CN107338224A - PD 1 knocks out the preparation of EGFRvIIICAR T cells - Google Patents
PD 1 knocks out the preparation of EGFRvIIICAR T cells Download PDFInfo
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Abstract
PD 1 knocks out the preparation of EGFRvIII CAR T cells, including EGFRvIII CAR plasmids and its transposase plasmids, the sgRNA plasmids for knocking out the genes of PD 1 and CAS9 plasmids are turned reagent with electricity and mixed, and adds in PBMC cells and carries out electrotransfection；Using the enrichment with magnetic bead CD3 positive T cells of coupling CD3 or CD3+CD28 antibody；And screen and cultivate the T cell after amplification transfection and knock out EGFRvIII CAR T cells to obtain PD 1.The present invention improves expression efficiency and the time of transfection efficiency and transgenosis from PiggyBac Transposon Systems, simplifies CART preparation procedure, enhances the load capacity of system.In addition, avoided during present invention knockout 1 genes of PD higher using viral vector, security.
Description
Technical field
It the present invention relates to the use of CRISPR-Cas9 systems and prepare PD-1 knockout EGFRvIII CAR-T cells.
Background technology
EGFR is 170kD transmembrane glycopeptide polymeric immunoglobulin receptor, in EGF (epidermal growth factor, epidermis life
The long factor) stimulate in the presence of can exercise the function of EGFR-TK, guide downstream passages such as MAPK, Akt and JNK activation
And promote genetic transcription.The imbalance of EGFR paths can cause the generation of a variety of human epithelium's malignant tumours.EGFR paths are caused to lose
The reason for tune the overexpression including EGFR, and itself mutation caused by sustained activation.In EGFR various mutation types, most
Commonly EGFRvIII (also referred to as de2-7EGFR or Δ EGFR).This saltant type has lacked normal EGFR the 2nd to the 7th
801 base-pairs between individual extron, that is, 267 amino acid of film outskirt are lost, are reduced into 145kD.Cut caused by so
Short acceptor no longer can with any ligand binding, but can continuous activation, result in the excessive activation of EGFR paths.
Think that EGFRvIII is a kind of membrane receptor of tumour-specific than more consistent viewpoint at present, and normally organizing
In be no.Major part is all to be conceived to glioblastoma multiforme (glioblastoma on its research work
Multiforme, GBM).GBM is the very high brain tumor of a kind of common and lethal, in World Health Organization WHO tumor grade
For IV levels.According to the data of 2014, the annual survival rate of patient one for being diagnosed as GBM was 43%, and mean survival time is 15 after examining
Individual month.Because EGFRvIII has expression in about 30% GBM, and on the 37%-86% cells in tumor tissues
There is expression, therefore, EGFRvIII is selected to a promising target of GBM immunization therapies.Early application EGFRvIII makees
Immunotherapy for target spot includes using vaccine, immunologic test point inhibitor and uses artificial Chimeric antigen receptor
(Chimeric antigen receptor, CAR) edited self T-cell, i.e. CAR-T, all achieve gratifying achievement.
CAR molecules mainly include extracellular antigen binding domain (single antibody variable chain, ScFv), across
Film area and intracellular signal area.First generation CAR intracellular signal area is made up of CD3 ζ (zeta) chains or Fc ε RI γ (gamma) chain.The
2nd, three generations CAR then introduces one or two costimulating factor domains (such as CD28,4- respectively on the basis of the first generation
1BB, ICOS etc.), significantly improve the multiplication capacity and antitumor action of CAR-T cells, extend CAR-T cells in vivo
Continue existence time.Two generation CAR Clinical practice is to anti-leukocythemia (EGFRVIII CAR) and neuroblastoma
(GD2CAR) significant effect is achieved on.
Nevertheless, CAR-T is generally unsatisfactory in the clinical effectiveness to solid tumor resisting.In numerous clinical trials,
CAR-T, which exposes, many includes propagation and sustainability is limited, tumour filtration ability, is suppressed in tumor microenvironment etc. and to ask
Topic.Wherein how in the tumor microenvironment of inhibition its propagation and continuous capability in vitro is kept for cell, continue to play
Its killing is particularly important with secreting function, and considers how to utilize CAR-T and the other treatment means such as phase such as antibody, vaccine
With reference to improvement local microenvironment, destroying tumour inherent structure turns into one of focus of research.
