CN106399375A - Method for constructing CD19 targeting CAR-T (chimeric antigen receptor-T) cells by knocking out PD-1 (programmed death 1) genes by virtue of CRISPR/Cas9 - Google Patents

Method for constructing CD19 targeting CAR-T (chimeric antigen receptor-T) cells by knocking out PD-1 (programmed death 1) genes by virtue of CRISPR/Cas9 Download PDF

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CN106399375A
CN106399375A CN201610796334.3A CN201610796334A CN106399375A CN 106399375 A CN106399375 A CN 106399375A CN 201610796334 A CN201610796334 A CN 201610796334A CN 106399375 A CN106399375 A CN 106399375A
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代红久
孙彬
李晓亮
张征
赵旭东
熊波
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Nanjing Kaidi Biotechnology Co Ltd
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Priority to PCT/CN2017/076513 priority patent/WO2018040537A1/en
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Abstract

The invention discloses a method for constructing novel CAR-T (chimeric antigen receptor-T) cells capable of targeting CD19 by knocking out PD-1 (programmed death 1) genes by virtue of a CRISPR/Cas9 technology. According to the method, the PD-1 genes are knocked out by synchronously infecting the CAR-T cells by virtue of virus liquid carrying human PD-1 gene sgRNA and Cas9-HF nuclease through the CRISPR/Cas9 technology, so that the novel CAR-T cells capable of targeting the CD19 are obtained. With the application of the novel CAR-T cells, a preparation for treating tumors can be prepared. The preparation method is simple in steps, and the obtained CAR-T cells have a high killing rate on tumor cells.

Description

Knock out people PD-1 gene constructed targeting CD19CAR-T cell using CRISPR/Cas9 Method
Technical field
The present invention relates to genetic engineering, immunology and oncology, especially relate to one kind and utilize CRISPR/Cas9 skill Art knocks out that people PD-1 is gene constructed can the method for targeting CD19 novel C AR-T cell and its application.
Background technology
All the time, cancer, as the number one killer of human health, causes the concern of countless scientists.Although operation is controlled The appearance of the therapeutic modalities such as treatment, chemotherapy, radiotherapy improves the state of an illness of cancer patient, but global countless cancer patient is still subject to The torment of cancer.2013, scientists were announced successfully to cure late period acute lymphoblastic using a kind of new immune cell therapy Chronic myeloid leukemia, brings new dawn to whole world cancer patient.This utilization Chimeric antigen receptor (Chimeric Antigen receptor, CAR) T cell immunotherapy method, specifically can identify tumor associated antigen, be finally reached Cure the purpose of tumor.
CAR-T immunotherapy is most to overturn one of emerging technology of begetting power at present, and American-European and countries in Asia are all long-pending The clinical trial of this therapy is carried out in pole, and the wherein U.S. carries out clinical trial project at most, reaches 41, occupies global CAR-T and treat The 74.5% of method clinical trial.Hospital of the University of Pennsylvania, The Children's Hospital of Philadelphia, american cancer academy (NCI), Fred Hutchinson DKFZ, souvenir Si Long Caitlin Cancer center and Seattle children's hospital etc. are to carry out CAR- earliest The research institution of T cell therapy.Emerging the world biopharmaceutical company such as Juno, Kite, Celgene, Cellectis, Bluebird and the tradition world pharmacy giant such as Novartis, GlaxoSmithKline PLC, Pfizer, Johnson ＆ Johnson etc. have all put into substantial contribution and people Power, in the research and development of CAR-T therapy, promotes the therapy of this great prospect to enter into clinic as early as possible, thus benefiting cancer and tumor Patients Patients (A service of the U.S.National Institute of Health.http:// clinicaltrials.gov).
