JP6783009B2 - Il6rがブロッキングされたcrsを緩和するためのcar−t遺伝子組換えベクターおよびその構築方法と使用 - Google Patents
Il6rがブロッキングされたcrsを緩和するためのcar−t遺伝子組換えベクターおよびその構築方法と使用 Download PDFInfo
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- Developmental Biology & Embryology (AREA)
Description
配列番号1で示される目的菌株の大量増幅のためのアンピシリン耐性遺伝子含有AmpR配列と、
配列番号2で示されるプラスミド複製のための原核レプリコンpUC Ori配列と、
配列番号3で示される真核細胞内における複製を増強するためのウイルスレプリコンSV40 Ori配列と、
配列番号11で示される遺伝子組換えの発現効率を増大させるためのeWPRE増強型ウッドチャックB型肝炎ウイルス転写後調節エレメントと、
配列番号12で示されるキメラ抗原受容体遺伝子の真核転写のためのヒトEF1αプロモーターと、
レンチウイルスパッケージングのためのレンチウイルスパッケージングのシスエレメントと、
配列番号21で示されるIL6RscFv1、または配列番号22で示されるIL6RscFv2、または配列番号23で示されるIL6RscFv3であるヒトIL6Rのヒト化一本鎖抗体と、
配列番号25で示されるタンパク質の共転写発現のためのIRESリボソーム結合配列と、
配列番号26で示されるIL6シグナルペプチドと、
配列番号27で示されるヒト由来抗体Fc断片と、
認識、伝達、開始が一体化した第二世代CARまたは第三世代CARを組み立てるためのキメラ抗原受容体と、
を含むベクターを提供する。
一.材料
1.レンチウイルス骨格プラスミドpLenti-3G Basic2、レンチウイルスパッケージングプラスミドpPac-GP、pPac-Rおよび膜タンパク質プラスミドpEnv-G、HEK293T/17細胞、相同組換え酵素はS&E (Shanghai) BIO-PHARMACEUTICAL TECHNOLOGY CO.,LTD.によって提供された。
EF1α-F:5’-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3’ (配列番号29)
EF1α-R:5’-TCACGACACCTGAAATGGAAGA-3’ (配列番号30)
CD8 leader-F:5’-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3’
(配列番号31)
CD8 leader-R:5’-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3’(配列番号32)
VL-F:5’-CACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATC-3’(配列番号33)
VL-R:5’-TGTGATCTCCAGCTTGGTCC-3’(配列番号34)
OLC-VH-F:5’-CAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCG
GGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCA-3’(配列番号35)
VH-R:5’-TGAGGAGACGGTGACTGAGGT-3’(配列番号36)
CD8 Hinge-F:5’-AGTCACCGTCTCCTCAACCACGACGCCAGCGCC-3’(配列番号37)
CD8 Hinge-R:5’-GTAGATATCACAGGCGAAGTCCA-3’(配列番号38)
CD8 Transmembrane-F:5’-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3’(配列番号39)
CD8 Transmembrane-R:5’-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG- 3’(配列番号40)
CD137-F:5’-AAACGGGGCAGAAAGAAACTC-3’(配列番号41)
CD137-R:5’-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3’(配列番号 42)
TCR-F:5’-AGAGTGAAGTTCAGCAGGAGCG-3’(配列番号43)
TCR-R:5’-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3’(配列番号44)
IRES-F:5’-GCCCTGCCCCCTCGCTAAGCCCCTCTCCCTCCCC-3’(配列番号45)