The content of the invention
An object of the present invention is to provide the preparation that a kind of safe and efficient PD-1 knocks out EGFRvIII CAR-T cells
Method.
According to the first aspect of the invention, there is provided a kind of (non-treatment purpose) prepares PD-1 and knock out EGFRvIIICAR-T
The method of cell, comprises the following steps：
Prepare PBMC cells；
Prepare EGFRvIII Chimeric antigen receptors (CAR), i.e. 139CAR in figure；
Prepare the transposase plasmids being used cooperatively with EGFRvIII CAR, it is thin for EGFRvIII CAR to be incorporated into PBMC
On the genome of born of the same parents；
Prepare the sgRNA plasmids for knocking out PD-1 genes；
Prepare to be used cooperatively to knock out the CAS9 plasmids of PD-1 genes with sgRNA plasmids；
By the EGFRvIII CAR plasmids and its transposase plasmids of preparation, the sgRNA plasmids for knocking out PD-1 genes and
CAS9 plasmids turn reagent with electricity and mixed, and add in the PBMC cells of preparation and carry out electrotransfection；
T cell after amplification transfection as T cell activation agent and is cultivated using the magnetic bead of coupling CD3 or CD3+CD28 antibody
EGFRvIII CAR-T cells are knocked out to obtain PD-1.
According to a preferred embodiment of the present invention, EGFRvIII CAR can utilize PiggyBac-transposon
Carrier is sequentially connected in series people EF1 α promoters, CSF2RA Chimerical receptors signal peptide, after birth exoantigen land, hinge area, intracellular letter
What number conducting region was prepared with the resistant gene puromycin that T 2A small peptides connect.
According to the present invention, the EGFRvIII that after birth exoantigen land is preferably used for combining EGFRvIII albumen is single-stranded anti-
Body (scFv, clones numbering 139, also referred to as 139CAR), is sequentially connected in series c-myc Epitope tags, CD8 Hinge Chimerical receptors hinge
Chain, CD8Transmembrane Chimerical receptor transmembrane regions.
Intracellular signal transduction area is preferably that CD28-4-1BB-CD3 ζ, CD28 and 4-1BB are Chimerical receptor costimulatory molecules.
Its Successful transfection CD3 positive T cell together with transposase plasmids, foreign aid's gene high effective integration in host cell is mediated, and it is high
The stable expression of effect, it is not high effectively to solve the problems, such as that EGFRvIII CAR-T are prepared into power.
It is possible in accordance with a preferred embodiment of the present invention to the sgRNA of two selectively targeted PD1 genes is connected simultaneously online
On the pGL3-U6-sgRNA plasmids of property, CD3 positive T cells are transfected together with pST1374-NLS-flag-Cas9-ZF plasmids
The knockout of PD1 genes can be achieved.It is of the invention therefore to realize quick, simple, efficient, specific knockdown PD1 strategy, effectively
Solves the EGFRvIII CAR-T inhibitory action that tumour cell passes through PD1-PDL1 paths in by tumor microenvironment in vivo.
According to the specific embodiment of the present invention, preparing PBMC cells includes：(1) peripheral blood, side are gathered with anticoagulant tube
Collection side is rocked and peripheral blood is sufficiently mixed with anti-coagulants, and with phosphate buffer PBS 1：1 dilution；(2) periphery hemodilution
Liquid mixes in equal volume with lymphocyte separation medium, slow to rise slow drop centrifugation, draws the tunica albuginea confluent monolayer cells after centrifugation；(3) will obtain
Tunica albuginea confluent monolayer cells centrifuge after being mixed with PBS or serum-free cell culture medium (such as RPMI 1640), continue to use PBS or 1640
Cleaning three times, PBMC cells are obtained after precipitation.
