JP6940406B2 - キメラ抗原レセプター分子の制御的発現のためのレンチウイルスベクター - Google Patents
キメラ抗原レセプター分子の制御的発現のためのレンチウイルスベクター Download PDFInfo
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Description
(i)パッケージングプラスミド(少なくともGag、Pol Rev、Tatおよび、幾つかの場合ではトランスファー構築物のパッケージングに必要な構造タンパク質および酵素タンパク質を発現する);
(ii)プロウイルストランスファープラスミド(発現カセットならびにパッケージング、逆転写、および組み込みに必要なHIVシス作用性因子を含有する);ならびに
(iii)エンベロープコードプラスミド(ほとんどの場合、幅広い種類の細胞、特に、DCを含む主要組織適合(MHC)抗原提示細胞(APC)を標的にすることができる混合粒子(偽型)の形成を可能にするタンパク質である、水疱性口内炎ウイルスの糖タンパク質(VSV.G))
によりHEK 293Tヒト培養細胞などを一過性トランスフェクションしたときに組換え技術により作製することができる。
最大の免疫応答を誘導するための、樹状細胞を含む抗原提示細胞における高発現;
誘導された免疫応答による排除に十分な他の形質導入細胞タイプにおける発現;および
挿入による影響を回避するためのエンハンサーエレメントの欠如。
キメラ抗原レセプター(CAR)をコードする種々のレンチベクターコンストラクトを、T細胞でのCARの発現のために作製した。どのプロモーターがヒトT細胞における発現に最も適しているのかは不明であった。プロモーターを比較するために、腎細胞株および樹状細胞株に、種々のプロモーターから緑色蛍光タンパク質(GFP)を発現するレンチウイルスベクターを形質導入した。ヒトEF1αプロモーターは、樹状細胞株BDCMにおいて最も強力なプロモーターであることが判明した(図1)。このプロモーターはまた、腎細胞株293Tにおいても非常に強力であった。様々なプロモーターの細胞特異的発現を、biogps.orgのデータセットを用いて分析した(図2)。このデータセットから、T細胞におけるEF1αプロモーターの発現は、BDCA+細胞における発現に類似している。実際、ヒトEF1αプロモーターは、活性化されたヒトT細胞においてCARを発現するために既に使用されていた。Buddeら、PLoS ONE 8(12):e82742(2013)。
本発明は、細胞外抗原結合ドメイン(結合ドメイン)、ヒンジおよび膜貫通ドメイン(膜貫通ドメイン);フック結合ドメイン;および細胞内シグナル伝達ドメイン(活性化ドメイン)を含むキメラ抗原レセプター(CAR)を包含する。CARは、これらドメインのそれぞれの1つ、2つ、3つまたはそれ以上を含むことができる。
MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR (配列番号3)
MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (配列番号4)
MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (配列番号5)
MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG (配列番号6)
MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI (配列番号:7)
MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (配列番号:8)
MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (配列番号:33)
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (配列番号:34)
本発明は、CARをコードする核酸を包含する。核酸は、一本鎖または二本鎖でありうる。核酸は、RNA分子またはDNA分子でありうる。好ましい核酸は、本明細書に詳述される配列番号の少なくとも1つのアミノ酸配列をコードする。本発明は、ベクターに挿入された本発明の単離された核酸を包含する。
HOOK:(CORE+HA TAG+他の配列)
ATGCACCGGAGGAGATCACGCTCTTGTAGGGAGGACCAGAAACCTGTCACCGGTGACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCATTACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTGGAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCCGAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGACGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACCGGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCTGAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGCAAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGAAACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAACAACGGCAATCCTCTGGATGCCGTCCAGCAGGTCGACTATCCGTACGACGTACCAGACTACGCAGTCGGACCGATGGACGATCAGAGGGACCTCATTAGCAACAACGAACAGCTGCCTATGCTGGGACGGCGACCTGGAGCCCCTGAATCCAAATGCTCTAGGGGAGCACTGTACACTGGCTTCTCCATTCTCGTGACACTGCTGCTGGCCGGGCAGGCTACTACTGCTTACTTCCTGTACCAGCAGCAGGGGCGGCTGGACAAACTCACTGTGACATCTCAGAACCTCCAGCTGGAAAATCTGAGGATGAAACTGCCCAAACCCCCTAAACCCGTGTCCAAAATGAGGATGGCCACACCTCTGCTCATGCAGGCACTGCCAATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACCATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAGAGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGACCAAGCAGGATCTGGGCCCAGTCCCCATGTGA (配列番号35)。
コアストレプトアビジン:
GACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCATTACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTGGAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCCGAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGACGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACCGGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCTGAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGCAAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGAAACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAACAACGGCAATCCTCTGGATGCCGTCCAGCAG (配列番号36)。
HA TAG:TATCCGTACGACGTACCAGACTACGCA (配列番号37)。
CAR_CD19 第2世代:1518bp
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCATGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCGGAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATATGGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACAAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCATTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTATGGCGTGATCCTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCTCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC (配列番号39)
CAR_CD19第3世代:1641bp
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTCGCTCTGCTGCTGCATGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCGGAAGTGGAGGGGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATATGGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGAGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCGGTCCAAGCGGAGCAGACTGCTGCACTCCGACTACATGAACATGACCCCCAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCCGGGACTTCGCCGCCTACAGAAGCAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTATGGCGTGATCCTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGCAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCAGCGGCGGAAGAACCCTCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC (配列番号40)
本発明は、CARおよびフック結合ドメインをコードするベクターを包含する。好ましいベクターは、核酸配列を含むか、または本明細書に詳述される配列番号の少なくとも1つのアミノ酸配列をコードする。
MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ (配列番号31)、または、
MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYTLTGRYDSAPATDGSGTALGWRVAWKNNYRNAHSATTWSGQYVGGAEARINTQWTLTSGTTEANAWKSTLRGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ (配列番号32)、または、
MHRRRSRSCREDQKPVTGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQVDYPYDVPDYAVGPMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM (配列番号42)。
