細胞エピトープを表1から選択してHPV CD4ポリエピトープを生成した。CD4+ポリエピトープは、ER(小胞体)シグナル配列をアミノ末端に、リソソームシグナル配列をカルボキシ末端に含む(図7Bを参照)。また、ポリエピトープペプチド構築物を設計して、CD8+及びCD4+の両方のT細胞エピトープを発現させた。この場合、ポリエピトープ配列は、配列内リボソーム進入部位(IRES;脳心筋炎ウイルスに由来する)により分離された(図7C及びDを参照)。HPV CD4+、CD8+又はCD8IRESCD4ポリエピトープをコードするDNA配列をAtum Bio社からpJ201クローニングベクター中で得た。ヌクレオチド配列は、5'Nhe1及び3'Kpn1(CD4+及びCD8+ポリエピトープ用)並びに5'Nhe1及び3'Not1(CD8IRESCD4ポリ用)制限酵素で分解することにより、クローニングベクターから放出させた。これらのポリエピトープヌクレオチド配列をpShuttle2ベクターにクローニングして、哺乳動物発現カセットを作製した。組換えpShuttle2ベクターを、I-CeuI及びPI-SceI制限酵素で分解して、HPV CD4+、CD8+又はCD8IRESCD4ポリエピトープをコードする哺乳動物発現カセットを放出させ、次いで、I-CeuI及びPI-SceI制限酵素で分解したAd5F35ベクターにライゲートした(図8を参照)。HPV CD4+、CD8+又はCD8IRESCD4ポリエピトープを発現する、すべての組換えアデノウイルスベクターをPCRにより確認し、次いで、ベクターをPacI制限酵素で直鎖化し、逆位末端反復を露出させて、Ad5F35DNAをパッケージングした。組換えアデノウイルスを生成するために、HEK293細胞に直鎖状DNAをトランスフェクトし、6日目にHEK293細胞を凍結融解サイクルで溶解することにより、組換えウイルスの一次株を回収した。高力価の組換えアデノウイルスを得るために、HEK293細胞に組換えウイルスの一次株を繰返し感染させた。HPV CD4+、CD8+又はCD8IRESCD4ポリエピトープヌクレオチド配列のカプシド形成されたアデノウイルスゲノムにおける存在をPCRにより評価した(図9を参照)。
組換えAdHPVCD8の免疫原性の評価
AdHPVCD8ポリの免疫原性を調査するために、細胞にAdHPVCD8ポリ組換えウイルスを1時間、パルスした。次いで、細胞を洗浄し、一晩インキュベートし、次いで、HPV特異的T細胞のパネルの活性化を、IFN-γの細胞内発現を測定することにより評価した。図10に示す代表的データは、HPV16-E2及びE6抗原由来のHPV16-E2 HLA A02:01制限エピトープ、並びにHPV16-E2抗原由来のHLA A01:01制限エピトープが、より効率的にプロセシングされ、HPV特異的CD8+T細胞に提示されたことを明らかに示す。HPV特異的CD8+の頻度は、HPVペプミックスをパルスしたJuSt線維芽細胞と類似した。
組換えAdHPVCD8を使用したHPV HNC患者由来のHPV特異的CD8+T細胞の増殖
AdHPVCD8ポリの免疫原性をさらに評価するために、HPV HNC患者由来のPBMCを、IL-2の存在下で、ウイルスにより14日間刺激した。次いで、HPV特異的CD8+T細胞の増殖を、IFN-γ分泌を測定することにより評価した。図11に示す代表的データは、HPV AdCD8ポリが、患者の大多数においてHPV特異的CD8+T細胞の増殖を誘導し、2つの異なるHPV株(HPV16及びHPV18)由来の複数の抗原(E2、E4、E5、E6及びE7)に対して増殖が観察されたことを示す。
AdHPVCD4ポリの免疫原性の評価
HPV AdCD4ポリの免疫原性を調査するために、HPV HNC患者のうちの1人由来のPBMCをAdHPVCD4ポリで刺激し、次いで、IL-2の存在下で14日間培養した。図12に示すデータは、HPVペプミックスによるPBMCの刺激及び複数のHPV16 E2、E6及びE7抗原に対するT細胞の増殖と比較した、HPV AdCD4ポリによるPBMCの刺激後のHPV特異的CD4+T細胞の安定な増殖を示す。興味深いことに、CD8+T細胞エピトープのより長いCD4+T細胞エピトープへの組込みの可能性として、HPV AdCD4ポリが、HPV16 E6及びE7抗原に対するHPV特異的CD8+T細胞の増殖を同時に刺激し、これらのCD8+T細胞の頻度は、HPVペプミックスで刺激したPBMCよりも劇的に高くなった(図12)。
AdCD8IRESCD4組換えウイルスの免疫原性の評価
