A chimeric antigen receptor, gene, recombinant vector, host cell, CAR-NK cell and preparation method and use thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a chimeric antigen receptor, a gene, a recombinant vector, a host cell, a CAR-NK cell and a preparation method and application thereof. The application provides a CAR-NK cell targeting CD19, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, the extracellular region comprises a signal peptide (CD8 alpha), an anti-CD19 single-chain antibody (CD19 scFv) and a hinge region (lgG4, lgG4(L) or CD8 alpha), the transmembrane region comprises CD28 TM or CD8 alpha TM, and the intracellular region comprises a costimulatory factor domain (CD28, 4-1BB, OX40 or 2B4) and a signal transduction domain (CD3 zeta). Compared with a traditional NK cell, the CAR-NK cell constructed in the application has a significant improvement in killing efficiency on CD19 positive tumor cells, and has a good application prospect in treating tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a chimeric antigen receptor, a gene, a recombinant vector, a host cell, a CAR-NK cell and a preparation method and application thereof. BACKGROUND

[0002] The chimeric antigen receptor (CAR) is a genetically engineered biological macromolecule with specific targeting, which can be expressed on the cell surface to realize the in vivo targeting of cells. The most typical carrier cells are T cells and NK cells. CAR-T has made a major breakthrough in leukemia research, bringing new hope to blood tumor patients. However, with the approval of various CAR-T drugs, the in-depth research and the increasing application of clinical treatment, it is found that CAR-T cell drugs can produce a series of adverse reactions, such as cytokine release syndrome (CRS) and neurotoxicity of CAR-T, resistance and tolerance of solid tumors, and tumor immunosuppressive microenvironment. It has important theoretical significance and clinical application value to inhibit or avoid the adverse reactions of CAR-T cell drugs and develop new effector cells with strong anti-tumor effect.

[0003] NK cells (natural killer cells) are a kind of lymphocytes with strong killing effect on tumor cells and MHC independence. The recognition of tumor cells mainly depends on the mutual cross-regulation of its surface activating receptors and inhibitory receptors. After recognizing tumor cells, NK cells kill tumor cells through multiple pathways such as releasing killing mediators perforin and granzyme to make target cells apoptosis, expressing membrane TNF family molecules to induce target cell apoptosis and antibody-dependent cellular cytotoxicity. It is expected to enhance the ability of CAR-modified NK cells to target and kill tumor cells and develop effector cells with strong anti-tumor effect.

[0004] Therefore, it is necessary to develop new CAR-NK cells with high killing activity to provide more options for tumor treatment. SUMMARY

[0005] In view of the defects of the prior art, the application provides a CAR-NK cell targeting CD19 and a preparation method and application thereof.

[0006] The application provides a chimeric antigen receptor, which has an extracellular region, a transmembrane region and an intracellular region connected in sequence; the extracellular region has a signal peptide, an anti-CD19 single-chain antibody and a hinge region connected in sequence, and the amino acid sequence of the anti-CD19 single-chain antibody is shown as SEQ ID NO. 2.

[0007] Preferably, the structure of the signal peptide is CD8a, and the structure of the hinge region is selected from lgG4, lgG4(L) or CD8a.

[0008] the structure of the transmembrane region is selected from CD28 TM or CD8a TM;

[0009] the structure of the intracellular region is a co-stimulatory factor domain and a signal transduction domain connected in sequence, the structure of the co-stimulatory factor domain is selected from CD28, 4-1BB, OX40 or 2B4, and the structure of the signal transduction domain is CD3 zeta.

[0010] Preferably, the amino acid sequence is shown as SEQ ID NO. 1.

[0011] The present application provides a gene encoding the chimeric antigen receptor described in any of the above.

[0012] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

[0013] The present application provides a recombinant vector comprising the gene.

[0014] The present application provides a host cell comprising the recombinant vector as described above.

[0015] The present application provides a CAR-NK cell expressing on the surface a chimeric antigen receptor as described in any of the above.

[0016] Preferably, it is prepared by transducing NK cells with a gene encoding the chimeric antigen receptor by a lentiviral transduction method.

[0017] The present application provides a method for preparing the CAR-NK cell as described in any of the above, which comprises transducing NK cells with a gene encoding the chimeric antigen receptor by a lentiviral transduction method.