ATGCACCGGAGGAGATCACGCTCTTGTAGGGAGGACCAGAAACCTGTCACCGGTGACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCATTACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTGGAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCCGAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGACGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACCGGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCTGAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGCAAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGAAACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAACAACGGCAATCCTCTGGATGCCGTCCAGCAGGTCGACTATCCGTACGACGTACCAGACTACGCAGTCGGACCGATGGACGATCAGAGGGACCTCATTAGCAACAACGAACAGCTGCCTATGCTGGGACGGCGACCTGGAGCCCCTGAATCCAAATGCTCTAGGGGAGCACTGTACACTGGCTTCTCCATTCTCGTGACACTGCTGCTGGCCGGGCAGGCTACTACTGCTTACTTCCTGTACCAGCAGCAGGGGCGGCTGGACAAACTCACTGTGACATCTCAGAACCTCCAGCTGGAAAATCTGAGGATGAAACTGCCCAAACCCCCTAAACCCGTGTCCAAAATGAGGATGGCCACACCTCTGCTCATGCAGGCACTGCCAATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACCATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAGAGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGACCAAGCAGGATCTGGGCCCAGTCCCCATGTGA (配列番号43)。
GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACGCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAA (配列番号44)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCATGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCGGAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATATGGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCACCGGTATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGAAGGACTGGCCGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACCCCCAGGGCCAGAGAGAGCCCAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTACGGCGTGATCCTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC (配列番号45)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITTGMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY (配列番号46)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCATGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCGGAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATATGGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACAAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAGCTGACCGGTATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGAAGGACTGGCCGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACCCCCAGGGCCAGAGGGAACCCCCCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTACGGCGTGATCCTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC (配列番号47)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTGMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY (配列番号48)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCATGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCGGAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAGGAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATATGGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACAAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTACGGCGTGATCCTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTACACCGGTATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGAAGGACTGGCCGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACCCCCAGGGCCAGAGGGAACCC (配列番号49)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYTGMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (配列番号50)
本発明は、T細胞、好ましくはヒトT細胞でのレンチベクターからのCARの高発現を促進するためのプロモーターの使用を包含する。好ましいプロモーターは、ヒトユビキチン、MHCクラスI、MHCクラスII、およびβ2ミクログロブリン(β2m)プロモーターである。
HLA−A2(MHC I):
attggggagtcccagccttggggattccccaactccgcagtttcttttctccctctcccaacctatgtagggtccttcttcctggatactcacgacgcggacccagttctcactcccattgggtgtcgggtttccagagaagccaatcagtgtcgtcgcggtcgcggttctaaagtccgcacgcacccaccgggactcagattctccccagacgccgagg (配列番号9)
HLA−B7(MHC I):
ggggaggcgcagcgttggggattccccactcccctgagtttcacttcttctcccaacttgtgtcgggtccttcttccaggatactcgtgacgcgtccccacttcccactcccattgggtattggatatctagagaagccaatcagcgtcgccgcggtcccagttctaaagtccccacgcacccacccggactcagag (配列番号10)
HLA−Cw5(MHC I):
cactggggaggcgccgcgttgaggattctccactcccctcagtttcacttcttctcccaacctgcgtcgggtccttcttcctgaatactcatgacgcgtccccaattcccactcccattgggtgtcgggttctagagaagccaatcagcgtctccgcagtcccggtctaaagtccccagtcacccacccggactcagattctccccagacgccgag (配列番号11)
HLA−E(MHC I):
taagaactgctgattgctgggaaactctgcagtttcccgttcctctcgtaacctggtcatgtgtccttcttcctggatactcatgacgcagactcagttctcattcccaatgggtgtcgggtttctagagaagccaatcagcgtcgccacgactcccgactataaagtccccatccggactcaagaagttctcaggactcagagg (配列番号12)
HLA−F(MHC I):
aggccccgaggcggtgtctggggttggaaggctcagtattgagaattccccatctccccagagtttctctttctctcccaacccgtgtcaggtccttcatcctggatactcataacgcggccccatttctcactcccattgggcgtcgcgtttctagagaagccaatcagtgtcgccgcagttcccaggttctaaagtcccacgcaccccgcgggactcatatttttcccagacgcggaggttggggtcatg (配列番号13)
aacatcacgagactctaagaaaaggaaactgaaaacgggaaagtccctctctctaacctggcactgcgtcgctggcttggagacaggtgacggtccctgcgggccttgtcctgattggctgggcacgcgtttaatataagtggaggcgtcgcgctggcgggcattcctgaagctgacagcattcgggccgag (配列番号14)
ggcctccgcgccgggttttgggcctcccgcgggcgcccccctcctcacggcgagcgctgccacgtcagacgaagggcgcagcgagcgtcctgatccttccgcccggacgctcaggacagcggcccgctgctcataagactcggccttagaaccccagtatcagcagaaggacattttaggacgggacttgggtgactctagggcactggttttctttccagagagcggaacaggcgaggaaaagtagtcccttctcggcgattctgcggagggatctccgtggggcggtgaacgccgatgattatataaggacgcgccgggtgtggcacagctagttccgtcgcagccgggatttgggtcgcggttcttgtttgtggatcgctgtgatcgtcacttggtgagtagcgggctgctgggctggccggggctttcgtggccgccgggccgctcggtgggacggaagcgtgtggagagaccgccaagggctgtagtctgggtccgcgagcaaggttgccctgaactgggggttggggggagcgcagcaaaatggcggctgttcccgagtcttgaatggaagacgcttgtgaggcgggctgtgaggtcgttgaaacaaggtggggggcatggtgggcggcaagaacccaaggtcttgaggccttcgctaatgcgggaaagctcttattcgggtgagatgggctggggcaccatctggggaccctgacgtgaagtttgtcactgactggagaactcggtttgtcgtctgttgcgggggcggcagttatggcggtgccgttgggcagtgcacccgtacctttgggagcgcgcgccctcgtcgtgtcgtgacgtcacccgttctgttggcttataatgcagggtggggccacctgccggtaggtgtgcggtaggcttttctccgtcgcaggacgcagggttcgggcctagggtaggctctcctgaatcgacaggcgccggacctctggtgaggggagggataagtgaggcgtcagtttctttggtcggttttatgtacctatcttcttaagtagctgaagctccggttttgaactatgcgctcggggttggcgagtgtgttttgtgaagttttttaggcaccttttgaaatgtaatcatttgggtcaatatgtaattttcagtgttagactagtaaattgtccgctaaattctggccgtttttggcttttttgttagaccgatc (配列番号38)
gtctagaagtcagattggggttaaagagtctgtccgtgattgactaacagtcttaaatacttgatttgttgttgttgttgtcctgtttgtttaagaactttacttctttatccaatgaacggagtatcttgtgtcctggaccctttgcaagaacccttcccctagcaacagatgcgtcatctcaaaatatttttctgattggccaaagagtaattgatttgcattttaatggtcagactctattacaccccacattctcttttcttttattcttgtctgttctgcctcactcccgagctc (配列番号41)
本発明は、本発明のCARをコードするベクターおよびレンチウイルスベクター粒子を含む、細胞、特に免疫系の細胞を包含する。好ましくは、細胞は、TαβおよびT?δ細胞またはNK細胞を含むT細胞である。
本発明は、レンチウイルスベクター粒子の製造方法を提供する。レンチウイルスベクター粒子(またはレンチウイルス粒子ベクター)は、ウイルスタンパク質と会合したレンチウイルスベクターを含む。ベクターは好ましくは組み込みベクターである。
このようなDNAプラスミドは、
−細菌複製起源(例えば、pUC ori)、
−選別のための抗生物質耐性遺伝子(例えば、KanR)、およびより特定すると
−β2m、Ubi、MHCクラスIIまたはMHCクラスIプロモーターに転写的に連結されたCARをコードする少なくとも1つの核酸を含むレンチウイルスベクター
を含みうる。
i)適切な宿主細胞にレンチウイルスベクターを核酸移入するステップと、
ii)前記宿主細胞に、レトロウイルス(好ましくはレンチウイルス)の少なくとも構造的およびポリメラーゼ(+インテグラーゼ)活性をコードするウイルスDNA配列を含有するパッケージングプラスミドベクターを核酸移入するステップと(このようなパッケージングプラスミドは当技術分野で記載されている(Dullら、1998、J Virol、72(11):8463〜71頁;Zuffereyら、1998、J Virol 72(12):9873〜80頁))、
iii)前記レンチウイルスベクターの発現およびレンチウイルスベクター粒子へのパッケージングを得るために、前記核酸移入した宿主細胞を培養するステップと、
iv)前記培養宿主細胞におけるステップiii)の発現およびパッケージングの結果生じるレンチウイルスベクター粒子を回収するステップと
を含む、本発明に係る組換えベクター粒子の作製が可能となる。
−cPPT/CTSポリヌクレオチド配列と、
−β2m、UbiまたはMHCIプロモーターの制御下でCARをコードする核酸、および場合により、上に記載されたさらなるエレメントの1つ。
本発明は、細胞、好ましくはT細胞、好ましくは増殖したT細胞においてCARを発現させるための方法を包含する。この方法は、CARの発現を可能にし、好ましくはT細胞を増殖させる条件下で、本発明のレンチウイルスベクターまたはレンチウイルス粒子ベクターで細胞を形質導入することを含む。
本発明は、さらに、ベクターにコードされたCARとの免疫学的反応の発生または改善を誘導または促進することができる治療用組成物またはワクチンの調製のための、本発明に係るレンチウイルスベクターの、特にレンチウイルスベクター粒子の形態での使用に関する。
pTRIPΔU3−CMV−GFP(15)のプロウイルス領域のPCR増幅は、SpeIおよびXbaI制限酵素部位をそれぞれ含む順方向(5'-CTTACTAGTTGGAAGGGCTAATTCACTCCCAAC-3';配列番号15)および逆方向(5'-CATTCTAGAACTGCTAGAGATTTTCCACACTG-3';配列番号16)オリゴヌクレオチドを用いて行った。得られた断片を消化し、MluI部位が欠失しているpVAX−1プラスミド(インビトロジェン、Lifetech)のSpeI部位とXbaI部位との間にクローニングした。得られたプラスミドをpFLAP−CMV−GFPと命名した。SV40配列は、pTRIPΔU3−CMV−GFPプラスミド(5'-TACCCCGGGCCATGGCCTCCAAAAAAGCCTCCTCACTACTTC-3'(配列番号17)および5'-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC-3'(配列番号18)オリゴヌクレオチドを使用)からPCRによって増幅し、pFLAP−CMV−GFPのPml1部位にクローニングし、得られたプラスミドをpFLAP−CMV−GFP−SVと命名した。