CN108531457A - A kind of method that Cas9/RNP knocks out T cell PD-1 and LAG3 gene and prepares CAR-T cells - Google Patents
A kind of method that Cas9/RNP knocks out T cell PD-1 and LAG3 gene and prepares CAR-T cells Download PDFInfo
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Abstract
The present invention relates to a kind of Cas9/RNP to prepare the dual-gene methods for knocking out CD19 CAR T cells of PD 1 and LAG3, it is mixed including sgRNA the and Cas9 albumen for being used to knock out PD 1 and LAG3 genes is turned reagent with electricity, is added in CD4+/CD8+ cells and carries out Electroporation Transformation；Using CD19 CAR slow-virus transfection CD4+/CD8+ positive cells；And it screens and cultivates the T cell after amplification transfects and obtained the dual-gene knockout CD19CAR T cells of PD 1 and LAG3.The present invention selects Cas9/RNP gene editing systems to improve expression efficiency and the time of transfection efficiency and transgenosis, simplifies the preparation flow of CAR T cells, increases the feasibility and load capacity of system.In addition, the present invention avoids using transfection reagent, safety higher when knocking out PD 1 and LAG3 dual-gene.
Description
Technical field
The present invention relates to immunocyte preparation fields, in particular it relates to which a kind of preparing PD-1 using Cas9/RNP
The CD19 CAR-T cells knocked out with LAG3.
Background technology
After operation, radiotherapy, chemotherapy, the immunotherapy of tumour has become effective therapeutic modality, receives in the recent period wide
General concern.Wherein Chimeric antigen receptor T cell technology (Cheimeric Antigen Receptors-T cell, CAR-T) is
A kind of emerging adoptive cell therapy (Adoptive cell transfer therapy, ACT), the technology is by patient T
Cell feeds back to patient's body after modifying, activate and being proliferated in vitro, the specific receptor expressed by T cell, targeting
Tumor cell, and show stronger killing activity and persistence.
Dead molecule -1 (Programmed death-1, PD-1, also referred to as CD279) belongs to the inhibition of T lymphocytes
Co-receptor, is a kind of important immunologic test point, and ligand (Programmed death ligand 1, PD-L1, also referred to as
B7-H1 or CD274) it can be in kinds of tumor cells surface expression.PD-1/PD-L1, which is combined, mediates negative immune to adjust access, has
Conducive to tumor microenvironment is formed, promote tumor immune escape.
Lymphocyte activation gene -3 (lymphocyte activation gene-3, LAG-3) is a kind of important exempts from
Epidemic disease checkpoint, effector T cell, regulatory T cells, natural killer cells, B cell and the Plasmacytoid being mainly expressed in after activation
Dendritic Cells etc. plays very delicate effect in regulatory T-cell immune response.More and more evidences show nothing
By in vivo or external, LAG-3 all can negative regulation effector T cell function (activation, cytokine release and cell killing),
The immune suppression function of regulatory T cells can be enhanced simultaneously.The mark that LAG-3 or T cell are exhausted, in tumour and chronic
It infects in environment, the expression of LAG-3 can obviously raise on T cell surface, by monoclonal antibodies block LAG-3, can enhance T
The proliferation and cytokine release of cell, and cell cycle arrest can be postponed, increase the ratio of memory T cell hypotype.
Find that PD-1 and LAG-3 are sent out during promoting immunologic escape in the preclinical study of chronic infection and tumour
Synergistic effect is waved, while PD-1 and LAG-3 signal paths being blocked T cell can be reversed to exhaust, it is anti-infective and anti-swollen to improve T cell
The ability of tumor.LAG-3 antibody is used alone at present or PD-1 antibody and LAG-3 Antybody therapies neoplastic hematologic disorder and entity is used in combination
The clinical test of tumor is carrying out (ClinicalTrials.gov, No.NCT02061761, NCT01968109).
