CN113005092A

CN113005092A - Preparation method and application of PD1 knockout LMP1 targeted CAR-T cell

Info

Publication number: CN113005092A
Application number: CN202110101793.6A
Authority: CN
Inventors: 廖兴华; 王君; 张慧敏; 项园; 李佳蓬; 李慧; 张同存
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE; Wuhan University of Science and Technology WHUST
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-06-22

Abstract

A method for preparing a PD1 knockout LMP1 targeted CAR-T cell, comprising the steps of: s1, constructing PD1 gene sgRNA plasmid pGL3-U6-hPD1-sgRNA and LMP1-CAR cell plasmid LMP1-CAR PiggyBac Transposon, mixing pGL3-U6-hPD1-sgRNA and LMP1-CAR PiggyBac Transposon plasmid with an electrotransfer buffer solution to prepare an electrotransfer mixed solution, and transfecting PBMC cells by using the electrotransfer mixed solution to obtain transfected PBMC cells; s2, transferring the transfected PBMC cells into a T cell culture medium, activating CD3 positive T cells by using magnetic beads, and culturing and amplifying the transfected PBMC cells to obtain the PD1 knocked-out LMP1-CAR-T cells. The invention adopts the combined treatment of targeted LMP1 CAR-T cell combined knock-out PD1 to knock out PD1 of the T cell of a patient, correct immune escape, recover the immune function of the patient and enhance the effect of CAR-T cell attacking lymphoma. The application of the compound in preparing EVB lymphoma diagnostic reagents or therapeutic drugs has important clinical significance.

Description

Preparation method and application of PD1 knockout LMP1 targeted CAR-T cell

Technical Field

The invention relates to the field of medical biology, in particular to a preparation method and application of PD1 knockout LMP1 targeted CAR-T cells.

Background

EBV viruses are important oncogenic viruses, which initiate a series of lymphoid malignant clones that are difficult to treat and are susceptible to relapse. CAR-T (chimeric antigen receptor T cell) treatment is an important method for treating refractory recurrent hematological tumors at present, and very good curative effect data are obtained aiming at B cell acute leukemia and lymphoma. However, there are some problems such as off-target effects due to lack of tumor specific target antigens and severe CRS (cytokine release syndrome), and therefore the selection of targets is an important research content for CAR-T cell therapy.

Tumor immune escape in patients at the end stage of lymphoma, mainly manifested by up-regulation of patient T cell PD1 molecule and up-regulation of lymphoma PDL1, resulted in the failure of approximately 30% of patients to obtain sufficient numbers and functions of autologous T cells to undergo CAR-T cell therapy. The majority of EBV lymphomas have up-regulation of PD1 and PDL1 due to LMP1 regulation, so that knockout T cell PD1 corrects tumor immune escape and is critical for CAR-T cell therapy in patients at the end of lymphoma.

Disclosure of Invention

In view of the above, the invention provides a preparation method and application of a PD 1-knocked LMP 1-targeted CAR-T cell capable of specifically knocking out PD1 without generating serious CRS.

The technical scheme of the invention is realized as follows: the invention provides a preparation method and application of PD1 knockout LMP1 targeted CAR-T cells.

A method for preparing a PD1 knockout LMP1 targeted CAR-T cell, comprising the steps of:

s1, constructing PD1 gene sgRNA plasmid pGL3-U6-hPD1-sgRNA and LMP1-CAR cell plasmid LMP1-CAR PiggyBac Transposon, mixing pGL3-U6-hPD1-sgRNA and LMP1-CAR PiggyBac Transposon plasmid with an electrotransfer buffer solution to prepare an electrotransfer mixed solution, and transfecting PBMC cells by using the electrotransfer mixed solution to obtain transfected PBMC cells;

s2, transferring the transfected PBMC cells into a T cell culture medium, activating CD3 positive T cells by using magnetic beads, and culturing and amplifying the transfected PBMC cells to obtain the PD1 knocked-out LMP1-CAR-T cells.

Based on the above technical solution, preferably, the PD1 gene sequence of the pGL3-U6-hPD1-sgRNA plasmid is selected from one of 5' -GGN (19) GG, 5' -GN (20) GG and 5' -N (21) GG.

On the basis of the technical scheme, preferably, the construction method of the pGL3-U6-hPD1-sgRNA plasmid comprises the following steps:

s1, adding CCGG to 5 'of sgRNA nucleotide chain sequence of PD1 gene to synthesize forward oligonucleotide, and adding CACG to 5' of corresponding complementary chain sequence to synthesize reverse oligonucleotide;

s2, performing paired denaturation and annealing on the synthesized 1 pair of complementary sgRNA oligonucleotides to form double-stranded sgRNA oligonucleotides which can be connected into a U6 eukaryotic expression vector;

s3, connecting the annealed sgRNA oligonucleotide double strand with a linearized pGL3-U6-sgRNA plasmid to obtain a pGL3-U6-hPD1-sgRNA plasmid.

