CN109971715A - A kind of cultural method of specific amplification CAR-T cell - Google Patents
A kind of cultural method of specific amplification CAR-T cell Download PDFInfo
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- CN109971715A CN109971715A CN201711458709.6A CN201711458709A CN109971715A CN 109971715 A CN109971715 A CN 109971715A CN 201711458709 A CN201711458709 A CN 201711458709A CN 109971715 A CN109971715 A CN 109971715A
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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
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- 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
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- 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 invention discloses a kind of cultural methods of specific amplification CAR-T cell.The cultural method of specific amplification CAR-T cell disclosed by the invention, comprising: 1) add the antigen that immunocyte is identified into the cultivating system of immunocyte, obtain co-culture system；2) co-culture system is cultivated, realizes the amplification of immunocyte；Immunocyte is the T cell for expressing Chimeric antigen receptor；The proportion of immunocyte and antigen is 10 in co-culture system6It is a: 1~100ng.It is demonstrated experimentally that the positive rate of the CAR-T cell obtained using the cultural method of specific amplification CAR-T cell of the invention can be improved 4-6 times, and operation is easy and efficient, high specificity for this method, practical.
Description
Technical field
The present invention relates in field of biomedicine, a kind of cultural method of specific amplification CAR-T cell.
Background technique
Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is based on the manually modified of T cell receptor
Fusion protein identifies that structural domain and Cellular Signaling Transduction Mediated structural domain form by extracellular antigen.Chimeric antigen receptor T cell
(CAR-T cell) is the portion intracellular that will identify the antigen-binding portion and CD3- ζ chain or Fc ε RI γ of the antibody of certain tumour antigen
Dividing coupling in vitro is a chimeric protein, and the T cell of patient is transfected by the method for gene transfer, its is made to express chimeric antigen
Receptor (CAR).Chimeric antigen receptor (CAR) by the extracellular antigen binding domain (scFv) of T cell receptor, hinge area, centre across
Film area and 4, intracellular signal area part form.It is identified currently, existing kinds of tumors antigen can be used for extracellular region, including CD19,
CD20, EGF-R ELISA (EGFR) and ErbB-2 (HER-2) etc..Hinge area have CD8Hinge,
CD28Hinge and longer IgG1Fc or IgG4Fc etc..Intermediate transmembrane region has CD4, CD8 and CD28 etc., by CAR Structure anchor
In on T cell film.Intracellular domain generally comprises CD3 ζ, CD28,4-1BB or OX40, for increasing T cell activation.Heredity
Modification is to express the antigen that the T cell of CAR can be targeted with Direct Recognition CAR, and then the tumour of CAR specific antigen is expressed in triggering
The T cell activation of cell, proliferation, cytokine secretion and cytotoxicity.Currently, the T cell of CAR modification is in treatment blood cancer
It is achieved in terms of disease, including lymthoma, chronic lymphocytic leukemia and acute lymphatic leukemia (ALL) significant
Success.It is reported that CD19 targets the complete remission rate that CAR-T cell has 70% to 90% in all patients.As it can be seen that CAR-
T cell having a extensive future in immunotherapy of tumors, market value is huge.
It is main to be carried out by special molecular of the Ag-Ab recognition mode to tumor cell surface in CAR-T cell therapy
Identification, is then activated, is proliferated and played cell killing function by its signal transduction intracellular.Therefore, this CAR is special
Property kill tumour cell mechanism, need in CAR-T preparation process, it is obtained after CAR gene transfer patient T cells
CAR-T specific cell is The more the better, i.e., in total T cell, the ratio (positive rate) of the CAR-T cell for CAR gene of having transduceed
The higher the better, and to reach better tumour cell recognition reaction, activation target cell kills mechanism.