The early studies in man that CAR-T cell therapy is late carried out in Refractory Leukemia and Lymphoma has shown Very soul-stirring result is shown.University of Pennsylvania's research worker October in this year is in the top medical journal in the world《New English Glan medical journal》On clinical test results delivering show, receive CTL019 infusion (targeting CD19 antigen CAR-T therapy) 30 recurrents or intractable ALL experimenter in, treatment has 27 patients to obtain complete incidence graph for latter 1 month, Wherein even include 15 patients having accepted Bone Marrow Stem Cells Transplantation, when 6 months, no survival rate is 67% (20 repeatedly People), overall survival is 78% (23 people).Still continued complete remission (Grupp SA when in addition also having 1 patient follow-up in 2 years et al,N Engl J Med 2013,368(16):1509-18).The clinical trial that NCI carries out also presents good As a result, this tests into having organized 20 ALL infants, and after accepting CAR-T cell therapy, 14 have reached complete incidence graph；12 trouble Immature leukaemia has been can't detect, wherein 10 receive stem cell transplantation again, so far no cancer life in the bone marrow of youngster Deposit.Result of study is it is also shown that CAR-T cell therapy can help the unresponsive patient of chemotherapy successfully excessively to arrive bone marrow transplantation controls Treat.Commemorate this Long Kaitelin Cancer center and also achieve similar result with 1 clinical trial phase that NCI carries out in adult patient (Mazzarella L.2013American Society of Haematology meeting[J]2014,8:390).
In addition to leukemia, CAR-T therapy also show gratifying curative effect to lymphoma, and the CAR-T cell of NCI leader faces 15 Adult human subjects have been recruited in bed test, and the overwhelming majority (9) therein is suffering from late period diffusivity large B cell lymphoid tumor (DLBCL).According to the colleague of doctor Kochenderfer and NCI, the Most patients of this clinical trial have all reached alleviation, 4 acquisition complete incidence graph in 7 appreciable DLBCL experimenters, the persistent period was from 9 months to 22 months (Kochenderfer JN et al,Blood 2012,119(12):2709-20).
But cancerous cell have also been developed a set of escape mechanism of itself in long-term evolutionary process, can escape T thin The destruction of born of the same parents, and then unbridled expand.Research worker finds that there is important " joint " molecule on T cell surface, they It is referred to as " programmed death receptor -1 " or PD-1 (Programmed Death 1), under normal condition, it can shrewd " enemy I ".But when " PD-L1 " (Programmed Death Like 1) in cancer cell surfaces, situation just becomes.PD-L1 Albumen is the part of PD-1, it two once combine will to T cell transmit a kind of negative regulation signal, inducing T cell enters in meditation Breath state, allows its None- identified cancerous cell, and so that T cell its own amplification is reduced or apoptosis, and the immunity effectively releasing body is anti- Should, therefore cancerous cell can without lifting an eyebrow just " passing through the hall into the inner chamber ".It is noted that many cancer cell surfaces all exist this PD-L1 albumen, including breast carcinoma, pulmonary carcinoma, gastric cancer, intestinal cancer, the esophageal carcinoma, ovarian cancer, cervical cancer, renal carcinoma, bladder cancer, cancer of pancreas, Glioma, melanoma etc..Then, scientists start to think the combination how stoping PD-1 and PD-L1, help T Cell activity recovery, the normal threat identifying cancerous cell, and then attack cancerous cell with all strength.Ge great world pharmacy giant is numerous and confused at present It is devoted to clinical research and the exploitation of PD-1 inhibitor and antibody drug.
Up-to-date quick, easy, efficient, the selectively targeted knockout gene of genome editing technique CRISPR-Cas9 energy, base In important function during tumor immune escape for the PD-1 molecule, we utilize CRISPR/Cas9 in the CAR-T of targeting CD19 By PD-1 gene knockout in cell, the escape and the inhibitory action that block cancerous cell to T cell are so that CAR-T cell can more be increased The identification of effect ground and attack cancerous cell.
Content of the invention
First purpose of the present invention be to provide a kind of knock out that people PD-1 is gene constructed using CRISPR/Cas9 technology can The method of targeting CD19 novel C AR-T cell.
