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

Abstract

The present invention relates to a CAR-T cell preparation and a preparation method, which consists of the following steps: i. Using genetic engineering technology to design a combination of multiple tumor antigens, the combination of tumor antigens includes a. CD19; and b. CD20, CD138, One or both of CD123, ii. Insert the combination of tumor antigens between positions 4513 and 4514 of the carrier sequence to form a CAR, the base sequence of the carrier sequence is shown in SEQ ID NO.1; iii. Carry out viral packaging of the CAR ; iv. Transfect CAR T cells with virus; v. Culture through culture medium; vi. Mix prepared CAR T cells with human serum albumin and normal saline, wherein the concentration of CAR T cells is 0.5～1× 10 ⁶ cells/mL, the CAR positive rate was above 25%, and the activity rate was above 95%. Compared with the prior art, the present invention can improve the recognition ability of tumor cells and ensure the killing efficiency of T cells to tumor cells by establishing CAR-T cells with different combinations of tumor antigens and multi-target recognition.

Description

Multi-target combined CAR-T cell preparation and preparation method

[technical field]

The invention relates to a CAR-T cell preparation and a preparation method.

[Background technique]

Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell therapy (CAR-T) technology is a new means of tumor adoptive immunotherapy developed rapidly in recent years. CAR is an artificially constructed transmembrane molecule encoded by a fusion gene, which consists of three parts: extracellular region, intracellular region and transmembrane region. The extracellular region is responsible for the recognition of tumor antigens, the intracellular region is responsible for signal transduction, and the transmembrane region connects the extracellular region and the intracellular region. The single-chain variable fragment (scFv) and the intracellular signal domain of the antibody that recognizes the tumor-associated antigen are genetically recombined in vitro to generate a recombinant plasmid, which is packaged with the virus and then transfected into the patient's T cells. The patient's T cells express tumor antigen receptors, and the purified and massively expanded T cells after transfection are CART cells. CAR can endow T cells with the ability to recognize tumor antigens in an HLA-independent manner. The choice of target tumor antigen is a key determinant for the specificity and effectiveness of CAR, as well as the safety of genetically modified T cells themselves. At present, although CART that recognizes the target of CD19 can successfully cure B-lymphocytic leukemia and B-cell lymphoma, there are still cases of treatment failure. The specificity of the spot is not strong, so that CAR-T cells cannot correctly recognize tumor cells. Therefore, develop a combination of CARs with multiple tumor antigens, select CARs according to the expression of tumor antigens in the peripheral blood of patients, and prepare CAR-T cells. Through multi-target recognition, it can improve the ability to recognize tumor cells and ensure that T cells Killing efficiency of tumor cells.

[Content of the invention]

The invention improves the recognition and killing efficiency of T cells to tumor cells.

In order to achieve the above objectives, a multi-target combined CAR-T cell preparation and a preparation method thereof are provided, which consist of the following steps:

i. Using genetic engineering technology to design a combination of multiple tumor antigens, the combination of tumor antigens includes a. CD19; and b. one or both of CD20, CD138, and CD123,

ii. A combination of tumor antigens is inserted between positions 4513 and 4514 of the carrier sequence to form a CAR, and the base sequence of the carrier sequence is shown in SEQ ID NO.1;

iii. Carrying out viral packaging of CAR;

iv. Transfect T cells with CAR through virus;

v. Cultivate by culture medium;

vi. Mix the prepared CAR-T cells with human serum albumin and normal saline, in which the concentration of CAR-T cells is 0.5-1× ¹⁰⁶ cells/ml, the positive rate of CAR is above 25%, and the activity rate is above 95%. .

Two different CD19s are included, said CD19 is from human and/or murine origin.

Two different CD123 sequences are included.

The tumor antigen combinations are CD19-CD20, CD19-CD138, CD19-CD123, CD19-CD20-CD138, CD19-CD20-CD123, CD19-CD138-CD123.

Compared with the prior art, the present invention can improve the recognition ability of tumor cells and ensure the killing efficiency of T cells to tumor cells by establishing CAR-T cells with different combinations of tumor antigens and multi-target recognition.

[detailed description]

Hereinafter, the present invention will be further described in conjunction with the examples. It should be understood that the examples are only for illustration and not for limiting the protection scope of the invention.

The preparation of the CAR-T cell preparation in this embodiment is achieved through the following steps:

a. Use genetic engineering technology to design a combination of multiple tumor antigens. These antigens involve CD19, CD20, CD138, and CD123. The antigen combination is CD19 plus another antigen combination and CD19 plus two other antigen combinations, namely CD19- CD20, CD19-CD138, CD19-CD123, CD19-CD20-CD138, CD19-CD20-CD123, CD19-CD138-CD123, which are selected according to the expression of tumor antigens in the peripheral blood of patients, CD19, CD20, CD138, The gene of CD123 is clear to those skilled in the art, such as

Two sequences can be selected for CD19, one is mouse-derived (CD19-1, as shown in SEQ ID NO.2), and the other is human-derived (CD19-2, as shown in SEQ ID NO.3), therefore, the choice of CD19 Can be either of the above two;

The base sequence of CD20 is shown in SEQ ID NO.4.