Currently, the method that CAR gene transfer enters T cell is mainly had: electroporation, change in CAR-T cell preparation process
Learn mediated method (such as calcium phosphate precipitation, lipofection), virus-mediated methods.Wherein electroporation is to utilize high impulse
Voltage damages cell membrane potential makes nucleic acid import cell by the aperture formed on film.This method applicability is wide, transducible DNA
Or RNA, disadvantage is however that required nucleic acid and cell dosage are big, and cell lethality is high, is not ideal CAR channel genes T
Cellular processes.In chemistry mediation method, calcium phosphate method is using calcium phosphate DNA compound adherent cell film by cell endocytic, behaviour
Make easy but poor repeatability, and is poorly suitable for the transfection of this primary cell of patient's T cell；Liposome method is to utilize band just
The electronegative phosphate group of liposome and nucleic acid of electricity forms compound, the saliva on liposome on remaining charge and cell membrane
The negative electrical charge of sour residue combines or so that nucleic acid is entered cell by endocytosis, and this method uses simply, but has certain thin
Cellular toxicity, and strong anti-inflammatory response can be induced in vivo, therefore largely also limits its application.Virus-mediated
Rotaring dyeing technology, can be by exogenous origin gene integrator into cellular genome, and transfection efficiency is high, and exogenous origin gene integrator is more stable, is current
Most common CAR-T cell transduction mode.In the preparation of CAR cell, retrovirus or slow virus, wherein slow virus sense are usually used
Dye method can not only infect division cells but also can infect nondividing phase cell, in CAR-T clinical test preparation the most often
With.Above-mentioned CAR-T transfection method, purpose be that CAR gene is transferred to T cell, it is expected to obtain at high proportion or high positive rate
CAR-T cell, however in practical operation, there are still many challenges and obstacles, it is difficult to obtain more high positive rate and specificity
CAR-T cell.On the one hand, electroporation and chemistry mediation method are uncomfortable because cell dosage is big or lethality is high, has certain toxicity etc.
In the preparation of clinical CAR-T cell；On the other hand, retrovirus or slow virus packaging step are cumbersome, and time-consuming, workload
Greatly, generally requiring veteran professional technician can just efficiently accomplish, and recombinant virus hardly results in higher titre,
This becomes restraining factors crucial in the preparation of CAR-T cell；Secondly, transfecting used tool when because of different batches virus packaging
Cell state difference, different technologies personnel's difference in operation etc., the slow virus titre of packaging is usually very unstable, it is poor between batch to cause
Different larger, these become the main speed limit link of CAR-T cell preparation and application study.
Therefore, as can a kind of method for finding high-efficient simple, can effectively improve the specificity and positive rate of CAR-T cell,
The efficiency that clinical CAR-T cell preparation can then be greatly improved is that the therapeutic effect of CAR-T cell improves guarantee.
Summary of the invention
It is of the invention that the technical problem to be solved is that the specificity and positive rate that how to improve CAR-T cell.
In order to solve the above technical problems, present invention firstly provides the methods of amplification immunocyte, which comprises
1) antigen that the immunocyte is identified is added into the cultivating system of immunocyte, obtains co-culture system；
2) co-culture system is cultivated, realizes the amplification of the immunocyte.
In the above method, the immunocyte can be the immunocyte of expression Chimeric antigen receptor.
The immunocyte concretely expresses the T cell of Chimeric antigen receptor.
In the above method, the antigen can be tumour antigen.
Further, the antigen can be B cell maturation antigen (BCMA).
The immunocyte can be the CAR-T cell of anti-BCMA.
In the above method, the proportion of immunocyte described in the co-culture system and the antigen can be 106It is a: 1~
100ng.Further, the proportion of immunocyte described in the co-culture system and the antigen can be 106It is a: 5~50ng.
In the above method, the number for the antigen that the immunocyte is identified is added into the cultivating system of immunocyte
And/or the time can adjust as the case may be, as long as being able to achieve the positive rate and/or specificity for improving the immunocyte,
It belongs to the scope of protection of the present invention.
The application of the method for the amplification immunocyte in medicine preparation, also belongs to protection scope of the present invention.
The drug can be used for treating tumour, such as Huppert's disease.
For the present invention in the preparation of existing CAR-T cell, gained CAR-T cell positive rate is low and influences its validity and spy
The opposite sex provides a kind of method of amplification immunocyte for improving CAR-T cell positive rate, immune thin using amplification of the invention
The positive rate for the CAR-T cell that the method for born of the same parents obtains can be improved 4-6 times, and operation is easy and efficient, high specificity for this method,
It is practical.