HPV AdCD8IRESCD4ポリの免疫原性を評価するために、6人の異なるHPV HNC患者由来のPBMCを、IL-2の存在下で、ウイルスにより14日間刺激した。同一の患者由来の別のPBMCのセットをAdHPVCD4ポリ及びAdHPVCD8ポリで刺激して、HPV特異的CD4+及びCD8+T細胞を増殖させるAdCD8IRESCD4ポリの能力と比較した。HPV特異的CD4+及びCD8+T細胞の増殖を、IFN-γ分泌を測定することにより判定した。図13A及びBに示す代表的データは、AdCD8IRESCD4ポリが、2人のドナー由来のHPV16 E1、E6及びE7に対するHPV特異的CD4+T細胞の増殖を誘導し、増殖したT細胞の頻度が、HPV AdCD4ポリ及びHPV AdCD8ポリにより増殖したPBMCと比較して明白なパターンを示さなかったことを示す。加えて、図13C及びDに示すデータは、AdCD8IRESCD4ポリがまた、HPV16 E5、E6及びE7抗原に対する、複数のドナー由来のHPV特異的CD8+T細胞の増殖を誘発したことを実証し、これは、HPV HNC患者由来のHPV特異的CD4+及びCD8+T細胞を増殖させる単一組換えウイルスとしてのHPV AdCD8IRESCD4ポリの能力を示す。
本明細書で述べられる全ての刊行物、特許、特許出願及び配列アクセッション番号は、各刊行物、特許又は特許出願が参照により、特に、個別に組み込まれたことを示したものとして、その全体を参照として本明細書に組み込む。競合する場合、本出願が、本明細書でのいかなる定義も含めて、優先されることになる。
当業者は、日常的な実験の程度を用いて、本明細書に記載される発明の具体的な実施形態に対する多くの均等物を認識し、又は確認することができる。そのような均等物は、以下の特許請求の範囲に包含されることを意図する。
Claims (91)
- 配列番号1〜24に記載の細胞傷害性T細胞エピトープアミノ酸配列から選択されるアミノ酸配列を有する細胞傷害性T細胞エピトープからなる単離されたペプチド。
- 表1に記載のT細胞エピトープアミノ酸配列から選択される複数のT細胞エピトープアミノ酸配列を含む単離されたポリエピトープペプチド。
- 配列番号34、36、及び38のいずれか1つに記載のアミノ酸配列を含む、請求項2に記載の単離されたポリエピトープペプチド。
- 配列番号1〜24から選択されるアミノ酸配列を有する細胞傷害性T細胞エピトープからなるペプチドをコードするヌクレオチド配列を含む単離された核酸。
- 発現ベクターである、請求項4に記載の核酸。
- 発現ベクターがウイルスベクターである、請求項5に記載の核酸。
- ウイルスベクターがアデノウイルスに基づく発現ベクターである、請求項6に記載の核酸。
- 請求項1から3のいずれか1項に記載のペプチド又は請求項4から7のいずれか1項に記載の核酸を含むワクチン組成物。
- ペプチド及びアジュバントを含む、請求項8に記載のワクチン組成物。
- 配列番号1〜24に記載のHPV CTLアミノ酸配列から選択されるアミノ酸配列を有する複数の単離されたHPV CTLエピトープを含む単離されたポリペプチド。
- 配列番号34及び38のいずれか1つに記載のアミノ酸配列を含む、請求項9に記載の単離されたポリペプチド。
- 対象におけるCTLの誘導における使用のための組成物を調製する方法であって、表1に記載のアミノ酸配列から本質的になる少なくとも1つのペプチドを、薬学的に許容される担体、希釈剤又は賦形剤と混合することを含む、方法。
- クラスI MHC上に提示される表1に列挙された1つ以上のエピトープペプチドを含むHLAクラスI又はHLAクラスII分子に結合した1つ以上のHPVエピトープペプチドに特異的に結合するT細胞受容体(TCR)を含む細胞傷害性T細胞(CTL)を含む医薬組成物を対象に投与することを含む、対象においてがんを処置する方法。
- クラスI MHC上に提示される表1に列挙された1つ以上のエピトープペプチドを含むHLAクラスI又はHLAクラスII分子に結合した1つ以上のHPVエピトープペプチドに特異的に結合するT細胞受容体(TCR)を含む細胞傷害性T細胞(CTL)を含む医薬組成物を対象に投与することを含む、対象において前癌状態を処置する方法。
- クラスI MHC上に提示される表1に列挙された1つ以上のエピトープペプチドを含むHLAクラスI又はHLAクラスII分子に結合した1つ以上のHPVエピトープペプチドに特異的に結合するT細胞受容体(TCR)を含む細胞傷害性T細胞(CTL)を含む医薬組成物を対象に投与することを含む、対象においてヒトパピローマウイルス(HPV)感染を処置する方法。