Chimeric antigen receptor (CAR) is made of extracellular antigen-binding site, hinge area, transmembrane region intracellular signal transduction area etc.,
Generally by viral transduction or electroporation, CAR developed by molecule is made to form CAR-T cells on T cell surface.The design of CAR molecules
The development of four generations is experienced according to the difference in intracellular signal area, wherein first generation signaling zone only includes CD3 ζ；Second generation signaling zone exists
A costimulating factor, such as CD28,4-1BB, OX40 are added on the basis of the first generation；The third generation has added two on the basis of the first generation
A concatenated costimulating factor；Forth generation includes the cell factor that IL12, IL7, CCL19 etc. can improve microenvironment.Target blood
The CAR-T cells of the CD19 in tumour direction, overall relief rate is up to 90% or more during part is studied.However in part neoplastic hematologic disorder and
Due to the inhibition of tumor microenvironment in most of entity tumor, effect is less desirable, and immunity inspection point is to release immune suppression
One of key link of system, so knockout of the immunologic test point in T cell has important clinical value.
Before adoptive cell infusion, pass through Cas9/RNP (ribonucleoproteins) and single-stranded guiding RNA in vitro
(single-guide RNAs, sgRNA) target gene editing technique permanently knocks out the immunologic test point receptor on CAR-T cells
Such as PD-1 and LAG-3, the function of CAR-T can be improved, promote to can recognize that tumour antigen and generates the T cells of specific reaction
Proliferation, plays the effect of its killing tumor cell.Studies have reported that T can be enhanced by knocking out PD-1 in T cell or CAR-T cells
Cell or CAR-T cell anti-tumor functions, but there is presently no the reports that PD-1 and LAG-3 is knocked out about T or CAR-T cells.
The present invention carries out the knockout of PD-1 and LAG-3 using CRISPR-Cas9 systems in T cell and CAR-T cells for the first time, and sees
Examine T and CAR-T Immunophenotypings and changes of function after gene editing.
Invention content
Invention content
It is an object of the invention to overcome bottleneck in the prior art, a kind of Cas9/RNP preparations PD-1 and LAG3 is provided
The dual-gene method for knocking out CD19 CAR-T cells, and demonstrate its potential application in oncotherapy.Specifically, invention
A kind of CRISPR/Cas9 is provided and edits T cell or the method for CAR-T in vitro, is T cell or CAR-T cell tables by editor
The expression of the PD-1 and LAG3 albumen in face is lowered or is disappeared, and immunosupress signal is released, and is assigned the stronger tumour of T cell and is killed
Hinder ability.
To achieve the above object, the present invention adopts the following technical scheme that：
The first purpose of the invention is to provide a kind of methods of CD4+/CD8+ T cells extraction, wherein CD4+ T cells and CD8
+ T cells are operated by corresponding specification by EasySeq kits (STEMCELL) extract respectively respectively.
It is external that second object of the present invention is to provide a kind of Cas9/RNP of targeting knock out T cell PD-1 and LAG3 gene
The method of editor, including the synthesis of sgRNA and Cas9 albumen, and by the two electroporation transfection into T cell；Further
Ground, the sgRNA are made of crRNA two parts of the fixed tracrRNA of sequence and targeting DNA, wherein the sequence of targeting PD-1
Such as NO.1~2 SEQ, sequence such as NO.3~4 SEQ of LAG3 are targeted；
Further, the Cas9 is S.pyogenes Cas9 albumen, and there are one HA labels and two cores in its c-terminus
Positioning signal (NLS) sequence such as NO.5~6 SEQ；
Further, tracrRNA and crRNA presses concentration ratio 1:1 is mixed into sgRNA, then by sgRNA and Cas9 albumen by dense
Spend ratio 1:1 is mixed into Cas RNP；
Further, by 3 × 105A CD4+/CD8+ T cells and 20 μ L Amaxa electroporation transfection seminal plasma fructose detection kits P3
It is mixed into cell suspension, 3 μ L 20mM Cas9/RNP are mixed with cell suspension, is turned by 4D-Nuclelfecter electroporation electricity
Program EH-115 carries out electroporation transfection.
