JP5597335B2 - 樹状細胞の微粒子ベースのトランスフェクションおよび活性化 - Google Patents
樹状細胞の微粒子ベースのトランスフェクションおよび活性化 Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Description
本願は、特許出願番号60/146391(1999年7月29日出願)に関する。この出願は本明細書中でその全体が参考として援用される。
本発明は、例えば、ウイルスまたは腫瘍と共に免疫治療のための樹状細胞を提供する組成物および方法に関する。詳細には、本発明は、ウイルスおよび腫瘍に対する養子細胞免疫治療において使用するため、例えば、多数のウイルスまたは腫瘍抗原特異的T細胞の活性化および拡大を可能にするトランスフェクションによって、抗原提示樹状細胞を生成するための方法に関する。
免疫応答の発生は、抗原提示細胞との相互作用による、ヘルパー(CD4+)(TH)および細胞傷害性(CD8+)(CTL)T細胞サブセットの感作を包含する。抗原提示細胞は、抗原性フラグメント(すなわち、細胞表面上における提示のためのMHCIおよびMHCIIに結合する抗原由来の特異的アミノ酸配列)に関連する主要な組織適合性(MHC)クラスIまたはクラスII分子を発現する。ヒトにおけるMHCはまた、HLA(ヒト白血球抗原)複合体と称される。感作CD4+T細胞は、B細胞および様々なT細胞サブセットの活性化に関与するリンホカインを産生する。感作CD8+T細胞は、リンホカインに対する応答数を増加し、そして適合性MHCコード化クラスI分子に関連する特定の抗原性フラグメントを発現する細胞を破壊するように作用する。腫瘍またはウイルス感染の過程において、細胞傷害性T細胞は、腫瘍またはウイルス関連抗原を発現する細胞を根絶する。
本発明は、非ウイルス方法による樹状細胞のトランスフェクションのための有効な方法を提供する。本発明は、樹状細胞をインキュベートすることによるこの利点、および特定のトランスフェクション剤を提供する。このトランスフェクション剤は、ポリヌクレオチドおよび微粒子を含み、この微粒子は、生分解性ポリマーおよびカチオン性界面活性剤からなる。樹状細胞およびトランスフェクション剤は、ポリヌクレオチドでこの樹状細胞をトランスフェクトするのに十分な時間インキュベートされる。
本発明の実施は、他に示されない限り、化学、ポリマー化学、生化学、分子生物学、免疫学および薬理学の当該分野の技術内の通常の方法を使用する。このような技術は、文献に十分に記載されている。例えば、Remington’s Pharmaceutical Sciences,第18版(Easton,Pennsylvania:Mack Publishing Company,1990);Methods In Enzymology(S.Colowick and N.Kaplan編、Academic Press,Inc);Handbook of Experimental Immunology,Vols.I−IV(D.M.Weir および C.C.Blackwell編、1986、Blackwell Scientific Publications);Sambrookら,Molecular Cloning:A Laboratory Manual(第2版、1989);Handbook of Surface and Colloidal Chemistry(Birdi,K.S.編、CRC Press,1997)およびSeymour/Carraher’s Polymer Chemistry(第4版、Marcel Dekker Inc.,1996を参照されたい)。
本発明を記載する際に、以下の用語が使用され、そして以下に例示されるように定義されることを意図する。
本発明において、目的の抗原を含むポリヌクレオチドは、ポリマーおよびカチオン性界面活性剤から形成される微粒子に吸収される。吸収剤の微粒子の表面へのポリヌクレオチドの吸収は、結合−相互作用機構(限定しないが、イオン結合、水素結合、共有結合、ファンデルワールス結合、および親水性/疎水性相互作用を介する結合を含む)によって生じる。当業者は、本発明のために適切なカチオン性界面活性剤を容易に選択し得る。上記のように、公知のカチオン性界面活性剤としては、限定しないが、セチルトリメチルアンモニウムブロマイド(CTAB)、セトリミド(少量のドデシルトリメチルアンモニウムブロマイドおよびCTABと一緒に、主にテトラデシルトリメチルアンモニウムブロマイドからなる混合物)、ベンザルコニウムクロライド、DDA(ジメチルジオクトデシルアンモニウムブロマイド)、DOTAPなどが挙げられる。CTABが特に好ましい。カチオン性界面活性剤(例えば、CTAB)を用いて製造される微粒子(例えば、CTAB−PLG微粒子)は、陰性に荷電したポリヌクレオチドを容易に吸収する。