On the basis of the technical scheme, preferably, the construction method of the LMP1-CAR PiggyBac Transposon plasmid comprises the following steps:

s1, connecting the light chain of anti-LMP1 single-chain antibody, the heavy chain of anti-LMP1 single-chain antibody, CD8 alpha hinge region, CD28 transmembrane region and intracellular region, 4-1BB intracellular region and CD3 zeta intracellular region in turn, and synthesizing gene sequence segment;

s2, inserting the gene sequence fragment into a Piggybac Transposon vector, and extracting plasmids by using a column centrifugation method to obtain the plasmid LMP1-CARPiggyBac Transposon capable of expressing LMP1 target point CAR-T.

Based on the above technical solution, preferably, the light chain of the anti-LMP1 single-chain antibody in step S1 further includes a signal peptide, and a light-heavy chain spacer region is further included between the light chain of the anti-LMP1 single-chain antibody and the heavy chain of the anti-LMP1 single-chain antibody.

Based on the above technical solution, preferably, the magnetic beads are magnetic beads coupled with CD3/CD28 antibodies.

On the basis of the above technical scheme, preferably, the PBMC cell preparation method is as follows:

s1, collecting peripheral blood, diluting anticoagulated blood with PBS, adding into a centrifuge tube filled with lymphocyte separation liquid, and slowly rising and slowly falling for centrifugation;

s2, sucking the centrifuged white membrane layer, transferring the white membrane layer into a centrifuge tube, adding PBS, slowly lifting, slowly lowering, centrifuging, discarding the supernatant, retaining the precipitate, and repeating the steps of adding PBS and centrifuging for 2 times to obtain PBMC.

Based on the technical scheme, preferably, pGL3-U6-hPD1-sgRNA plasmid, pST1374-NLS-flag-Cas9-ZF plasmid, LMP1-CAR PiggyBac Transposon plasmid and Super PiggyBac Transposase plasmid are mixed with an electrotransfection buffer solution in a T cell nuclear transfection kit to obtain an electrotransfection mixed solution containing the four plasmids, and 1-2 × 107 PBMC cells are suspended by the electrotransfection mixed solution for electrotransformation.

Based on the above technical solution, preferably, the T cell culture medium in step S2 is RPMI-1640 medium containing 10% FBS.

Still further preferably, the application of the PD1 knockout LMP1 targeted CAR-T cell in preparing EVB lymphoma diagnostic reagents or therapeutic drugs is also included.

Compared with the prior art, the preparation method and the application of the PD1 knockout LMP1 targeted CAR-T cell have the following beneficial effects:

(1) the invention adopts the combined treatment of targeted LMP1 CAR-T cell combined knock-out PD1 to knock out PD1 of the T cell of a patient, corrects immune escape, recovers the immune function of the patient, enhances the effect of CAR-T cell attacking lymphoma, and has important clinical significance in the application of preparing EVB lymphoma diagnostic reagents or therapeutic drugs.

(2) The PD1-KO-LMP1-CAR-T cell provided by the invention can be specifically combined with tumor cells, and Hodgkin lymphoma cells infected by EB virus with specific target expression can promote the expansion of the T cells in a patient body, generate a killing effect on the tumor cells, and avoid accidental injury aiming at B cells or T cells.

(3) Compared with LMP1-CAR-T cells and T cells, the PD1-KO-LMP1-CAR-T cells provided by the invention have a very high killing effect, and when the effective-target ratio is 1:10, the killing efficiency of the PD1-KO-LMP1-CAR-T cells can reach 81.5%.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the effect of the PD1 knockout of LMP1-CAR-T cells in example four of the present invention;

FIG. 2 is a TNF- α cytokine profile of PD1-KO-LMP1-CAR-T cells of example five of the present invention, with different capital letters indicating that the inter-treatment differences reached very significant levels (P < 0.01);

FIG. 3 is a cytokine profile of IL-2 from PD1-KO-LMP1-CAR-T cells of example five of the invention, with different capital letters indicating that the inter-treatment differences reached very significant levels (P < 0.01).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

EXAMPLE one PBMC cell preparation

Peripheral blood of a healthy person (or patient) is collected by an anticoagulation tube. Diluting anticoagulated blood (1: 1) with PBS, slowly adding into 50ml centrifuge tube containing equal volume of lymphocyte separation liquid (Ficoll), and slowly rising and falling under centrifugal force of 350g for 30 min;

after the centrifugation is finished, carefully absorbing the tunica albuginea layer above the lymphocyte separation liquid, transferring the tunica albuginea layer into a new 50ml centrifuge tube, adding PBS, slowly rising and falling for centrifugation for 15min at the centrifugal force of 300g, discarding the supernatant, and keeping the cell sediment at the bottom of the centrifuge tube; adding PBS again, slowly rising and slowly falling under the centrifugal force of 160g for centrifugation for 15min, and discarding the supernatant; and finally adding PBS, slowly rising and slowly falling under the centrifugal force of 300g for centrifugation for 10min, and removing supernatant to obtain PBMC.

EXAMPLE two preparation of CAR-T cells

1. Plasmid construction

1.1 construction of the plasmid pGL3-U6-hPD1sg

1.1.1 design of sgrnas targeting PD1 gene:

since in vitro transcription is not used, it is only made in the manner of constructing a common vector. Therefore, unless otherwise specified, the sgRNA sequence in this context refers to the sgRNA-corresponding DNA sequence.