It is a further object of the present invention to provide it is a kind of be used for prepare PD-1 knock out EGFRvIII CAR-T cells device, its
Including：
PBMC cell preparation systems；
Amaxa electricity turns kit；
Electrotransfection system, receive from Amaxa electricity turn kit electricity turn reagent and by it with EGFRvIIICAR plasmids and
Mixture is formed after its transposase plasmids, the sgRNA plasmids for knocking out PD-1 genes and the mixing of CAS9 plasmids；And receive and come from
The PBMC cells of PBMC cell preparation systems and the mixture is added wherein carry out electroporation transfection；
CD3 positive T cells stimulate enrichment system, include the magnetic bead for being coupled CD3 or CD3+CD28 antibody；And
Training system is sieved, EGFRvIII CAR-T are knocked out to obtain PD-1 for screening and cultivating the T cell after amplification transfects
Cell.
It is yet another object of the invention to provide a kind of T cell of separation obtained according to the above method or cell line or secondly
For culture.
The present invention improves expression efficiency and the time of transfection efficiency and transgenosis, letter from PiggyBac Transposon Systems
Change CART preparation procedure, enhance the load capacity of system.Carried in addition, the present invention avoids when knocking out PD-1 genes using virus
Body, security are higher.
The equipment of gene knockout and CAR is placed in same step by the present invention to be completed, and improves the preparation of CAR-T cells
Efficiency and survival rate, reduce the cost of multi-pass operation；The cultivation cycle of CAR-T cells in vitro is shortened simultaneously, ensure that
Its activity expanded in vitro.
Brief description of the drawings
Fig. 1 is the gene structure figure according to the PB-EGFRvIII CAR-BBZ-puro expression vectors of the present invention；
Fig. 2 is CAR tables in PD1-KO EGFRvIII CAR-T cells and EGFRvIII CAR-T cells according to the present invention
Up to the comparing result figure of rate；
Fig. 3 is PD-1 in PD1-KO EGFRvIII CAR-T cells and EGFRvIII CAR-T cells according to the present invention
Knockout result figure；
Fig. 4 be according to the present invention PD1-KO EGFRvIII CAR-T cells and EGFRvIII CAR-T cells, respectively with
After being overexpressed EGFRvIII human glioma cells U251 (U251-OE) and U251 (U251-WT) co-cultivations of wild type, cell
The secretory volume column diagram of factor IFN-γ, GM-CSF, IL-2 and TNF-α；
Fig. 5 be according to the present invention PD1-KO EGFRvIII CAR-T cells and EGFRvIII CAR-T cells, respectively with
After being overexpressed EGFRvIII human glioma cells U251 (U251-OE) and U251 (U251-WT) co-cultivations of wild type, cell
The comparison diagram of toxicity detection.
Embodiment
It is prepared by PBMC cells
Healthy People or peripheral blood in patients are gathered with anticoagulant tube, is rocked in collection so that peripheral blood is fully mixed with anti-coagulants
Close, and with phosphate buffer PBS 1：1 dilution.Peripheral blood dilution is slowly added to isometric separation of lymphocytes is housed
In the 50ml centrifuge tubes of liquid (Ficoll), 450g is slow to rise slow drop centrifugation 25min；
After centrifugation terminates, the careful tunica albuginea layer drawn above lymphocyte separation medium, a new 50ml centrifuge tube is transferred to
In, PBS, 300g are added, it is slow to rise slow drop centrifugation 10min, supernatant is abandoned, retains the cell precipitation of centrifugation bottom of the tube；Add again
PBS, 160g, it is slow to rise slow drop centrifugation 15min, abandon supernatant；PBS, 300g are eventually adding, it is slow to rise slow drop centrifugation 10min, supernatant is abandoned,
Obtain PBMC.
Prepare CAR-T cells
1. carry out electricity after PBMC is slightly cultivated into 1 to 2 hour with the culture mediums of RPMI 1640 containing 10%FBS to turn, respectively
Transfection is following two groups：
(1) by pGL3-PD1sg (1+2), pST1374-NLS-flag-Cas9-ZF, PB-EGFRvIIICAR-BBZ-puro
The electricity that (shown in Fig. 1 and referring to sequence table 1) and each 5 μ g of four plasmids of transposase turn with Amaxa electricity in kit turns reagent and mixed,
Obtain the about 110 μ l electricity comprising four kinds of plasmids and turn mixed liquor；
(2) to include original plasmid pGL3-U6-sgRNA, pST1374-NLS-flag-Cas9-ZF, PB-EGFRvIII
The electricity of four plasmids of CAR-puro and transposase turns mixed liquor as control；
A part of cell is separately stayed to turn without electricity.
