JP6452118B2 - T細胞の機能増強方法 - Google Patents
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Description
[1]T細胞のProgrammed Death−1 Ligand 1(PD−L1)及び/又はProgrammed Death−1 1igand 2(PD−L2)の発現を抑制することを特徴とするT細胞の機能増強方法、
[2]T細胞が細胞障害性T細胞又は調節性T細胞である[1]記載のT細胞の機能増強方法、
[3]調節性T細胞がヘルパーT細胞、制御性T細胞又はTh17細胞である[2]記載のT細胞の機能増強方法、
[4]機能増強がT細胞インターフェロン−γ(IFN−γ)産生増強である[1]記載のT細胞の機能増強方法、
[5]PD−L1及び/又はPD−L2に対するsiRNAにより、PD−L1及び/又はPD−L2の発現を抑制する[1]記載のT細胞の機能増強方法、
[6]PD−L1及び/又はPD−L2の発現が抑制された機能増強T細胞、
[7]T細胞が細胞障害性T細胞又は調節性T細胞である[6]記載の機能増強T細胞、
[8]調節性T細胞がヘルパーT細胞、制御性T細胞又はTh17細胞である[7]記載の機能増強T細胞、
[9]機能増強がT細胞インターフェロン−γ(IFN−γ)産生増強である[6]記載の機能増強T細胞、
[10]PD−L1及び/又はPD−L2に対するsiRNAにより、PD−L1及び/又はPD−L2の発現が抑制された[6]記載の機能増強T細胞、ならびに
[11][6]〜[10]いずれか記載の機能増強T細胞を有効成分として含有する、前記T細胞に感受性を示す疾患の治療剤、
に関する。
本発明のT細胞の機能増強方法は、T細胞のPD−L1及び/又はPD−L2の発現を抑制する工程を含むことを特徴とする方法である。また、本発明の機能増強T細胞は、本発明のT細胞の機能増強方法により得られる細胞である。本発明のT細胞の機能増強方法により、T細胞の増殖率の向上、サイトカイン産生量の増加又は細胞傷害性の向上を含むT細胞のエフェクター機能が向上する。更に、アネルギー状態、休止状態などのT細胞の抑制状態を解除又は抑制し、外部の刺激に対するT細胞の感受性を向上させる。こうして得られる本発明の機能増強T細胞は、がん、感染疾患又は自己免疫疾患の治療又は予防に有用である。
本発明の治療方法又は予防方法は、前記(1)の本発明の機能増強方法により調製されたT細胞を生体に投与することを特徴とする。すなわち本発明の治療方法又は予防方法は、(c)工程(b)で得られた機能増強T細胞を生体に投与する工程、を包含する。本発明の機能増強T細胞が投与される疾患としては、当該T細胞に感受性を示す疾患であればよく、特に限定はないが、例えば、がん(白血病、固形腫瘍など)、肝炎、インフルエンザ、HIVなどのウイルス、細菌、真菌が原因となる感染性疾患、例えば結核、MRSA、VRE、深在性真菌症などが例示される。また、本発明の細胞は、骨髄移植又は放射線照射後の感染症予防、あるいは再発白血病の寛解を目的としたドナーリンパ球輸注などにも利用できる。又、制御性T細胞の機能を向上させ自己免疫疾患の予防又は治療に利用できる。
(3)本発明の機能増強T細胞を含有する疾患の治療剤
前記(1)の本発明の機能増強方法により調製された機能増強T細胞、ならびに当該T細胞を有効成分として含有する組成物は、当該T細胞に感受性を有する疾患、例えば前記のがん、肝炎又は感染性疾患の治療剤として有用である。
本発明の治療剤は製薬分野で公知の方法に従い、例えば、本発明の方法により調製されたT細胞を有効成分として、例えば公知の非経口投与に適した有機又は無機の担体、賦形剤、安定剤等と混合することにより調製できる。また、その使用は前記(2)の本発明の治療方法に準じればよい。
国際公開第2007/032255号パンフレットに従い、HLA−A24拘束性MAGE−A4143−151特異的な細胞傷害性CD8陽性T細胞クローン2−28(Miyahara Y 他13名 クリニカル キャンサー リサーチ(Clin. Cancer Res.)、第11巻、第5581−5589頁(2005)以下、CD8陽性T細胞クローン2−28と呼ぶ)とMAGE−A4143−151ペプチドとを調製した。CD8陽性T細胞クローン2−28を、80GlyのX線を照射した自己リンパ芽球様細胞株(lymphoblastoid cell line:LCL)にMAGE−A4143−151ペプチドをパルスしたものを、37℃で共培養し、0日、1日後、2日後及び3日後の細胞表面のPD−L1を、抗ヒトPD−L1抗体(BD bioscience社製)にて染色を行いフローサイトメーターで解析した。図1にフローサイトメーターによる解析結果を示す。培養1日後においてPD−L1の発現は最も高かった。
配列番号1記載の配列を有するPD−L1に特異的なsiRNA、配列番号2記載の配列を有するPD−L2に特異的なsiRNA、もしくはネガティブコントロールsiRNA(いずれもInvitrogen社製)を、CD8陽性T細胞クローン2−28にエレクトロポレーションにより導入した。
ネガティブコントロールsiRNA、配列番号3記載の配列を有するPD−1に特異的なsiRNA(Invitrogen社製)、配列番号1記載の配列を有するPD−L1に特異的なsiRNA、又は配列番号2記載の配列を有するPD−L2に特異的なsiRNAを、CD8陽性T細胞クローン2−28にエレクトロポレーションにより導入した。3日間培養した後、この細胞を自己LCLにMAGE−A4143−151ペプチドをパルスしたものと、4:1もしくは2:1の割合で混合して37℃で共培養し、翌日、各培養上清中のIFN−γ濃度をELISA法で測定した。
