CN106822184A - 多靶点联合的car‑t细胞制剂与制备方法 - Google Patents

多靶点联合的car‑t细胞制剂与制备方法 Download PDF

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CN106822184A
CN106822184A CN201611158116.3A CN201611158116A CN106822184A CN 106822184 A CN106822184 A CN 106822184A CN 201611158116 A CN201611158116 A CN 201611158116A CN 106822184 A CN106822184 A CN 106822184A
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tumour antigen
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梁爱斌
刘劼
孙毅
张文君
汪俊帮
李萍
唐晓晨
陈彩琴
朱益辉
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Abstract

本发明涉及CAR‑T细胞制剂与制备方法，由以下步骤构成：i.采用基因工程技术设计多个肿瘤抗原的组合，所述的肿瘤抗原的组合包括a.CD19；以及b.CD20、CD138、CD123中的一种或两种，ii.将肿瘤抗原的组合***到载体序列的4513和4514位置之间构成CAR，载体序列的碱基序列如SEQIDNO.1所示；iii.将CAR进行病毒包装；iv.将CAR通过病毒转染T细胞；v.通过培养基进行培养；vi.将制备的CAR‑T细胞与人血白蛋白、生理盐水混合，其中CAR‑T细胞浓度为0.5～1×106个/毫升，CAR阳性率在25％以上，活率在95％以上。本发明同现有技术相比，通过建立多种不同肿瘤抗原组合的CAR‑T细胞以及多靶点识别，能提高对肿瘤细胞的识别能力，确保T细胞对肿瘤细胞的杀伤效率。

