CN106822184A - The united CAR T cells preparation of Mutiple Targets and preparation method - Google Patents

The united CAR T cells preparation of Mutiple Targets and preparation method Download PDF

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Publication number
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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China
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car
preparation
cell
cell preparations
tumour antigen
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Inventor
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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Individual
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

The present invention relates to CAR T cells preparation and preparation method, it is made up of following steps：I. the combination of multiple tumour antigens is designed using technique for gene engineering, the combination of described tumour antigen includes a.CD19；And one or two in b.CD20, CD138, CD123, be inserted between 4513 and 4514 positions of carrier sequence for the combination of tumour antigen and constitute CAR by ii., and the base sequence of carrier sequence is as shown in SEQIDNO.1；Iii. CAR is carried out into viral packaging；Iv. CAR is passed through into virus transfection T cell；V. cultivated by culture medium；Vi. the CAR T cells for preparing are mixed with human serum albumin, physiological saline, wherein CAR T cells concentration is 0.5~1 × 106Individual/milliliter, more than 25%, motility rate is more than 95% for CAR positive rates.The present invention compared with the existing technology, by setting up CAR T cells and the Mutiple Targets identification that various different tumour antigens are combined, can improve the recognition capability to tumour cell, it is ensured that killing-efficiency of the T cell to tumour cell.