樹状細胞は、それらが存在する任意の組織(皮膚の表皮(ランゲルハンス細胞)のような非リンパ組織ならびに脾臓、骨髄、リンパ節および胸腺のようなリンパ組織、ならびに血液(血液樹状細胞)、例えば、末梢血および臍帯血、およびリンパ(ベール細胞(veiled cell))を含む循環系を含む)から得られる。
発現され得る選択された抗原としては、脊椎動物の感染因子または癌の、1以上の選択された抗原が含まれ、そしてこれらは、構造タンパク質または非構造タンパク質のいずれかに対応することができる。本明細書中に記載される本発明は、このような抗原の、樹状細胞の表面のMHC分子との会合を提供し、その結果、目的の抗原に対する免疫応答が、マウントされ得る。
本発明に従って、1以上のポリヌクレオチドが、樹状細胞にエキソビボで挿入され、その結果、1以上の選択された抗原が、その樹状細胞の表面上に有効量で提示される。「有効量」によって、提示が、樹状細胞に免疫応答を誘発させ得るのに十分であることを意味する。
目的のポリヌクレオチドを目的の微粒子と会合させるために、微粒子を、例えば、適切な緩衝液中で、ポリヌクレオチドを単純に混合する。得られた処方物は、使用前に凍結乾燥化し得る。一般に、ポリヌクレオチドは、約0.0001:1〜0.25:1のポリヌクレオチド:微粒子、好ましくは、0.001:1〜0.1、より好ましくは、0.01〜0.05の重量比を有する、吸着されたポリヌクレオチドを有する微粒子を生じるように、微粒子に添加される。微粒子のポリヌクレオチド含量は、標準的な技術を使用して決定され得る。
一旦、樹状細胞およびポリヌクレオチド/微粒子を調製すると、これらを、トランスフェクションが生じるに十分な時間および温度で、溶液中でインキュベートする。好ましい実施形態に従って、樹状細胞およびポリヌクレオチド/微粒子は、加湿CO2インキュベーター中で、37℃で24時間インキュベートする。
本発明の実施形態に従って、本明細書中に記載の方法のいずれかを使用してポリヌクレオチド/微粒子でトランスフェクトした樹状細胞を使用して、T細胞をインビトロで活性化する。T細胞またはT細胞のサブセットは、種々のリンパ組織から得られ得る。このような組織としては、脾臓、リンパ節および末梢血が挙げられるが、これらに限定されない。
(実施例1)
(プラスミドおよびDNA処方物)HIVp55gagタンパク質をコードするpCMVgagプラスミドをサイトメガロウイルス初期プロモーターの制御下でQiagen Endo Free Giga Kitを使用してイオン交換クロマトグラフィーにより精製し、そしてエンドとキシンがない(<2.5EU/ml)ことを決定した。取り込みおよびレセプター遺伝子発現実験のために、BガラクトシダーゼをコードするローダミンPNAクランププラスミドをGene Therapy Systems(San Diego,CA)から購入した。
(RNA単離およびRT−PCR)BMDCを培養の6日目でRPMI+GM−CSF中0.5×106細胞/mlの濃度でプレートした。細胞をネガティブコントロールとして未処理のまま残すか、または1μg/ml pCMV−gagDNA(単独(裸の)またはPLG−CTAB微粒子上に処方されるかのいずれか)の存在下で培養するかのいずれかとした。24時間のインキュベーション後、2×105の細胞を取り除き、冷たいPBS(Life Technologies)で2回洗浄し、次いでmRNA Captureキット(Roche)についての製造者の説明書により溶解し、−80℃で凍結した。サンプルをRNaseを含有しないDNaseおよびRNaseインヒビター(Roche)を添加して氷上で解凍した。次に、ストレプトアビジンPCRチューブ中のビオチンハイブリダイズmRNAの単離のためにmRNA単離プロトコルに従った。プロメガ逆転写システム(Madison,WI)を製造者の説明書に従ってcDNA合成のために利用し、そして反応を45℃で45分間行い、次に99℃で5分間、熱不活化した。PCR制御チューブを上記のように処理するが、続く混入プラスミドDNA存在決定のためにAMV逆転写酵素は添加しなかった。PCR増幅のために、サンプルをHIVgag遺伝子の300bp領域、またはBアクチンをポジティブコントロールとして一般的なPCR条件を使用して増幅するようにセットした。
