CAR-T cell targeting CD38 and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a CD38-targeted CAR-T cell and a preparation method and application thereof, the method comprises the step of infecting a T cell with a recombinant lentivirus, and the recombinant lentivirus contains a nucleic acid molecule encoding CD38-CAR or is prepared through an expression vector containing the nucleic acid molecule. The CD38-targeted CAR-T cell disclosed by the invention can express CD38-CAR, and the killing capability of the T cell on tumor cells can be enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a CD38-targeted CAR-T cell, a preparation method thereof, and an application thereof. Background Art

[0002] Multiple Myeloma (MM) is a plasma cell malignancy, which is caused by the monoclonal proliferation of plasma cells, leading to the production of monoclonal antibodies and damage to terminal organs such as the bone marrow, further resulting in cytopenia and bone fragility, etc. (Padala et al., 2021). Although emerging drug therapies have effectively improved the prognosis and extended the survival of MM patients, MM is still incurable at present. Therefore, there is an urgent need to develop new treatment strategies (Kumaret al., 2008).

[0003] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) is an emerging adoptive immunotherapy. By means of genetic engineering techniques, chimeric antigen receptors are expressed on the surface of T cells, enabling them to specifically target, recognize, and eliminate cells expressing specific target antigens (Sterner and Sterner, 2021). CAR-T therapy has shown durable and effective curative effects clinically, and CAR-T products targeting CD19 have been approved for the treatment of B-lymphocyte acute leukemia and diffuse large B-cell lymphoma (Fournieretal., 2017).

[0004] B-cell maturation antigen (BCMA) is selectively expressed on tumor plasma cells and is involved in the survival and proliferation of MM cells. Therefore, it is considered an excellent target for targeted therapy of multiple myeloma. Anti-BCMA-CAR-T cell therapy has significant curative effects on multiple myeloma, and its objective remission rate ranges from 30% to 100% (Rajeetal., 2019). However, multiple experiments have also reported that some patients have treatment failures or relapses within a short time after treatment, mainly due to tumor cell antigen escape. Therefore, it has become particularly important to find new targets and more effective CAR-T cell treatment regimens (Manier et al., 2022).

[0005] CD38 is a single-chain type II transmembrane glycoprotein that participates in regulating the adhesion of lymphocytes to endothelial cells and is a surface marker of human leukemia and myeloma cells (Deaglio et al., 1998, Deaglio et al., 2001, Malavasi et al., 2008). CD38 is highly expressed on the surface of MM cells and is considered an effective immunotherapeutic target for MM. Anti-CD38 monoclonal antibodies exert anti-tumor effects through multiple mechanisms of action and prolong the survival of patients. Multiple CD38 antibodies have been approved for clinical treatment (Munshi et al., 2021). However, there are currently no CAR-T cells targeting CD38 for the clinical treatment of MM. Summary of the Invention

[0006] In view of this, the present invention is based on the modification of anti-CD38 monoclonal antibodies to prepare CAR-T cells targeting CD38, aiming to improve the efficacy against multiple myeloma and provide an effective alternative approach for the treatment of cancers such as human leukemia and myeloma.

[0007] Therefore, on the one hand, the present invention provides a chimeric antigen receptor targeting CD38 (CD38-CAR) comprising the following elements:

[0008] 1) a signal peptide region, 2) an anti-CD38 antibody region; 3) a hinge region; 4) a transmembrane region; 5) an intracellular co-stimulatory region; and 6) an intracellular stimulatory region,

[0009] wherein the amino acid sequence of the anti-CD38 antibody region is shown as any one of SEQ ID NO: 4-13.

[0010] In an embodiment of the present invention, the signal peptide region can be the signal peptide of any mammalian cell surface protein. Preferably, the signal peptide is the human albumin signal peptide, the human insulin signal peptide, or the murine IgGkappa signal peptide, and its amino acid sequence is shown as SEQ ID NO: 1, 2, or 3.

[0011] In an embodiment of the present invention, the hinge region can be the hinge region of human CD8α or human CD28. Preferably, the hinge region is the hinge region of human CD8α, and its amino acid sequence is shown as SEQ ID NO: 14.

[0012] In an embodiment of the present invention, the transmembrane region can be the transmembrane region of human CD8α or human CD28. Preferably, the transmembrane region is the transmembrane region of human CD8α, and its amino acid sequence is shown as SEQ ID NO: 15.