(1) The PD1 gene may be selected from the 5'-GGN (19) GG sequence, or the 5' -GN (20) GG or the 5'-N (21) GG sequence may be excluded if the 5' -GGN (19) GG sequence is not present.

(2) The sgRNA targets a site on the PD1 gene at an exon of the gene.

(3) The sgRNA targets sites on the PD1 gene on different consensus exons of various splicing forms.

(4) Whether the target sequence of the sgRNA is unique is determined in the UCSC database using BLAT or in the NCBI database using BLAST.

1.1.2 selection of sgrnas targeting PD1 gene:

(1) the gene can not be too close to the ATG initiator, so that the situation that another downstream ATG starts after the transcription to generate a truncated gene form is prevented, and the complete inactivation of the gene cannot be ensured;

(2) the targeting site of the sgRNA on the PD1 gene is located in the first half of the entire gene, particularly preferably in the functional domain of the gene;

(3) sites paired at a certain distance (10-30 bp) are selected. This is beneficial for the formation of specific fragment deletion and also for the reduction of off-target effects;

1.1.3 construction of oligonucleotide double strands of sgRNA

Depending on the sgRNA chosen, adding CCGG at its 5 'end gives a Forward oligonucleotide (Forward oligo) (if the sequence itself already has 1 or 2 Gs at the 5' end, the corresponding omission of 1 or 2 Gs); based on the selected sgRNA, the complementary strand of its corresponding DNA is obtained, and AAAC is added 5' thereto to obtain a Reverse oligonucleotide (Reverse oligo). Synthesizing the Forward oligonucleotide and the Reverse oligonucleotide respectively, pair-denaturing the Forward oligo and Reverse oligo of the synthesized sgRNA oligonucleotide, annealing, and forming a double strand which can be connected into a U6 eukaryotic expression vector after annealing, wherein the Forward oligo and the Reverse oligo are as follows:

Forward oligo:5'-CCGGNNNNNNNNNNNNNNNNNN

Reverse oligo:NNNNNNNNNNNNNNNNNNCAAA-5'

the Forward oligo nucleotide sequence table is shown as SEQ ID NO. 3; the Reverse oligo nucleotide sequence table is shown in SEQ ID NO. 4.

1.1.4 construction of sgRNA oligonucleotide plasmids

(1) The linearized pGL3-U6-sgRNA plasmid has a nucleotide sequence shown in SEQ ID No. 2.

(2) The annealed sgRNA oligonucleotide duplex was ligated with the linearized pGL3-U6-sgRNA plasmid to obtain the pGL3-U6-hPD1sg plasmid.

(3) Transform and coat Amp + plates.

(4) The method of sequencing the universal primer U6 identified positive clones.

(5) The cells were shaken overnight at 37 ℃ and pGL3-U6-hPD1sg Plasmid was extracted with AxyPrep Plasmid Miniprep Kit (AP-MN-P-250).

1.2 construction of LMP1-CAR piggyBac Transposon plasmid

1.2.1A signal peptide, the light chain of a single-chain antibody against LMP1, a spacer, the heavy chain of a single-chain antibody against LMP1, the CD8 a hinge region, the CD28 transmembrane and intracellular region, the 41BB intracellular region, and the CD3 zeta intracellular region are sequentially linked, and a gene sequence fragment is synthesized. All sequences were humanized and the gene fragments were synthesized by Kingchi organisms.

The nucleotide sequence of the signal peptide is shown as SEQ ID NO. 5;

the nucleotide sequence of the light chain of the LMP 1-resistant single-chain antibody is shown in SEQ ID NO. 6;

the nucleotide sequence of the spacer is shown as SEQ ID NO. 7;

the nucleotide sequence of the heavy chain of the LMP 1-resistant single-chain antibody is shown in SEQ ID NO. 8;

the nucleotide sequence of the CD8 alpha hinge region is shown as SEQ ID NO. 9;

the nucleotide sequences of the CD28 transmembrane region and the intracellular region are shown as SEQ ID NO. 10;

the nucleotide sequence of the 41BB intracellular domain is shown as SEQ ID NO. 11;

the nucleotide sequence of the intracellular domain of CD3 ζ is shown in SEQ ID NO. 12.

1.2.2 the above synthesized fragment was inserted into a commercial PiggyBac Transposon vector to obtain the plasmid LMP1-CAR PiggyBac Transposon capable of expressing the CAR of LMP1 target. This linking step is done by Jinzhi Biometrics.

1.3 extraction of LMP1-CAR PiggyBac Transposon plasmid

1.3.1A puncturing bacterium of the synthesized LMP1-CAR PiggyBac Transposon recombinant plasmid was inoculated into 250mL LB medium containing 100. mu.g/mL ampicillin and cultured overnight at 37 ℃ and 200 rpm. The overnight-cultured bacterial suspension was put into a 50ml centrifuge tube on average, centrifuged at 13000rpm (16200 Xg) for 5 minutes to collect the bacteria, and the whole supernatant was discarded as much as possible.