CMVプロモーターは、MluIおよびBamHI部位をそれぞれ含む順方向(5'-TACACGCGTGGAGTTCCGCGTTACATAACTTACGG-3';配列番号19)および逆方向(5'-CGTGGATCCGATCGCGGTGTCTTCTATGGAGGTCAAAAC-3';配列番号20)オリゴヌクレオチドで増幅した。得られた断片を、pFlap−CMV−GFP−SVのMluIとBamHI部位との間にクローニングして戻し、レンチウイルスベクター内のプロモーターの置換を容易にした。次いでプロモーターを、β2mプロモーターのための5'-GCCGGCGCGCCGAGAAACCCTGCAGGGAATTCCC-3'(配列番号21)および5'-CGTGGATCCGATCGCTCGGCCCGAATGCTGTCAGCTTCAGG-3'(配列番号22)にてHEK293T細胞DNAからPCRにより増幅し、pFLAP−CMV−GFP−SVのMluIとBamH1部位との間にクローニングしてpFlap−β2m−SVを作製した。増幅されたβ2mプロモーター配列は以下の通りである。GAGAAACCCTGCAGGGAATTCCCCAGCTGTAGTTATAAACAGAAGTTCTCCTTCTGCTAGGTAGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTCGAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCGGGGGCACCATTAGCAAGTCACTTAGCATCTCTGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGTCCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCACCCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCACCCAGTCTAGTGCATGCCTTCTTAAACATCACGAGACTCTAAGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAACCTGGCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCCTGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATATAAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGACAGCATTCGGGCCGAG (配列番号23)。CARは合成して、pFlop−β2m−SVのBamHIとXhoI部位との間に、GFP遺伝子の代わりにクローニングすることができる。
R&D生産物:ベクターは、以前に記載されているように(25)、HEK293Tの一時的なリン酸カルシウムトランスフェクションによって製造されうる。HEK293T(ヒト胚性腎細胞株、ATCC CRL−11268(Grahamら、1977))細胞を、10%ウシ胎仔血清(FBS、PAA)、1%L−グルタミン(ユーロビオ)、1%ペニシリン−ストレプトマイシン(ライフテクノロジーズギブコ)および1%ピルビン酸ナトリウム(ライフテクノロジーズギブコ)を添加したダルベッコ変法イーグル培地(DMEM/高変法、ハイクローン)中で維持した。細胞株は37℃、5%CO2の加湿した大気中の保温器に維持した。標準的なリン酸カルシウム沈殿プロトコールを用いてHEK293T細胞の一過性形質転換によりレンチウイルスベクターを製造した。10mlの完全培養培地を含む10cm2組織培養皿(BDファルコン)に7×106のHEK293T細胞を播き、5%CO2の加湿大気、37℃の恒温槽に24時間維持し、接着させた。各ベクターの製造のために、一つの組織培養皿は以下のように形質転換する:レンチウイルス主鎖プラスミドpFlap−プロモーター−CAR_CD19(10μg)、pThV−Env1コード化エンベローププラスミド(2μg)およびpThV−GPパッケージ化プラスミド(10μg)を、353μlの滅菌水(ライフテクノロジーズギブコ)および125μlのCaCl2(フルカ)と混合した。次いで、DNA混合液を500μlの37℃に予め温めたHBS2×、pH=7.3に少しずつ加え、得られた1mlの沈殿物を細胞の培養培地に加えた。次いで形質転換した細胞を37℃、5%CO2でインキュベートした。培地は、形質転換から24時間後に、血清を含まない回収培地7mlと交換し、さらに24時間後にウイルス上清を回収し、2500rpmで5分間遠心分離により浄化した。収穫された浄化バルク(210mL)を、MgCl2(シグマアルドリッチ)の存在下でDNアーゼ(ロッシュ)にて30分間処理して、残りのトランスフェクションDNAを避け、22000rpm、4℃で1時間遠心分離して超濃縮した。各ベクターペレットを70μlのPBS−ラクトース(40mg/L)に再懸濁し、プールし、30μLを等分し、−70℃±10℃で保存する。
qPCR反応:HEK293T細胞を培養培地を含む6ウェルプレート(BDファルコン)に播種し、37℃、5%CO2、加湿で4時間インキュベートする。細胞は、レンチウイルスベクターの3連続希釈物で形質導入される。インキュベートの72時間後、細胞を回収し、形質導入されたHEK293T細胞ペレットを生成する。形質導入された細胞ペレットから全ゲノムDNAをQIAGEN QIAamp DNAミニキットハンドブックに基づく方法を用いて抽出する。プロウイルス定量は、Taqman qPCRを用いて行う。増幅は、Master Mix(Fermentas Thermo Scientific)、Flap A(CCCAAGAACCCAAGGAACA;配列番号26)およびFlap S(AGACAA GATAGAGGAAGAGCAAAAC;配列番号27)プライマーおよびLENTI TMプローブ(6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ;配列番号52)にて行った。アクチン遺伝子(同じMix、アクチンA-CGGTGAGGATCTTCATGAGGTAGT-(配列番号28)、アクチンS -AACACCCCAGCCATGTACGT-(配列番号29)プライマーおよびHUMURA?? ACT TMプローブ-6FAM-CCAGCCAGGTCCAGACGCAGGA-BBQ-(配列番号30)の定量により正規化を行った。両方の反応は、MasterCycler Ep Realplex S(エッペンドルフ、50℃で2分、95℃で10分、そして95℃で15秒および63℃で1分を40サイクル)で達成する。分析はMasterCycler Ep Realplexソフトウエアで行った。