Third object of the present invention is to provide a kind of CD19 CAR slow virus carriers；
Further, the CAR molecules are to be sequentially connected in series people EF1 α, CSF2RA Chimerical receptors signal peptide, born of the same parents using pCDH carriers
The screening-gene CopGFP that outer antigen-binding site, hinge area, transmembrane region intracellular signal transduction area are connected with T2A small peptides prepare and
At；
Further, it is Chimerical receptor costimulatory molecules that intracellular signal area, which is OX40-4-1BB-CD3 ζ, OX40 and 4-1BB,；
Preferably, the hinge area is the regions CD8hinge, and the transmembrane region is CD8 α；
Further, CD19 CAR pCDH vector plasmids exist in helper plasmid pSPA2 and vsv-G envelope plasmid pMD2.G
In the case of, it waits for that cell is grown to 80-90%, cotransfection HEK293T cells, collects supernatant, can be made into high quality after concentration
Slow virus with CAR molecules.
Finally, 4th purpose of the invention is to provide a kind of coding CD19 CAR genes knocked out containing PD-1 and LAG3
The host cell of recombinant expression carrier；
Further, the host cell is T cell or the cell mass containing T cell；
Further, the moi values of the slow virus and host cell with CD19 CAR molecules are 1~5；
Further, it is activated using anti-CD3+CD28 antibody magnetic beads after the host cell infection, for screening and cultivating expansion
Increase the T cell after transfection to obtain the CD19 CAR-T cells that PD-1 and LAG3 is knocked out.
In one embodiment, by CD19 CAR-T cells and expression CD19 (+) PD-1 (-) LAG3 (-), CD19 (+)
The tumor cell line K652 of PD-1 (+) LAG3 (+) is incubated altogether, as a result shows that CD19 CAR-T cells can be in CD19 (+) PD-1
(-) LAG3 (-) target cell largely generates IFN-γ in the case of stimulating；But it cannot be with CD19 (+) PD-1 (+) LAG3
(+) target cell generates IFN-γ when being incubated altogether；The T cell of non-CD19 targetings can not be in the case where CD19 (+) target cell stimulates simultaneously
Generate IFN-γ.The case study on implementation illustrates that the CAR-T cells of the dual-gene knockouts of PD-1 and LAG3 have good targeting, energy
It is enough to activate T cell killing ability under the stimulation of CD19 target spots, and PD-1 and LAG-3 immunosuppressor pair can be released
Immune inhibition.
In another implementation, it was demonstrated that in certain effect target ratio (CAR-T cells：Target cell) under, CD19 CAR-T are thin
Born of the same parents or the CD189 CAR-T cells for knocking out PD-1 and LAG3, can kill the tumour cell of the CD19 positives, and regardless of whether strike
Go out PD-1 and LAG3, the capacity variance of the two killing is not notable；But the T cell or PD-1 and LAG3 of non-targeted CD19 knock out
T cell, cannot kill the tumour cell of CD19 feminine genders.The example illustrates that CD19 CAR-T cells can effectively kill CD19
Positive tumour cell, and it is smaller to the activity influence of CD19 CAR-T cells to knock out PD-1 and LAG3.
Compared with prior art, the invention has the advantages that：
Currently, CAR-T technologies are in the application in solid tumor field and unsuccessful, an important reason is exactly exempting from for immune microenvironment
Epidemic disease depression effect knocks out PD-1 and LAG3 genes by external Cas9/RNP, can be by the decrease of immunity inspection point to solve
Except the inhibition function of immune microenvironment；Cas9/RNP is the S.pyogenes Cas9 eggs that core sequence is inducted by carrying in vitro
White and synthesis PD-1 and LAG3 sgRNA compositions, interference of the external sequence to cell, increases and faces when avoiding carrier transfection
The safety of bed application.It is of the present invention using Cas9/RNP by the CAR-T cells preparation side of PD-1 and LAG3 gene knockouts
Method, is capable of the inhibition of the immune microenvironment of specificity releasing, and differential high efficient kills targets neoplastic cells.