1.3.2 Add 600. mu.l Buffer P1 to the tube containing the pellet (please first check if RNase A had been added).

1.3.3, fully and uniformly mixing by using a pipettor or a vortex oscillator, and suspending bacteria for precipitation; adding 600 μ l of Buffer P2 into the centrifuge tube, gently inverting and mixing for 8-10 times to fully crack the thallus, and standing at room temperature for 3-5 min. At the moment, the solution becomes clear and viscous; 600. mu.l of Buffer E3 was added to the tube and mixed immediately by turning it upside down for 8-10 times, whereupon a white flocculent precipitate appeared and left to stand at room temperature for 5 minutes. The supernatant was centrifuged at 13000rpm for 5 minutes, and the supernatant was aspirated and applied to a filtration column (Endo-Remover FM) (which was filled into a collection tube), centrifuged at 13000rpm for 1 minute for filtration, and the filtrate in the collection tube was transferred to a centrifuge tube.

1.3.4 to the filtrate was added 600. mu.l of isopropanol, and the mixture was inverted upside down and mixed.

1.3.5 column equilibration: to an adsorption column (Spin Columns DL) loaded into the collection tube was added 200. mu.l Buffer PS, centrifuged at 13000rpm for 2 minutes, the waste liquid in the collection tube was discarded, and the adsorption column was replaced into the collection tube.

1.3.6 transferring the mixed solution of the filtrate and the isopropanol in the step 1.3.4 to an adsorption column (filled in a collecting pipe) with good balance. Centrifuge at 13000rpm for 1min, remove waste from the collection tube and replace the adsorption column back into the collection tube.

1.3.7 Add 750. mu.l Buffer PW (please check if absolute ethanol has been added) to the adsorption column, centrifuge at 13000rpm for 1min, and discard the tube.

1.3.8 the column was replaced in the collection tube and centrifuged at 13000rpm for 1 minute.

1.3.9 the adsorption column was placed in a new centrifuge tube (self-contained), 150. mu.l of Endo-Free Buffer EB was added to the middle of the adsorption membrane, left at room temperature for 2-5 minutes, centrifuged at 13000rpm for 2 minutes, and the plasmid was stored at-20 ℃.

2. The PBMC were slightly cultured in T cell medium (RPMI1640 medium containing 10% FBS) for 1 to 2 hours and then electroporated.

3. pGL3-U6-hPD1-sgRNA plasmid, pST1374-NLS-flag-Cas9ZF plasmid, LMP1-CAR PiggyBac Transposon plasmid and Super PiggyBac Transposase plasmid each 5. mu.g were mixed with an electrotransfer buffer in a human T cell nuclear transfection kit (Lonza, VPA-1002) to obtain approximately 110. mu.L of an electrotransfer mixture containing the four plasmids.

The nucleotide sequence of the pST1374-NLS-flag-Cas9-ZF plasmid is shown as SEQ ID NO. 1.

An electrotransfer mixture containing four plasmids, namely original plasmids pGL3-U6-sgRNA, pST1374-NLS-flag-Cas9-ZF, LMP1-CAR PiggyBac Transposon and Super PiggyBac Transposase, was used as a control, and a part of cells were left without electrotransfer.

4. With reference to the instructions of the human T cell nuclear transfection kit (Lonza, VPA-1002), suspending 1-2 × 107 PBMC cells with the two sets of electrotransformation mixed liquor for electrotransformation, and immediately transferring the cells into a preheated T cell culture medium after the electrotransformation is finished; the medium was changed again after 2 hours.

5. After 16-18 hours of culture, a T cell medium containing 100IU/ml IL-2 was added and CD 3-positive T cells were activated by stimulation with magnetic beads conjugated with CD3/CD28 antibody (Thermo Fisher SCIENTIFIC, 11141D).

6. The T cell medium containing 100IU/ml of IL-2 was replaced every 2 to 3 days while observing the cell expansion status. The stimulation duration is determined according to the proliferation status, but the stimulation duration does not exceed one week.

7. After removing the magnetic beads, 1. mu.g/ml puromycin was added and the culture was continued for 5 to 7 days. A small number of cells were harvested for testing, and the remaining cells were further expanded in bulk using rapid expansion to yield PD-1 gene-silenced LMP 1-targeted CAR-T cells (i.e., PD1-KO-LMP1-CAR-T cells) and traditional LMP 1-targeted CAR-T cells (LMP1-CAR-T cells).

Example three detection of expression of CAR using flow cytometry

105 amplified CAR-T cells were resuspended in 100. mu.l FACS buffer (PBS containing 2mmol/l EDTA and 0.5% BSA), incubated with Goat anti-H. mu.man IgG Antibody, FITC conj. mu.gate for 30 minutes at room temperature, washed twice with physiological saline, and FITC fluorescence signal was detected by flow cytometry to measure the FITC positive cell ratio, which reflects the ratio of CAR-T cells in total cells, as shown in Table 1.

TABLE 1 transduction efficiency of PD1 knockout LMP1-CAR-T cells

Transduction type	Efficiency of transduction
		LMP1-CAR-T cells	35.8％
PD1-KO-LMP1-CAR-T cells	37.2％

Note: PD1-KO-LMP1-CAR-T cells represent PD1 knockout LMP1-CAR-T cells.