ネガティブコントロールsiRNA、配列番号3記載の配列を有するPD−1に特異的なsiRNA、配列番号1記載の配列を有するPD−L1に特異的なsiRNA、又は配列番号2記載の配列を有するPD−L1に特異的なsiRNAを、MAGE−A446−66特異的なCD4陽性T細胞クローン5にエレクトロポレーションにより導入し、3日間培養した。この細胞を自己LCLにMAGE−A446−66ペプチドをパルスしたものと、2:1で混合して37℃で培養し、翌日、各培養上清中のIFN−γ濃度をELISA法で測定した。
LCLに配列番号8、9記載の配列を有するPD−L1に特異的なsiRNA(sh831、sh832)、もしくは配列番号10、11記載の配列を有するPD−L2に特異的なsiRNA(sh833、sh834)(いずれもタカラバイオ社製)、もしくは実施例2で使用したネガティブコントロールsiRNAをエレクトロポレーションにより導入し、37℃で2日培養したのち、それぞれの細胞からトータルRNAを抽出して、QuanTitect SYBR PCR Kit(キアゲン社製)を用いてリアルタイムRT−PCRでPD−L1とPD−L2の発現を測定し、発現抑制力の強いsiRNAを選択した。
配列番号12、13にPD−L1の発現測定に使用したリアルタイムRT−PCR用プライマー配列を、配列番号14、15にPD−L2の発現測定に使用したリアルタイムRT−PCR用プライマー配列を、配列番号16、17に発現測定の対象としたヒトGAPDH(グリセルアルデヒド3リン酸脱水素酵素)のリアルタイムRT−PCR用プライマー配列をそれぞれ示す。
図6にPD−L1のリアルタイムRT−PCRの結果を、図7にPD−L2のリアルタイムRT−PCRの結果を示す。図中、縦軸はヒトGAPDHの発現に対するPD−L1、PD−L2の発現比率を示す。この結果より、PD−L1に特異的なsiRNAとして配列番号9に示すsh832、PD−L2に特異的なsiRNAとして配列番号11に示すsh834を選択した。
国際公開第2008/153029号パンフレットに従い、pMS−Ma2及びpMS−Pb2を作製した。これらのベクターはMSCV(Murine Stem Cell Virus)をベースとするレトロウイルスベクターシステムを含む。更に、それぞれ腫瘍抗原MAGE−A4を認識するTCRのα鎖及びβ鎖をコードする遺伝子を含み、これらの遺伝子は後述するTCRに対するsiRNAによるRNA干渉により発現が抑制されないようにコドンを変換している(以下、コドンを変換したものをコドン変換型と呼ぶ)。
実施例6で作製したpMS−Ma2から、MluI及びBglIIによりコドン変換型TCRα遺伝子を切り出し、TCRα−MluI/BglIIを調製した。配列番号4に示すsiRNAクラスター、PDL1−PDL2−TCRα−TCRβ人工合成遺伝子をBglII及びNotIで消化し、TCRα−MluI/BglIIと共に、pMS−Pb2のMluI−NotIサイトにクローニングすることにより、MS−aPb1−siPDL_1/2_siTCRベクター(ベクター1)を調製した。ベクター1は、コドン変換型TCRを発現し、配列番号9、11、18及び19に示すPD−L1、PD−L2、TCRα及びTCRβに対するsiRNAを発現することができる。このうち、TCRα及びTCRβに対するsiRNAは、内在性の野生型TCRの発現を抑制し、コドン変換型TCRの発現は抑制しない。
実施例7で調製したベクター1〜4により大腸菌JM109を形質転換し、プラスミドDNAをQIAGEN Plasmid Midi Kit(キアゲン社製)を用いて精製し、トランスフェクション用DNAとして供した。
レトロネクチン(登録商標、タカラバイオ社製)を用いて取扱説明書に従って、ヒト末梢血より分離した末梢血単核球(PBMC)に、実施例8で作製したレトロウイルス溶液を2回感染させ、コドン変換型TCR及びsiRNA共発現導入末梢血単核球を作製した。2回目ウイルス感染3日後に細胞を回収し、FastPure DNA Kit (タカラバイオ)によりゲノムを抽出し、Proviral Copy Number Detection Primer Set (タカラバイオ社製)及びCycleavePCR Core Kit (タカラバイオ社製)にて各細胞ゲノム中の平均プロウイルスコピー数を算出した。ベクター1〜4を含むウイルス溶液を感染させたPBMCのプロウイルスコピー数は、それぞれ4.6copies/cell、2.2copies/cell、3.2copies/cell、4.5copies/cellであった。次に、2回目のウイルス感染の5日後に細胞を回収し、HLA−A2402 MAGE−A4 テトラマー−PE(MBL社製)及びHuman CD8−FITC CONJUGATE(ベクトン・ディッキンソン社製)により染色し、フローサイトメーターによりCD8陽性であって、かつテトラマー陽性である細胞の割合を測定した。
SEQ ID NO:2: siRNA specific for PD-L2
SEQ ID NO:3: siRNA specific for PD-1
SEQ ID NO:4:siRNA cluster PDL1-PDL2-TCR alpha-TCR beta
SEQ ID NO:5:siRNA cluster PDL1-PDL2