CARをコードするレンチウイルスベクターを作製した。CAR_CD19第2および第3世代配列(配列番号39および配列番号40)は、GeneArt(Lifetech)から購入し、必要とするプロモーターに応じてpFlap−ΔU3−β2m−GFP、pFlap−ΔU3−HLA−A2−GFP、pFlap−ΔU3−HLA−DRα−GFPまたはpFlap−ΔU3−UBC−GFPのBamHIとXhoI制限部位との間のGFP遺伝子を置換してクローニングした。
末梢血単核細胞(PBMC)をフィコール(Ficoll)上の密度勾配遠心分離によって末梢血から精製した。PBMC洗浄後、Pan T細胞単離キット(ミルテニー)を用いて、陰性磁気選択(すなわち、不要な細胞を磁気的に標識し、T細胞をそのままにしたもの)でCD3+T細胞を精製した。この工程は、高度に精製されたT細胞集団を得るために必要とされる。この単離工程後に得られた収量に基づき、ヒトT細胞(TexMACS培地、ミルテニー)の培養および増殖のために開発された最適化無血清細胞培養培地中で107〜108T細胞を2.5×106/mlで培養した。これらのT細胞は、ミルテニーのT細胞活性化/増殖キットによって活性化される。このキットは、抗ビオチンMACSiBead粒子およびヒトCD2、CD3およびCD28に対するビオチン化抗体からなる。ビオチン化抗体を負荷した抗ビオチンMACSiBead粒子を用いて抗原提示細胞を模倣し、T細胞を活性化する。T細胞の最適な活性化は、2つの細胞あたり1つの負荷された抗ビオチンMACSiBead粒子を使用することによって達成される。T細胞を3日間活性化する。次いで活性化T細胞の形質導入を4のMOIで行う。このMOI値は1つのT細胞が4つのレンチウイルス粒子形質導入単位と共にインキュベートされることを意味する。CAR発現は、ビオチン化ヤギ抗マウスIgGでマウスCD19結合ドメインを染色し、続いてフィコエリトリンにコンジュゲートしたストレプトアビジンにて染色することにより、フローサイトメトリーの48または72時間のレンチウイルス粒子添加により評価した。T細胞亜集団は、CD3、CD4およびCD8の染色によって特徴付けた。これにより、T細胞の表面上のCARの特異的検出が可能になった。T細胞の生存および増殖を可能にするTexMACS培地を用いて全プロセスを実施した。
適格な血液は、フランスの血液機関(Etablissement Francais du Sang(ランジス、フランス))から入手した。末梢血単核細胞(PBMC)をフィコール(リンパ球分離培地、ユーロビオ)密度勾配分離によって精製した。次いで、T細胞を、Pan T細胞単離キット(ミルテニー)を用いた磁気単離によってPBMCから精製した。T細胞は製造者の指示に従って分離した。T細胞を、TexMACS培地(ミルテニー)中37℃/5%CO2で2.5×106細胞/mlの濃度で培養物に入れ、ミルテニーのT細胞活性化/増殖キット(製造業者の指示によって調製した抗CD2/CD3/CD28ナノ粒子)によりビーズ:T細胞比1:2で3日間活性化した。活性化後、T細胞を回収し、計数し、37℃/5%CO2で24ウェルプレート中のTexMACS培地中で培養した。形質導入は、異なるMOIのレンチウイルスベクターをウェルに直接添加することによって行った。試験した種々のレンチウイルスベクターは、(i)4−1BBおよびCD3ζ細胞内ドメインを含む第2世代の抗CD19 CAR;(ii)3つの異なる位置(CAR−SBP1、CAR−SBP2、CAR−SBP3)のストレプトアビジン結合タンパク質を含む同じベクターとした。
適格な血液は、フランスの血液機関(Etablissement Francais du Sang(ランジス、フランス))から入手した。末梢血単核細胞(PBMC)をフィコール(リンパ球分離培地、ユーロビオ)密度勾配分離によって精製した。次いで、T細胞を、Pan T細胞単離キット(ミルテニー)を用いた磁気単離によってPBMCから精製した。T細胞は製造者の指示に従って分離した。T細胞を、TexMACS培地(ミルテニー)中37℃/5%CO2で2.5×106細胞/mlの濃度で培養物に入れ、ミルテニーのT細胞活性化/増殖キット(製造業者の指示によって調製した抗CD2/CD3/CD28ナノ粒子)によりビーズ:T細胞比1:2で3日間活性化した。
適格な血液は、フランスの血液機関(Etablissement Francais du Sang(ランジス、フランス))から入手した。末梢血単核細胞(PBMC)をフィコール(リンパ球分離培地、ユーロビオ)密度勾配分離によって精製した。次いで、T細胞を、Pan T細胞単離キット(ミルテニー)を用いた磁気単離によってPBMCから精製した。T細胞は製造者の指示に従って分離した。T細胞を、TexMACS培地(ミルテニー)中37℃/5%CO2で2.5×106細胞/mlの濃度で培養物に入れ、ミルテニーのT細胞活性化/増殖キット(製造業者の指示によって調製した抗CD2/CD3/CD28ナノ粒子)によりビーズ:T細胞比1:2で3日間活性化した。