Table 1 the results are shown: the CAR-T cells released granzyme and perforin, thereby killing tumor cells, and the results in table 1 show that the PD1 knockout LMP1-CAR-T cells (PD1-KO-LMP1-CAR-T) cells released granzyme and perforin higher than those of LMP1-CAR-T cells, showing higher lethality.

Example four-cell genome extraction and PD1 knock-out Effect analysis

1. Cells (0.1-1X 106) were digested with proteinase K-containing cell lysates and CAR-T cell genomic DNA was extracted by phenol chloroform.

2. The target fragment was PCR amplified using the detection on-target primers hPDl test For and hPD1 test Rev. The reaction system is as follows: mu.l 10 XBuffer, 12. mu.l dNTP (0.5mmol/l), 500ng genomic DNA, 0.6. mu.l hPD1 test For (0.5. mu. mol/l), 0.6. mu.l hPD1 test Rev (0.5. mu. mol/l), 3U Super-Taq, and make up water to 120. mu.l system. The reaction procedure is as follows: 5min at 95 ℃; 30cycles at 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃; 4 ℃ forever. And (3) purifying and recovering by using an Axygen PCR product recovery kit.

3. The PCR-annealed product was cleaved using the enzyme T7EN1, and the cleavage products were analyzed by electrophoresis on a 3% agarose gel.

4. The PCR product was ligated into pMD19-T vector (TAKARA, 3271), the ligation was performed according to the instructions of pMD19-T vector, the ligation product was transformed into DH 5. alpha. competent bacteria (TransGen, CD201), 20 or more single colonies were randomly picked, and sequencing was performed using the universal primer M13-47 to evaluate the base change and cleavage efficiency after the target fragment was cleaved, and the results are shown in FIG. 1.

FIG. 1 shows the results: in PD1-KO-LMP1-CAR cells, there were deletion and insertion mutations in the PD1 gene. Furthermore, in the case of the PD1-KO-LMP1-CAR cells stimulated by target cells for 3 days, a large amount of up-regulation of PD1 receptors cannot be detected on the surface, and the effect of correcting tumor immune escape is achieved.

Example five cytokine assays

Snk6, snt8 and raji cells were plated at 1 × 105 per well in 96-well plates, 2 × 105 PD-1 knockout LMP1-CAR-T cells were mixed with target cells in a total volume of 200 μ l of culture medium (RPMI1640 medium + 10% FBS) at 37 ℃ 5% CO₂The cells were co-cultured in an incubator for 24 hours. Each group of 3 multiple wells. After that, 10. mu.l of the supernatant was centrifuged and the cytokine concentration levels of TNF-. alpha.and IL-2 were measured by ELISA kit, and the results are shown in FIG. 2 and FIG. 3.

The results of fig. 2 and 3 show: the secretion amounts of cytokines IL-2 and IFN-gamma of PD1-KO-LMP1-CAR-T cells are statistically different from those of LMP1-CAR-T cells, PD1-KO cells and CAR-T cells, and show stronger lethality to EB virus infected Hodgkin lymphoma cells.

Example detection of six-granular enzyme and perforin

Snk6, snt8 and raji cells were plated at 1 × 105 per well in 96-well plates, 2 × 105 PD1-KO-LMP1-CAR-T cells were mixed with target cells in a total volume of 200 μ l of culture medium (RPMI1640 medium + 10% FBS) at 37 ℃ in 5% CO concentration₂The cells were co-cultured in an incubator for 24 hours. Each group of 3 multiple wells. And measuring OD values by using an ELISA double-antibody sandwich method, and calculating the concentrations of the granular enzyme and the perforin of the cell supernatants of each group according to the concentrations of the standard substances. Further, cell proteins can be extracted, and expression of granzyme and perforin can be detected by Western Blot, and the results are shown in Table 2.

TABLE 2 detection results of granzyme and perforin

Different capital letters after the same column of data indicate that the inter-treatment differences reached a very significant level (P < 0.01).

The CAR-T cells released granzyme and perforin, killing tumor cells, and the results in table 2 show that both granzyme and perforin released by PD1-KO-LMP1-CAR-T cells were significantly higher than LMP1-CAR-T cells and CAR-T cells, showing higher lethality to lymphoma cells.

Example seven PD1-KO-LMP1-CAR-T cell killing effect assessment

1. Appropriate amounts of snk6, snt8 and raji cells were taken as target cells, Calcein-acetohydroxy-methyl ester (Calcein-AM) was added to a cell suspension of 1 × 106/mL (PBS, 5% fetal bovine serum) to a final concentration of 25 μ M, and incubated in an incubator for 30 min.

2. After washing twice at room temperature, the cells were resuspended to 1.5X 105 mL. Adding PD1-KO-LMP1-CAR-T cells, LMP1-CAR-T cells and T cells according to different effect target ratios, centrifuging at 200g for 30 seconds, and incubating at 37 ℃ for 2-3 hours. After the incubation, the supernatant was taken, the fluorescence intensity of calcein was measured, and the percentage of target cell lysis was calculated from the spontaneous release control and the maximum release control, and the results are shown in table 3.