Description of the drawings
Fig. 1 is to target the sgRNA targetings sequences of PD-1 and LAG3 genes to illustrate schematic diagram；
Fig. 2 is that the gene structure of slow virus pCDH carriers illustrates schematic diagram；
Fig. 3 is the knockout PCR result schematic diagrams that flow cytometer detection PD-1 and LAG3 knock out T cell；
Fig. 4 is the knockout T7EH1 digestion result schematic diagrams that flow cytometer detection PD-1 and LAG3 knock out T cell；
Fig. 5 is the schematic diagram of flow cytometer detection CD19 CAR expressions；
Fig. 6 be normal CAR-T cells and PD-1 (-) LAG3 (-) CD19 (+) CAR-T cells, respectively with K562 Leukemia Cell Lines
After being co-cultured with Raji lymphoma cells, the schematic diagram of secretion of gamma-IFN situation；
Fig. 7 A and Fig. 7 B are that normal CAR-T cells and PD-1 (-) LAG3 (-) CD19 (+) CAR-T cells in vitro kill CD19+
The result schematic diagram of K652 cells.
Specific implementation mode
The present invention provides a kind of method that Cas9/RNP prepares the dual-gene knockout CD19 CAR-T cells of PD-1 and LAG3,
It is prepared including CD4+/CD8+ T cells, sgRNA the and Cas9 albumen that PD-1 and LAG3 are knocked out prepares, Cas9/RNP and T is thin
The electroporation transfection of born of the same parents, the transfection of CD19 CAR slow virus, anti-CD3+CD28 antibody magnetic beads are activated and are obtained by screening and culturing
Obtain CD19 CAR-T cells.
The preparation of CD4+/CD8+T：
Healthy human peripheral blood is acquired with EDTA anticoagulant tubes, isometric phosphate buffer PBS dilutions will be entered, peripheral blood dilution is added
Enter into the 50ml centrifuge tubes of isometric lymphocyte separation medium (Ficoll), 400g 30min centrifugations；It is careful to draw lymph point
Tunica albuginea confluent monolayer cells in chaotropic are transferred in new 50ml centrifuge tubes, are washed 3 times with PBS centrifugations 300g 10min, you can obtain
PBMC cells；By PBMC cells respectively according to EasySeq kits (EasySepTMHuman CD4+/CD8+ T Cell
Enrichment Kit, STEMCELL) specification operation can be obtained CD4+ and CD8+ T cells；
T cell is stored in the RPIM culture mediums containing 20% people AB serum and 10%DMSO；T cell recovery after, with containing
The T cell medium culture of 30U/ml is stayed overnight；T cell culture medium (T cell culture medium, TCM) is made up of：
X-Vivo15 (Lonza), 5% people AB serum (Valley Biomedical), 55 μM of beta -mercaptoethanols, 10mM N- acetyl L-
Cysteine.
The preparation of the sgRNA and Cas9 albumen of Cas9/RNP mediate T cell gene editings：
Cas9/RNP is made of the sgRNA of target gene and Cas9 albumen two parts for shearing gene；SgRNA is by solid
Surely the crRNA of the tracrRNA and targeting target gene that form are constituted, the target of the crRNA for knocking out PD-1 and LAG3 genes
Expire biochemistry by Shanghai Ji to sequence (NO.1~2 SEQ and No.3~4 SEQ) to synthesize, schematic diagram is shown in Fig. 1；Cas9 is
S.pyogenes Cas9 albumen has one in order to monitor Cas9 albumen and enter core conducive to Cas9 albumen in its c-terminus
A HA labels and two nuclear localization signal (NLS) sequences (NO.5~6 SEQ), are synthesized by Shanghai offshore science and technology.