Second object of the present invention is to provide a kind of novel C AR-T cell.
Third object of the present invention is to provide the application of above-mentioned novel C AR-T cell.
For realizing above-mentioned first purpose, the present invention adopts herein below：
A kind of utilization CRISPR/Cas9 technology knock out people PD-1 gene constructed can targeting CD19 novel C AR-T cell side Method, the method utilizes CRISPR/Cas9 technology by using the virus of carrier PD-1 gene sgRNA and Cas9-HF nuclease Liquid synchronously infects CAR-T cell to knock out people's PD-1 gene, and obtaining can targeting CD19 novel C AR-T cell；Wherein, people PD-1 base People's PD-1 gene extron shown in SEQ ID No.22 to SEQ ID No.26 for the mRNA sequence of cause forms, targeting sgRNA People's PD-1 gene target sgRNA sequence PD-1-Tn shown in SEQ ID No.27 to SEQ ID No.32 for the sequence forms； Cas9-HF-NLS-HA nucleotide sequence shown in sequence table SEQ ID No.33 to SEQ ID No.36 for the Cas9-HF nuclease Composition.
Further, the method comprising the steps of：
1) build carrier's PD-1 gene sgRNA oligonucleotide viral vector
Target sequence on PD-1 gene for the described sgRNA meets the series arrangement rule of 5 '-N (20) NGG, and described Target sequence on PD-1 gene for the sgRNA is located at the exon of gene；Target sequence on PD-1 gene for the described sgRNA is unique , and people PD-1 shown in SEQ ID No.27 to SEQ ID No.32 for the target site sequence on PD-1 for the described sgRNA Gene target sgRNA sequence PD-1-Tn forms；Synthesis sgRNA double strand oligonucleotide, and anneal, slow with linearizing afterwards Viral vector connects, and obtains and carries PD-1 gene sgRNA oligonucleotide viral vector；
2) build and carry Cas9-HF nuclease viral vector
The mutation in 1-4 site is carried out on Cas9 nucleic acid enzyme amino acid sequence, including N497A, R661A, Q695A successively And Q926A, obtain high-fidelity Cas9-HF nuclease, clone high-fidelity Cas9-HF into slow virus carrier afterwards, obtain Cas9- HF nuclease viral vector；
3) transduction T cell
The virus liquid carrying PD-1 gene sgRNA and Cas9-HF nuclease is synchronously infected by the value according to MOI=1-10 CAR-T cell, completes to knock out people's PD-1 gene, obtains the T cell knocking out PD-1 gene and expression targeting CD19CAR molecule, I.e. novel C AR-T cell.
For realizing above-mentioned second purpose, the present invention adopts herein below：
A kind of novel C AR-T cell, in this T cell, PD-1 gene is knocked and expresses the CAR molecule of targeting CD19, should Sequence shown in sequence table SEQ ID No.1 to SEQ ID No.20 for the CAR molecule forms；
Wherein, SEQ ID No.1 to SEQ ID No.2 is homing sequence；
SEQ ID No.3 to SEQ ID No.10 is scFv sequence；
SEQ ID No.11 to SEQ ID No.12 is CD8 hinge region；
SEQ ID No.13 to SEQ ID No.14 is CD8 transmembrane region；
SEQ ID No.15 to SEQ ID No.16 is 4-1BB intracellular domain；
SEQ ID No.17 to SEQ ID No.20 is CD3 ζ domain.
Described CAR molecular cloning is entered slow virus carrier, builds the total length CAR sequence expression cassette of single encoder block, utilize EF1alpha promoter (sequence table SEQ ID No.21) is expressed.
For realizing above-mentioned 3rd purpose, the invention also discloses above-mentioned can prepare targeting CD19 novel C AR-T cell For treating the application in the preparation of tumor, described tumor is CD19 positive tumor cell, CD20 positive tumor cell, CD30 sun Property tumor cell, CD33 positive tumor cell, CD138 positive tumor cell, preferably CD19 positive tumor cell.
The present invention has advantages below：
Present invention application CRISPR/Cas9 technology, by the people's PD-1 gene knockout in CAR-T cell, blocks tumor cell pair The escape of T cell and inhibitory action, obtain can targeting CD19 novel C AR-T cell, this novel C AR-T cell can more effectively target To attacking tumor cell, can be used to prepare the preparation for treating tumor, the especially preparation of CD19 positive tumor cell.This The killing rate of the CAR-T cells against tumor cells that bright preparation method step is simple, obtain is high.
Brief description
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in further detail.
Fig. 1 is that PD-1 knocks out CAR design drawing.
Fig. 2 is the expression of KD-019CAR molecule.
Fig. 3 is PD-1 and Cas9-HF protein expression analysis.
Fig. 4 is the killing rate to Raji cell for the KD-019CAR-T cell.
Specific embodiment
In order to be illustrated more clearly that the present invention, with reference to preferred embodiment, the present invention is described further.Ability Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, and should not limit this with this The protection domain of invention.
Embodiment
A kind of novel C AR-T cell of the gene constructed targeting CD19 of utilization CRISPR/Cas9 specific knockdown people PD-1 Method, comprises the following steps：
1) carry the structure of PD-1 gene sgRNA oligonucleotide viral vector
Target sequence on PD-1 gene for the sgRNA meets the series arrangement rule of 5 '-N (20) NGG, and sgRNA is in PD-1 Target sequence on gene is located at the exon of gene；Target sequence on PD-1 gene for the sgRNA is unique, and sgRNA exists People's PD-1 gene target sgRNA sequence shown in SEQ ID No.27 to SEQ ID No.32 for the target site sequence on PD-1 PD-1-Tn forms；
People's PD-1 gene extron shown in SEQ ID No.22 to SEQ ID No.26 for the mRNA sequence of people's PD-1 gene Son composition；
Synthesis sgRNA double strand oligonucleotide (Sangon Biotech's synthesis), mix after Anneal 5 minutes for 95 DEG C, be connected with linearizing slow virus carrier lentiGuide-Puro afterwards, obtain and carry PD-1 gene SgRNA oligonucleotide viral vector；
2) carry the structure of Cas9-HF nuclease viral vector
The mutation in 1-4 site is carried out on Cas9 nucleic acid enzyme amino acid sequence, including N497A, R661A, Q695A successively And Q926A, obtain high-fidelity Cas9-HF nuclease, the Cas9- shown in sequence table SEQ ID No.33 to SEQ ID No.36 HF-NLS-HA nucleotide sequence forms, and clones high-fidelity Cas9-HF into slow virus carrier lentiCas9-Blast afterwards, obtains Obtain Cas9-HF nuclease viral vector；
3) transduction T cell
The virus liquid carrying PD-1 gene sgRNA and Cas9-HF nuclease is synchronously infected target by the value according to MOI=1-10 To the CAR-T cell (being designated as KD-019CAR-T cell) of CD19, complete knock out people's PD-1 gene, obtain knock out PD-1 gene with And the CAR molecule T cell of expression targeting CD19, that is, obtain the novel C AR-T cell after knocking out gene.
This special CAR molecule, is designated as KD-019CAR molecule, shown in sequence table SEQ ID No.1 to SEQ ID No.20 Sequence composition；Wherein, SEQ ID No.1 to SEQ ID No.2 is homing sequence；SEQ ID No.3 to SEQ ID No.10 For scFv sequence；SEQ ID No.11 to SEQ ID No.12 is CD8 hinge region；SEQ ID No.13 to SEQ ID No.14 is CD8 transmembrane region；SEQ ID No.15 to SEQ ID No.16 is 4-1BB intracellular domain；SEQ ID No.17 to SEQ ID No.20 is CD3 ζ domain.CAR molecular cloning is entered slow virus carrier, builds the total length CAR sequence expression of single encoder block Frame, is expressed using EF1 alpha promoter (sequence table SEQ ID No.21).