Detailed description of the invention
Fig. 1 is the cultural method flow chart of specific amplification CAR-T cell.
Fig. 2 is the testing result of anti-BCMA CAR-T cell positive rate, and the abscissa of A-F is CD3 positive cell number, indulges and sits
Mark indicates CAR positive cell number, and Q1+Q2 is the total positives rate of CAR, and Q2+Q3 is the total positives rate of CD3, and Q4 is double-negative rate, Q2
It is double positive rates.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
293T and 293FT cell in following embodiments is one hundred Biotechnology Co., Ltd's product of Nanjing section.
Embodiment 1, the method for expanding CAR-T cell
The present embodiment is to identify the anti-of B cell maturation antigen (B Cell Maturation Antigen, BCMA, CD269)
The method for improving CAR-T cell amplification efficiency is specifically described for BCMA CAR-T cell, flow chart is shown in Fig. 1, specific steps
It is as follows:
1, the preparation of plasmid
Slow virus envelope plasmid pMD2.G, psPAX2 and base containing CAR are extracted using the big extraction reagent kit of endotoxin-free plasmid
The slow virus expression plasmid (pWPXLd-CAR-BCMA-EGFP plasmid) and control plasmid pWPXLd-EGFP of cause are stand-by.Wherein,
PMD2.G, psPAX2, pWPXLd-EGFP plasmid are addgene product (http://www.addgene.org)；Base containing CAR
The slow virus expression plasmid (pWPXLd-CAR-BCMA-EGFP) of cause is built-up by following manner:
DNA fragmentation between the PmeI of pWPXLd-EGFP and SpeI identification sequence is replaced with into SEQ ID NO.1 in sequence table
Shown in DNA fragmentation, obtain recombinant vector pWPXLd-CAR-BCMA-EGFP, pWPXLd-CAR-BCMA-EGFP can be expressed
The fusion protein that anti-BCMA scfv, CD8hinge, 4-1BB, CD3 ζ are formed, the sequence of the fused protein are in sequence table
SEQ ID NO.2。
2, the preparation of slow virus
Slow virus is packed by 293T or 293FT cell, concrete operations are as follows:
1. adding FBS (mass percent of FBS in the medium is 10%) into DMEM high glucose medium to cultivate afterwards
293T cell is in 10cm culture dish, when 293T cell confluency degree reaches 70-90%, using lipo2000 (or PEI) by pWPXLd-
Tri- CAR-BCMA-EGFP plasmid, pMD2.G and psPAX2 plasmids convert 293T cell, each 10cm ware conversion 12ug's
PWPXLd-CAR-BCMA-EGFP plasmid, lipo2000 (or the 72 μ l of the pMD2.G of the psPAX2 of 7.8ug, 4.2ug, 48 μ l
PEI)；
2. step 1. after the completion of, the system after conversion is normally cultivated, respectively culture for 24 hours, 48h and 72h collect three
The culture supernatant of period；The culture supernatant of three periods is centrifuged 5 minutes through 400g respectively, abandons precipitating, then will
It is centrifuged three kinds of obtained supernatants and passes through 0.45 μm of filter filtering respectively, it is after filtering that filtrate is admixed together, obtain slow disease
Venom；Slow virus liquid is placed in ultra centrifugal pipe respectively to 25000rpm, 4 DEG C of centrifugation 2h keep virus heavy on Ultracentrifuge
It forms sediment, abandons supernatant, PBS the or RPMI-1640 free serum culture of 4 DEG C of 100 μ l pre-coolings is then added into each ultracentrifugation pipe
Base weight is outstanding to get to the slow virus suspension for having transfected pWPXLd-CAR-BCMA-EGFP plasmid, places -80 DEG C for use.
According to the method described above, pWPXLd-CAR-BCMA-EGFP plasmid is replaced with into control plasmid pWPXLd-EGFP, other
Step is constant, has been transfected the slow virus suspension of pWPXLd-EGFP, places -80 DEG C for use.