The Electroporation Transformation of Cas9 RNP and T cell：
After people's CD4+ or CD8+T cell thaws, for 24 hours with TCM cultures；Then trained with the TCM culture mediums containing 30U/ml IL2
It supports, while 48h is stimulated with anti-CD3 (10 μ g/mL) and anti-CD28 (5 μ g/mL)；
The crRNA of chemically synthesized tracrRNA, targeting PD-1 or LAG3 exons 1s are suspended in 10mM Tris-HCl respectively
PH7.4 obtains 80 μM of tracrRNA and 80 μM of crRNA；CrRNA and tracrRNA is according to 1:1 ratio mixing, is placed in 37
DEG C 30min, obtains 40 μM of crRNA:The sgRNA of tracrRNA mixing；By 40 μM of isometric sgRNA and 40 μM of spCas9
Albumen (freezing in 20mM HEPES, 150mM KCl, 10% glycerine, 1mM TCEP) mixes, and obtains 20 μM of Cas9 RNP；CD4
+ or CD8+T cells be suspended in P3 buffer solutions (Amaxa P3Primary Cell Kit) make its cell concentration be~3 ×
105, obtain T cell suspension；3 μ L, 20 μM of Cas9 RNP are added in 20 μ L cell suspensions, mixing；4D-
Nucleofecter (Lonza) programs EH-115 carries out Electroporation Transformation；
Then extraction T cell genomic DNA, and using designed PD-1 primers (5 '-tttcccttccgctcacctc-3 ' and
5 '-cacctacctaagaaccatcctg-3 ') and LAG3 primers (5 '-attccggcctctggtcatcc-3 ' and 5 '-
Ggagaggcttcactaggtgag-3 '), carry out PCR amplification, target fragment purifying recycling；It is detected and is mutated using T7EN1 digestions
Body, 3% agarose gel electrophoresis detect digestion as a result, as shown in Fig. 3 gel electrophoresis figures.By PCR product pMD18T carriers
In, TOP10 competent escherichia coli cells are transformed into, random picked clones are sequenced, alkali after assessment target fragment is cut
Base changes and cutting efficiency.
CAR molecules are built to slow virus carrier pCDH：
CAR molecules are to be sequentially connected in series people EF1 α, CSF2RA Chimerical receptors signal peptide, extracellular antibody using pCDH carriers in the present invention
The screening-gene CopGFP that combined area, hinge area, transmembrane region intracellular signal transduction area are connected with T2A small peptides is prepared, letter
Number domain is OX40-4-1BB-CD3 ζ, and molecular structure is shown in Fig. 2 slow virus pCDH structural schematic diagrams；
First in chemical synthesis CD19 FMC63 nucleic acid fragments to pUC57 carriers, Not I/Xba I double digestions obtain FMC63 pieces
Section；Not I/Xba I double digestion slow virus carriers pCDH；Linear pCDH carriers and FMC63 segments are used into T4 ligase chains
It connects, room temperature 1~2 hour is then transformed into stb13 competent cells, and after being incubated overnight, picking positive monoclonal amplification cultivation is simultaneously
Plasmid, its sequence of sequence verification are extracted, correct plasmid is named as pCDH-EF1 α-CSF2RA-CD19scFv (FMC63)-CD8
Hinge-CD8 α-OX40-4-1BB-CD3 ζ-T2A-CopGFP (hereinafter abbreviated as pCDH-CD19scFv).
