Application of CAR-NK cells in targeted killing of TRBC1 positive tumors

Abstract

The invention discloses a novel method for treating tumors by taking peripheral blood NK (Natural Killer) cells as CAR (Chimeric Antigen Receptor) carrier cells. The CAR-NK modified by the chimeric antigen receptor can be used as an effective tool, so that the CAR-NK has higher killing capability on tumor cells. The CAR-NK can accurately identify and attack tumor cells, so that the growth and diffusion of tumors are effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and specifically refers to an application of CAR-NK cells in targeted killing of TRBC1 positive tumors. Background Art

[0002] CAR-NK cell therapy has become one of the most popular and effective adjuvant treatments for tumors in recent years. Peripheral blood NK cells are a type of natural killer cells, which are important cells involved in the body's inherent immunity. CAR-NK can also be used as an effective therapeutic tool. The principle is to use genetic engineering methods to couple the single-chain variable region (Scfv) of an antibody that can recognize a certain tumor antigen with the intracellular region of the CD3ζ chain in vitro as a chimeric protein, and transfect the peripheral blood NK cells that have been expanded and cultured in vitro by gene transduction to express the chimeric antibody receptor (CAR). After the peripheral blood NK cells are "reprogrammed", a large number of lethal CAR-NK cells are generated, which can specifically target tumor cells. Compared with traditional immunotherapy, CAR-MOLT4 has significant advantages such as more precise treatment, higher infection efficiency, higher CAR expression efficiency, more extensive tumor killing, and reduced probability of cytokine storm.

[0003] T cell-related blood tumors are a group of blood tumors that are common in children, and the incidence in adults is lower than that in children. With the continuous update of medical methods, the treatment methods for T cell-related blood tumors have also been updated. In particular, the addition of immunotherapy methods and treatment targeting different targets have also made the treatment of patients more individualized. Studies have shown that TRBC1 + T cells show monoclonal proliferation in T-ALL, which makes TRBC1 a good target for diagnosis and treatment. However, CAR-T cell therapy often has adverse reactions such as cytokine storm, which greatly interferes with the treatment. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for treating TRBC1-positive tumors using CAR-NK as a tool, wherein CARNK cells carrying ScFv sequences that broadly recognize various TRBC1-positive tumors can effectively kill any tumor cells expressing TRBC1 on their surface.

[0005] The present invention provides a chimeric antigen receptor, which comprises a signal peptide, a single-chain antibody ScFv, a Strep tag II, a CD8 hinge, a CD28 transmembrane region and an intracellular domain in sequence from the N-terminus to the C-terminus, wherein the intracellular domain comprises an intracellular signal stimulation domain, and the single-chain antibody ScFv recognizes and binds to TRBC1 on the surface of tumor cells.

[0006] Furthermore, the intracellular domain of the chimeric antigen receptor also includes a co-stimulatory domain, and the co-stimulatory domain is selected from one or more of CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, NKG2D, DAP10, and 2B4, preferably NKG2D and 2B4; the intracellular signal stimulation domain is selected from one of CD3ζ, CD3γ, CD3δ, CD3ε, CD8, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, and CD79a, preferably CD3ζ.

[0007] In some embodiments of the present invention, the single-chain antibody ScFv of the chimeric antigen receptor is composed of the heavy chain variable region V H , connecting polypeptide Linker, light chain variable region V L Preferably, the heavy chain variable region V H The amino acid sequence of the light chain variable region V L The amino acid sequence of is shown in A2 above, preferably, the heavy chain variable region V H The nucleotide sequence of the light chain variable region V L The nucleotide sequence is shown in A4 above.

[0008] The second object of the present invention is to provide a gene vector of a recombinant chimeric antigen receptor, using a lentivirus, adenovirus, adeno-associated virus, retrovirus, transposon vector or non-viral transient expression vector as a backbone, into which the above-mentioned chimeric antigen receptor nucleotide sequence is inserted; preferably, using a viral vector PTK881-EF1α as a backbone, into which the above-mentioned chimeric antigen receptor nucleotide sequence is inserted.

[0009] The third object of the present invention is to provide a peripheral blood NK cell expressing a chimeric antigen receptor, which is obtained by transfecting peripheral blood NK cells with the above-mentioned chimeric antigen receptor nucleotide sequence or the above-mentioned recombinant chimeric antigen receptor gene vector; the single-chain antibody ScFv of the chimeric antigen receptor binds to TRBC1 on the tumor surface; preferably, CRISPR and RNA interference technology are combined with the expression of the chimeric antigen receptor element.