2. two groups of electricity are turned into mixed liquor adds 1-2 × 107Electricity is carried out in individual PBMC cells to turn, and liquid is changed after 2 hours；
100IU/ml IL-2 RPMI 1640 culture mediums are added after 3.12 hours, have CD3/CD28 antibody with coupling
Magnetic bead (Life Technologies, 11132D) stimulates activation CD3 positive T cells；
4. every 2 to 3 day half amount changes nutrient solution, while observes cell amplification situation.Its thorn is determined according to its proliferative conditions
Swash duration, but stimulation time is no more than one week.
5. separate except after beads, continue culture 5 to 7 days.Collect a small amount of cell to be used to detect, remaining cell can continue
Further largely expanded using rapid amplification.
Utilize Flow cytometry CAR expression
To expand obtained CAR-T cells be resuspended in afterwards in FACS liquid twice with PBS (containing 0.1% sodium azide and
0.5%BSA PBS), by Alexa647Conjugate Myc-Tag (9B11) Mouse mAb add cell suspension
In, 4 DEG C are incubated 10 minutes, and rear BD Fortessa are used to obtain staining cell cleaning cell twice, and FlowJo, which is used to analyze, to be tied
Fruit.As shown in Fig. 2 CAR expression can reach more than 90%.
Cellular genome is extracted to be analyzed with cutting effect
Use cell pyrolysis liquid vitellophag (0.1-1 × 10 containing Proteinase K6), and it is thin with phenol chloroform method extraction CAR-T
Born of the same parents' genomic DNA.And detect on-target primers (primer is unlisted) well with designed in advance, with high-fidelity Taq enzyme (such as
Takara PrimerStar) PCR amplification purpose fragments, purified and reclaimed using Axygen PCR primers QIAquick Gel Extraction Kit.Use
T7EN1 digestions detect PCR annealed products, 3% agarose gel electrophoresis detection and analysis digestion, as a result such as Fig. 3 gel electrophoresis figures
It is shown.PCR primer is connected into pMD19-T carriers, converted into DH5 α competence bacteriums, the list that random picking is more than 20
Bacterium colony, it is sequenced using universal primer M13-47, assesses base change and cutting efficiency after target fragment is cut.Such as Fig. 3
Shown, PD-1 knockout efficiency can reach more than 59%.
PD-1 knocks out EGFRvIII-CAR-T cytokine release outcome evaluations
U251-OE cells, U251-WT cells and effector cell EGFRvIII CAR-T and PD-1 knockout are cultivated respectively
EGFRvIII CAR-T cells.
By target cell U251-OE and U251-WT respectively with every hole 1x 105Kind is in 96 orifice plates, by effector cell
EGFRvIII CAR-T and PD1-KO；EGFRvIII CAR-T are respectively with 2x 105Mixed with target cell, with the μ l's of cumulative volume 200
Nutrient solution (culture medium+10%FBS of RPMI 1640) is in 37 DEG C of 5%CO224 hours are co-cultured in incubator.Every group 3 multiple
Hole.Afterwards the μ l of centrifuging and taking supernatant 10 with PerkinElmer AlphaLISA assay reagents series box (AL208, AL216,
AL217, AL221) to detect TNF-α, GM-CSF, IFN-γ and IL-2 cytokine concentrations respectively horizontal.As shown in figure 4,
Although TNF-α, GM-CSF, IFN-γ first three factor level do not have significant difference, IL-2 horizontal knockout group substantially secretes water
Ping Genggao, it means that knockout group cell is more beneficial for surviving.