Description

多靶点联合的CAR-T细胞制剂与制备方法
[技术领域]
本发明涉及CAR-T细胞制剂与制备方法。
[背景技术]
嵌合抗原受体(chimeric antigen receptor，CAR)T细胞治疗(CAR-T)技术是近年来迅速发展起来的一种肿瘤过继免疫治疗的新手段。CAR是人工构建的融合基因编码的跨膜分子，由胞外区、胞内区和跨膜区三部分构成。胞外区负责肿瘤抗原的识别，胞内区负责信号的转导，跨膜区连接胞外区和胞内区。通过将识别肿瘤相关抗原的抗体的单链可变区(single-chain variable fragment，scFv)和胞内信号域在体外进行基因重组，生成重组质粒，采用病毒包装后再转染至患者T细胞，使患者的T细胞表达肿瘤抗原受体，转染后经过纯化和大量扩增后的T细胞，即为CART细胞。CAR能够赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力。目标肿瘤抗原的选择对于CAR的特异性、有效性以及基因改造T细胞自身的安全性都是关键的决定因素。目前，尽管识别CD19靶点的CART可以成功治愈B淋巴细胞白血病和B细胞淋巴瘤，但仍然存在治疗失败的病例，根本原因是对于有的B淋巴细胞白血病和B细胞淋巴瘤患者，CD19作为靶点的特异性不强，使CAR-T细胞不能正确识别肿瘤细胞。因此，开发多种肿瘤抗原组合的CAR，根据患者外周血中的肿瘤抗原的表达情况选择CAR，制备的CAR-T细胞，通过多靶点识别，能提高对肿瘤细胞的识别能力，确保T细胞对肿瘤细胞的杀伤效率。
[发明内容]
本发明提高T细胞对肿瘤细胞的识别和杀伤效率。
为了实现上述目的，提供一种多靶点联合的CAR-T细胞制剂及其制备方法，由以下步骤构成：
i.采用基因工程技术设计多个肿瘤抗原的组合，所述的肿瘤抗原的组合包括a.CD19；以及b.CD20、CD138、CD123中的一种或两种，
ii.将肿瘤抗原的组合***到载体序列的4513和4514位置之间构成CAR，载体序列的碱基序列如SEQ ID NO.1所示；
iii.将CAR进行病毒包装；
iv.将CAR通过病毒转染T细胞；
v.通过培养基进行培养；
vi.将制备的CAR-T细胞与人血白蛋白、生理盐水混合，其中CAR-T细胞浓度为0.5～1×106个/毫升，CAR阳性率在25％以上，活率在95％以上。
包括两种不同的CD19，所述的CD19来自人源和/或鼠源。
包括两种不同的CD123序列。
所述的肿瘤抗原组合为CD19-CD20、CD19-CD138、CD19-CD123、CD19-CD20-CD138、CD19-CD20-CD123、CD19-CD138-CD123。
本发明同现有技术相比，通过建立多种不同肿瘤抗原组合的CAR-T细胞以及多靶点识别，能提高对肿瘤细胞的识别能力，确保T细胞对肿瘤细胞的杀伤效率。
[具体实施方式]
以下，结合实施例对于本发明做进一步说明，应当理解实施例仅用于解释说明而不用于限定发明的保护范围。
本实施例中的CAR-T细胞制剂的制备通过以下步骤实现：
a.采用基因工程技术设计多个肿瘤抗原的组合，这些抗原涉及CD19、CD20、CD138、CD123，抗原组合的方式是CD19加上另外一个抗原组合和CD19加上另外二个抗原组合，即CD19-CD20,CD19-CD138,CD19-CD123，CD19-CD20-CD138，CD19-CD20-CD123，CD19-CD138-CD123，这是根据患者外周血中的肿瘤抗原的表达而选择的，CD19、CD20、CD138、CD123的基因对于本领域的技术人员是清楚的，如
CD19可选择两个序列，一条为鼠源的(CD19-1，如SEQID NO.2所示)，一条为人源的(CD19-2，如SEQ ID NO.3所示)，因此，CD19的选择可以是上述两个中的任意一个；
CD20的碱基序列如SEQ ID NO.4所示。
CD123有两个序列，标记为CD123-1(如SEQ ID NO.5所示)，CD123-2(如SEQ IDNO.6所示)，CD123的选择可以是上述两个中的任意一个。
CD20的碱基序列如SEQ ID NO.7所示。
b.载体的序列为CARVec，如SEQ ID NO.1所示，将上面a.组合的序列***到载体序列的4513和4514位置之间；
c.将CAR进行病毒包装；
d.将CAR通过病毒转染T细胞；
e.通过培养基进行培养；
f.将制备的CAR-T细胞与人血白蛋白、生理盐水混合，其中CAR-T细胞浓度为0.5～1×106个/毫升，CAR阳性率在25％以上，活率在95％以上。
<110>梁爱斌，刘劼，孙毅，张文君，汪俊帮，李萍，唐晓晨，陈彩琴，朱益辉
<120>多靶点联合的CAR-T细胞制剂与制备方法
<160>7
<210>1
<211>7145
<212>DNA
<213>人工序列
<220>
<223>载体序列，在位置4513和4514之间***抗原序列
<400>1
caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60
attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120
aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180
tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 240
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300
gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 360
cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 420
agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 480
taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 540
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 600
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 660
acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 720
ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 780
cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 840
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 900
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 960
agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1020
tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1140
tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1200
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1260
tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1320
agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380
taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1440
caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1500
agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1560
aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1620
gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740
gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1800
ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1860
ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1920
aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980
aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2040
atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2100
tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2160
acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 2220
gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 2280
cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 2340
tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgga 2400
ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg 2460
ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc 2520
taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 2580
tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 2640
tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc 2700
tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 2760
tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 2820
gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag 2880
ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat 2940
tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc 3000
tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa 3060
ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga 3120
tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga 3180
cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta 3240
aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa 3300
aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact 3360
atgggcgcag cctcaatgac gctgacggta caggccagac aattattgtc tggtatagtg 3420
cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca 3480
gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat 3540
caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct 3600
tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg 3660
gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg 3720
caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg 3780
tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta 3840
ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg 3900
cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg 3960
cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg 4020
aacggatctc gacggtatcg gttaactttt aaaagaaaag gggggattgg ggggtacagt 4080
gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 4140
caaattacaa aaattcaaaa ttttatcgat cacgagacta gcctcgagaa gcttgatcga 4200
tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 4260
gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 4320
tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 4380
agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg 4440
acgcggatcc atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca 4500
tgcggcgcgt ccg 4513
accacca ccccggcgcc gcgtccgccg accccggcgc cgaccattgc 4560
gagccagccg ctgagcctgc gtccggaagc gtgccgtccg gcggcgggcg gcgcggtgca 4620
tacccgtggc ctggattttg cgtgcgatat ttatatttgg gcgccgctgg cgggcacctg 4680
cggcgtgctg ctgctgagcc tggtgattac cctgtattgc aaacgtggcc gtaaaaaact 4740
gctgtatatt tttaaacagc cgtttatgcg tccggtgcag accacccagg aagaagatgg 4800
ctgcagctgc cgttttccgg aagaagaaga aggcggctgc gaactgcgtg tgaaatttag 4860
ccgtagcgcg gatgcgccgg cgtatcagca gggccagaac cagctgtata acgaactgaa 4920
cctgggccgt cgtgaagaat atgatgtgct ggataaacgt cgtggccgtg atccggaaat 4980
gggcggcaaa ccgcgtcgta aaaacccgca ggaaggcctg tataacgaac tgcagaaaga 5040
taaaatggcg gaagcgtata gcgaaattgg catgaaaggc gaacgtcgtc gtggcaaagg 5100
ccatgatggc ctgtatcagg gcctgagcac cgcgaccaaa gatacctatg atgcgctgca 5160
tatgcaggcg ctgccgccgc gttaagtcga cctcgaggga attccgataa tcaacctctg 5220
gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 5280
tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 5340
ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 5400
aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 5460
gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 5520
gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 5580
aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc 5640
acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 5700
cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 5760
cagacgagtc ggatctccct ttgggccgcc tccccgcatc gggaattcga gctcggtacc 5820
tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 5880
gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg 5940
gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 6000
gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 6060
tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 6120
tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga 6180
gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 6240
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 6300
caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 6360
cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 6420
gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc 6480
gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 6540
tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 6600
cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 6660
tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg 6720
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 6780
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 6840
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 6900
attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 6960
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 7020
ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 7080
aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 7140
tttcc 7145
<210>2
<211>726
<212>DNA
<213>鼠源的CD19
<400>2
gatattcaga tgacccagac caccagcagc ctgagcgcga gcctgggcga tcgtgtgacc 60
attagctgcc gtgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120
gatggcaccg tgaaactgct gatttatcat accagccgtc tgcatagcgg cgtgccgagc 180
cgttttagcg gcagcggcag cggcaccgat tatagcctga ccattagcaa cctggaacag 240
gaagatattg cgacctattt ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300
ggcaccaaac tggaaattac cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360
ggcagcgaag tgaaactgca ggaaagcggc ccgggcctgg tggcgccgag ccagagcctg 420
agcgtgacct gcaccgtgag cggcgtgagc ctgccggatt atggcgtgag ctggattcgt 480
cagccgccgc gtaaaggcct ggaatggctg ggcgtgattt ggggcagcga aaccacctat 540
tataacagcg cgctgaaaag ccgtctgacc attattaaag ataacagcaa aagccaggtg 600
tttctgaaaa tgaacagcct gcagaccgat gataccgcga tttattattg cgcgaaacat 660
tattattatg gcggcagcta tgcgatggat tattggggcc agggcaccag cgtgaccgtg 720
agcagc 726
<210>3
<211>750
<212>DNA
<213>人源的CD19
<400>3
gaactggtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgtgcgacc 60
attagctgca aagcgagcca gagcgtggat tatgatggcg atagctatct gaactggtat 120
cagcagattc cgggccagcc gccgaaactg ctgatttatg atgcgagcaa cctggtgagc 180
ggcattccgc cgcgttttag cggcagcggc agcggcaccg attttaccct gaacattcat 240
ccggtggaaa aagtggatgc ggcgacctat cattgccagc agagcaccga agatccgtgg 300
acctttggcg gcggcaccaa actggaaatt aaacgtcgta gcggcggcgg cggcagcggc 360
ggcggcggca gcggcggcgg cggcagccag gtgcagctgc tggaaagcgg cgcggaactg 420
gtgcgtccgg gcagcagcgt gaaaattagc tgcaaagcga gcggctatgc gtttagcagc 480
tattggatga actgggtgaa acagcgtccg ggccagggcc tggaatggat tggccagatt 540
tggccgggcg atggcgatac caactataac ggcaaattta aaggcaaagc gaccctgacc 600
gcggatgaaa gcagcagcac cgcgtatatg cagctgagca gcctgcgtag cgaagatagc 660
gcggtgtata gctgcgcgcg tcgtgaaacc accaccgtgg gccgttatta ttatgcgatg 720
gattattggg gccagggcac caccgtgacc 750
<210>4
<211>666
<212>DNA
<213> CD20
<400>4
gatattcagc tgacccagag cccggcgatt ctgagcgcga gcccgggcga aaaagtgacc 60
atgacctgcc gtgcgagcag cagcctgagc tttatgcatt ggtatcagca gaaaccgggc 120
agcagcccga aaccgtggat ttatgcgacc agcaacctgg cgagcggcgt gccggcgcgt 180
tttagcggca gcggcagcgg caccagctat agcctgacca ttagcaccgt ggaagcggaa 240
gatgcggcga gctatttttg ccatcagtgg agcagcaacc cgctgacctt tggcgcgggc 300
accaaactgg aaattagcag cggcggcggc ggcagcggcg gcggcggcag cggcgatgtg 360
atgggcgtgg atagcggcgg cggcctggtg cagccgggcg gcagccgtaa actgagctgc 420
gcggcgccgg gctttacctt tagcagcttt ggcatgcatt gggtgcgtca ggcgccggaa 480
aaaggcctgg aatgggtggc gtatattagc agcccgagca gcaccctgca ttatgcggat 540
cgtgtgaaag gccgttttac cattagccgt gataacccga aaaacaccct gtttctgcag 600
atgaaactgc cgagcctgtg ctatggcctg ctgggcccgc gtgatcatgt gcatcgtctg 660
ctgaaa 666
<210>5
<211>732
<212>DNA
<213> CD123-1
<400>5
cagattcagc tggtgcagag cggcccggaa ctgaaaaaac cgggcgaaac cgtgaaaatt 60
agctgcaaag cgagcggcta tatttttacc aactatggca tgaactgggt gaaacaggcg 120