[0010] The fourth object of the present invention is to provide the above-mentioned nucleotide sequence expressing chimeric antigen receptor, the above-mentioned recombinant chimeric antigen receptor gene vector, and the use of the above-mentioned chimeric antigen receptor expressing NK cell, including the preparation of a drug or kit for treating, preventing, and diagnosing tumors. Preferably, the tumor is a TRBC1-positive tumor.

[0011] The fifth object of the present invention is to provide a combination of the peripheral blood NK cells expressing chimeric antigen receptors, comprising:

[0012] 1) Combination with anti-tumor drugs, antibodies and / or oncolytic viruses, wherein the anti-tumor drugs are selected from one or more of glucocorticoids, cytarabine, asparaginase (PEG-ASP), vincristine, daunorubicin, methotrexate, cyclophosphamide, doxorubicin, vincristine and prednisone, anthracycline antibiotics cyclophosphamide, cyclosporine A (CyA) and etoposide; the antibodies are selected from CD52 antibodies, CD30 antibodies, CTLA-4 antibodies, and the oncolytic viruses are selected from reoviruses, adenoviruses, herpes simplex viruses, vesicular stomatitis viruses, measles viruses, vaccinia viruses, and polio viruses.

[0013] 2) Other targets combined with dual-target or multi-target TRBC1 peripheral blood NK cells expressing chimeric antigen receptors, wherein the other targets are selected from CD30, CD52, and CTLA-4 antibodies.

[0014] The beneficial effects of the present invention are as follows: 1. The immune cells provided by the present invention use TRBC1 as the target, and use NK cells with immune killing ability but causing minimal cytokine release syndrome as carrier cells. The modified NK cells can be used to treat tumors that express TRBC1 positively. 2. The combined use of TRBC1-CAR-NK and CHOP treatment regimens produces a synergistic effect on the lysis of TRBC1 positive target cells, achieving an effect of 1+1>2. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram of the DNA fragment of TRBC1-CAR;

[0016] Figure 2 This is the plasmid map of PTK881-EF1α-TRBC1;

[0017] Figure 3 This is a schematic diagram of the TRBC1-CAR-NK cell transduction efficiency test results;

[0018] Figure 4 This is a schematic diagram of the results of the in vitro tumor killing experiment of TRBC1-CAR-NK cells;

[0019] Figure 5 Schematic diagram of cytokine release after TRBC1-CAR-NK cells were co-incubated with target cells in vitro. DETAILED DESCRIPTION

[0020] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0021] Example 1: Construction of PTK881-EF1α-MSLN plasmid

[0022] 1. Artificially synthesize the A1 sequence, the linker nucleotide sequence, and the nucleotide fragment shown in A2 in the above text to form SP-TRBC1. The linker has an amino acid sequence of GGGGSGGGGSGGGGS, and a nucleotide sequence of

[0023] GGCGGTGGCGGTAGCGGTGGCGGTGGCTCTGGTGGTGGTGGTGGCAGC. 2. Using the human cDNA library as a template, primers were designed to amplify the fragments CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB, and CD3ζ respectively, and Strep tagⅡ and short fragments were obtained by primer complementation. Overlap PCR technology was used to sequentially amplify SPTRBC1 and fragments Strep tagⅡ, CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB, and CD3ζ to form TRBC1-CAR with restriction sites EcoRI and BamH I. The structural diagram is shown in the figure. Figure 1 shown.

[0024] Among them, TRBC1-CAR is a single-chain antibody ScFv with a signal peptide that can recognize MSLN on the surface of tumor cells. Single-chain antibody ScFv: SPTRBC1: heavy chain variable region V H The amino acid sequence of the light chain variable region V L The amino acid sequence of the heavy chain variable region V H The nucleotide sequence of the light chain variable region V L The nucleotide sequence is the A4 sequence described above.

[0025] The nucleotide sequence of the signal peptide is:

[0026] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCAC GCCGCCATCCCC, the nucleotide sequence of Strep tag II is:

[0027] The nucleotide sequence of AACTGGAGCCACCCCCAGTTCGAGAAG, CD8 hinge is: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTC GCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT, and the nucleotide sequence of CD28 is TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC. The nucleotide sequence of 4-1BB is:

[0028] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG

[0029] ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG, and the nucleotide sequence of CD3ζ is: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0030] 3. The plasmid PTK881-Kan was double-digested with EcoR I and BamH I restriction endonucleases. The products were subjected to 0.8% agarose gel electrophoresis and cut and recovered in Eppendorf tubes. The corresponding fragments were recovered using Axygen's agarose gel recovery kit, and the purity and concentration of the products were determined.