(T細胞刺激)6日間GM−CSFの存在下で分化した骨髄細胞を、細胞表面表現型(CD11c+、CD11b+、H−dKd+、I−Ad(low)、CD80(low)、およびCD86(low))のFACS分析により決定して未成熟と分類し、そして9日目で成熟と分類した(CD11c+、CD11b+、H−2Kd+、I−Ad(bright)、CD80+、CD86+)。(R.C.Fields,J.J.O.、J.A.Fuller、E.K.Thomas、P.J.Geraghty、およびJ.J.Mule’、1998。Comparative analysis of murine dendritic cells derived from spleen and bone marrow.J.Immunother.21:323)。未熟のBMDCおよび成熟BMDCの両方をPLG−CTAB−pCMVgagDNAまたは裸のpCMVgagDNAで24時間刺激した。コントロールは、未処理の細胞、微粒子のみ、または非特異的プラスミドDNA(pCMVルシフェラーゼ)で処方された微粒子、および非特異的裸のDNAで処方された微粒子を含む。T細胞ハイブリドーマ12.2(HIVp55gagのp7gエピトープ(AMQMLKETI)に特異的なd制限T細胞ハイブリドーマ)をU底マイクロタイタープレートの96ウェルのウエル毎に1×105細胞でプレートした。変化させた数のBMDCを、培養物総容量200μl中のハイブリドーマとともにプレートした。個々の各実験を2連で行った。24時間の培養期間後、プレートを遠心分離し、上清を取り除き、そしてIL−2産生についてのさらなるアッセイまで−80℃で保存した。培地中に分泌されたIL−2レベルについてアッセイするために、培養上清を室温で解凍し、前処理したマウスIL−2ELISAマイクロタイタープレート上にプレートし、製造者の説明書(Endogen)によって分析した。比色基質の顕色に続いて、マイクロタイタープレートをMolecular Devices νmaxキネティクプレートリーダーにより読み、SoftMaxソフトウェアで解析した。
【図面の簡単な説明】
【図1】
図1は、標的遺伝子発現の検出のためのRT−PCR産物のアガロースゲル電気泳動を示す。レーンの命名は、以下の通りである:1)500bp DNAラダー、2−4)未処理のプラスミドDNA、およびPLG−CTAB−DNAで処理した骨髄由来の樹状細胞(BMDC)由来のβアクチンコントロールRT−PCR反応物、5)100bpのDNAラダー、6)pCMV−gag DNAのコントロールスパイクで調製した未処理のmRNA、8−10)未処理のプラスミドDNA、およびPLG−CTAB−DNAで処理したBMDC由来のp55gag RT−PCR、11−13)未処理のプラスミドDNA、およびPLG−CTAB−DNAで処理したBMDC由来のPCRネガティブコントロール、14)pCMV−gagDNA PCRポジティブコントロール。
【図2】
図2は、骨髄由来の樹状細胞(BMDC)を用いてMHCクラスI T細胞ハイブリドーマを刺激した後、IL−2の産生レベルを例示する。未成熟(6日)BMDCおよび成熟(9日)BMDCの両方を、試験した。各成熟群内でのBMDC処理は、以下(左から右)の通りである:未処理、PLG−CTABで処理、PLG−CTAB−pCMV gag DNAで処理、PLG−CTAB−luc DNAで処理、裸のpCMV gag DNAで処理、および裸のlucDNAで処理。
【図3】
図3は、PLG−CTAB微粒子上で形成された種々の濃度のpCMV−gagプラスミドDNAとともにインキュベートした、BMDCを用いてT細胞ハイブリドーマを刺激した後のIL−2産生レベル(左 y−軸、バーシリーズ)を例示する。図3はまた、PLG−CTAB微粒子上で形成された種々の濃度のpCMV−gagプラスミドDNAとともにインキュベートした、BMDCの生存%(右 y−軸、ラインシリーズ)を例示する。適切なデータポイントは、2つ組サンプルの平均値および標準誤差を示す。
【図4】