Fig. 1 is that PD-1 knocks out CAR design drawing.
External activity analysis is carried out to the KD-019CAR-T cell obtaining
1) T cell separation and Culture
Fresh peripheral blood lymphocytes (PBMC, peripheral blood is separated by density gradient centrifugation mononuclear cell)；Using the paramagnetic beads being coupled anti-CD3 and anti-CD28 antibody (Dynabeads ClinExVivo CD3/CD28, Invitrogen, Camarillo, CA, USA) is enriched with CD3+ cell, magnetic bead It is 3 with cell proportion：1；It is (10-30) × 10 that cell is diluted to TNC (total nucleated cell) concentration6/ mL, in training It is incubated 2-3 hour with magnetic bead altogether in room temperature in foster ware；Using Magnetic particles concentrator (MPC) (Invitrogen) it is enriched with CD3+ cell；Cell containing CD3+ is resuspended in culture fluid (OpTmizer in culture dishTMCTSTM T-Cell Expansion SFM, Life Technologies) in, final concentration of 1 × 106cells/mL.In 37 DEG C, 5%CO2 Cultivate 2 days in incubator.
2) preparation of virus liquid
KD-019CAR molecule, people PD-1 gene sgRNA and high-fidelity Cas9-HF nuclease DNA fragmentation are cloned respectively Enter slow viruss shuttle vector；Package carrier is carried out according to Lenti-X Packaging Single Shots (Takara) description Preparation.All carriers carry out high-purity endotoxin-free extracting, cotransfection Lenti-X 293T cell, and after transfection, 6h is replaced by completely Culture medium, after culture 48h and 72h, collects the cell supernatant rich in lentiviral particle, vial supernatant passes through ultracentrifugation respectively Concentrating virus.
3) knock out the preparation of the KD-019CAR-T cell of people's PD-1 gene
Culture T cell to concentration is (1-2) × 106/ml；Cell in good condition is inoculated into 24 orifice plates, makes cell dense Spend for 1 × 105/ ml cell, inoculating cell quantity is slightly different because of the speed of growth of cell, is typically to ensure that second day and carries out When virus infection, cell confluency rate is between 50-70%；Appropriate was carried KD-019CAR molecule and people in second day After the virus liquid of PD-1 gene sgRNA together adds Tissue Culture Flask with the virus liquid carrying high-fidelity Cas9-HF nuclease, envelope Good mouth, after putting into straight angle centrifuge, low speed (500g-1000g/min) is centrifuged 1h, is then placed in 37 DEG C of cultures in incubator.Sense The functional experiment of next step can be carried out after contaminating latter 48 hours.
4) flow cytometry is utilized to detect the expression of CAR molecule
Centrifuge cell, is resuspended in FACS liquid with PBS twice afterwards (containing 0.1% Hydrazoic acid,sodium salt and 0.4%BSA PBS)；By Biotin-labeled polyclonal goat anti-mouse-F (ab) 2 antibody (anti-Fab, Jackson Immunoresearch) add in cell suspension, 4 DEG C are incubated 30 minutes；Cleaning cell twice after, add phycoerythrin (PE)-labeled streptavidin (BD Pharmingen), room temperature 30 minutes；BD FacsCanto II is used for obtaining dye Cytochrome, FlowJo is used for analysis result.As shown in Fig. 2 matched group is the T cell of infection empty carrier virus liquid, almost detect Expression less than CAR molecule；And in the T cell of infection KD-019 CAR molecule virus liquid, the expression rate of CAR molecule reaches 44.7%.