[0031] 4. Add the above vector recovery fragment and TRBC1-CAR in a 1:2 molar ratio to an Eppendorf tube, add ExnaseⅡ ligase (Vazyme) and homologous recombinase 5×CEⅡbuffer, and react at 37℃ for 0.5 hours; take out 10μL of the ligation solution and add 100μL DH5α competent cells to ice bath for 30min and then heat shock at 42℃ for 90s. After completion, add 500μL soc medium and culture at 37℃ and 220rpm for 2 hours; after 2 hours, centrifuge the Eppendorf tube at 4000g for 1min to remove 400μL of excess liquid. Spread the remaining liquid on the LB plate and culture at 37℃ for 12 hours; pick single colonies on the plate and inoculate them into 5mL LB liquid culture medium and culture at 37℃ and 220rpm for 12 hours.

[0032] 5. Use Axygen Mini-Extraction Kit to extract the plasmid, obtain the plasmid PTK881-EF1α-MSLN, and send it to Shanghai Biotech Co., Ltd. for first generation sequencing verification. Then, the DH5α strain containing the plasmid PTK881-EF1α-TRBC1 is preserved. The complete map of PTK881-EF1α-TRBC1 is shown in the figure below. Figure 3 shown.

[0033] Example 2: Preparation and sequencing of plasmids

[0034] 1. Preparation of plasmid

[0035] The plasmid PTK881-EF1α-TRBC1 was inoculated into 250 mL of LB culture medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 220 rpm. The culture medium was centrifuged at 6000 g for 20 min at 4°C and the supernatant was discarded.

[0036] Take out Buffer P1 from EndoFree plasmid mega kit (Qiagen), add 120 mL of pre-cooled Buffer P1 to the E. coli precipitate obtained by centrifugation, cover the centrifuge bottle cap, and shake the centrifuge bottle vigorously to completely disperse the E. coli precipitate in Buffer P1.

[0037] Add 120 mL of Buffers P2 to the centrifuge bottle, cover the bottle cap and place it on a roller mixer. Slowly increase the speed to 50 rpm, mix thoroughly and place at room temperature for 5 minutes.

[0038] Add 120 mL of Buffers P3 to the centrifuge bottle, cover the bottle cap and place it on a roller mixer, slowly increase the speed to the maximum speed of 70 rpm, and mix thoroughly until a white, non-sticky, fluffy mixture is obtained. Centrifuge at 9000 g for 15 min at 4°C.

[0039] Pour 50 mL of Buffer FW into the QIAfilter Cartridge, pour the supernatant obtained by centrifugation into the QIAfilter Cartridge, and gently stir to mix. Filter the mixture into the corresponding labeled glass bottle.

[0040] Add 20 mL of Buffer ER to each glass bottle, mix by inverting 6 times, and incubate at -20°C for 30 min.

[0041] Place the marked mega columns in the corresponding racks, add 35 mL of Buffers QBT to each mega column for balance, and let gravity drain.

[0042] Pour all the liquid in the glass bottle into the corresponding marked mega column in batches. After the liquid in the column has drained out, add 200mL Buffer QC to each mega column in batches for cleaning. After the liquid in the column has drained out, pour the waste liquid in the waste liquid collection tray into a 50mL clean centrifuge tube.

[0043] Add 40 mL of Buffer QN to each mega column, collect the effluent in a 50 mL clean centrifuge tube, mix by inverting the tube 6 times, and dispense 20 mL into another clean, labeled 50 mL centrifuge tube.

[0044] Add 14 mL of isopropanol (room temperature) to each 50 mL centrifuge tube, mix by inverting 6 times, and centrifuge at 15,000 g for 50 min at 4°C.

[0045] Aspirate the supernatant in the clean bench and add 3.5 mL Endotoxin-free water to each tube for rinsing. Do not disperse the bottom precipitate. Centrifuge at 15000g for 30 min at 4°C. Preheat the Buffer TE in the EndoFree plasmid mega kit in an oven.

[0046] Aspirate the supernatant after centrifugation in the clean bench and blow dry in the clean bench (to evaporate the residual anhydrous ethanol, the time is about 10 minutes).