- CTLが対象に対して自家である、請求項13から15のいずれか1項に記載の方法。
- CTLが対象に対して自家ではない、請求項13から15のいずれか1項に記載の方法。
- CTLがCTLのライブラリー又はバンクから得られる、請求項17に記載の方法。
- HPV特異的細胞傷害性T細胞(CTL)の増殖を誘導する方法であって、CTLと、表1に列挙された1つ以上のエピトープを含む1つ以上のHPVペプチドを提示する抗原提示細胞(APC)とを含むサンプルをインキュベートし、これによりサンプルにおいてペプチド特異的CTLの増殖を誘導することを含む方法。
- サンプルが1つ以上のサイトカインをさらに含む、請求項19に記載の方法。
- APCがB細胞である、請求項19又は20に記載の方法。
- APCが抗原提示T細胞である、請求項19又は20に記載の方法。
- APCが樹状細胞である、請求項19又は20に記載の方法。
- APCがaK562細胞である、請求項20又は21に記載の方法。
- サンプルが末梢血単核球細胞(PBMC)を含む、請求項19から23のいずれか1項に記載の方法。
- HPVペプチドが20アミノ酸長以下である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが15アミノ酸長以下である、請求項26に記載の方法。
- HPVペプチドが10アミノ酸長以下である、請求項26に記載の方法。
- HPVペプチドがHPV16-E1抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号5〜8、及び20のいずれか1つに記載の配列を含む、請求項29に記載の方法。
- HPVペプチドがHPV16-E2抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号1、9、10、26、及び27のいずれか1つに記載の配列を含む、請求項31に記載の方法。
- HPVペプチドがHPV16-E4抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドがWPTTPPRPI(配列番号11)の配列を含む、請求項33に記載の方法。
- HPVペプチドがHPV18-E6抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号2、20及び21のいずれか1つに記載の配列を含む、請求項35に記載の方法。
- HPVペプチドがHPV18-E5抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドがSPATAFTVY(配列番号3)の配列を含む、請求項37に記載の方法。
- HPVペプチドがHPV16-E5抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号4及び12のいずれか1つに記載の配列を含む、請求項39に記載の方法。
- HPVペプチドがHPV16-E6抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号13〜19、及び28〜30のいずれか1つに記載の配列を含む、請求項41に記載の方法。
- HPVペプチドがHPV16-E7抗原である、請求項19から25のいずれか1項に記載の方法。
- HPVペプチドが配列番号22、23、及び24のいずれか1つに記載の配列を含む、請求項43に記載の方法。
- 表1に列挙されたアミノ酸配列を含むペプチドであって、HPVタンパク質の30超の連続したアミノ酸を含まないペプチド。
- 表1に列挙されたアミノ酸配列が、LQDVSLEVYL、TVLELTEVFEF、SPATAFTVY又はSAFRCFIVYである、請求項45に記載のペプチド。
- 表1に列挙された2つ以上の配列を含む、請求項45又は46に記載のペプチド。
- 請求項45から47のいずれか1項に記載のペプチドを含むワクチン組成物。
- アジュバントをさらに含む、請求項48に記載のワクチン組成物。
- 対象においてがんを処置及び/又は予防する方法であって、請求項48又は49に記載の1つ以上のワクチン組成物を対象に投与することを含む方法。
- 請求項48又は49に記載の1つ以上のワクチン組成物を対象に投与することを含む、対象において前癌状態を処置及び/又は予防する方法。
- 請求項48又は49に記載の1つ以上のワクチン組成物を対象に投与することを含む、対象においてHPV感染を処置及び/又は予防する方法。