5) checking of the expression of Cas9-HF and PD-1 gene knockout
Have detected the expression of Cas9-HF nuclease first with western blotting.Cell warp after infection virus Cross centrifugation, cleaning cracks 1 hour in the RIPA lysate containing protease inhibitor later, the cracking obtaining after high speed centrifugation Supernatant is carried out after being mixed with 4x LDS sample-loading buffer point in 10%Bis-Tris pre-prepared colloid (Life technologies) From then by electricity turn by protein delivery to pvdf membrane.Pvdf membrane is closed one hour in the TBST containing 5% defatted milk powder, 1：4 DEG C of overnight incubation in 1000 rabbit anti-HA antibody.Clean film 3 times with TBST, use 1：The goat anti-rabbit igg of 5000 coupling HRP Two anti-incubated at room 1 hour.After being incubated 5 minutes with ECL Plus luminescent solution, expose in Tanon 9500 machine.As Fig. 3 institute Show, for the comparison T cell not infecting virus liquid, have very in the T cell of infection Cas9-HF nuclease virus liquid Strong Cas9-HF protein expression.
Next western blotting is utilized to detect expression in CAR-T cell for the PD-1 albumen.Infection disease Cell after poison cracks 1 hour after centrifugation, cleaning in the RIPA lysate containing protease inhibitor, high speed centrifugation The cracking supernatant obtaining afterwards mix with 4x LDS sample-loading buffer after in 10%Bis-Tris pre-prepared colloid (Life Technologies carry out in) separating, then pass through electricity and turn on protein delivery to pvdf membrane.Pvdf membrane is containing 5% defatted milk Close one hour in the TBST of powder, 1：4 DEG C of overnight incubation in 1000 rabbit anti-human PD-1 antibody.Clean film 3 times with TBST, use 1：The goat anti-rabbit igg two anti-incubated at room of 5000 coupling HRP 1 hour.After being incubated 5 minutes with ECL Plus luminescent solution, in Tanon Expose in 9500 machines.As shown in figure 3, relative comparison group, the expressing quantity of PD-1 significantly decreases in knockout group.
6) KD-019CAR-T cell killing test
Assess killing of KD-019CAR-T cells against tumor cells Raji using 7-AAD/CFSE cytotoxicity test test kit Overstrain.After CSFE marked tumor cell, with every hole 2 × 104Cell be laid in culture plate, respectively with 10：1、3：1 and 1：1 KD-019 CAR-T cell is added in tumor cell by ratio, and culture is centrifuged after 20 hours and removes supernatant, after cell precipitation cleaning Dyeed with 7AAD, BD FacsCanto II is used for obtaining staining cell, FlowJo is used for analysis result.As shown in figure 4, relatively In the matched group not infecting virus and infection empty carrier virus liquid, KD-019CAR-T cell has to target cell Raji and significantly kills Wound acts on, and knockout people's PD-1 gene can significantly improve this killing rate in KD-019CAR-T cell.
The CAR molecule of the present invention can the positive tumor cell of selectively targeted identification CD19.The Cas9-HF nucleic acid of the present invention Enzyme can effectively reduce the phenomenon of missing the target in CRISPR/Cas9 genome editing process.Knock out PD-1 gene using the inventive method The targeting CD19 novel C AR-T cell obtaining afterwards can be used for assessing the target killing power to leukaemia cancer cell, to normal cell Side effect, and be used for all kinds of hematologic cancers such as leukemia and other lymphatic cancers, lymphoma.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not right The restriction of embodiments of the present invention, for those of ordinary skill in the field, also may be used on the basis of the above description To make other changes in different forms, all of embodiment cannot be exhaustive here, every belong to this Obvious change that bright technical scheme is extended out or change the row still in protection scope of the present invention.