IRES-R:5’- CCAGGGAGAAGGCAACTGGACCGAAGGCGCTTGTGGAGAAGGAGTTC
ATGGTGGCATTATCATCGTGTTTTTCAAAGGA -3’(配列番号46)
IL6Rs1-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGACATCCAGATGACCCAGAG -3’(配列番号47)
IL6Rs1-R:5’- GCAGCTTTTCGGTTCTGAGGAGACTGTGACGAGGCT -3’(配列番号48)
IL6Rs2-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGAAATTGTGATGACCCAGAG -3’(配列番号49)
IL6Rs2-R:5’-GCAGCTTTTCGGTTCGCTGCTCACGGTCACGGTGGT -3’(配列番号50)
IL6Rs3-F:5’- GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGATATTCAGATGACCCAGAG -3’(配列番号51)
IL6Rs3-R:5’- GCAGCTTTTCGGTTCGCTGCTCACGGTCACGGTGGT -3’(配列番号52)
s0-F:5’- GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCATTGTTCTGGATTCCTGCTTCCA -3’(配列番号53)
s0-R:5’- GCAGCTTTTCGGTTCTGCAGAGACAGAGACCAGAGT -3’(配列番号54)
Fc-F:5’- GAACCGAAAAGCTGCGATAAAAC -3’(配列番号55)
Fc-R:5’- CTAGCAATCTAGAGGTTATTTGCCCGGGCTCAGGCTCA -3’(配列番号56)
WPRE-QPCR-F:5’-CCTTTCCGGGACTTTCGCTTT-3’(配列番号57)
WPRE-QPCR-R:5’-GCAGAATCCAGGTGGCAACA-3’(配列番号58)
アクチン-QPCR-F:5’-CATGTACGTTGCTATCCAGGC-3’(配列番号59)
アクチン-QPCR-R:5’-CTCCTTAATGTCACGCACGAT-3’(配列番号60)
CAR-QPCR-F:5’-GACTTGTGGGGTCCTTCTCCT-3’(配列番号61)
CAR-QPCR-R:5’-GCAGCTACAGCCATCTTCCTC-3’(配列番号62)
IL6-QPCR-F:5’-GGATTCAATGAGGAGACTT-3’(配列番号63)
IL6-QPCR-R:5’-ATCTGTTCTGGAGGTACT-3’(配列番号64)
CRP-QPCR-F:5’- GACATTGGAAATGTGAACATGT-3’(配列番号65)
CRP-QPCR-R:5’- CACAGCTGGGGTTTGGTGA-3’(配列番号66)
Fc-QPCR-F:5’- GACATTGGAAATGTGAACATGT-3’(配列番号67)
Fc-QPCR-R:5’- CACAGCTGGGGTTTGGTGA-3’(配列番号68)
19.LDH検出キットはpromega社から購入した。
(1)完全培地:加熱しておいた新鮮な培地を取り、10%FBS +5ml Pen-Srepを入れ、上下反転で均一に混合した。
(8)上記2〜6個の培養シャーレにおける残留細胞を培地瓶に移し、培地でもう一回培養シャーレを洗った。
(17)一台目のインキュベーターのCO2濃度設定値を5%に戻した。
一.イオン交換クロマトグラフィーによる組換えレンチウイルスベクターの精製(図7に示す):
(1)収集された上清液をThermo真空ポンプで、0.22μm〜0.8μmのPESフィルターで吸引ろ過し、不純物を除去した。
(1)24ウェルプレートを取って293T細胞を接種した。各ウェルの細胞は5×104個で、加えられた培地の体積は500μlで、異なる種類の細胞の生長速度には差異があり、ウイルス感染時の細胞融合率は40%〜60%であった。
2×TaqMan Master Mix 25 μl × n
フォワードプライマー(100 pmol ml-1) 0.1μl × n
リバースプライマー(100 pmol ml-1) 0.1μl × n
プローブ(100 pmol ml-1) 0.1μl × n
H2O 19.7μl × n
2×TaqMan Master Mix 25 μl × n
10×RNasePプライマー/プローブミックス 2.5 μl × n
H2O 17.5μl × n
IU ml-1 = (C ×N× D×1000)/V
ただし、
C = 1ゲノムあたりに組み込まれたウイルスのコピー数
N = 感染時の細胞数(約1×105)
D = ウイルスベクターの希釈倍数
V = 入れた希釈ウイルスの体積数