- 請求項45から47のいずれか1項に記載の1つ以上のペプチドを対象に投与することを含む、対象においてがんを処置及び/又は予防する方法。
- 請求項45から47のいずれか1項に記載の1つ以上のペプチドを対象に投与することを含む、対象において前癌状態を処置及び/又は予防する方法。
- 請求項45から47のいずれか1項に記載の1つ以上のペプチドを対象に投与することを含む、対象においてHPV感染を処置及び/又は予防する方法。
- クラスI MHC上に提示される、請求項45から47のいずれか1項に記載のペプチドを含む抗原提示細胞(APC)。
- 抗原提示T細胞である、請求項56に記載のAPC。
- 樹状細胞である、請求項56に記載のAPC。
- B細胞である、請求項56に記載のAPC。
- 人工APCである、請求項56に記載のAPC。
- 人工APCがaK562細胞である、請求項56に記載のAPC。
- 請求項45から47のいずれか1項に記載の1つ以上のペプチド、又は請求項45から47のいずれか1項に記載の1つ以上のペプチドをコードする1つ以上の核酸とともに、抗原提示細胞をインキュベートすることを含む、1つ以上のHPVペプチドを提示する抗原提示細胞(APC)を生成する方法。
- APCが抗原提示T細胞である、請求項62に記載の方法。
- APCが樹状細胞である、請求項62に記載の方法。
- APCがB細胞である、請求項62に記載の方法。
- APCが人工APCである、請求項62に記載の方法。
- 人工APCがaK562細胞である、請求項62に記載の方法。
- 対象においてがんを処置又は予防する方法であって、請求項56から61のいずれか1項に記載のAPCを対象に投与することを含む方法。
- APCが対象に対して自家である、請求項68に記載の方法。
- APCが対象に対して自家ではない、請求項68に記載の方法。
- 対象において前癌状態を処置又は予防する方法であって、請求項56から61のいずれか1項に記載のAPCを対象に投与することを含む方法。
- APCが対象に対して自家である、請求項71に記載の方法。
- APCが対象に対して自家ではない、請求項71に記載の方法。
- 請求項56から61のいずれか1項に記載のAPCを対象に投与することを含む、対象においてHPV感染を処置又は予防する方法。
- APCが対象に対して自家である、請求項74に記載の方法。
- APCが対象に対して自家ではない、請求項74に記載の方法。
- 請求項45から47のいずれか1項に記載のペプチドをコードする核酸。
- 発現ベクターである、請求項77に記載の核酸。
- 発現ベクターがウイルスベクターである、請求項78に記載の核酸。
- ウイルスベクターがアデノウイルスに基づく発現ベクターである、請求項79に記載の核酸。
- 請求項77から80のいずれか1項に記載の核酸を含むワクチン組成物。
- 対象においてがんを処置及び/又は予防する方法であって、請求項81に記載のワクチン組成物を対象に投与することを含む方法。
- 請求項81に記載のワクチン組成物を対象に投与することを含む、対象において前癌状態を処置及び/又は予防する方法。
- 請求項81に記載のワクチン組成物を対象に投与することを含む、対象においてHPV感染を処置又は予防する方法。
- 表1に列挙されたHPVエピトープと結合する抗体又はその抗原結合フラグメント。
- 全長免疫グロブリン分子、
scFv、
Fabフラグメント、
Fab'フラグメント、
F(ab')2、
Fv、
ラクダ抗体、
ジスルフィド結合Fv、
設計アンキリンリピートタンパク質(Designed Ankyrin Repeat Protein)(DARPin)
である、請求項85に記載の抗体又はその抗原結合フラグメント。 - 対象においてがんを処置する方法であって、請求項85又は86に記載の抗体又はその抗原結合フラグメントを対象に投与することを含む方法。
- 対象において前癌状態を処置する方法であって、請求項85又は86に記載の抗体又はその抗原結合フラグメントを対象に投与することを含む方法。
- 請求項85又は86に記載の抗体又はその抗原結合フラグメントを対象に投与することを含む、対象においてHPV感染を処置する方法。
- 主要組織適合複合体(MHC)上に提示される表1に列挙された1つ以上のエピトープを含む1つ以上のペプチドに結合するT細胞受容体(TCR)を発現するT細胞。
- 細胞傷害性T細胞(CTL)である、請求項90に記載のT細胞。
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