PD-1 knocks out EGFRvIII-CAR-T Cell killing efficacies and assessed
Collect stable expression luciferase target cell (U251-OE-luc, U251-WT-luc) and effector cell
(CAR-T cells), 300g is slow to rise slow drop centrifugation 10min, abandons supernatant；Target cell is resuspended respectively with 1ml PBS solutions and effect is thin
Born of the same parents, 300g, 10min is centrifuged, it is slow to rise slow drop, abandon supernatant；Repeated washing is once；With the nutrient solution (culture mediums+10% of RPMI 1640
FBS it is 2x 10 that target cell to concentration), which is resuspended,6/ ml, resuspension effector cell to concentration is 3.2x 107/ml。
Use the method for doubling dilution that effect target ratio is set for 1:1、2:1、4:1、8:1 experimental port, and control group is set, it is empty
White group (every group of 3 multiple holes, cultivates 16 to 24h in 37 DEG C of 5%CO2 incubators；All cells, 500g centrifugations 5min are drawn in digestion；
Cell is resuspended with 100 μ l PBS, adds in the 96 hole detection plates at opaque bottom, and adds 100 μ l D- in every hole rapidly
Luciferin substrate solutions (lucifuge operation), ELIASA detection 560nm absorbances, as a result as shown in figure 5, PD1- is knocked out
EGFRvIII CAR-T cells in U251-OE target cells under the conditions of different effect target ratios killing-efficiency apparently higher than not knocking out group.
Assessed in Mice Body into knurl and Experiment on therapy
2x 105U251-OE-luc cell is implanted into advance in immunodeficient mouse NSG encephalic, uses living imaging instrument
Its stable situation into knurl was observed every 2-3 days.Be implanted into latter week, in-situ injection 2x 106EGFRvIII CAR-T or
PD1-KO；EGFRvIII CAR-T cell, and set not electricity and turn group NT as control.Every injection in 3 days once, co-injection 3 times.It
Mouse survival state is observed afterwards and records survivorship curve, as a result as shown in Figure 6.Knockout group can be obviously prolonged the mouse survival longevity
Life.
The present invention uses PiggyBac Transposon Systems, so as to which A, preparation technology are simple, it is not necessary to required for prepared by virus
Various complex conditions；B, PiggyBac system loads can exceed 14KB, and virus system loads no more than 8KB, otherwise
It cannot get satisfactory virus；C, for the metainfective effective expression time window of virus system at two weeks or so, this was virus system
One of the reason for mediated therapy easily recurs；And PiggyBac systems can mediate long-term expression, extend CAR expression time,
Preferably meet the requirement of T cell treatment.
In addition, the method that the present invention is turned using electricity, does not both need the complicated technology of virus packaging, avoids virus infection band again
The potential safety hazard come.
In addition, the present invention uses the EF1 α promoters of people itself, enable CAR genes in human body long lasting for table
Reach；The specific knockdown of people's PD-1 genes, enhance the anti-tumor capacity of T cell.
Sequence table
<110>Chengdu Chris wins bio tech ltd
<120>PD-1 knocks out the preparation of EGFRvIII CAR-T cells
<160> 1
<210> 1
<211> 7827
<212> DNA
<213>Artificial sequence PB-EGFRvIII CAR-BBZ-puro
<400> 1
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatgctg 1920
ctgctcgtta caagtctgct gctgtgtgaa ctgcctcatc ccgctttcct gctgatccca 1980
gacatccaga tgactcagag tcccagcagt ctcagcgcta gtgtgggcga cagagtgacc 2040
atcacttgca gggccagcca gggaatcagg aataacctcg cctggtatca acagaagcca 2100
gggaaagctc caaagcggct catctatgcc gcttcaaatc tgcaatctgg agtgcccagc 2160
agattcactg gctcagggtc aggaaccgag tttaccctga ttgtgtcatc tctgcaacct 2220
gaggactttg ctacctacta ctgcctccaa catcatagct acccactcac ttccggtggt 2280
gggaccaaag tggagattaa gggctctact tccgggagcg gcaaaccagg ctctggagaa 2340
gggagcgaag tccaagtcct cgaaagcggt ggaggcctgg tgcagccagg cggttctctg 2400
aggctgtctt gtgcagcctc aggtttcacc tttagcagct acgcaatgtc ctgggtcagg 2460
caggctccag gtaaaggact ggaatgggtg tctgctatat ctggtagcgg agggtccacc 2520
aactatgcag acagtgtcaa gggtagattc accatctcaa gagacaactc caagaacaca 2580
ctgtacctgc aaatgaatag cctgagagca gaggatactg ctgtctatta ctgcgctggt 2640
tctagtggct ggtcagagta ctggggtcag ggcaccctgg tcactgtcag ttcagctgct 2700
gggtctgaac agaagctcat aagcgaagaa gatctgttcg tccccgtgtt cctgcctgcc 2760
aagccaacaa ctacccctgc tccacgacca cctactccag cacctaccat cgcaagtcag 2820
cccctgtcac tgcgacctga ggcttgccgg ccagcagctg gaggagcagt gcacacccga 2880
ggcctggact tcgcatgcga tatctacatt tgggcaccac tggctggaac ctgtggggtc 2940
ctgctgctga gcctggtcat caccctgtat tgtaaccaca gaaataggag caaacgctcc 3000
cgactgctgc attccgacta catgaacatg acacctcgga gaccaggccc cactagaaag 3060
cattaccagc catatgcccc acccagggat ttcgcagcct atcggagccg gttcagcgtc 3120
gtgaaaaggg ggcgcaagaa actgctgtac atcttcaagc agccttttat gcgcccagtg 3180
cagacaactc aggaggaaga cggatgctct tgtcggttcc cagaggagga ggaaggaggc 3240
tgcgagctga gagtgaagtt cagccggagc gccgatgcac cagcatatca gcagggacag 3300
aatcagctgt acaacgagct gaatctgggc aggcgcgagg aatatgacgt gctggataag 3360
cgacgaggac gggaccccga aatgggagga aaacccagaa ggaagaaccc tcaggagggg 3420
ctgtataatg aactgcagaa agacaagatg gctgaggcat acagcgaaat tggaatgaaa 3480
ggagagcgcc gacgggggaa gggacacgat gggctgtacc agggactgtc aaccgccact 3540
aaagatacct acgacgcact gcacatgcag gctctgcccc caagagaatt cgaaggatcc 3600