[0018] The present application provides the use of the chimeric antigen receptor as described in any of the above, the gene as described in any of the above, the recombinant vector as described above, the host cell as described above, the CAR-NK cell as described in any of the above in the preparation of a medicament for treating anti-CD19 positive tumor diseases.

[0019] The application provides a CAR-NK cell targeting CD19 by screening sequences and structures, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, the extracellular region comprises a signal peptide (CD8 alpha), an anti-CD19 single-chain antibody (CD19 scFv) and a hinge region (lgG4, lgG4(L) or CD8 alpha), the structure of the transmembrane region comprises CD28 TM or CD8 alpha TM, and the intracellular region comprises a costimulatory factor domain (CD28, 4-1BB, OX40 or 2B4) and a signal transduction domain (CD3 zeta). The CAR-NK cell targeting CD19 constructed in the application can exert cell killing effect by combining with CD19 antigens on the surface of tumor cells, and the killing efficiency on tumor cells is obviously improved compared with traditional NK cells, and the CAR-NK cell targeting CD19 has a good application prospect in treating tumors.

[0020] Obviously, according to the above content of the application, according to the ordinary technical knowledge and means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0021] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology achieved based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Structure diagram of the chimeric antigen receptor targeting CD19;

[0023] Figure 2 Flow analysis result diagram of NK cells before and after D0 sorting, wherein, Figure 2 A is a flow analysis scatter diagram of NK cells before D0 sorting, the horizontal axis represents the fluorescence intensity of CD56-FITC, and the vertical axis represents the fluorescence intensity of CD3-APC-Cy7, Figure 2 B is a flow histogram of NK cells before D0 sorting, the horizontal axis represents the fluorescence intensity of CD3-APC-Cy7, Figure 2 C is a flow analysis scatter diagram of NK cells after D0 sorting, the horizontal axis represents the fluorescence intensity of CD56-FITC, and the vertical axis represents the fluorescence intensity of CD3-APC-Cy7, Figure 2 D is a flow histogram of NK cells after D0 sorting, the horizontal axis represents the fluorescence intensity of CD3-APC-Cy7; it is found by flow cytometry analysis that after CD3 negative selection, the proportion of CD3+CD56- cells decreases from 61.87% to 0.33%, and after NK cell (CD3-CD56+) sorting, the proportion increases from 14.53% to 19.56%;

[0024] Figure 3Figure A is a detection result chart of the CAR1-NK cells, and the CAR positive rate is 48.01%, Figure 3 A is a detection result chart of the CAR1-NK cells, Figure 3 B is a detection result chart of the CAR2-NK cells, Figure 3 C is a detection result chart of the CAR3-NK cells; the horizontal axis in the chart represents the fluorescence intensity of CD19 CAR-APC.

[0025] Figure 4 Figure A is a detection result chart of the NK cells (CD3-CD56+) cultured to the 19th day, and the NK cell (CD3-CD56+) ratio is 99.24%, and the CD3+CD56-cell ratio is 0.2%, Figure 4 A is a detection result chart of the NK cells cultured to the 19th day in Example 1, and the NK cell (CD3-CD56+) ratio is 99.24%, and the CD3+CD56-cell ratio is 0.2%, Figure 4 B is a detection result chart of the NK cells cultured to the 19th day in Comparative Example 2; the horizontal axis in the chart represents the fluorescence intensity of CD56-FITC, and the vertical axis represents the fluorescence intensity of CD3-APC-Cy7.

[0026] Figure 5 Figure A is a detection result chart of the CAR1-NK cells, and the CAR positive rate is 48.01%, Figure 5 A is a detection result chart of the CAR1-NK cells, and the CAR positive rate is 48.01%, Figure 5 B is a detection result chart of the CAR2-NK cells, and the CAR positive rate is 40.43%, Figure 5 C is a detection result chart of the CAR3-NK cells, and the CAR positive rate is 27.26%; the horizontal axis in the chart represents the fluorescence intensity of CD19 CAR-APC.

[0027] Figure 6 Figure A is a detection result chart of the CAR1-NK cells, and the CAR positive rate is 48.01%, DETAILED DESCRIPTION

[0028] In the following examples and experimental examples, the reagents and materials not specifically stated are commercially available.