SEQ ID NO:6:siRNA cluster PDL1
SEQ ID NO:7:siRNA cluster PDL2
SEQ ID NO:8:siRNA for PD-L1 (sh831)
SEQ ID NO:9:siRNA for PD-L1 (sh832)
SEQ ID NO:10:siRNA for PD-L2 (sh833)
SEQ ID NO:11:siRNA for PD-L2 (sh834)
SEQ ID NO:12:real time PCR F-primer for PD-L1
SEQ ID NO:13:real time PCR R-primer for PD-L1
SEQ ID NO:14:real time PCR F-primer for PD-L2
SEQ ID NO:15:real time PCR R-primer for PD-L2
SEQ ID NO:16:real time PCR F-primer for GAPDH
SEQ ID NO:17:real time PCR R-primer for GAPDH
SEQ ID NO:18:siRNA for TCR alpha
SEQ ID NO:19:siRNA for TCR beta
Claims (12)
- T細胞のProgrammed Death−1 Ligand 1(PD−L1)及び/又はProgrammed Death−1 1igand 2(PD−L2)の発現を、PD−L1及び/又はPD−L2に対する直接T細胞に導入されたsiRNA又はT細胞内で転写されたsiRNAによりエクス・ビボで抑制することを特徴とする、T細胞インターフェロン−γ(IFN−γ)産生増強方法であって、該siRNAが、配列番号1及び2からなる群より選択される塩基配列からなるアンチセンス鎖を含むsiRNAである、方法。
- T細胞が細胞障害性T細胞又は調節性T細胞である請求項1記載の方法。
- 調節性T細胞がヘルパーT細胞、制御性T細胞又はTh17細胞である請求項2記載の方法。
- さらにT細胞に所望の抗原を認識するTCRをコードする遺伝子を導入する、請求項1〜3のいずれか1項記載の方法。
- TCRをコードする遺伝子がベクターによりT細胞に導入され、該ベクターに、PD−L1及び/又はPD−L2に対するsiRNAを転写する核酸構築物が組み込まれている、請求項4記載の方法。
- PD−L1及び/又はPD−L2に対する直接T細胞に導入されたsiRNA又はT細胞内で転写されたsiRNAによりPD−L1及び/又はPD−L2の発現が抑制されたIFN−γ産生増強T細胞であって、該siRNAが、配列番号1及び2からなる群より選択される塩基配列からなるアンチセンス鎖を含むsiRNAである、T細胞。
- T細胞が細胞障害性T細胞又は調節性T細胞である請求項6記載のT細胞。
- 調節性T細胞がヘルパーT細胞、制御性T細胞又はTh17細胞である請求項7記載のT細胞。
- さらにT細胞に所望の抗原を認識するTCRをコードする遺伝子が導入された、請求項6〜8のいずれか1項記載のT細胞。
- TCRをコードする遺伝子がベクターによりT細胞に導入され、該ベクターに、PD−L1及び/又はPD−L2に対するsiRNAを転写する核酸構築物が組み込まれている、請求項9項記載のT細胞。
- 請求項6〜10いずれか1項記載のIFN−γ産生増強T細胞を有効成分として含有する、前記T細胞に感受性を示す疾患の治療剤。
- さらに、PD−L1及び/又はPD−L2の発現が抑制されたT細胞をエクス・ビボで刺激する工程を含む、請求項1〜5のいずれか1項記載の方法。
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CN107090437A (zh) * | 2009-03-06 | 2017-08-25 | 国立大学法人三重大学 | 用于增强t细胞功能的方法 |
JP2013523162A (ja) * | 2010-04-06 | 2013-06-17 | アルナイラム ファーマシューティカルズ, インコーポレイテッド | Cd274/pd−l1遺伝子の発現を阻害するための組成物および方法 |
EP2663316A2 (en) * | 2011-01-12 | 2013-11-20 | TiGenix, S.A.U. | Adipose-derived mesenchymal stem cells for intralymphatic administration in autoimmune and inflammatory diseases |
PL2855667T3 (pl) * | 2012-05-25 | 2024-03-25 | Cellectis | Sposoby uzyskiwania metodami inżynierii allogenicznych i opornych na immunosupresję limfocytów t do immunoterapii |
KR102158924B1 (ko) | 2013-03-15 | 2020-09-22 | 제넨테크, 인크. | Pd-1 및 pd-l1 관련 상태를 치료하기 위한 바이오마커 및 방법 |
RU2715038C2 (ru) | 2014-07-11 | 2020-02-21 | Дженентек, Инк. | Антитела анти-pd-l1 и способы их диагностического применения |