TABLE 3 killing efficiency of PD-1 knock-out LMP1-CAR-T cells

Different capital letters after the same row of data indicate that the inter-treatment differences reach a very significant level (P < 0.01).

Table 3 the results are shown: when the effective target ratio is 1:1, 1:2, 1:5 and 1:10, the killing efficiency of the PD1-KO-LMP1-CAR-T cell is obviously higher than that of the LMP1-CAR-T cell and the T cell, and the result proves that the PD1-KO-LMP1-CAR-T cell has specific killing activity on lymphoma cells.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhan university of science and technology

<120> preparation method and application of PD1 knockout LMP1 targeted CAR-T cell

<130> 2021

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gcctccatga taaaacgcta tgatgaacac catcaagacc tgactctgct caaagccctc 1980

gtgaggcaac agctgccaga gaagtacaaa gagatattct tcgaccagag caagaatgga 2040

tatgccggat acatcgatgg cggagcatca caggaagaat tttacaagtt catcaaacca 2100

atcctcgaga agatggacgg tactgaagag ctgctggtga agctgaacag ggaggacctg 2160

ctgaggaagc agaggacctt tgataatggc tccattccac atcagataca cctgggagag 2220

ctgcatgcaa tcctccgcag gcaggaggat ttctatcctt tcctgaagga taaccgggag 2280

aagatagaga agatcctgac cttcaggatc ccttattacg tcggccctct ggctagaggc 2340

aactcccgct tcgcttggat gaccaggaaa tctgaggaga caattactcc ttggaacttc 2400

gaagaggtcg tggataaggg cgcaagcgcc cagtcattca tcgaacggat gaccaatttc 2460

gataagaacc tgcccaacga gaaggtcctg cccaaacatt cactcctgta cgagtatttc 2520

accgtctata acgagctgac taaagtgaag tacgtgaccg agggcatgag gaagcctgcc 2580

ttcctgtccg gagagcagaa gaaggctatc gttgatctgc tcttcaagac taatagaaag 2640

gtgaaagtga agcagctcaa ggaggattac tttaagaaga tcgaatgctt tgactcagtg 2700

gaaatctctg gcgtggagga ccgctttaat gccagcctgg gcacttacca tgatctgctg 2760

aagataatca aagacaaaga tttcctcgat aatgaggaga acgaggacat cctggaagat 2820

atcgtgctga ccctgactct gttcgaggat agagagatga tcgaagagcg cctgaagacc 2880

tatgcccatc tgtttgacga taaagtcatg aaacagctca agcggcggcg ctacactggg 2940

tggggtagac tctccaggaa actcataaac ggcatccgcg acaaacagag cggaaagacc 3000

atcctggatt tcctgaaatc cgacggattc gctaacagga acttcatgca actgattcac 3060

gatgactctc tgacatttaa agaggacatc cagaaggcac aggtgagcgg tcaaggcgac 3120

agcctgcacg agcacatcgc caacctcgct ggatcacccg ccataaagaa gggaatactg 3180

cagacagtca aggtcgtgga cgaactcgtc aaagtgatgg gtcggcacaa gccagagaat 3240

atcgttatcg aaatggcaag ggagaaccaa accacccaga agggccagaa gaactctcgg 3300

gaacggatga aaagaatcga agagggaatt aaggagctgg gatctcagat actgaaggag 3360

caccctgtgg agaatacaca gctccagaac gagaaactct acctgtacta cctccagaac 3420

gggcgggaca tgtacgttga ccaggaactc gacatcaacc ggctgtccga ttatgacgtg 3480

gaccatattg ttccacagtc cttcctcaaa gatgactcca ttgacaacaa ggtgctgacc 3540

agatccgata agaatcgcgg taagtctgac aatgttccat cagaagaggt ggtcaagaag 3600

atgaagaatt actggcggca gctcctcaac gccaaactga tcacccagcg gaagtttgac 3660

aatctgacta aggcagaaag aggaggtctg agcgaactcg acaaggccgg ctttattaag 3720

aggcaactgg tcgaaacacg ccagattacc aaacacgtgg cacaaatcct cgactctagg 3780

atgaacacta agtacgatga gaacgataag ctgatcaggg aagtgaaagt gataactctg 3840

aagagcaagc tggtgtctga cttccggaag gactttcaat tctacaaagt tcgcgaaata 3900

aacaattacc atcatgctca cgatgcctat ctcaatgctg tcgttggcac cgccctgatc 3960

aagaaatacc ctaaactgga gtctgagttc gtgtacggtg actataaagt ctacgatgtg 4020

aggaagatga tagcaaagtc tgagcaagag attggcaaag ccaccgccaa gtacttcttc 4080

tactctaata tcatgaattt ctttaagact gagataaccc tggctaacgg cgaaatccgg 4140

aagcgcccac tgatcgaaac aaacggagaa acaggagaaa tcgtgtggga taaaggcagg 4200

gacttcgcaa ctgtgcggaa ggtgctgtcc atgccacaag tcaatatcgt gaagaagacc 4260

gaagtgcaga ccggcggatt ctcaaaggag agcatcctgc caaagcggaa ctctgacaag 4320

ctgatcgcca ggaagaaaga ttgggaccca aagaagtatg gaggtttcga ttcccctaca 4380

gtggcttatt ccgttctggt cgtggcaaaa gtggagaaag gcaagtccaa gaaactcaag 4440

tctgttaagg agctgctcgg aattactatt atggagagat ccagcttcga gaagaatcca 4500

atcgatttcc tggaagctaa gggctataaa gaagtgaaga aagatctcat catcaaactg 4560

cccaagtact ctctctttga gctggagaat ggtaggaagc ggatgctggc ctccgccgga 4620

gagctgcaga aaggaaacga gctggctctg ccctccaaat acgtgaactt cctgtatctg 4680

gcctcccact acgagaaact caaaggtagc cctgaagaca atgagcagaa gcaactcttt 4740

gttgagcaac ataaacacta cctggacgaa atcattgaac agattagcga gttcagcaag 4800

cgggttattc tggccgatgc aaacctcgat aaagtgctga gcgcatataa taagcacagg 4860

gacaagccaa ttcgcgaaca agcagagaat attatccacc tctttactct gactaatctg 4920

ggcgctcctg ctgccttcaa gtatttcgat acaactattg acaggaagcg gtacacctct 4980

accaaagaag ttctcgatgc caccctgata caccagtcaa ttaccggact gtacgagact 5040

cgcatcgacc tgtctcagct cggcggcgac cctaagaaaa agaggaaggt gtaggggccc 5100

ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtcat 5160

catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 5220

cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 5280

actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 5340

attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 5400

catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct 5460

agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 5520

cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 5580

tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta 5640

gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 5700

tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 5760

ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 5820

tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt 5880

taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 5940

ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 6000

aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 6060

tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 6120

tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 6180

gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 6240

tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca 6300

attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca 6360

tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca 6420

acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc 6480

gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg 6540

tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa 6600

atgagaacag gggcatcttg agcccctgcg gacggtgtcg acaggtgctt ctcgatctgc 6660

atcctgggat caaagcgata gtgaaggaca gtgatggaca gccgacggca gttgggattc 6720

gtgaattgct gccctctggt tatgtgtggg agggctaagc acttcgtggc cgaggagcag 6780

gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 6840

ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 6900

ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6960

atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 7020

ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 7080

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 7140

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 7200

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 7260

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 7320

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 7380

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 7440

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 7500