(1)内毒素使用標準品は15EU/本であった。
(1)実験前の3日は、細胞サンプルを無抗生物質培地で培養した。
(7)サンプルを95℃で5min加熱した。
一.CAR遺伝子の細胞レベルの発現検出:
(1)組換えレンチウイルスベクターlvCAR19-IL6RscFv1、lvCAR19-IL6RscFv2、lvCAR19-IL6RscFv3、lvCAR19-scFv0および対照ウイルスMockをPBMC細胞に感染させた後、細胞を収集してRT-PCRによってCAR遺伝子およびscFv遺伝子のmRNA転写レベルの検出を行った。CAR遺伝子およびscFv遺伝子の発現を検証したが、CAR遺伝子およびscFv遺伝子のmRNA転写レベルが向上すると、CAR遺伝子およびscFv遺伝子の転写レベルの発現に成功したことになる。
(1)それぞれCD19+K562細胞とPBMC細胞を培養した。
殺傷効率= (実験ウェル−エフェクター細胞ウェル−標的細胞ウェル)/(標的細胞最大ウェル−標的細胞ウェル)×100%
(1)それぞれCD19+K562細胞とPBMC細胞を培養した。
(2)実験開始前の4日は、それぞれMOI=15のlvCAR19-IL6RscFv1、lvCAR19-IL6RscFv2、lvCAR19-IL6RscFv3、lvCAR19-scFv0のウイルスでPBMC細胞に感染させ、72-96h培養した後、実験を開始した。
Claims (10)
- IL6RがブロッキングされたCRSを緩和するためのCAR-T遺伝子組換えベクターであって、
配列番号1で示される目的菌株の大量増幅のためのアンピシリン耐性遺伝子含有AmpRに対応するヌクレオチド配列と、
配列番号2で示されるプラスミド複製のための原核レプリコンpUC Ori配列と、
配列番号3で示される真核細胞内における複製を増強するためのウイルスレプリコンSV40 Ori配列と、
配列番号11で示される遺伝子組換えの発現効率を増大させるためのeWPRE増強型ウッドチャックB型肝炎ウイルス転写後調節エレメントと、
配列番号12で示されるキメラ抗原受容体遺伝子の真核転写のためのヒトEF1αプロモーターと、
レンチウイルスパッケージングのためのレンチウイルスパッケージングのシスエレメントと、
配列番号21で示されるIL6RscFv1、または配列番号22で示されるIL6RscFv2、または配列番号23で示されるIL6RscFv3のヒトIL6Rに結合するヒト化一本鎖抗体に対応するヌクレオチド配列と、
配列番号25で示されるタンパク質の共転写発現のためのIRESリボソーム結合配列と、
配列番号26で示されるIL6シグナルペプチドに対応するヌクレオチド配列と、
配列番号27で示されるヒト由来抗体Fc断片に対応するヌクレオチド配列と、
および認識、伝達、開始が一体化した第二世代CARまたは第三世代CARを組み立てるためのキメラ抗原受容体に対応するヌクレオチド配列と、
を含むことを特徴とするベクター。 - 前記ヒトIL6Rに結合するヒト化一本鎖抗体は、対応するヌクレオチド配列が配列番号21で示されるIL6RscFv1であることを特徴とする請求項1に記載のベクター。
- 前記レンチウイルスパッケージングのシスエレメントは、第二世代のレンチウイルスベクターを使用し、配列番号5で示されるレンチウイルス5'末端LTR、配列番号6で示されるレンチウイルス3'末端自己不活性化LTR、配列番号7で示されるGagシスエレメント、配列番号8で示されるRREシスエレメント、配列番号9で示されるenvシスエレメント、配列番号10で示されるcPPTシスエレメントを含むことを特徴とする請求項1に記載のベクター。
- 前記レンチウイルスパッケージングのシスエレメントは、第三世代のレンチウイルスベクターを使用し、配列番号5で示されるレンチウイルス5'末端LTR、配列番号6で示されるレンチウイルス3'末端自己不活性化LTR、配列番号7で示されるGagシスエレメント、配列番号8で示されるRREシスエレメント、配列番号9で示されるenvシスエレメント、配列番号10で示されるcPPTシスエレメント、および配列番号4で示されるRSVプロモーターを含むことを特徴とする請求項1に記載のベクター。
- 前記認識、伝達、開始が一体化した第二世代CARを組み立てるためのキメラ抗原受容体に対応するヌクレオチド配列は、配列番号13で示されるCD8 leaderキメラ受容体シグナルペプチドに対応するヌクレオチド配列、配列番号14で示されるCD19一本鎖抗体軽鎖VLに対応するヌクレオチド配列、配列番号15で示されるOptimal Linker Cに対応するヌクレオチド配列、配列番号16で示されるCD19一本鎖抗体重鎖VHに対応するヌクレオチド配列、配列番号17で示されるCD8 Hingeキメラ受容体ヒンジに対応するヌクレオチド配列、配列番号18で示されるCD8 Transmembraneキメラ受容体膜貫通領域に対応するヌクレオチド配列、配列番号19で示されるCD137キメラ受容体共刺激因子に対応するヌクレオチド配列、配列番号20で示されるTCRキメラ受容体T細胞活性化ドメインに対応するヌクレオチド配列を含む、又は、