Description

The united CAR-T cell preparations of Mutiple Targets and preparation method
[technical field]
The present invention relates to CAR-T cell preparations and preparation method.
[background technology]
Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell treatment (CAR-T) technology is in recent years Come the new tool of a kind of adoptive immunotherapy for developing rapidly.CAR be artificial constructed fusion coding across Membrane molecule, is made up of extracellular region, intracellular region and the part of transmembrane region three.Extracellular region is responsible for the identification of tumour antigen, and intracellular region is responsible for The transduction of signal, transmembrane region connection extracellular region and intracellular region.By the single-stranded variable region of the antibody by tumor associated antigen is recognized (single-chain variable fragment, scFv) and intracellular signal domain carry out genetic recombination, generation restructuring in vitro Plasmid, using virus packaging after transfect again to patient T cells, make patient T cell express tumour antigen acceptor, after transfection by T cell after purifying and a large amount of amplifications, as CART cells.The mode that CAR can assign T cell HLA non-dependent recognizes tumour The ability of antigen.Specificity of the selection of target tumor antigen for CAR, validity and the genetic modification T cell peace of itself Full property is all crucial determinant.At present, although the CART of identification CD19 target spots can successfully cure bone-marrow-derived lymphocyte leukaemia And B cell lymphoma, but still there is the case of Endodontic failure, basic reason is thin for the bone-marrow-derived lymphocyte leukaemia and B that have Born of the same parents' Lymphoma, CD19 is not strong as the specificity of target spot, prevents CAR-T cells from correct tumor cell.Therefore, open The CAR of hair kinds of tumors antigen combination, the expression selection CAR of the tumour antigen in peripheral blood in patients, preparation CAR-T cells, are recognized by Mutiple Targets, can improve the recognition capability to tumour cell, it is ensured that killing of the T cell to tumour cell Efficiency.
[content of the invention]
The present invention improves identification and killing-efficiency of the T cell to tumour cell.
To achieve these goals, there is provided a kind of united CAR-T cell preparations of Mutiple Targets and preparation method thereof, by following Step is constituted：
I. the combination of multiple tumour antigens is designed using technique for gene engineering, the combination of described tumour antigen includes a.CD19；And one or two in b.CD20, CD138, CD123,
Ii. the combination of tumour antigen is inserted between 4513 and 4514 positions of carrier sequence and constitutes CAR, carrier sequence Base sequence as shown in SEQ ID NO.1；
Iii. CAR is carried out into viral packaging；
Iv. CAR is passed through into virus transfection T cell；
V. cultivated by culture medium；
Vi. the CAR-T cells for preparing are mixed with human serum albumin, physiological saline, wherein CAR-T cell concentrations are 0.5 ~1 × 106Individual/milliliter, more than 25%, motility rate is more than 95% for CAR positive rates.
Including two kinds of different CD19, described CD19 comes from people source and/or mouse source.
Including two kinds of different CD123 sequences.
Described tumour antigen be combined as CD19-CD20, CD19-CD138, CD19-CD123, CD19-CD20-CD138, CD19-CD20-CD123、CD19-CD138-CD123。
The present invention compared with the existing technology, by setting up CAR-T cells and many targets that various different tumour antigens are combined Point identification, can improve the recognition capability to tumour cell, it is ensured that killing-efficiency of the T cell to tumour cell.
[specific embodiment]
Hereinafter, be described further for the present invention in conjunction with the embodiments, it should be understood that embodiment be only used for illustrate and It is not used in the protection domain for limiting invention.
The preparation of the CAR-T cell preparations in the present embodiment is realized by following steps：
A. design the combination of multiple tumour antigens using technique for gene engineering, these antigens be related to CD19, CD20, CD138, CD123, the mode of antigen combination is that CD19 adds other two antigen combinations plus another antigen combination and CD19, i.e., CD19-CD20, CD19-CD138, CD19-CD123, CD19-CD20-CD138, CD19-CD20-CD123, CD19-CD138- CD123, this is the expression of the tumour antigen in peripheral blood in patients and selects, the base of CD19, CD20, CD138, CD123 Because will readily suggest themselves to those skilled in the art, such as
CD19 may be selected two sequences, and one is (CD19-1, as shown in SEQID NO.2) in mouse source, and one is people source (CD19-2, as shown in SEQ ID NO.3), therefore, the selection of CD19 can be any one in above-mentioned two；
The base sequence of CD20 is as shown in SEQ ID NO.4.
CD123 has two sequences, labeled as CD123-1 (as shown in SEQ ID NO.5), CD123-2 (such as SEQ ID Shown in NO.6), the selection of CD123 can be any one in above-mentioned two.
The base sequence of CD20 is as shown in SEQ ID NO.7.
B. the sequence of carrier is CARVec, as shown in SEQ ID NO.1, the sequence that a. above is combined is inserted into carrier sequence Between 4513 and 4514 positions of row；
C. CAR is carried out into viral packaging；
D. CAR is passed through into virus transfection T cell；
E. cultivated by culture medium；
F. the CAR-T cells for preparing are mixed with human serum albumin, physiological saline, wherein CAR-T cell concentrations for 0.5~ 1×106Individual/milliliter, more than 25%, motility rate is more than 95% for CAR positive rates.
<110>Liang Aibin, Liu's Jie, Sun Yi, Zhang Wenjun, Wang Junbang, Li Ping, Tang Xiaochen, Chen Caiqin, Zhu Yihui
<120>The united CAR-T cell preparations of Mutiple Targets and preparation method
<160>7
<210>1
<211>7145
<212>DNA
<213>Artificial sequence
<220>
<223>Carrier sequence, antigen sequence is inserted between position 4513 and 4514
<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>The CD19 in mouse source
<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>The CD19 in people source
<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. the preparation method of the united CAR-T cell preparations of a kind of Mutiple Targets, it is characterised in that be made up of following steps：
I. the combination of multiple tumour antigens is designed using technique for gene engineering, the combination of described tumour antigen includes a.CD19； And one or two in b.CD20, CD138, CD123,
Ii. the combination of tumour antigen is inserted between 4513 and 4514 positions of carrier sequence and constitutes CAR, the alkali of carrier sequence Basic sequence is as shown in SEQ ID NO.1；
Iii. CAR is carried out into viral packaging；
Iv. CAR is passed through into virus transfection T cell；
V. cultivated by culture medium；
Vi. the CAR-T cells for preparing are mixed with human serum albumin, physiological saline, wherein CAR-T cell concentrations for 0.5~1 × 106Individual/milliliter, more than 25%, motility rate is more than 95% for CAR positive rates.
2. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that including two kinds of different CD19, Described CD19 comes from people source and/or mouse source.
3. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that including two kinds of different CD123 Sequence.
4. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD20。
5. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD138。
6. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD123。
7. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD20-CD138。
8. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD20-CD123。
9. the preparation method of CAR-T cell preparations as claimed in claim 1, it is characterised in that described tumour antigen is combined as CD19-CD138-CD123。
10. a kind of united CAR-T cell preparations of Mutiple Targets, are prepared into by any described preparation method of claim 1~9 Arrive.
CN201611158116.3A 2016-12-15 2016-12-15 The united CAR T cells preparation of Mutiple Targets and preparation method Pending CN106822184A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220247A (en) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 A kind of double CAR-T cells and its preparation method and application
WO2020108643A1 (en) * 2018-11-30 2020-06-04 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd19-and cd70-based combined car-t immunotherapy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105384825A (en) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof
CN106086077A (en) * 2016-07-05 2016-11-09 北京普瑞金科技有限公司 The slow virus carrier prepared for CAR T and construction method thereof and application
CN106086078A (en) * 2016-07-08 2016-11-09 宜明细胞生物科技有限公司 A kind of CAR T cell toxicity instruction carrier

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105384825A (en) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 Bispecific chimeric antigen receptor based on variable domains of heavy chain of heavy-chain antibody and application thereof
CN106086077A (en) * 2016-07-05 2016-11-09 北京普瑞金科技有限公司 The slow virus carrier prepared for CAR T and construction method thereof and application
CN106086078A (en) * 2016-07-08 2016-11-09 宜明细胞生物科技有限公司 A kind of CAR T cell toxicity instruction carrier

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220247A (en) * 2018-03-20 2018-06-29 杭州史迪姆生物科技有限公司 A kind of double CAR-T cells and its preparation method and application
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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