Claims (6)

1. a kind of utilization CRISPR/Cas9 technology knock out people PD-1 gene constructed can targeting CD19 novel C AR-T cell method, It is characterized in that, methods described utilizes CRISPR/Cas9 technology by using carrier PD-1 gene sgRNA and Cas9-HF core The virus liquid of sour enzyme synchronously infects CAR-T cell to knock out people's PD-1 gene, and obtaining can targeting CD19 novel C AR-T cell；Its In, the mRNA sequence of the people's PD-1 gene people's PD-1 gene extron subgroup shown in SEQ ID No.22 to SEQ ID No.26 Become, people's PD-1 gene target sgRNA sequence PD- shown in SEQ ID No.27 to SEQ ID No.32 for the targeting sgRNA sequence 1-Tn forms；Cas9-HF-NLS-HA shown in sequence table SEQ ID No.33 to SEQ ID No.36 for the Cas9-HF nuclease Nucleotide sequence forms.
2. according to claim 1 a kind of knock out that people PD-1 is gene constructed using CRISPR/Cas9 technology can targeting CD19 The method of novel C AR-T cell is it is characterised in that methods described is to knock out people's PD-1 gene structure using CRISPR/Cas9 technology Build the novel C AR-T cell of targeting CD19.
3. according to claim 2 a kind of knock out that people PD-1 is gene constructed using CRISPR/Cas9 technology can targeting CD19 The method of novel C AR-T cell is it is characterised in that the method comprising the steps of：
1) build carrier's PD-1 gene sgRNA oligonucleotide viral vector
Target sequence on PD-1 gene for the described sgRNA meets the series arrangement rule of 5 '-N (20) NGG, and described sgRNA exists Target sequence on PD-1 gene is located at the exon of gene；Target sequence on PD-1 gene for the described sgRNA is unique, and People's PD-1 gene target shown in SEQ ID No.27 to SEQ ID No.32 for the target site sequence on PD-1 for the described sgRNA SgRNA sequence PD-1-Tn forms；Synthesis sgRNA double strand oligonucleotide, and anneal, afterwards with linearizing slow virus carrier Connect, obtain and carry PD-1 gene sgRNA oligonucleotide viral vector；
2) build and carry Cas9-HF nuclease viral vector
The mutation in 1-4 site is carried out successively on Cas9 nucleic acid enzyme amino acid sequence, including N497A, R661A, Q695A and Q926A, obtains high-fidelity Cas9-HF nuclease, clones high-fidelity Cas9-HF into slow virus carrier afterwards, obtains Cas9-HF Nuclease viral vector；
3) transduction T cell
The virus liquid carrying PD-1 gene sgRNA and Cas9-HF nuclease is synchronously infected targeting by the value according to MOI=1-10 CD19 people's CAR-T cell, completes to knock out people's PD-1 gene, obtains the T knocking out PD-1 gene and expression targeting CD19CAR molecule Cell, i.e. novel C AR-T cell.
4. a kind of novel C AR-T cell it is characterised in that in this T cell PD-1 gene be knocked and express targeting CD19's CAR molecule, sequence shown in sequence table SEQ ID No.1 to SEQ ID No.20 for the described CAR molecule forms；
Wherein, SEQ ID No.1 to SEQ ID No.2 is homing sequence；
SEQ ID No.3 to SEQ ID No.10 is scFv sequence；
SEQ ID No.11 to SEQ ID No.12 is CD8 hinge region；
SEQ ID No.13 to SEQ ID No.14 is CD8 transmembrane region；
SEQ ID No.15 to SEQ ID No.16 is 4-1BB intracellular domain；
SEQ ID No.17 to SEQ ID No.20 is CD3 ζ domain.
5. described method as arbitrary in claim 1-3 prepare, as claimed in claim 4 can targeting CD19 novel C AR- T cell is used for the application in the preparation treating tumor in preparation, and described tumor is CD19 positive tumor cell, CD20 positive tumor Cell, CD30 positive tumor cell, CD33 positive tumor cell, CD138 positive tumor cell.
6. application according to claim 5, described tumor is CD19 positive tumor cell.
CN201610796334.3A 2016-08-31 2016-08-31 Method for constructing CD19 targeting CAR-T (chimeric antigen receptor-T) cells by knocking out PD-1 (programmed death 1) genes by virtue of CRISPR/Cas9 Pending CN106399375A (en)

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