[0013] In an embodiment of the present invention, the amino acid sequence of the intracellular co-stimulatory region is shown as SEQ ID NO: 16.

[0014] In an embodiment of the present invention, the amino acid sequence of the intracellular stimulating region is as shown in SEQ ID NO: 17.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CD38-CAR) targeting CD38.

[0016] In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule encoding a chimeric antigen receptor (CD38-CAR) targeting CD38.

[0017] In a fourth aspect, the present invention provides a recombinant lentivirus comprising the nucleic acid molecule of the second aspect or prepared by the expression vector of the third aspect.

[0018] In a fifth aspect, the present invention provides a method for preparing a recombinant lentivirus, which co-transfects a mammalian host cell with the expression vector of the third aspect and an auxiliary plasmid to obtain a recombinant lentivirus.

[0019] In an embodiment of the fifth aspect, the auxiliary plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0020] In an embodiment of the fifth aspect, the mammalian host cell can be but is not limited to HEK293 cells, human PER.C6 cells, human Hela cells, murine CHO cells, etc., preferably HEK293 cells.

[0021] In a sixth aspect, the present invention provides a CAR-T cell targeting CD38 (CD38-CAR-T cell), which expresses a chimeric antigen receptor (CD38-CAR) targeting CD38.

[0022] In a seventh aspect, the present invention provides a method for preparing the CD38-CAR-T cell of the sixth aspect, including infecting T cells with the recombinant lentivirus of the fifth aspect.

[0023] In a specific embodiment of the seventh aspect, the method for preparing the CD38-CAR-T cell specifically includes the following steps:

[0024] (1) Construct a recombinant lentiviral expression vector carrying a nucleotide sequence encoding CD38-CAR;

[0025] (2) Transfect a host cell with the recombinant lentiviral expression vector and an auxiliary plasmid to prepare a recombinant lentivirus capable of infecting T cells;

[0026] (3) Isolate PBMC from the peripheral blood provided by a donor, and use magnetic beads to isolate and activate T cells;

[0027] (4) Infect T cells with the recombinant lentivirus obtained in step (2) to generate T cells expressing CD38-CAR.

[0028] In a further embodiment, the helper plasmids can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0029] In a further embodiment, the method may further include the following steps after step (4):

[0030] (5) Cultivate the cells obtained in step (4) in vitro;

[0031] (6) Greatly expand the cells obtained in step (5); and

[0032] (7) Collect T cells expressing CD38-CAR.

[0033] In an eighth aspect, the present invention provides a disease treatment drug, which comprises the nucleic acid molecule encoding CD38-CAR of the present invention, an expression vector containing the nucleic acid molecule encoding CD38-CAR, the recombinant lentivirus of the fourth aspect, or the CD38-CAR-T cells of the sixth aspect.

[0034] In a ninth aspect, the present invention provides the use of the above nucleic acid molecule, the above expression vector, the above recombinant lentivirus, or the above CD38-CAR-T cells in the preparation of a disease treatment drug.

[0035] In the embodiments of the present invention, the disease refers to a disease characterized by high expression of CD38 molecules on the cell surface. In a specific embodiment, the disease can be multiple myeloma, lymphoma, leukemia, systemic lupus erythematosus, Sjogren's syndrome, etc.

[0036] In the embodiments of the present invention, CAR represents a chimeric antigen receptor, CD38-CAR represents a chimeric antigen receptor targeting CD38, CAR-T cells represent chimeric antigen receptor T cells, and CD38-CAR-T cells represent T cells expressing a chimeric antigen receptor targeting CD38.

[0037] The present invention provides CAR-T cells targeting CD38 (CD38-CAR-T cells), which express a chimeric antigen receptor targeting CD38 (CD38-CAR). The chimeric antigen receptor comprises a signal peptide region, an anti-CD38 antibody region, a hinge region, a transmembrane region, an intracellular co-stimulatory region, and an intracellular stimulatory region, and thus has a unique structural design. The CD38-CAR-T cells of the present invention have an extremely high CD38-CAR positive rate and have a good killing effect on multiple myeloma MM.1S. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of the structure of CD38-CAR.