CN107106604B (zh) * | 2014-09-18 | 2021-04-06 | 财团法人卫生研究院 | 提升及抑制免疫调节细胞表达之方法 |
US20180265874A1 (en) * | 2014-10-10 | 2018-09-20 | Global Biopharma, Inc. | Methods for treating and/or preventing a tumor growth, invasion and/or metastasis |
MX2017015239A (es) * | 2015-05-29 | 2018-02-19 | Juno Therapeutics Inc | Composicion y metodos para regular interacciones inhibitorias en celulas geneticamente modificadas. |
PE20181131A1 (es) | 2015-09-02 | 2018-07-17 | Alnylam Pharmaceuticals Inc | COMPOSICIONES DE ARNi PARA LIGANDO 1 DE MUERTE CELULAR PROGRAMADA 1 (PD-L1) Y METODOS DE USO DE LAS MISMAS |
KR20230136689A (ko) * | 2016-03-14 | 2023-09-26 | 에프. 호프만-라 로슈 아게 | Pd-l1 발현의 감소를 위한 올리고뉴클레오티드 |
WO2018170766A1 (zh) * | 2017-03-22 | 2018-09-27 | 深圳市博奥康生物科技有限公司 | 一种人程序性死亡配体2基因的siRNA及其应用 |
CN107201380B (zh) * | 2017-07-11 | 2019-11-08 | 深圳华云生物技术有限公司 | 抗原特异性t细胞及其制备方法和应用 |
WO2020026591A1 (ja) | 2018-08-01 | 2020-02-06 | 本田技研工業株式会社 | クラッチ制御装置 |
US20220143089A1 (en) * | 2019-03-21 | 2022-05-12 | Onk Therapeutics Limited | Modified immune effector cells with increased resistance to cell death |
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US20060276422A1 (en) * | 2001-05-18 | 2006-12-07 | Nassim Usman | RNA interference mediated inhibition of B7-H1 gene expression using short interfering nucleic acid (siNA) |
CA2388441A1 (en) * | 2002-06-10 | 2003-12-10 | Wei-Ping Min | Immunomodulation using rna interference |
ATE481985T1 (de) * | 2002-07-03 | 2010-10-15 | Ono Pharmaceutical Co | Immunpotenzierende zusammensetzungen |
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JP2009011236A (ja) * | 2007-07-04 | 2009-01-22 | Shizuoka Prefecture | 1細胞レベルでのt細胞抗原レセプター遺伝子の解析・同定方法 |
CN107090437A (zh) * | 2009-03-06 | 2017-08-25 | 国立大学法人三重大学 | 用于增强t细胞功能的方法 |
JP2013523162A (ja) * | 2010-04-06 | 2013-06-17 | アルナイラム ファーマシューティカルズ, インコーポレイテッド | Cd274/pd−l1遺伝子の発現を阻害するための組成物および方法 |
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JPWO2010101249A1 (ja) | 2012-09-10 |
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CN102428179A (zh) | 2012-04-25 |
US9603874B2 (en) | 2017-03-28 |
US9181525B2 (en) | 2015-11-10 |
EP2404997A1 (en) | 2012-01-11 |
EP2404997A4 (en) | 2013-06-26 |
CN107090437A (zh) | 2017-08-25 |
JP6709549B2 (ja) | 2020-06-17 |
JP2018145205A (ja) | 2018-09-20 |
US20110318839A1 (en) | 2011-12-29 |
JP2017035090A (ja) | 2017-02-16 |
US20160015751A1 (en) | 2016-01-21 |
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