3, the preparation of T cell
The mononuclearcell in Healthy People or multiple myeloma patients blood is isolated by Ficoll density gradient method
(PBMC)。
After PBMC separation, the RPMI-1640 culture medium culture PBMC 12h for being added to 10%FBS is first used within first day, then
Use the CD28 antibody of the OKT3 antibody (R&D system, MAB100), 50ng/ml that are added to 10%FBS, 50ng/ml instead
The RPMI-1640 culture medium of (MILTENYI, 130-093-375) and 10ng/ml IL-2 (SIGMA, SPR3085) activate T cell
And continue to cultivate 48h.
Post-stimulatory T cell is resuspended and 400g is centrifuged 5min, supernatant removed, with polybrene containing 10ug/ml by third day
RPMI1640 culture medium be resuspended and bed board into six orifice plates, every hole 2ml, contain 2 × 106A cell, then in different Kong Zhongjia
Enter a kind of slow virus that step 2 obtains, according to slow virus titre, MOI is set as 20, and cell is then placed in 37 DEG C of 5%CO2Culture
It is cultivated in case for 24 hours, is resuspended cell, 400g 5min centrifugation is gone after supernatant to change fresh containing 10%FBS, 50ng/ml IL-2
With the RPMI-1640 culture medium of 50ng/ml IL-7,37 DEG C of 5%CO2Culture tentatively obtained anti-BCMA to the 7th day in incubator
During which CAR-T cell every other day measures T cell half and changes culture solution and count, can Fluirescence observation and flow cytometer detection during this
The expression rate of EGFP and CAR.
4, antigenic stimulus expands anti-BCMA CAR-T cell
After tentatively obtaining anti-BCMA CAR-T cell, step 3 is connect with containing 10%FBS, 50ng/ml IL-2 and 50ng/ml
The RPMI-1640 culture medium of IL-7 continues to cultivate cell, during which every other day, adds respectively into cultivating system in cultivating system
In concentration be 10ng/ml BCMA antigen (ACRO BIOSYSTEMS, BCA-H522y), keep cell culture density be 1 ×
106A/ml, progress cell count in every 2~3 days and half amount change liquid, cultivate the 14th day, harvest CAR-T cell and use fluidic cell
Instrument detects CAR-T cell positive rate, and agents useful for same is the CD3 antibody (EBIOSCIENCE, 17-0036-72) of APC label, biology
The sheep anti mouse Fab antibody (Jackson Immunoresearch, 115-066-072) of elementization, sheep IgG antibody (ABCAM,
37373), biotinylated sheep IgG antibody (ABCAM, 37376), mouse IgG antibody (the green skies, A7028), the chain of PE label
Mould Avidin (BD, 554061).Addition antigen assay sets two repetitions and tests, and is denoted as first group and second group respectively.
The anti-BCMA CAR-T cell tentatively obtained using BCMA antigen incubation step 3 is not added, culture is to the 14th heaven-made
Not add antigen control experiment.
As a result as shown in Fig. 2, A is the positive for not adding the 5th day anti-BCMA CAR-T cell in antigen control experiment in Fig. 2
Rate (expresses the percentage of the total CAR-T cell of CAR-T cell Zhan of anti-BCMA), and B is not add the 14th in antigen control experiment
The positive rate of its anti-BCMA CAR-T cell, C, E are the positive rate for adding the 5th day anti-BCMA CAR-T cell in antigen assay,
D, F is the positive rate for adding the 14th day anti-BCMA CAR-T cell in antigen assay.The results show that not adding antigen control experiment
In, anti-BCMA CAR-T cell is 4.49% in the 5th day positive rate, is 10.5% in the 14th day positive rate；Add antigen
In experiment, first group, anti-BCMA CAR-T cell was 4.49% (C in Fig. 2) in the 5th day positive rate, in the 14th day positive
Rate is 46.8% (D in Fig. 2)；Second group, anti-BCMA CAR-T cell positive rate of (the 5th day) before antigenic stimulus is 12.2%
(E in Fig. 2), it is 4-6 times without antigenic stimulus that the positive rate of (the 14th day), which is 68.2% (F in Fig. 2), after antigenic stimulus.Table
It is bright, the positive rate of CAR-T cell can be improved using the method for amplification CAR-T cell of the invention, illustrate having for CAR-T cell
Effect property and specificity are greatly improved.CAR-T cell after amplification can be used for inside and outside Function detection, kill tumor effect and comment
Estimate a grade clinical application.