[0039] Figure 2 , Figure 3 , Figure 4 It is a detection chart of the CAR positive rate of CD38-CAR-T cells on the 7th and 9th days after virus transfection.

[0040] Figure 5 and Figure 6 It is a comparison chart of the killing results of CD38-CAR-T cells. The horizontal axis is the effector-to-target ratio, that is, the number ratio of T cells or CAR-T cells to target cells MM.1S. Specific implementation mode

[0041] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as the Molecular Cloning Experimental Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or according to the conditions recommended by the manufacturer's instructions.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained through commercial channels.

[0043] Materials and methods

[0044] 1. Design and construction of plasmid encoding CD38-CAR

[0045] The plasmid encoding CD38-CAR was synthesized by Nanjing Biaode Biotechnology Co., Ltd. This plasmid contains nucleotide sequences encoding the following elements in the 5' to 3' direction of the CAR structure: signal peptide (SEQ ID NO: 1, 2 or 3), anti-CD38 antibody region (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), hinge region (SEQ ID NO: 14), transmembrane region (SEQ ID NO: 15), intracellular co-stimulatory region (SEQ ID NO: 16) and intracellular stimulatory region CD3ζ (SEQ ID NO: 17). This plasmid was cloned into the lentiviral backbone plasmid pMSGV-IRES (Shanghai New Bio-Tech Co., Ltd., product number: V000092) to obtain a plasmid encoding CD38-CAR, which is called pMSGV-CD38 and is a recombinant lentiviral expression vector in virus packaging.

[0046] 2. Preparation of recombinant lentivirus expressing CD38-CAR

[0047] The recombinant lentivirus expression vector pMSGV-CD38 and three helper plasmids pMDLG-pRRE-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0374), pMD2G-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0648), and pRSV-REV-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0370) were co-transfected into the WayneLVPro HEK293 cell line (Suspension Adapted) (Zhongshan Kangsheng Biotechnology Co., Ltd., product number A23109, hereinafter referred to as 293T cells), and the recombinant lentivirus that can infect T cells can be prepared. The specific operation steps are as follows:

[0048] 2.1. Adjust the density of 293T cells to 0.5×10 6 cells / mL, and culture them in SMM 293-TII Expression Medium (Sino Biological Inc., product number: M293TII) at 37°C, 5% CO 2 , in a shaker at 160 rpm for 48 h, and count to 4×10 6 cells / mL.

[0049] 2.2. Take 144 μL of PEI with a concentration of 1 mg / mL (Merck Biotechnology Co., Ltd., 937762), add it to 1 mL of OPTI-MEM (Thermo Fisher Scientific Inc., product number: 11058021), mix well, and let it stand at room temperature for 20 min.

[0050] 2.3. Add 16 μg of the recombinant lentivirus expression vector pMSGV-CD38 and 12 μg of pMDLG-pRRE-Kana, 4 μg of pMD2G-Kana, and 4 μg of pRSV-REV-Kana helper plasmids to 1 mL of SMM 293-TII Expression Medium, and mix well.

[0051] 2.4. Add the mixed solution of PEI and 1 mL of OPTI-MEM in step 2 to the solution in step 3, mix well, and let it stand at room temperature for 20 min.

[0052] 2.5. Take the mixed solution in step 4, add it to the cells prepared in step 1, and mix well. After 6.6 - 8 h, add 2% by volume of LVFeed 04 supplement (Zhongshan Kangsheng Biotechnology Co., Ltd., product number: QRD003).

[0053] After 2.6.48 h, the cell supernatant was collected and centrifuged at 5000 g for 10 min to remove cells. The supernatant containing the recombinant lentivirus was collected and filtered through a 0.22-μm filter membrane (Thermo Fisher Scientific, catalog number: 4115-2800). The filtered supernatant was stored frozen at -80 °C in a refrigerator for later use.

[0054] 3. Detection of recombinant lentivirus titer

[0055] Jurkat cells (purchased from ATCC: MD130) after 2 passages of resuscitation culture were centrifuged at 300 g for 10 min, and resuspended in RPMI1640 (GE Healthcare Life Sciences, catalog number: SH30809-01) medium to 2×10 6 cells / mL. 0.1 mL of the cell suspension was seeded into each well of a 48-well plate. 100 μL of the virus solution diluted 200-fold with RPMI1640 was added to the Jurkat cells and mixed well. After 6 h, 800 μL of RPMI 1640 complete medium containing 10% FBS (Thermo Fisher Scientific, catalog number: A5670701) was supplemented and cultured in a 37 °C, 5% CO 2 cell incubator. Flow cytometry was performed after 48 h.