前記認識、伝達、開始が一体化した第三世代CARを組み立てるためのキメラ抗原受容体に対応するヌクレオチド配列は、配列番号13で示されるCD8 leaderキメラ受容体シグナルペプチドに対応するヌクレオチド配列、配列番号14で示されるCD19一本鎖抗体軽鎖VLに対応するヌクレオチド配列、配列番号15で示されるOptimal Linker Cに対応するヌクレオチド配列、配列番号16で示されるCD19一本鎖抗体重鎖VHに対応するヌクレオチド配列、配列番号17で示されるCD8 Hingeキメラ受容体ヒンジに対応するヌクレオチド配列、配列番号18で示されるCD8 Transmembraneキメラ受容体膜貫通領域に対応するヌクレオチド配列、配列番号19で示されるCD137キメラ受容体共刺激因子に対応するヌクレオチド配列、配列番号20で示されるTCRキメラ受容体T細胞活性化ドメインに対応するヌクレオチド配列、および配列番号28で示されるCD28キメラ受容体共刺激因子に対応するヌクレオチド配列を含む、ことを特徴とする請求項1に記載のベクター。 - 前記eWPRE増強型ウッドチャックB型肝炎ウイルス転写後調節エレメントは、6つのヌクレオチドの突然変異を含み、g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>Tであることを特徴とする請求項1〜5のいずれかに記載のベクター。
- 請求項1〜6のいずれかに記載のIL6RがブロッキングされたCRSを緩和するためのCAR-T遺伝子組換えベクターの構築方法であって、以下の工程を含むことを特徴とする方法:
(1)配列番号1で示されるアンピシリン耐性遺伝子含有AmpRに対応するヌクレオチド配列、配列番号2で示される原核レプリコンpUC Ori配列、配列番号3で示されるウイルスレプリコンSV40 Ori配列、レンチウイルスパッケージングのためのレンチウイルスパッケージングのシスエレメント、配列番号11で示されるeWPRE増強型ウッドチャックB型肝炎ウイルス転写後調節エレメントをレンチウイルス骨格プラスミドに組み込む;
(2)配列番号12で示されるヒトEF1αプロモーター、認識、伝達、開始が一体化した第二世代CARまたは第三世代CARを組み立てるためのキメラ抗原受容体に対応するヌクレオチド配列を第二世代CARまたは第三世代CARの設計態様に組み立て、酵素切断、連結、組換え反応を経てレンチウイルス骨格プラスミドにクローニングし、第二世代CARまたは第三世代CARの設計の組換えレンチウイルスプラスミドを得る;
(3)ヒトIL6Rに結合するヒト化一本鎖抗体IL6RscFv1、IL6RscFv2、またはIL6RscFv3に対応するヌクレオチド配列、IRESリボソーム結合配列、IL6シグナルペプチドに対応するヌクレオチド配列およびヒト由来抗体Fc断片に対応するヌクレオチド配列をそれぞれ組換えレンチウイルスプラスミドにクローニングし、IL-6R遮断組換えレンチウイルスプラスミドを得る;
(4)得られた組換えレンチウイルスプラスミドをそれぞれレンチウイルスパッケージングプラスミドpPac-GP、pPac-Rおよび膜タンパク質プラスミドpEnv-GとHEK293T/17細胞に共形質移入させ、HEK293T/17細胞において遺伝子の転写・発現を行わせた後、パッケージングに成功した組換えレンチウイルスベクターが細胞培養上清に放出され、組換えレンチウイルスベクターを含む上清液を収集する;
(5)得られた組換えレンチウイルス上清を吸引ろ過、吸着、溶離のカラム精製手段によって精製し、それぞれ組換えレンチウイルスベクターを得る。 - 工程(3)により得られる組換えレンチウイルスプラスミドは、ヒトEF1αプロモーターによってCAR遺伝子全体の発現が開始し、CARタンパク質は細胞膜の表面に局在化し、CD19抗原を認識し、T細胞の増殖およびサイトカインの分泌を刺激し、下流シグナル経路の発現を活性化させ、scfv領域がCD19抗原に結合すると、シグナルがキメラ受容体を介して細胞内に伝達されることで、T細胞の増殖、サイトカインの分泌の増加、抗アポトーシスタンパク質の分泌の増加、細胞死亡の遅延、標的細胞の分解といった一連の生物学的効果が生じ、IRESリボソーム結合配列によってIL6RscFvとFcの融合タンパク質が共発現され、そしてIL6シグナルペプチドの誘導によって細胞外に分泌され、IL6Rとの結合によって、IL-6とIL6Rの結合が遮断されることで、IL6のシグナル経路を遮断して、CRSを抑制する効果を実現させる機能を有することを特徴とする請求項7に記載の方法。