gcggccgctg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 3660
ccttccggga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 3720
agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 3780
gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 3840
gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc 3900
acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 3960
ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg 4020
cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag 4080
ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc 4140
gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc 4200
gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 4260
ggtgcctgaa tctaggtcga caatcaacct ctggattaca aaatttgtga aagattgact 4320
ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 4380
tatcatgcgt taactaaact tgtttattgc agcttataat ggttacaaat aaagcaatag 4440
catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 4500
actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat tagtctatca 4560
gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat ttaaacagca 4620
gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc agggtttcct 4680
gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc cccggaattg 4740
actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc cccaggtgtc 4800
tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc 4860
gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga 4920
gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct 4980
gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat atataataag 5040
ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa 5100
gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca 5160
gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt 5220
cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg 5280
catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata 5340
tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg 5400
aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg 5460
atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag 5520
gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt 5580
cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga 5640
acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg 5700
cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat 5760
ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc 5820
tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 5880
taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 5940
cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 6000
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 6060
ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 6120
agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 6180
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 6240
caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 6300
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6360
cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 6420
tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 6480
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 6540
cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 6600
tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 6660
tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 6720
caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 6780
aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 6840
cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 6900
ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 6960
tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 7020
atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 7080
tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 7140
aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 7200
catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 7260
gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 7320
ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 7380
aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 7440
atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 7500
cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 7560
gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 7620
agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 7680
gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 7740
caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 7800
ggcgacacgg aaatgttgaa tactcat 7827
Claims (7)
1. a kind of method for preparing PD-1 and knocking out EGFRvIII CAR-T cells, comprises the following steps：
Prepare PBMC cells；
Prepare EGFRvIII Chimeric antigen receptors (CAR)；
Prepare the transposase plasmids being used cooperatively with EGFRvIII CAR, for EGFRvIII CAR to be incorporated into PBMC cells
On genome；
Prepare the sgRNA plasmids for knocking out PD-1 genes；
Prepare to be used cooperatively to knock out the CAS9 plasmids of PD-1 genes with sgRNA plasmids；
By the EGFRvIII CAR plasmids and its transposase plasmids of preparation, sgRNA plasmids and-CAS9 for knocking out PD-1 genes
Plasmid turns reagent with electricity and mixed, and adds in the PBMC cells of preparation and carries out electrotransfection；
The magnetic bead of coupling CD3 or CD3+CD28 antibody is used as T cell activation agent and cultivates the T cell after amplification transfection to obtain
Obtain PD-1 and knock out EGFRvIII CAR-T cells.