<110>Shenzhen Hua Da life science institute
<120>a kind of cultural method of specific amplification CAR-T cell
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1449
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
atggccctgc ctgtcaccgc tctgttgctg ccactggctc tgttgctgca cgctgctaga 60
ccagaggtgc agctgctgga atctggcgga ggactggtgc agcctggcgg ctctctgaga 120
ctgtcttgtg ccgccagcgg cttcaccttc agcagctacc ctatgagctg ggtgcgccag 180
gcccctggta aaggtttgga atgggtttct gctattggtg gttcaggtgg ttggagttat 240
tatgccgatt ctgttaaggg tagattcacc atttctagag acaactctaa gaacaccttg 300
tacttgcaaa tgaactcctt gagagctgaa gatactgctg tttattactg tgctagatac 360
tggccaatgg attcttgggg tcaaggtact ctggtcaccg tctcctcagg tggaggaggt 420
agcggaggag gcgggagcgg tggaggtggc tctgagatcg tgctgacaca gagccctggc 480
accctgagcc tgtctcctgg tgaaagagct actttgtctt gttggttgtc tcaatctgtt 540
tcctctactt acttggcttg gtatcaacaa aaaccaggtc aagctccaag attattgatc 600
tacgatgctt cttctagagc accaggtatt ccagatagat tttctggttc tggttccggt 660
actgatttca ctttgactat ctctagattg gaaccagaag atttcgctgt ttactactgc 720
caacaatact ctgagtggcc attgactttt ggtcaaggta caaaggttga aatcaaaacc 780
acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 840
ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 900
ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 960
tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 1020
caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1080
ccagaagaag aagaaggagg atgtgaactg cgcgtgaagt tcagcaggag cgcagacgcc 1140
cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1200
gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1260
aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1320
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1380
cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1440
cctcgctaa 1449
<210> 2
<211> 482
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 2
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Ser Tyr Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Trp Ser Tyr
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Tyr Trp Pro Met Asp Ser Trp Gly Gln
115 120 125
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly
145 150 155 160
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Trp Leu
165 170 175
Ser Gln Ser Val Ser Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
180 185 190
Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala Pro
195 200 205
Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
210 215 220
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
225 230 235 240
Gln Gln Tyr Ser Glu Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
245 250 255
Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
260 265 270
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
275 280 285
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
290 295 300
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
305 310 315 320
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
325 330 335
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
340 345 350
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
355 360 365
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
Claims (10)
1. the method for expanding immunocyte, comprising:
1) antigen that the immunocyte is identified is added into the cultivating system of immunocyte, obtains co-culture system；
2) co-culture system is cultivated, realizes the amplification of the immunocyte.
2. according to the method described in claim 1, it is characterized by: the immunocyte is to express being immunized for Chimeric antigen receptor
Cell.
3. method according to claim 1 or 2, it is characterised in that: the immunocyte is the T for expressing Chimeric antigen receptor
Cell.
4. method according to claim 1 to 3, it is characterised in that: the antigen is tumour antigen.
5. method according to any one of claims 1-4, it is characterised in that: the antigen is B cell maturation antigen.
6. method according to claim 5, it is characterised in that: the immunocyte is anti-B cell maturation antigen
CAR-T cell.
7. any method in -6 according to claim 1, it is characterised in that: immunocyte described in the co-culture system
Proportion with the antigen is 106It is a: 1~100ng.
8. any method in -6 according to claim 1, it is characterised in that: immunocyte described in the co-culture system
Proportion with the antigen is 106It is a: 5~50ng.
9. the application of any the method in medicine preparation in claim 1-8.
10. application according to claim 9, it is characterised in that: the drug is used for tumour.
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