適格な血液は、フランスの血液機関(Etablissement Francais du Sang(ランジス、フランス))から入手した。末梢血単核細胞(PBMC)をフィコール(リンパ球分離培地、ユーロビオ)密度勾配分離によって精製した。次いで、T細胞を、Pan T細胞単離キット(ミルテニー)を用いた磁気単離によってPBMCから精製した。T細胞は製造者の指示に従って分離した。T細胞を、TexMACS培地(ミルテニー)中37℃/5%CO2で2.5×106細胞/mlの濃度で培養物に入れ、ミルテニーのT細胞活性化/増殖キット(製造業者の指示に従って調製した抗CD2/CD3/CD28ナノ粒子)によりビーズ:T細胞比1:2で3日間活性化した。
Claims (17)
- キメラ抗原レセプターであって、
結合ドメイン;
膜貫通ドメイン;
ストレプトアビジン結合ペプチドを含むフック結合ドメイン;および
配列番号3、配列番号4、配列番号5、配列番号7、配列番号8、配列番号33または配列番号34の少なくとも100アミノ酸のT細胞活性化断片、または配列番号6の少なくとも90アミノ酸のT細胞活性化断片を含む活性化ドメイン
を含み、
前記フック結合ドメインが、配列番号1のアミノ酸配列を含み、キメラ抗原レセプターの細胞質内領域に位置している、キメラ抗原レセプター。 - レンチウイルスベクターであって、
結合ドメイン;
膜貫通ドメイン;
ストレプトアビジン結合ペプチドを含むフック結合ドメイン;および
配列番号3、配列番号4、配列番号5、配列番号7、配列番号8、配列番号33または配列番号34の少なくとも100アミノ酸のT細胞活性化断片、または配列番号6の少なくとも90アミノ酸のT細胞活性化断片を含む活性化ドメイン
を含み、
前記フック結合ドメインが、配列番号1のアミノ酸配列を含み、キメラ抗原レセプターの細胞質内領域に位置している、キメラ抗原レセプターを発現する、レンチウイルスベクター。 - 核酸ベクターであって、
結合ドメイン;
膜貫通ドメイン;
ストレプトアビジン結合ペプチドを含むフック結合ドメイン;および
配列番号3、配列番号4、配列番号5、配列番号7、配列番号8、配列番号33または配列番号34の少なくとも100アミノ酸のT細胞活性化断片、または配列番号6の少なくとも90アミノ酸のT細胞活性化断片を含む活性化ドメイン
を含み、
前記フック結合ドメインが、配列番号1のアミノ酸配列を含み、キメラ抗原レセプターの細胞質内領域に位置している、キメラ抗原レセプターをコードする、核酸ベクター。 - 前記結合ドメインが、単鎖Fv抗体またはナノボディを含む、請求項1に記載のキメラ抗原レセプター、または請求項2に記載のレンチウイルスベクター、または請求項3に記載の核酸ベクター。
- 前記ベクターが、配列番号31または配列番号32のアミノ酸配列を含むフックをコードする、請求項2に記載のレンチウイルスベクター、または請求項3に記載の核酸ベクター。
- 前記ベクターが、β2−ミクログロブリン、ユビキチン、MHCIまたはMHCIIプロモーターを含む、請求項2から5のいずれか一項に記載のベクター。
- 前記ベクターがDNAである、請求項2から6のいずれか一項に記載のベクター。
- レンチウイルスベクター粒子であって、
結合ドメイン;
膜貫通ドメイン;
ストレプトアビジン結合ペプチドを含むフック結合ドメイン;および
配列番号3、配列番号4、配列番号5、配列番号7、配列番号8、配列番号33または配列番号34の少なくとも100アミノ酸のT細胞活性化断片、または配列番号6の少なくとも90アミノ酸のT細胞活性化断片を含む活性化ドメイン
を含み、
前記フック結合ドメインが、配列番号1のアミノ酸配列を含み、キメラ抗原レセプターの細胞質内領域に位置している、キメラ抗原レセプターをコードする、レンチウイルスベクター粒子。 - 前記結合ドメインが、単鎖Fv抗体またはナノボディを含む、請求項8に記載のレンチウイルスベクター粒子。
- 前記レンチウイルスベクター粒子が、配列番号31または配列番号32のアミノ酸配列を含むフックをコードする、請求項8に記載のレンチウイルスベクター粒子。
- 前記レンチウイルスベクター粒子が、水疱性口内炎ウイルス糖タンパク質を含む、請求項8から10のいずれか一項に記載のレンチウイルスベクター粒子。
- 前記レンチウイルスベクター粒子が、HIV−1サブタイプD GagおよびPolタンパク質を含む、請求項8から10のいずれか一項に記載のレンチウイルスベクター粒子。
- 請求項2から7のいずれか一項に記載のベクターを含む単離された細胞。
- ヒトにおいて免疫応答を誘導するための、請求項1に記載のキメラ抗原レセプターまたは請求項2に記載のレンチウイルスベクターまたは請求項3に記載の核酸ベクターまたは請求項8に記載のレンチウイルスベクター粒子。
- 配列番号45、配列番号47または配列番号49のいずれかのヌクレオチド配列を含む、請求項2に記載のレンチウイルスベクター、または請求項3に記載の核酸ベクター。
- 前記キメラ抗原レセプターが、配列番号46、配列番号48または配列番号50のいずれかのアミノ酸配列を含む、請求項1に記載のキメラ抗原レセプター。
- 配列番号42のアミノ酸配列をさらにコードするか、または配列番号43および/または配列番号44の核酸配列を含む、請求項15に記載のレンチウイルスベクターまたは核酸ベクター。
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HK1248720A1 (zh) | 2018-10-19 |
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US20170166623A1 (en) | 2017-06-15 |
JP7341110B2 (ja) | 2023-09-08 |
EP3511413B1 (en) | 2022-09-07 |
EP3172228B1 (en) | 2019-02-27 |
JP2021019590A (ja) | 2021-02-18 |
CN107531800A (zh) | 2018-01-02 |
CN113846062A (zh) | 2021-12-28 |
EP3511413A1 (en) | 2019-07-17 |
US20210009653A1 (en) | 2021-01-14 |
CN107531800B (zh) | 2021-08-10 |
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