[0029] The CD3 nano magnetic beads are purchased from Meitian Nanometer Magnetic Beads, and the item number is 130-097-043.

[0030] IL21 NK cell expansion reagent (nurse cells): manufacturer: Zhongying Biology, item number: ZY-NKJ-1000.

[0031] The exponential method counting method used by the present application when indicating the cell density is, for example, when the cell density is 5E7 / ml, it indicates that the cell density is 5x10 7

[0032] ​In the following examples, recombinant vectors are constructed according to conventional experimental methods in the art.

[0033] Example 1: A CAR-NK cell targeting CD19 and a preparation method thereof

[0034] The present example provides a CAR-NK cell targeting CD19, wherein the chimeric antigen receptor is CAR1, and a structural schematic diagram of CAR1 is shown in Figure 1 The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region. The extracellular region comprises a signal peptide, an anti-CD19 single-chain antibody (CD19 scFv), and a hinge region structure of IgG4 Hinge. The transmembrane region has a structure of CD28 TM. The intracellular region comprises a costimulatory factor domain (CD28) and a signal transduction domain (CD3 zeta), which is composed of an amino acid sequence shown as SEQ ID NO. 1. The intracellular domain transmits the information of CD19 CAR binding to human CD19 to the inside of the immune effector cell to trigger effector cell functions (such as activation, cytokine production, proliferation, and cytotoxic activity).

[0035] SEQ ID NO. 1 (CAR1):

[0036] MALPVTALLLPLALLLHAARPDIQMTQSPSSVSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSLSDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0037] SEQ ID NO. 2 (CD19 scFv):

[0038] DIQMTQSPSSVSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSLSDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

[0039] CAR-NK cells targeting CD19 are prepared by the following method:

[0040] 1. Synthesis of gene sequence encoding CAR1 chimeric antigen receptor molecule

[0041] The gene encoding the CAR1 chimeric antigen receptor molecule is a fusion gene fragment, which is synthesized by GenScript Biotech Corporation (Shanghai) Limited, and the nucleotide sequence of the fusion gene fragment is shown in SEQ ID NO. 3:

[0042]

[0043] 2. Preparation of pseudovirus particles of CARl lentivirus vector containing nucleic acid molecule encoding CARl chimeric antigen receptor protein

[0044] Lentivirus is a gene therapy vector developed on the basis of human immunodeficiency virus (HIV), which has the ability to infect both dividing and non-dividing cells and can be expressed in cells for a long period of time. The lentivirus used in this embodiment is a "suicide" virus, i.e., the virus will not infect other cells after infecting the target cells, nor will it produce new virus particles using host cells.

[0045] (1) Plating on day 0: 0.25% trypsin-digested 293T cells were plated in a 10 cm culture dish containing 10 ml DMEM (containing 10% FBS) and cultured in a 37°C, 5% CO2 incubator to ensure that the cell confluence reached 90-95% after 24 h.

[0046] (2) Transfection of 293T cells: 2 hours before transfection, the 293T cells were changed. The CD19 chimeric antigen receptor lentivirus vector (pCDH-EF1) and the helper vector (pspax2 and BaEv) were mixed with PEI, blown evenly, and incubated at room temperature for 15 min to obtain the DNA / PEI mixture. The prepared DNA / PEI mixture was added dropwise to the 293T cells, and the culture was continued for 4-6 hours, and the medium was changed.

[0047] (3) Pseudovirus concentration and purification: The cell supernatants of the above-mentioned 293T cell transfection at 24 and 48 hours were collected, filtered with a 0.45 μM filter, and then concentrated by ultracentrifugation, i.e., the cell supernatant containing lentivirus was filtered with a 0.45 μM filter, ultracentrifuged at 25,000 rpm for 120 min, and finally dissolved in PBS, aliquoted, and stored at -80°C to obtain CD19 chimeric antigen receptor virus pseudovirus particles.

[0048] 3. Isolation of peripheral blood PBMC

[0049] Freshly collected peripheral blood (provided by healthy volunteers) was placed in a sterile heparin sodium collection tube, and whole blood was slowly added to the lymphocyte separation medium at a ratio of 1:1. PBMC was separated by density gradient centrifugation, and the layering was as clear as possible. After separation, the white membrane layer was aspirated, and mononuclear cells PBMC were collected. Red blood cell lysis solution was used to resuspend the cells, and they were mixed gently. The residual red blood cells in the PBMC were removed. The treated PBMC was frozen at a density of 5E7 / ml.