[0047] Take out Buffer TE from the oven, add 1 mL of Buffer TE to each tube in the clean bench, blow 10 times with a gun, and put it into a 65°C oven. During this time, tap the tube wall continuously to completely dissolve the precipitate. Centrifuge at 4000g for 1 min at 4°C to shake the liquid on the tube wall to the bottom of the tube, and then blow and mix.

[0048] Transfer all the liquid to the corresponding labeled EP tubes without endotoxin, pyrogen, or nuclease in the clean bench. Aspirate 2 μL, measure the plasmid concentration with a micro-spectrophotometer, and mark it on the corresponding EP tube to obtain plasmids PTK881-EF1α-C3-1, PTK881-EF1α-C3-2, and PTK881-EF1α-C4.

[0049] 2. Target gene sequencing

[0050] Take 20 μL (500 ng) of plasmid DNA respectively and send it for sequencing. According to the original seed sequence, check whether the target gene of the product produced by the plasmid has changed. Under a stable process, the working seed will not change during the fermentation and amplification process, and can be used for the next stage of production and correct protein expression.

[0051] Example 3. Preparation of Lenti3-TRBC1-CAR lentiviral vector and detection of live droplets

[0052] 1. Preparation of Lentiviral Vector

[0053] In a multilayer cell culture flask (Hyperflask), 130.0-140.0×10 6 293T cells (Takara) of the same number were added to 560 mL of DMEM complete medium (50 mL of fetal bovine serum, 5 mL of Antibiotic-Antimycotic (100×)), and cultured in a 37°C incubator containing 5% CO2 for 24 hours. DMEM basal medium mixed with 320 μg of plasmid (PTK881-EF1α-MSLN: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid = 12:10:5:6) was added to 960 μg of PEI tubes, vortexed, and equilibrated at room temperature for 10 minutes. The above 35 mL of PEI and plasmid mixture was mixed with 525 mL of DMEM complete medium and replaced into the above multilayer cell culture flask. The multilayer cell culture flask was placed in a 37°C incubator containing 5% CO2 for 3 days, and the cell culture supernatant was collected.

[0054] After centrifuging the supernatant at 4000rpm (or 3000g) for 30min, cryonase (Takara) was added to the supernatant after centrifugation and placed at 4°C. After 6 hours, the lentiviral supernatant was filtered using a 0.22μm filter membrane and centrifuged at 30000g for 2.5h at 4°C. The supernatant was removed and 1Ml PBS was added to resuspend the precipitate. After resuspension, 20μL was retained for viral activity titer detection, and the remaining lentiviral concentrate was packaged, labeled as Lenti3-TRBC1-CAR and stored at -80°C for later use.

[0055] 2. Lentiviral vector activity titer detection

[0056] Principle: The anti-Strep tag Ⅱ antibody is labeled with fluorescein, and the anti-Strep tag Ⅱ antibody can specifically bind to Strep tag Ⅱ in CAR. The fluorescent signal detected by flow cytometry indirectly reflects the expression of CAR in 293T cells.

[0057] Method: 5.0×10 5 293T cells / well were added, and 0.1μL, 0.5μL, and 1μL of lentiviral concentrate were added to each well, respectively, and a negative control was set up. Cultured in a 37°C incubator containing 5% CO2. Three days later, 293T cells were collected with Versene solution (Gibco) and sent to flow cytometry to detect the proportion of CAR-positive 293T cells, and the activity titer of Lenti3-TRBC1-CAR lentiviral concentrate was converted.

[0058] The current activity titer of lentiviral concentrate is 1×10 8 ~10×10 8 The detection and analysis results are shown in Table 1.

[0059] Table 1 Lenti3-TRBC1-CAR lentivirus activity titer detection analysis results

[0060] Sample No. Activity titer (TU / mL) Lenti3-TRBC1-CAR <![CDATA[4.3×10 8 ]]>

[0061] Example 4. Preparation of TRBC1-CAR-MOLT4 cells

[0062] 1. Preparation of CAR-NK cell preparations:

[0063] The Lenti3-TRBC1-CAR lentiviral vector was transduced into NK cells at an MOI of 10, mixed and incubated in a CO2 incubator, and an appropriate amount of 1640 complete medium was added for culture after 4 hours.

[0064] 24 hours after lentiviral transduction, the transduced TRBC1-CAR-NK cells were replaced with fresh 1640 cell complete culture medium and the live cell density was adjusted to 4.0-5.0×10 5 / mL, continue to culture and expand for 10 to 20 days, observe and count every day, and replenish fluid and expand the culture according to the counted cell number.