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 7560

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 7620

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 7680

caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 7740

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 7800

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 7860

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7920

actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7980

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 8040

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 8100

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 8160

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 8220

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 8280

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 8340

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 8400

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 8460

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 8520

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 8580

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 8640

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 8700

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 8760

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 8820

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 8880

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 8940

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 9000

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 9060

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 9120

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 9180

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 9240

gtc 9243

<210> 2

<211> 4952

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60

cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300

tgtggaaagg acgaaacacc ggtgagaccg agagagggtc tcagttttag agctagaaat 360

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 420

ttttttaaag aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga 480

aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac 540

atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac 600

agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca 660

aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag 720

cggcgccgac cctgggactc gcacattctt cacgtccgtt cgcagcgtca cccggatctt 780

cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg 840

ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac 900

cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc 960

caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga 1020

ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg 1080

caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc 1140

tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc 1200

cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 1260

cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga 1320

actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 1380

cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 1440

cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 1500

cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 1560

gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 1620

cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc tccccttcta 1680

cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 1740

gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga 1800

aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt 1860

agatcttagc cactttctag agtcggggcg gccggccgct tcgagcagac atgataagat 1920

acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 1980

aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca 2040

acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa 2100

gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg 2160

agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 2220

cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc 2280

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 2340

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 2400

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2460

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2520

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2580

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2640

gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2700

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2760

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2820

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2880

cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2940

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3000

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3060

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3120

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3180

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 3240

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 3300

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc 3360

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 3420

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 3480

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3540

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3600

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3660

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3720

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3780

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3840

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3900

aaaactctca aggatcttac agctgttgag atccagttcg atgtaaccca ctcgtgcacc 3960

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 4020

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 4080

cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 4140

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 4200

acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4260

gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 4320

cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 4380

atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 4440

tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 4500

tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 4560

tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 4620

atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct 4680

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct 4740

accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata 4800

tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac 4860

gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 4920

agtgcaggtg ccagaacatt tctctatcga ta 4952

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccggcgtgac ttccacatga gcg 23

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaaccgctca tgtggaagtc acg 23

<210> 5

<211> 63

<212> DNA

<213> Signal peptide sequence (Signal peptide sequence)

<400> 5

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 6

<211> 321

<212> DNA

<213> Light chain of anti-LMP1 Single chain antibody (Light chain aginst LMP1 single chain antibody)