図4は、ナイーブ(未処理)のBMDCまたはPLG−CTAB−pCMVgagDNAで処理したBMDCのいずれかを用いてgag特異的T細胞ハイブリドーマを刺激した後、および過剰の合成ペプチドエピトープを用いてパルスした後のIL−2の生成レベルを例示する。抗原提示細胞(すなわち、BMDC)に対するT細胞の比を変えて、全ての場合においてT細胞の数を一定に保持したままで研究した。データポイントは、一連の希釈によりアッセイされた2つ組サンプルの平均値±誤差を示す。
Claims (31)
- 樹状細胞をトランスフェクトする方法であって、以下:
トランスフェクション剤を提供する工程であって、該トランスフェクション剤は、ポリヌクレオチドおよび微粒子を含み、該微粒子は、生分解性ポリマーおよびカチオン性界面活性剤を含む、工程;
該ポリヌクレオチドを該微粒子上に吸着させる工程;ならびに
該樹状細胞およびトランスフェクション剤を、該樹状細胞を該ポリヌクレオチドでトランスフェクトするのに十分な時間インキュベートする工程、
を包含する、方法。
- 前記樹状細胞が、骨髄に由来する、請求項1に記載の方法。
- 前記樹状細胞が、血液に由来する、請求項1に記載の方法。
- 前記樹状細胞が、脊椎動物被験体に由来する、請求項1に記載の方法。
- 前記樹状細胞が、ヒト被験体に由来する、請求項1に記載の方法。
- 前記カチオン性界面活性剤が、CTABを含む、請求項1に記載の方法。
- 前記カチオン性界面活性剤が、セトリミドを含む、請求項1に記載の方法。
- 前記ポリマーが、ポリ(α−ヒドロキシ酸)である、請求項1に記載の方法。
- 前記ポリマーが、ポリ(ラクチド)である、請求項1に記載の方法。
- 前記ポリマーが、D,L−ラクチドとグリコリドまたはグリコール酸とのコポリマーである、請求項1に記載の方法。
- 前記ポリマーが、ポリ(D,L−ラクチド−co−グリコリド)である、請求項1に記載の方法。
- 前記ポリマーが、D,L−ラクチドとカプロラクトンとのコポリマーである、請求項1に記載の方法。
- 前記樹状細胞が、トランスフェクションの前に約5日間培養される、請求項1に記載の方法。
- 前記樹状細胞が、トランスフェクションの前に約10日間培養される、請求項1に記載の方法。
- 前記樹状細胞およびトランスフェクション剤が、約24時間インキュベートされる、請求項1に記載の方法。
- 前記ポリヌクレオチドが、プラスミドの形態で提供される、請求項1に記載の方法。
- 前記ポリヌクレオチドが、ウイルスに関連する抗原または腫瘍に関連する抗原をコードする、請求項1に記載の方法。
- 前記抗原が、HIV、髄膜炎A、髄膜炎Bまたは髄膜炎Cに関連する、請求項17に記載の方法。
- 免疫応答を生成するための組成物であって、
請求項17に記載の方法によって産生された樹状細胞の有効量
を含む、組成物。
- 前記樹状細胞が、脊椎動物被験体に由来する、請求項19に記載の組成物。
- 前記樹状細胞が、前記脊椎動物被験体に対してMHC適合性の健常な脊椎動物被験体に由来する、請求項19に記載の組成物。
- 前記樹状細胞が、非経口的に投与するために適していることを特徴とする、請求項19に記載の組成物。
- 前記樹状細胞が、罹患組織内への直接注入によって投与するために適していることを特徴とする、請求項19に記載の組成物。
- 免疫応答を必要とする脊椎動物被験体において免疫応答を生成するための組成物であって、
T細胞
を含み、該T細胞が、請求項17に記載の方法によって産生された樹状細胞に供することによって活性化されることを特徴とする、組成物。
- 前記樹状細胞およびT細胞が、前記脊椎動物被験体に由来する、請求項24に記載の組成物。
- 前記樹状細胞およびT細胞が、前記脊椎動物被験体に対してMHC適合性の健常な脊椎動物被験体に由来する、請求項24に記載の組成物。
- 前記T細胞が、非経口的に投与するために適していることを特徴とする、請求項24に記載の組成物。
- 前記T細胞が、罹患組織内への直接注入によって投与するために適していることを特徴とする、請求項24に記載の組成物。
- 請求項17に記載の方法によって作製された、抗原提示樹状細胞。
- 前記トランスフェクション剤が、約1ミクロンの直径を有する、請求項1に記載の方法。
- 前記トランスフェクション剤が、約1% w/wのポリヌクレオチドを含む、請求項1に記載の方法。
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ATE350015T1 (de) | 2000-09-28 | 2007-01-15 | Novartis Vaccines & Diagnostic | Mikropartikelzusammensetzungen und verfahren zu ihrer herstellung |