[0065] 2. CAR-NK cell transduction efficiency detection

[0066] Take 5×10 5 After transduction, NK cells were incubated with 1ug / mL FITC-Protein-L at room temperature for 30 minutes, washed twice with saline, and then the FITC fluorescence signal was detected by flow cytometry to measure the ratio of FITC-positive cells, which reflects the ratio of CAR-NK cells in total cells. The results of TRBC1-CAR-NK cell transduction efficiency test are as follows: Figure 3 shown. Figure 3 This indicated that TRBC1-CAR-NK cells were successfully prepared.

[0067] Example 5. In vitro functional detection of CAR-NK cells

[0068] 1. In vitro tumor killing detection:

[0069] The fluorescein sodium salt method was used to detect the in vitro tumor killing function of TRBC1-CAR-NK cells. The OV-TRBC1 positive cell JURKAT cell line was selected for the in vitro CAR-NK tumor killing experiment.

[0070] Take an appropriate amount of OV-TRBC1-JURKAT-Luci target cells and resuspend the cells to 0.5×10 5 / mL, 0.5×10 5 cells, cultured in 200ul culture medium, added TRBC1-CAR-NK cells at effector-target ratios of 1:1, 2:1, 5:1, and 10:1, and incubated at 37°C for 24 hours. After incubation, add 2ul of fluorescein sodium salt and incubate at 37°C for 15min before testing on the instrument. The target cell lysis percentage results are as follows: Figure 4 As shown, the results showed that TRBC1-CAR-NK cells could significantly promote the lysis of OV-TRBC1-JURKAT.

[0071] In parallel with the above experimental operation, take an appropriate amount of OV-TRBC1-JURKAT-Luci target cells and resuspend the cells to 0.5×10 5 / mL, 0.5×10 5cells, cultured in 200ul culture medium, added NK cells and TRBC1-CAR-NK cells at an effector-target ratio of 4:1, and added glucocorticoids to make the final concentration of glucocorticoids 2×10 -3 ug / μl, incubate at 37℃ for 24 hours. After the incubation, add 2ul of fluorescein sodium salt and incubate at 37℃ for 15min before testing on the machine, measure the fluorescence intensity of fluorescein sodium salt, and calculate the target cell JURKAT-Luci lysis percentage, which are 88.1% and 89.8% respectively.

[0072] From the above results, it can be seen that the combined use of TRBC1-CAR-NK and glucocorticoids produced a synergistic effect on the lysis of target cells OV-TRBC1-JURKAT-Luci, achieving an effect of 1+1>2.

[0073] 2. In vitro cytokine detection:

[0074] Take an appropriate amount of the above JURKAT target cells and add 1×10 6 / mL cell suspension (PBS, 5% fetal bovine serum) at room temperature, wash twice and resuspend the cells to 0.5×10 5 / mL, 0.05×10 5 / mL target cells, add JURKAT and TRBC1-CAR-JURKAT at a 4:1 effector-target ratio, centrifuge at 200g for 30 seconds, and incubate at 37°C for 18 hours. After incubation, take the supernatant and measure the concentration of IFN-γ. The results of IFN-γ concentration of NK and TRBC1-CAR-NK cells on OV-TRBC1-JURAT cell line are as follows Figure 5 shown.

[0075] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that The signal peptide, the single-chain antibody ScFv that recognizes and binds to TRBC1 on the surface of tumor cells, Strep tag II, CD8 hinge, CD28 transmembrane region, and the intracellular domain are sequentially spliced ​​from the N-terminus to the C-terminus, and the intracellular domain includes a signal stimulation domain.

2. The chimeric antigen receptor according to claim 1, characterized in that The intracellular domain also includes a co-stimulatory domain, which is selected from one or more of CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, NKG2D, DAP10, and 2B4, preferably NKG2D and 2B4; the intracellular signal stimulation domain is selected from one of CD3ζ, CD3γ, CD3δ, CD3ε, CD8, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, and CD79a, preferably CD3ζ.