ccgggcaaaa gctttaaatg gatgggctgg attaacacct ataccggcga aagcacctat 180
agcgcggatt ttaaaggccg ttttgcgttt agcctggaaa ccagcgcgag caccgcgtat 240
ctgcatatta acgatctgaa aaacgaagat accgcgacct atttttgcgc gcgtagcggc 300
ggctatgatc cgatggatta ttggggccag ggcaccagcg tgaccgtgag cagcggcggc 360
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg atattgtgct gacccagagc 420
ccggcgagcc tggcggtgag cctgggccag cgtgcgacca ttagctgccg tgcgagcgaa 480
agcgtggata actatggcaa cacctttatg cattggtatc agcagaaacc gggccagccg 540
ccgaaactgc tgatttatcg tgcgagcaac ctggaaagcg gcattccggc gcgttttagc 600
ggcagcggca gccgtaccga ttttaccctg accattaacc cggtggaagc ggatgatgtg 660
gcgacctatt attgccagca gagcaacgaa gatccgccga cctttggcgc gggcaccaaa 720
ctggaactga aa 732
<210>6
<211>711
<212>DNA
<213> CD123-2
<400>6
caggtgcagc tgcagcagcc gggcgcggaa ctggtgcgtc cgggcgcgag cgtgaaactg 60
agctgcaaag cgagcggcta tacctttacc agctattgga tgaactgggt gaaacagcgt 120
ccggatcagg gcctggaatg gattggccgt attgatccgt atgatagcga aacccattat 180
aaccagaaat ttaaagataa agcgattctg accgtggata aaagcagcag caccgcgtat 240
atgcagctga gcagcctgac cagcgaagat agcgcggtgt attattgcgc gcgtggcaac 300
tgggatgatt attggggcca gggcaccacc ctgaccgtga gcagcggcgg cggcggcagc 360
ggcggcggcg gcagcggcgg cggcggcagc gatgtgcaga ttacccagag cccgagctat 420
ctggcggcga gcccgggcga aaccattacc attaactgcc gtgcgagcaa aagcattagc 480
aaagatctgg cgtggtatca ggaaaaaccg ggcaaaacca acaaactgct gatttatagc 540
ggcagcaccc tgcagagcgg cattccgagc cgttttagcg gcagcggcag cggcaccgat 600
tttaccctga ccattagcag cctggaaccg gaagattttg cgatgtatta ttgccagcag 660
cataacaaat atccgtatac ctttggcggc ggcaccaaac tggaaattaa a 711
<210>7
<211>2031
<212>DNA
<213> CD138
<400>7
gatattcaga tgacccagag caccagcagc ctgagcgcga gcctgggcga tcgtgtgacc 60
attagctgca gcgcgagcca gggcattaac aactatctga actggtatca gcagaaaccg 120
gatggcaccg tggaactgct gatttattat accagcaccc tgcagagcgg cgtgccgagc 180
cgttttagcg gcagcggcag cggcaccgat tatagcctga ccattagcaa cctggaaccg 240
gaagatattg gcacctatta ttgccagcag tatagcaaac tgccgcgtac ctttggcggc 300
ggcaccaaac tggaaattct gcgtaccgtg gcggcgccga gcgtgtttat ttttccgccg 360
agcgatgaac agctgaaaag cggcaccgcg agcgtggtgt gcctgctgaa caacttttat 420
ccgcgtgaag cgaaagtgca gtggaaagtg gataacgcgc tgcagagcgg caacagccag 480
gaaagcgtga ccgaacagga tagcaaagat agcacctata gcctgagcag caccctgacc 540
ctgagcaaag cggattatga aaaacataaa gtgtatgcgt gcgaagtgac ccatcagggc 600
ctgagcagcc cggtgaccaa aagctttaac cgtggcgaat gcggcggcgg cggcagcggc 660
ggcggcggca gcggcggcgg cggcagccag gtgcagctgc agcagagcgg cagcgaactg 720
atgatgccgg gcgcgagcgt gaaaattagc tgcaaagcga ccggctatac ctttagcaac 780
tattggattg aatgggtgaa acagcgtccg ggccatggcc tggaatggat tggcgaaatt 840
ctgccgggca ccggccgtac catttataac gaaaaattta aaggcaaagc gacctttacc 900
gcggatatta gcagcaacac cgtgcagatg cagctgagca gcctgaccag cgaagatagc 960
gcggtgtatt attgcgcgcg tcgtgattat tatggcaact tttattatgc gatggattat 1020
tggggccagg gcaccagcgt gaccgtgagc agcgcgagca ccaaaggccc gagcgtgttt 1080
ccgctggcgc cgtgcagccg tagcaccagc gaaagcaccg cggcgctggg ctgcctggtg 1140
aaagattatt ttccggaacc ggtgaccgtg agctggaaca gcggcgcgct gaccagcggc 1200
gtgcatacct ttccggcggt gctgcagagc agcggcctgt atagcctgag cagcgtggtg 1260
accgtgccga gcagcagcct gggcaccaaa acctatacct gcaacgtgga tcataaaccg 1320
agcaacacca aagtggataa acgtgtggaa agcaaatatg gcccgccgtg cccgagctgc 1380
ccggcgccgg aatttctggg cggcccgagc gtgtttctgt ttccgccgaa accgaaagat 1440
accctgatga ttagccgtac cccggaagtg acctgcgtgg tggtggatgt gagccaggaa 1500
gatccggaag tgcagtttaa ctggtatgtg gatggcgtgg aagtgcataa cgcgaaaacc 1560
aaaccgcgtg aagaacagtt taacagcacc tatcgtgtgg tgagcgtgct gaccgtgctg 1620
catcaggatt ggctgaacgg caaagaatat aaatgcaaag tgagcaacaa aggcctgccg 1680
agcagcattg aaaaaaccat tagcaaagcg aaaggccagc cgcgtgaacc gcaggtgtat 1740
accctgccgc cgagccagga agaaatgacc aaaaaccagg tgagcctgac ctgcctggtg 1800
aaaggctttt atccgagcga tattgcggtg gaatgggaaa gcaacggcca gccggaaaac 1860
aactataaaa ccaccccgcc ggtgctggat agcgatggca gcttttttct gtatagccgt 1920
ctgaccgtgg ataaaagccg ttggcaggaa ggcaacgtgt ttagctgcag cgtgatgcat 1980
gaagcgctgc ataaccatta tacccagaaa agcctgagcc tgagcctggg caaa 2034