CD123 has two sequences, marked as CD123-1 (as shown in SEQ ID NO.5) and CD123-2 (as shown in SEQ ID NO.6). The choice of CD123 can be any one of the above two.

The base sequence of CD20 is shown in SEQ ID NO.7.

b. The sequence of the vector is CARVec, as shown in SEQ ID NO.1, insert the sequence combined in the above a. between positions 4513 and 4514 of the vector sequence;

c. Carry out viral packaging of CAR;

d. Transfect T cells with CAR through virus;

e. Cultivate by culture medium;

f. Mix the prepared CAR-T cells with human serum albumin and normal saline, in which the concentration of CAR-T cells is 0.5-1× ¹⁰⁶ cells/ml, the positive rate of CAR is above 25%, and the activity rate is above 95%. .

<110> Liang Aibin, Liu Jie, Sun Yi, Zhang Wenjun, Wang Junbang, Li Ping, Tang Xiaochen, Chen Caiqin, Zhu Yihui

<120> Multi-target combined CAR-T cell preparation and preparation method

<160>7

<210>1

<211>7145

<212>DNA

<213> Artificial sequence

<220>

<223> Vector sequence, insertion of antigen sequence between positions 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 ccaccctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggcctt 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 accactgct 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 aagcaggggag 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 taggttagg 3900

cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg 3960

cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattatagtg 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 tttattatg 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 cgattagtg ctttacggca cctcgacccc aaaaaacttg 6900

attagggtga tggttcacgt agtggggccat cgccctgata gacggttttt cgccctttga 6960

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 7020

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 7080

aaaatgagct gattttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 7140

tttcc 7145

<210>2

<211>726

<212>DNA

<213> murine 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 ccttagcaa 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 atttattaaag ataacagcaa aagccaggtg 600

tttctgaaaa tgaacagcct gcagaccgat gataccgcga tttaattattg cgcgaaacat 660

tattattatg gcggcagcta tgcgatggat tattggggcc agggcaccag cgtgaccgtg 720

agcagc 726

<210>3

<211>750

<212>DNA

<213> Human CD19

<400>3

gaactggtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgtgcgacc 60

attagctgca aagcgagcca gagcgtggat tatgatggcg atagctatct gaactggtat 120

cagcagattc cgggccagcc gccgaaactg ctgattatg 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 tgattatcg tgcgagcaac ctggaaagcg gcattccggc gcgttttagc 600

ggcagcggca gccgtaccga ttttaccctg accattaacc cggtggaagc ggatgatgtg 660

gcgacctatt attgccagca gagcaacgaa gatccgccga cctttggcgc gggcaccaaa 720

ctgga actga 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 ttaccccagag cccgagctat 420

ctggcggcga gcccgggcga aaccattacc attaactgcc gtgcgagcaa aagcattagc 480

aaagatctgg cgtggtatca ggaaaaaccg ggcaaaacca acaaactgct gatttatagc 540

ggcagcaccc tgcagagcgg cattccgagc cgttttagcg gcagcggcag cggcaccgat 600

tttaccctga ccttagcag 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 ccttagcaa 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. A method for preparing a multi-target combined CAR-T cell preparation, characterized in that it consists of the following steps: i. Using genetic engineering technology to design a combination of multiple tumor antigens, the combination of tumor antigens includes a. CD19; and b. one or both of CD20, CD138, and CD123, ii. A combination of tumor antigens is inserted between positions 4513 and 4514 of the carrier sequence to form a CAR, and the base sequence of the carrier sequence is shown in SEQ ID NO.1; iii. Carrying out viral packaging of CAR; iv. Transfect T cells with CAR through virus; v. Cultivate by culture medium; vi. Mix the prepared CAR-T cells with human serum albumin and normal saline, in which the concentration of CAR-T cells is 0.5-1× ¹⁰⁶ cells/ml, the positive rate of CAR is above 25%, and the activity rate is above 95%. . 2. The preparation method of the CAR-T cell preparation according to claim 1, characterized in that it comprises two different CD19s, and the CD19s are from human source and/or mouse source. 3. The preparation method of the CAR-T cell preparation according to claim 1, characterized in that it comprises two different CD123 sequences. 4. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of the tumor antigens is CD19-CD20. 5. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of the tumor antigens is CD19-CD138. 6. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of the tumor antigens is CD19-CD123. 7. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of the tumor antigens is CD19-CD20-CD138. 8. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of the tumor antigens is CD19-CD20-CD123. 9. The preparation method of CAR-T cell preparation according to claim 1, characterized in that the combination of tumor antigens is CD19-CD138-CD123. 10. A multi-target combined CAR-T cell preparation prepared by the preparation method according to any one of claims 1-9.