[0056] 4. T cell isolation and activation

[0057] Under sterile conditions, 15 mL of healthy human donor peripheral blood was taken into a heparin sodium anticoagulant tube and left to stand at room temperature for 30 min; centrifuged at 400 g for 30 min. After stratification, the upper plasma was aspirated and reserved. The lower cell supernatant was diluted and mixed with PBS at a ratio of 1:1, and then the bottom blood dilution was carefully added to the upper layer of lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd., catalog number: LTS1077) at a ratio of 1:2. Centrifuged at 300 g for 10 min. After centrifugation, the white film layer above the separation medium was aspirated into a new centrifuge tube, and the white film layer was washed with PBS and centrifuged at 300 g for 10 min. The supernatant was discarded to obtain PBMC (Peripheral Blood Mononuclear Cell).

[0058] T lymphocytes were sorted from PBMC according to the CD3 Microbeads (Human) (Miltenyi Biotec, catalog number: 130-050-101) magnetic bead instructions and cell counting was performed. Take the cell supernatant containing 9×10 6 sorted CD3 + T cells, centrifuge at 300 g for 10 min at 4 °C, discard the supernatant, and use X-VIVO containing 40 IU / mL human IL-2 (Suzhou Novoprotein Science & Technology Co., Ltd., catalog number: GMP-CD66) TMResuspend the cell pellet with 15% serum-free medium (Lonza Investment (China) Co., Ltd., product number: DL-102), and adjust the cell concentration to 1×10 6 cells / mL. Activate T cells according to the instructions of Dynabeads TM Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific Inc., product number 11452D), and seed the T cells at a concentration of 1×10 6 cells / mL, 1 mL per well, in a 24-well cell culture plate; and culture in a cell incubator for 48 h.

[0059] 5. Preparation of recombinant T cells

[0060] 5.1. Take a 24-well plate. After 48 h of T cell isolation and activation, add 30 μL of 1 mg / mL protamine (Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: P4020) to each well, and pipette to mix evenly.

[0061] 5.2. Calculate the required virus amount according to MOI = 3. The calculation formula is as follows: required virus amount (mL) = (MOI * number of cells) / virus titer. According to the calculation result, add the recombinant lentivirus prepared in "2. Preparation of recombinant lentivirus expressing CD38-CAR" above to the 24-well plate and mix well.

[0062] 5.3. Centrifuge the T cells in the 24-well plate at 1000 g for 30 min, and culture in a 37 °C, 5% CO 2 cell incubator.

[0063] 5.4. Stain all samples with FITC-CD38 (Beijing Sino Biological Inc., product number: 10818-H08H-F) antigen, and detect CAR expression by flow cytometry.

[0064] 6. In vitro killing experiment of CAR-T cells against target cells

[0065] Introduce GFP protein into MM.1S cells (Cell Bank of the Chinese Academy of Sciences, product number: SCSP-5017) to transform them into MM.1S-GFP cells as target cells. Use CD38-CAR-T cells and empty vector T cells as effector cells. Mix the effector cells and target cells at effector-to-target ratios of 0.2 and 0.4 for killing. In the killing experiment, the group containing empty vector T cell killing is the blank control group. The killing activity is measured as follows:

[0066] 6.1. Adjust the target cell state to the logarithmic growth phase and perform two consecutive passages before the experiment.

[0067] 6.2. Adjust the target cell density to 1×10 6 cells / mL and inoculate 1 mL per well into a 6-well plate.

[0068] 6.3. Experimental group: Adjust the CD38-CAR-T cell density to 1×10 6 cells / mL and add 0.2 mL and 0.4 mL of CD38-CAR-T cells to the wells containing the target cells above according to the effector-to-target ratios of 0.2 and 0.4, respectively, and mix well.

[0069] 6.4. Blank control group: Adjust the density of empty vector T cells to 1×10 6 cells / mL and add 0.2 mL and 0.4 mL of T cells to the wells containing the target cells above according to the effector-to-target ratios of 0.2 and 0.4, respectively, and mix well.