Claims (10)

1.一种多靶点联合的CAR-T细胞制剂的制备方法，其特征在于由以下步骤构成：
i.采用基因工程技术设计多个肿瘤抗原的组合，所述的肿瘤抗原的组合包括a.CD19；以及b.CD20、CD138、CD123中的一种或两种，
ii.将肿瘤抗原的组合***到载体序列的4513和4514位置之间构成CAR，载体序列的碱基序列如SEQ ID NO.1所示；
iii.将CAR进行病毒包装；
iv.将CAR通过病毒转染T细胞；
v.通过培养基进行培养；
vi.将制备的CAR-T细胞与人血白蛋白、生理盐水混合，其中CAR-T细胞浓度为0.5～1×106个/毫升，CAR阳性率在25％以上，活率在95％以上。
2.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于包括两种不同的CD19，所述的CD19来自人源和/或鼠源。
3.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于包括两种不同的CD123序列。
4.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD20。
5.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD138。
6.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD123。
7.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD20-CD138。
8.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD20-CD123。
9.如权利要求1所述的CAR-T细胞制剂的制备方法，其特征在于所述的肿瘤抗原组合为CD19-CD138-CD123。
10.一种多靶点联合的CAR-T细胞制剂，由权利要求1～9任一所述的制备方法制备得到。
CN201611158116.3A 2016-12-15 2016-12-15 多靶点联合的car‑t细胞制剂与制备方法 Pending CN106822184A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220247A (zh) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 一种双car-t细胞及其制备方法和应用
WO2020108643A1 (en) * 2018-11-30 2020-06-04 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd19-and cd70-based combined car-t immunotherapy

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Publication number Priority date Publication date Assignee Title
CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
CN106086077A (zh) * 2016-07-05 2016-11-09 北京普瑞金科技有限公司 用于car‑t制备的慢病毒载体及其构建方法和应用
CN106086078A (zh) * 2016-07-08 2016-11-09 宜明细胞生物科技有限公司 一种car‑t细胞毒性指示载体

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CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
CN106086077A (zh) * 2016-07-05 2016-11-09 北京普瑞金科技有限公司 用于car‑t制备的慢病毒载体及其构建方法和应用
CN106086078A (zh) * 2016-07-08 2016-11-09 宜明细胞生物科技有限公司 一种car‑t细胞毒性指示载体

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220247A (zh) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 一种双car-t细胞及其制备方法和应用
WO2020108643A1 (en) * 2018-11-30 2020-06-04 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd19-and cd70-based combined car-t immunotherapy

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