2. according to the method for claim 1, wherein EGFRvIII CAR be using PiggyBac-transposon carriers according to
Secondary series connection people EF1 α promoters, CSF2RA Chimerical receptors signal peptide, after birth exoantigen land, hinge area, intracellular signal transduction
What the resistant gene puromycin that area connects with T 2A small peptides was prepared.
3. according to the method for claim 2, wherein after birth exoantigen land is for combining EGFRvIII albumen
EGFRvIII single-chain antibodies (scFv), it is sequentially connected in series c-myc Epitope tags, CD8 Hinge Chimerical receptors hinge, CD8
Transmembrane Chimerical receptor transmembrane regions.
4. according to the method for claim 3, wherein intracellular signal transduction area is CD28-4-1BB--CD3 ζ, CD28 and 4-
1BB is Chimerical receptor costimulatory molecules.
5. according to the method for claim 1, wherein preparing PBMC cells includes：(1) peripheral blood, Bian Cai are gathered with anticoagulant tube
Collection side, which is rocked, makes peripheral blood be sufficiently mixed with anti-coagulants；(2) peripheral blood cells mix in equal volume with lymphocyte separation medium, slow to rise
Slow drop centrifugation, draws the tunica albuginea confluent monolayer cells after centrifugation；(3) by obtained tunica albuginea confluent monolayer cells and PBS or serum-free cell culture
Centrifuged after base mixing, continue to be cleaned three times with PBS or serum-free cell culture medium, PBMC cells are obtained after precipitation.
6. a kind of be used to prepare the device that PD-1 knocks out EGFRvIII CAR-T cells, including：
PBMC cell preparation systems；
Amaxa electricity turns kit；
Electrotransfection system, receive from Amaxa electricity turn kit electricity turn reagent and by it with EGFRvIII CAR plasmids and its
Mixture is formed after transposase plasmids, the sgRNA plasmids for knocking out PD-1 genes and the mixing of CAS9 plasmids；And receive and come from
The PBMC cells of PBMC cell preparation systems and the mixture is added wherein carry out electrotransfection；
CD3 positive T cells stimulate enrichment system, include the magnetic bead for being coupled CD3 or CD3+CD28 antibody；And sieve training system
System, EGFRvIII CAR-T cells are knocked out to obtain PD-1 for screening and cultivating the T cell after amplification transfects.
7. the T cell or cell line or its subculture of the separation that the method according to one of claim 1-5 is obtained.
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| CN112458116A (en) * | 2020-10-19 | 2021-03-09 | 广州重磅生物科技有限公司 | Construction method of PD-1 knockout CD19CAR-T cell |
| CN113005092A (en) * | 2021-01-26 | 2021-06-22 | 武汉科技大学 | Preparation method and application of PD1 knockout LMP1 targeted CAR-T cell |
| CN113637636A (en) * | 2021-09-10 | 2021-11-12 | 依科赛生物科技（太仓）有限公司 | Method for improving proportion of initial T cells cultured in vitro |
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| CN109837292A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | A kind of Chimeric antigen receptor T cell of targeting EGFR and its preparation method and application knocking out PD1 |
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| CN109652380A (en) * | 2019-01-25 | 2019-04-19 | 苏州茂行生物科技有限公司 | The CAR-T cell and its preparation method and application of LewisY is targeted based on base editor |
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| CN112458116B (en) * | 2020-10-19 | 2023-04-14 | 广州重磅生物科技有限公司 | Construction method of PD-1 knockout CD19CAR-T cell |
| CN113005092A (en) * | 2021-01-26 | 2021-06-22 | 武汉科技大学 | Preparation method and application of PD1 knockout LMP1 targeted CAR-T cell |
| CN113637636A (en) * | 2021-09-10 | 2021-11-12 | 依科赛生物科技（太仓）有限公司 | Method for improving proportion of initial T cells cultured in vitro |
| CN113637636B (en) * | 2021-09-10 | 2023-09-05 | 苏州依科赛生物科技股份有限公司 | Method for improving initial T cell proportion of in-vitro culture |
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