<400> 6

gaactgcaga tgacacaaag cccctcctcc ctctccgcca gcgtgggaga cagagtcgct 60

atcacatgta gagctagcca gtccatctcc tcccatctga actggtatca gcagaaaccc 120

ggcaaggccc ccaaactgct gatttatgcc gccagcagcc tccaatccgg cgtgcctagc 180

agattcagcg gctccggcag cggaacagac tttacactca ccatcagctc tctgcagccc 240

gaagacttcg ccacctacta ctgtcagcag tcctactcca caccttacac attcggccaa 300

ggcaccaagc tggagatcaa a 321

<210> 7

<211> 45

<212> DNA

<213> light and heavy chains (Linker sequence)

<400> 7

ggtggcggtg gctctggtgg cggtggctcc ggtggcggtg gctca 45

<210> 8

<211> 360

<212> DNA

<213> Heavy chain of anti-LMP1 single-chain antibody (Heavy chain of anti-LMP1 single chain antibody)

<400> 8

gaagtccaac tggtcgagag cggaggagga ctcgtccaac ccggcggctc tctgaggctg 60

agctgcgctg cttccggctt cacattctcc tcctactgga tgagctgggt gagacaagct 120

cccggcaagg gactcgaatg ggtggccaat atcaagcaag acggcagcga gaagtactac 180

gtggactccg tgaagggaag attcacaatc tccagagaca acgccaagaa ctctctgtat 240

ctgagaatga actctctgag agccgaagac acagccgtgt attactgcgc tagggcttgg 300

ggctactcca gctactactt cgattactgg ggacaaggca ccctcgtcac agtcagcccc 360

<210> 9

<211> 204

<212> DNA

<213> CD8 alpha hinge region (CD8 alpha hinge area)

<400> 9

ttttgggtcc tcgtggtggt gggaggagtg ctggcttgct attctctgct cgtgaccgtc 60

gcctttatca tcttttgggt gaggtccaaa aggagcagag gcggacacag cgattatatg 120

aacatgacac ctagaagacc cggacctacc agaaaacact accaacccta tgctcctcct 180

agagactttg ccgcctacag atcc 204

<210> 10

<211> 123

<212> DNA

<213> transmembrane and intracellular domains of CD28 (CD28 transmembrane and intracellular domains)

<400> 10

agatccaaga gatctagact gctgcattcc gactacatga atatgacccc cagaaggccc 60

ggccctacaa ggaagcacta ccagccttac gcccctccta gggatttcgc cgcttataga 120

agc 123

<210> 11

<211> 126

<212> DNA

<213> 41BB intracellular region (41BB intracellular region)

<400> 11

aagagaggaa gaaagaaact gctctacatt ttcaagcagc cctttatgag acccgtccaa 60

accacacaag aggaggatgg ctgcagctgt agatttcccg aggaagaaga gggaggctgc 120

gagctg 126

<210> 12

<211> 42

<212> DNA

<213> CD3 ζ intracellular region (CD3 ζ intracellular region)

<400> 12

agagtgaagt tttctagatc cgccgacgct cccgcctatc aa 42

Claims

1. A method for preparing a PD1 knockout LMP1 targeted CAR-T cell, comprising the steps of:

2. The method for preparing a PD 1-knockout LMP 1-targeted CAR-T cell according to claim 1, wherein the PD1 gene sequence of the pGL3-U6-hPD1-sgRNA plasmid is selected from one of 5' -GGN (19) GG, 5' -GN (20) GG and 5' -N (21) GG.

3. The method for preparing a PD1 knockout LMP1 targeted CAR-T cell according to claim 2, wherein the pGL3-U6-hPD1-sgRNA plasmid is constructed by:

4. The method for preparing a PD1 knockout LMP1 targeted CAR-T cell according to claim 1, wherein the LMP1-CARPiggyBac Transposon plasmid is constructed by the following steps:

5. The method for preparing a PD1 knockout LMP 1-targeted CAR-T cell according to claim 4, wherein the light chain of the anti-LMP1 single-chain antibody of step S1 is preceded by a signal peptide, and a light-heavy chain spacer region is further included between the light chain of the anti-LMP1 single-chain antibody and the heavy chain of the anti-LMP1 single-chain antibody.

6. The method for preparing a PD1 knockout LMP1 targeted CAR-T cell according to claim 1, wherein the magnetic bead is a CD3/CD28 antibody-coupled magnetic bead.

7. The method of claim 1, wherein the PD1 knockout LMP1 targeted CAR-T cells are prepared by the following method:

8. The method for preparing LMP 1-targeted CAR-T cells with PD1 knockout function according to claim 1, wherein pGL3-U6-hPD1-sgRNA plasmid, pST1374-NLS-flag-Cas9-ZF plasmid, LMP1-CAR PiggyBac Transposon plasmid and Super PiggyBac Transposase plasmid are mixed with an electrotransfer buffer solution in a T cell nuclear transfection kit to obtain an electrotransfer mixture solution containing the four plasmids, and 1-2 x 107 PBMC cells are suspended by the electrotransfer mixture solution for electrotransfer.

9. The method for preparing a PD1 knockout LMP1 targeted CAR-T cell as claimed in claim 1, wherein the T cell culture medium of step S2 is RPMI-1640 medium containing 10% FBS.

10. Use of a PD1 knock-out LMP 1-targeted CAR-T cell of any one of claims 1-9 in the preparation of a diagnostic reagent or therapeutic medicament for EVB lymphoma.