[0050] 4. Isolation and culture of NK cells

[0051] Recovery of frozen PBMC, NK cells were sorted by CD3 nano magnetic beads negative selection, and then the sorted NK cells were expanded in the presence of irradiated K562 cells (4-1BBL+IL21) as feeder cells, using the NK expansion kit (5% autologous serum) and IL2 500U / ml, with a cell density of 2E6 / mL.

[0052] CD3+ cells were removed from human peripheral blood PBMC using the NK cell sorting kit from Meiti, and the efficiency of CD3+ cell removal was verified by flow cytometry immunofluorescence staining (anti-CD3-APC and anti-CD56-FITC), as shown in Figure 2 Then, irradiated K562 cells stably expressing 4-1BBL and IL21 were used as feeder cells (Feeder cell), and the ratio of feeder cells to sorted NK cells was 2:1. The culture medium could be selected from the NK expansion kit (5% serum substitute, IL2 100U / ml).

[0053] 4. Virus transduction

[0054] NK cell culture was performed on the fourth day for lentivirus transduction, with a MOI of 5. After adding the virus, the cells were mixed by pipetting and cultured in a 5% CO2 incubator.

[0055] 5. Expansion

[0056] After 24 hours of NK cell culture after virus transduction, the cells were centrifuged and the supernatant was discarded. After centrifugation at 400g for 5 minutes at room temperature, NK complete medium was added to the cell pellet. On the seventh day, the CAR positive rate was detected by flow cytometry, as shown in Figure 3 A, the CAR positive rate was 29.58%.

[0057] According to the number ratio of Feeder cell:NK=1:2, Feeder cells were added to the cells cultured to the seventh day, and the total cell (including Feeder cell) concentration was 0.5-1E6 cells / mL. When the culture volume was greater than 200mL, the cells were transferred to G-rex culture bottles for culture. From the ninth to thirteenth day, the cell state was observed every day, and the medium was supplemented according to the color of the medium and the cell density. On the nineteenth day, the cells were collected, and the collected cells (CD19-CAR-NK cells) were frozen. Before freezing, the collected cells (CD19-CAR-NK cells) were labeled with anti-CD56-FITC and anti-CD3-APC-Cy7, and the NK purity was determined by flow cytometry, as shown in Figure 4 A, the NK purity was higher than 99%. The collected cells (CD19-CAR-NK cells) were labeled with anti-CD19-APC, and the transduction efficiency was determined by flow cytometry, as shown inFigure 5 The CAR positive rate was 48.01%, as shown in Figure A.

[0058] In other embodiments, the optimal cell collection time can be adjusted in the range of 16-21 days.

[0059] Comparative Example 1: A CAR-NK cell targeting CD19

[0060] A CAR-NK cell targeting CD19 was prepared according to the method of Example 1, except that the chimeric antigen receptor was CAR2, the structural diagram of CAR2 was as shown in Figure B, and CAR2 was composed of an amino acid sequence as shown in SEQ ID NO. 4. Figure 1

[0061] SEQ ID NO. 4 (CAR2):

[0062] MALPVTALLLPLALLLHAARPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0063] wherein the amino acid sequence of the CD19 scFv is as shown in SEQ ID NO. 5:

[0064] ​EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI

[0065] The CAR positive rate of the NK cells cultured for 7 days was 59.35%, as shown in FIG. 1B; the CAR positive rate of the NK cells cultured for 16 days was 40.43%, as shown in FIG. 1B. Figure 3 Figure 5

[0066] Comparative Example 2: A CAR-NK cell targeting CD19

[0067] A CAR-NK cell targeting CD19 was prepared according to the method of Example 1, except that the chimeric antigen receptor was CAR3, and the structural diagram of CAR3 is shown in FIG. 2B, which is composed of the amino acid sequence shown as SEQ ID NO. 6. Figure 1

[0068] SEQ ID NO. 6 (CAR3):

[0069] ​​​MALPVTALLLPLALLLHAARPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0070] The amino acid sequence of the CD19 scFv is shown in SEQ ID NO. 5.