3. The chimeric antigen receptor according to claim 1, characterized in that The single-chain antibody ScFv is composed of the heavy chain variable region V H , connecting polypeptide Linker, light chain variable region V L Preferably, the heavy chain variable region V H The amino acid sequence is: EVRLOOSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKORPGOGLEWIGF INPYNDDIOSNERFRGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGA GYNFDGAYRFFDFWGOGTTLTVSS (for the convenience of expression, this sequence is represented by A1 below), the light chain variable region V L The amino acid sequence is DVVMTOSPLSLPWSLGDOASISCRSSORLVHSNGNTYLHWYLOKPGOSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSOSTHVP YTFGGGTKLEIKR (for the convenience of expression, this sequence is represented by A2 below), preferably, the heavy chain variable region V H The nucleotide sequence is: GAGGTAAGACTTCAGCAGTCTGGGCCCGACTTGATCAAACCAGGCGCGTCCGTGAAAATGTCCTGCAAGGCCAGTGGCTATACATTCACCGGATACGTGATGCATTGGGTTAAACAGAGGCCAGGGCAAGGGCTGGAATGGATTGGTTTTATCAATCCTTACAACGATGACATCCAAAGTAACGAGCGGTTTAGGGGTA AGGCAACCCTTACGTCTGACAAATCCAGTACCACAGCCTATATGGAGCTGTCCAGCCTGACATCAGAAGATTCTGCTGTGTATTGTGCGCGAGGTGCCGGATACAACTTCGATGGTGCATACCGGTTCTTCGACTTTTGGGGGCAGGGAACCACTCTTACTGTGAGCTCA (for convenience of expression, this sequence is represented by A3 below), the light chain variable region V L The nucleotide sequence is: GGTGGGGGTGGGAGTGGCGGAGGCGGATCTGGAGGAGGTGGTAGCGACGTTGTCATGACTCAGAGCCCTCTCAGTCTTCCCGTGTCCCTGGGGGACCAGGCTAGCATCTCCTGCCGGTCTCTCCAGAGACTCGTTCATTCCAATGGAAATACATACCTGCACTGGTATTTGCAGAAGCCAGGCCAGTCACCCAAGCTCCTC ATCTACCGCGTGTCTAACAGATTCCCAGGCGTACCAGATCGGTTTTCCGGATCAGGTTCCGGGACCGACTTTACCTTGAAGATCAGTCGAGTGGAAGCCGAAGACCTGGGGATCTACTTTTGCTCCCAGTCCACACATGTCCCATACACGTTCGGCGGGGGGACCAAACTGGAAATTAAGCGC (for convenience of expression, this sequence is represented by A4 below).

4. A gene vector of a recombinant chimeric antigen receptor, characterized in that: The method comprises using a lentivirus, adenovirus, adeno-associated virus, retrovirus, transposon vector or non-viral transient expression vector as a backbone, and inserting the chimeric antigen receptor encoding nucleotide sequence as described in claims 1-3; preferably, using the viral vector PTK881-EF1α as a backbone, and inserting the chimeric antigen receptor nucleotide sequence as described in claims 1-3.

5. A cell expressing a chimeric antigen receptor, characterized in that Obtained by transfecting cells with the nucleotide sequence of the chimeric antigen receptor according to claims 1-3 or the recombinant chimeric antigen receptor gene vector according to claim 4; the single-chain antibody ScFv of the chimeric antigen receptor binds to antigens on the surface of tumor cells; preferably, CRISPR and RNA interference technology are combined with the expression of chimeric antigen receptor components.

6. Use of the chimeric antigen receptor nucleotide sequence according to claims 1-3, the recombinant chimeric antigen receptor gene vector according to claim 4, and the chimeric antigen receptor-expressing immune cell according to claim 5, characterized in that: It includes preparing drugs or kits for treating, preventing and diagnosing tumors. Preferably, the tumor is a blood tumor such as T-ALL, LGL, AITL, PTCL, etc.

7. A combination of cells expressing a chimeric antigen receptor according to claim 7, characterized in that: include: Combination with anti-tumor drugs, antibodies and / or oncolytic viruses, wherein the anti-tumor drugs are selected from one or more of glucocorticoids, cytarabine, asparaginase (PEG-ASP), vincristine, daunorubicin, methotrexate, cyclophosphamide, doxorubicin, vincristine and prednisone, anthracycline antibiotics cyclophosphamide, cyclosporine A (CyA) and etoposide; the antibodies are selected from CD52 antibodies, CD30 antibodies, CTLA-4 antibodies, and the oncolytic viruses are selected from reoviruses, adenoviruses, herpes simplex viruses, vesicular stomatitis viruses, measles viruses, vaccinia viruses, and polio viruses.

8. The combination of immune cells expressing chimeric antigen receptor according to claim 8, characterized in that: include: The chimeric antigen receptor-expressing immune cells obtained by transforming peripheral blood NK cells with the chimeric antigen receptor according to claim 5 are used in combination with glucocorticoids.

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