19 CAR-T (the PD-1 of CD of PD-1 and LAG3 gene knockouts:LAG3-DKO CAR-T) cell structure：
The packaging of CAR slow virus：Carry plasmid kit extraction pCDH-CD19scFv and slow virus greatly using endotoxin-free first
System assists packaging plasmid pMD2.G and PSPAX2.The day before transfection is by 2.0~3.0*107HEK 293T cells spread to T75 and train
It supports in square vase, for transfecting after cell is grown to 80% or so.293T cell culture mediums are changed to 10ml serum-frees before transfection
Culture medium uses transfection reagentHQ (Polyplus, France) transfects 24 by plasmid co-transfection to 293T cells
Cell culture medium is changed to complete medium DMEM+10%FBS after hour.Turn then to collect supernatant after 48 hours, and is added
15ml complete mediums regather supernatant after 72 hours, discard cell.0.45 μm of membrane filtration of supernatant, 5000g centrifugations
1min removes impurity, and subsequent 40000g centrifuges 2 hours by viral pellet, virus is resuspended with 0.1ml PBS.- 80 DEG C of refrigerators are placed in,
It freezes spare；
PD-1 and LAG3 knocks out T cell transduction：The TCM of 80 μ L preheatings will be added in CD4+ or CD8+ T cells after electroporation
Culture medium, 37 DEG C of 30min；By cell and anti-CD3+CD28 (Invitrogen) antibody magnetic bead with 1:1 ratio mixing, and
And cell concentration is adjusted to 1 × 10 with TCM6/ ml cultivates 2-4h；The slow virus containing CD19 CAR is added, cultivates 24-
36h；Using Flow cytometry GFP expression to detect CAR molecule transduction efficiencies after 72h；Magnetic bead moves after stimulating 4-6 days
It removes；By CD4+PD-1 (-) LAG3 (-) CD19 CAR-T cells and CD8+PD-1 (-) LAG3 (-) CD19 CAR-T cells by etc. it is dense
Degree 1:1 ratio mixes, you can obtains the CD19 CAR-T cells of final PD-1 and LAG3 gene knockouts.
The 19 CAR-T cytoactive detections of CD of PD-1 and LAG3 gene knockouts：
PD-1 and LAG3 is knocked out into CD19 CAR-T cells and expression CD19 (+) PD-1 (-) LAG3 (-), CD19 (+) PD-1 (+)
The tumor cell line K652 of LAG3 (+) is incubated altogether, as a result shows that PD-1 and LAG3 knockout CD19 CAR-T cells can be in CD19
(+) PD-1 (-) LAG3 (-) target cell largely generates IFN-γ in the case of stimulating；But it cannot be with CD19 (+) PD-1
(+) LAG3 (+) target cell generates IFN-γ when being incubated altogether；The T cell of non-CD19 targetings can not be in CD19 (+) target cell simultaneously
Stimulation is lower to generate IFN-γ, as shown in Figure 5.The case study on implementation illustrates that the CAR-T cells of the dual-gene knockouts of PD-1 and LAG3 have
T cell killing ability can be activated under the stimulation of CD19 target spots, and can release PD-1 and LAG- by good targeting
3 immunosuppressor are to immune inhibition.
The ability of the 19 CAR-T cell killing CD19 target cells of CD of PD-1 and LAG3 gene knockouts：
Complete base fluorescein succinimido (CFSE) with 5- to dye target cell, PD-1 (-) LAG3 (-) CD19 CAR-T is thin
Born of the same parents do not knock out the CAR-T cells of PD-1 and LAG3 and K652, Raji cell are mixed by following effect target ratio：05:1,1:1,
2:1,5:1.After 4 hours co-culture, cell PI kits are dyed, while control group is set, the effect in control group
Cell is T lymphocytes, and any effector cell is not added in blank group.Cell killing is detected using flow cytometry.Knot
Fruit sees Fig. 7 A and Fig. 7 B.Regardless of whether knocking out PD-1 and LAG3, CAR-T cells can effectively kill K652 and Raji cells,
But the T cell in control group can not kill cell effectively.It is specific swollen that the case study on implementation illustrates that CD CAR-T have
Cytotoxic effect activity, while also indicating that Electroporation Transformation knocks out PD-1 and LAG3 and do not produced to the killing activity of CAR-T cells
Raw substantive influence.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, any to the equivalent modifications that carry out of the present invention and to replace
In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and repair
Change, all should be contained within the scope of the invention.