DE60239317D1 (de) | 2001-01-12 | 2011-04-14 | Novartis Vaccines & Diagnostic | Nukleinsäure mukosale immunisierung |
AR045702A1 (es) | 2001-10-03 | 2005-11-09 | Chiron Corp | Composiciones de adyuvantes. |
EP1531796B1 (en) | 2002-02-20 | 2016-09-28 | GlaxoSmithKline Biologicals SA | Microparticles with adsorbed polypeptide-containing molecules |
AU2003299994A1 (en) | 2002-12-27 | 2004-07-29 | Chiron Corporation | Immunogenic compositions containing phospholpid |
US20050191358A1 (en) * | 2003-01-14 | 2005-09-01 | Derek O' Hagan | Microparticles with adsorbed polynucleotide-containing species |
CA2528007C (en) | 2003-06-02 | 2012-03-27 | Chiron Corporation | Immunogenic compositions based on microparticles comprising adsorbed toxoid and a polysaccharide-containing antigen |
EP1991204A2 (en) | 2006-02-24 | 2008-11-19 | Novartis AG | Microparticles containing biodegradable polymer and cationic polysaccharide for use in immunogenic compositions |
KR20130121699A (ko) | 2010-05-28 | 2013-11-06 | 테트리스 온라인, 인코포레이티드 | 상호작용 혼성 비동기 컴퓨터 게임 기반구조 |
WO2013006837A1 (en) | 2011-07-06 | 2013-01-10 | Novartis Ag | Cationic oil-in-water emulsions |
US9655845B2 (en) | 2011-07-06 | 2017-05-23 | Glaxosmithkline Biologicals, S.A. | Oil-in-water emulsions that contain nucleic acids |
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EP0860167A1 (en) * | 1997-01-30 | 1998-08-26 | Robert Gurny | RNA-and DNA- based active agents in nanoparticles |
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CA2392071C (en) | 2012-01-10 |
DE60029243D1 (de) | 2006-08-17 |
CA2392071A1 (en) | 2001-05-25 |
EP1235900A1 (en) | 2002-09-04 |
AU1623501A (en) | 2001-05-30 |
DE60029243T2 (de) | 2007-05-31 |
JP2003514522A (ja) | 2003-04-22 |
JP2011062213A (ja) | 2011-03-31 |
WO2001036599A1 (en) | 2001-05-25 |
ATE332361T1 (de) | 2006-07-15 |
EP1235900B1 (en) | 2006-07-05 |
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