- 工程(5)では、前記吸引ろ過ステップは、上清体積を200ml〜2000mlに、真空度を-0.5MPA〜-0.9MPAに抑え、穴詰まりによるベクターの損失を防ぎ、前記吸着ステップは、溶液のpH値を6〜8に抑え、pHの変化によるベクターの不活性化を防止し、前記溶離ステップは溶離液のイオン強度を0.5M〜1.0Mに抑え、イオン強度の変化による溶離の不完全またはベクターの不活性化を防ぐことを特徴とする請求項7に記載の方法。
- CRSを緩和する薬物の製造における請求項1〜6のいずれかに記載のベクターの使用。
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PCT/CN2017/110654 WO2018103502A1 (zh) | 2016-12-05 | 2017-11-13 | 一种封闭il6r的用于缓解crs的car-t转基因载体及其构建方法和应用 |
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AU2017319702A1 (en) | 2016-09-02 | 2019-04-11 | The Regents Of The University Of California | Methods and compositions involving interleukin-6 receptor alpha-binding single chain variable fragments |
EP3684919A1 (en) * | 2017-10-19 | 2020-07-29 | Cellectis | Targeted gene integration of nk inhibitors genes for improved immune cells therapy |
AU2020205951A1 (en) * | 2019-01-07 | 2021-07-22 | Hunan Siweikang Therapeutics Co. Ltd | Modified immune cells co-expressing chimeric antigen receptor and il-6 antagonist for reducing toxicity and uses thereof in adoptive cell therapy |
CN110055224B (zh) * | 2019-04-03 | 2023-06-30 | 深圳市体内生物医药科技有限公司 | 一种基因修饰的免疫细胞及其制备方法和应用 |
CN110846344A (zh) * | 2019-11-18 | 2020-02-28 | 山东省齐鲁细胞治疗工程技术有限公司 | 一种表达il-6r阻断抗体的靶向cd19的嵌合抗原受体t细胞及制备方法、应用 |
CN110749735A (zh) * | 2019-11-27 | 2020-02-04 | 合肥中科干细胞再生医学有限公司 | 一种示踪剂 |
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CN105640990A (zh) | 2016-01-06 | 2016-06-08 | 奥思达干细胞有限公司 | 一种治疗乳腺癌的car-t细胞制剂及其制备方法 |
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CN105950662B (zh) * | 2016-05-17 | 2019-04-30 | 上海优卡迪生物医药科技有限公司 | 一种靶向cd22的复制缺陷性重组慢病毒car-t转基因载体及其构建方法和应用 |
CN105950664B (zh) * | 2016-05-17 | 2019-03-29 | 上海优卡迪生物医药科技有限公司 | 一种靶向cd123的复制缺陷性重组慢病毒car-t转基因载体及其构建方法和应用 |
AU2017319702A1 (en) * | 2016-09-02 | 2019-04-11 | The Regents Of The University Of California | Methods and compositions involving interleukin-6 receptor alpha-binding single chain variable fragments |
CN106636090B (zh) * | 2016-10-11 | 2019-08-09 | 上海优卡迪生物医药科技有限公司 | 人源白细胞介素6的siRNA、重组表达CAR-T载体及其构建方法和应用 |
CN107245500B (zh) * | 2017-05-27 | 2019-05-17 | 上海优卡迪生物医药科技有限公司 | 一种基于octs技术的淋系白血病car-t治疗载体及其构建方法和应用 |
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