Sequence table
<110>The Hangzhou bio tech ltd Rong Ze
<120>A kind of method that Cas9/RNP knocks out T cell PD-1 and LAG3 gene and prepares CAR-T cells
<130> 2018.03.29
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tccggtggtg gtggttctca agtgcaactg caacagagcg gagccgaact ggtgagaccc 480
ggaagcagcg tgaagatcag ctgcaaggct tccggctacg cctttagcag ctactggatg 540
aactgggtga agcagagacc tggacaggga ctggaatgga tcggccagat ttaccctgga 600
gacggcgaca caaactacaa cggcaagttc aagggccaag ctacactgac cgccgacaaa 660
agcagcagca ccgcctatat gcagctgagc ggactgacca gcgaagatag cgctgtgtac 720
ttctgcgcca gaaagaccat cagcagcgtg gtggacttct acttcgacta ctggggacaa 780
ggcaccaccc tgacagtgag cagcgaattc accacgacgc cagcgccgcg accaccaaca 840
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960
cccctggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcgcc 1020
ctgtacctgc tccggaggga ccagaggctg ccccccgatg cccacaagcc ccctggggga 1080
ggcagtttcc ggacccccat ccaagaggag caggccgacg cccactccac cctggccaag 1140
atcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1200
caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1260
tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1320
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1380
agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1440
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1560
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcggatc c 1611
Claims (8)
1. a kind of method for preparing PD-1 and LAG3 and knocking out CD19 CAR-T cells, includes the following steps：
Prepare CD4+/CD8+ T cells；
Prepare and tracrRNA, crRNA and Cas9 albumen for knocking out PD-1 and LAG3 genes；
TracrRNA, crRNA and Cas9 albumen of preparation are turned reagent with electricity to mix, is added in CD4+/CD8+ T cells and carries out
Electrotransformation；
Prepare CD19 Chimeric antigen receptor slow virus；
The slow virus of preparation is used to infect the CD4+/CD8+ T cells after electrotransformation, antiCD3+CD28 antibody to activate T cell
Amplification cultivation is tested by fluorescent screening to obtain the CD19 CAR-T cells that PD-1 and LAG3 is knocked out.
2. method as described in claim 1, wherein CD4+/CD8+ T cells are obtained by EasySeq kits illustratively
Book operates, and is prepared.
3. method as described in claim 1, which is characterized in that the sgRNA for knocking out PD-1 and LAG3 by tracrRNA and
CrRNA two parts form, and the crRNA of wherein PD-1 targets sequence as shown in No.1~2 SEQ ID, and the crRNA of LAG3 targets sequence
Row are as shown in No.3~4 SEQ.
4. method as described in claim 1, which is characterized in that the Cas9 albumen for knocking out PD-1 and LAG3 is external albumen,
There are one HA labels and two nuclear localization signal sequences such as SEQ No.5-6 for c-terminus.
5. the method as described in claim 1, wherein CD19 CAR are to utilize pCDH carriers series connection people EF1 α promoters, CSF2RA
The screening that Chimerical receptor signal peptide, extracellular antigen-binding site, hinge area, transmembrane region intracellular signal transduction area are connected with T2A small peptides
Gene C opGFP is prepared, wherein CSF2RA-CD19 scFv-CD8 α-OX40-4-1BB-CD3 ζ sequences such as SEQ No.7.
6. method as claimed in claim 5, wherein extracellular antigen binding domain is CD19 single-chain antibody scFv FMC63, hinge area
For CD8 Hinge, transmembrane region is CD8 α membrane-spanning domains, and signal transduction area is OX40-4-1BB-CD3 ζ, and wherein OX40 and 4-1BB are
Costimulating factor.
7. a kind of device for being used to prepare PD-1 and LAG3 and knocking out CD19 CAR-T cells, including
CD4+/CD8+ cell preparation systems；
External tracrRNA, crRNA and Ca9 protein systems of Cas9/RNP；
Amaxa Electroporation Transformation kits；
Coupling CD3+CD28 antibody magnetic beads for activating T cell；And
Screening and culturing system, for screening and cultivating the T cell after turning obtaining the CD19 CAR-T that PD-1 and LAG3 are knocked out
Cell.
8. the method that PD-1 and LAG3 knocks out CAR-T is prepared using Cas9/RNP as described in one of claim 1~7, and
The T cell or cell line or its subculture of the separation of acquisition.
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