[0071] The CAR positive rate of the NK cells cultured for 7 days was 31.76% (C). Figure 3 The purity of the NK cells cultured for 16 days was still higher than 99% (B), and the CAR positive rate was 27.26% (C). Figure 4 Figure 5 The CAR positive rate of the NK cells cultured for 7 days was 31.76% (C).

[0072] The technical solutions of the present application are further described below through experiments.

[0073] Experimental Example 1: In vitro killing effect of CAR-NK cells on NALM-6

[0074] I. Experimental methods

[0075] Flow cytometry was used to detect the expression level of CD19 antigen protein of NALM-6 tumor cells.

[0076] ​The CAR-NK cells prepared in Example 1 and Comparative Examples 1-2 were adjusted to a CAR+ ratio of 27.26%, and then co-cultured with target cells NALM-6 at an effector-target ratio of 0.5:1, after 4 hours, the cells were collected, and the ratio of 7-AAD and Annixin V positive cells was analyzed by flow cytometry to determine the killing efficiency of the CAR-NK cells on the target cells.

[0077] II. Experimental results

[0078] The results of the expression level of CD19 antigen protein of NALM-6 tumor cells are shown in Table 1, which shows that NALM-6 tumor cells highly express CD19 antigen. Figure 6

[0079] The anti-tumor results are shown in Table 1, compared with conventional NK cells, the killing efficiency of the CAR-NK cells prepared in Examples 1-3 of the present application on target cells NALM-6 is improved, in particular, the CAR1-NK cells have the best effect on improving the anti-tumor effect.

[0080] Table 1

[0081]

[0082] From the above examples and experimental examples, it can be seen that the present application provides a CAR-NK cell targeting CD19 by screening sequences and structures, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, the extracellular region comprises a signal peptide (CD8α), an anti-CD19 single-chain antibody (CD19 scFv) and a hinge region (lgG4, lgG4(L) or CD8α), the structure of the transmembrane region comprises CD28 TM or CD8αTM, and the intracellular region comprises a costimulatory factor domain (CD28, 4-1BB, OX40 or 2B4) and a signal transduction domain (CD3ζ). The CAR-NK cell targeting CD19 constructed by the present application can exert a cell killing effect by binding to the CD19 antigen on the surface of tumor cells, and compared with conventional NK cells, the killing efficiency on tumor cells is significantly improved, and has a good application prospect in the treatment of tumors.​

Claims

1. A chimeric antigen receptor, characterized in that: Its structure consists of an extracellular region, a transmembrane region, and an intracellular region connected in sequence; the extracellular region consists of a signal peptide, an anti-CD19 single-chain antibody, and a hinge region connected in sequence, and the amino acid sequence of the anti-CD19 single-chain antibody is shown in SEQ ID NO.

2.

2. The chimeric antigen receptor according to claim 1, characterized in that: The structure of the signal peptide is CD8α, and the structure of the hinge region is selected from lgG4 or CD8α. The structure of the transmembrane region is selected from CD28TM or CD8αTM; The intracellular region is structured as a costimulatory factor domain and a signal transduction domain connected in sequence. The costimulatory factor domain is selected from CD28, 4-1BB, OX40 or 2B4, and the signal transduction domain is CD3ζ.

3. The chimeric antigen receptor according to claim 2, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

4. A gene, characterized by: The gene encodes the chimeric antigen receptor as described in any one of claims 1-3.

5. The gene according to claim 4, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

3.

6. A recombinant vector, characterized in that: The recombinant vector includes the gene described in claim 4 or 5.

7. A host cell, characterized in that: It comprises the recombinant vector as described in claim 6.

8. A CAR-NK cell, characterized in that: Its surface expresses the chimeric antigen receptor as described in any one of claims 1-3.

9. The CAR-NK cells according to claim 8, characterized in that, It is prepared by transducing NK cells with the gene encoding the chimeric antigen receptor via lentiviral transduction.

10. The method for preparing CAR-NK cells according to claim 8 or 9, characterized in that, It includes: The gene encoding the chimeric antigen receptor was transduced into NK cells via lentiviral transduction.

11. Use of the chimeric antigen receptor according to any one of claims 1-3, the gene according to claim 4 or 5, the recombinant vector according to claim 6, the host cell according to claim 7, or the CAR-NK cell according to claim 8 or 9 in the preparation of a medicament for treating human B-lymphocytic leukemia.

Images