[0070] 6.5. Incubate the above 6-well plate in a 37°C, 5% CO 2 cell incubator for 24 h, and then calculate the volume of the medium in the wells and the cell concentration, respectively.

[0071] 6.6. Perform flow cytometry on all samples and detect the positive rate of target cells through the FITC channel.

[0072] Results and Discussion

[0073] According to the above method, a total of 30 chimeric antigen T cells targeting CD38, namely CD38-CAR-T cells, were constructed according to the different sequences of the signal peptide region and anti-CD38 antibody region in the CAR. Table 1 lists these 30 cells.

[0074] Table 1. 30 CD38-CAR-T cells constructed in the present invention

[0075]

[0076]

[0077] * The former represents the killing rate of MM.1s-GFP target cells at an effector-to-target ratio of 0.2 on the second day, and the latter represents the killing rate of MM.1s-GFP target cells at an effector-to-target ratio of 0.4 on the second day.

[0078] The experimental results show that the positive rates of CD38-CAR in these 30 CD38-CAR-T cells are all higher than 90%. Figure 2 、 Figure 3 and Figure 4The positive rates of CD38 in three kinds of CD38-CAR-T cells formed by the combination of anti-CD38 antibody 1 (SEQ ID NO: 4) and three signal peptides (SEQ ID NO: 1, 2 or 3) were all higher than 90% respectively.

[0079] In the in vitro killing experiment of CD38-CAR-T cells against target cells, these 30 kinds of CD38-CAR-T cells also showed similar killing effects. For example, CD38-CAR-T cells 1, 2 and 3 (see Table 1) had obvious killing effects on MM.1s-GFP target cells. Compared with the empty vector T cells, when the effector-to-target ratios were 0.2 and 0.4 respectively, on the first day, the killing rates of CD38-CAR-T cells against target cells were about 50% and 70% respectively, as Figure 5 shown; on the second day, the killing rates against target cells were 96% and 99% respectively, as Figure 6 shown.

[0080] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

[0081] References

[0082] DEAGLIO, S., MEHTA, K. & MALAVASI, F. (2001), "Human CD38: a(r)evolutionary story of enzymes and receptors", Leuk Res, Vol. 25 No. 1, pp. 1-12.

[0083] Deaglio, S., Morra, M., Mallone, R., Ausiello, C. M., Prager, E., Garbarino, G., Dianzani, U., Stockinger, H. & Malavasi, F. (1998), "Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member", J Immunol, Vol. 160 No. 1, pp. 395-402. Fournier, C., Martin, F., Zitvogel, L., Kroemer, G., Galluzzi, L. & Apetoh, L. (2017), "TrialWatch: Adoptively transferred cells for anticancer immunotherapy", Oncoimmunology, Vol. 6 No. 11, pp. e1363139.

[0084] Kumar, S. K., Rajkumar, S. V., Dispenzieri, A., Lacy, M. Q., Hayman, S. R., Buadi, F. K., Zeldenrust, S. R., Dingli, D., Russell, S. J., Lust, J. A., Greipp, P. R., Kyle, R. A. & Gertz, M. A. (2008), "Improved survival in multiple myeloma and the impact of novel therapies", Blood, Vol. 111 No. 5, pp. 2516-20.

[0085] Malavasi, F., Deaglio, S., Funaro, A., Ferrero, E., Horenstein, A. L., Ortolan, E., Vaisitti, T. & Aydin, S. (2008), "Evolution and function of the ADP ribosyl cyclase / CD38 gene family in physiology and pathology", Physiol Rev, Vol. 88 No. 3, pp. 841-86.

[0086] MANIER, S., INGENERE, T., ESCURE, G., PRODHOMME, C., NUDEL, M., MITRA, S. & FACON, T. (2022), "Current state and next-generation CAR-T cells in multiple myeloma", Blood Rev, Vol. 54 100929. MUNSHI, N.C., ANDERSON, L.J., SHAH, N., MADDURI, D., BERDEJA, J., LONIAL, S., RAJE, N., LIN, Y., SIEGEL, D., ORIOL, A., MOREAU, P., YAKOUB-AGHA, I., DELFORGE, M., CAVO, M., EINSELE, H., GOLDSCHMIDT, H., WEISEL, K., RAMBALDI, A., REECE, D., PETROCCA, F., MASSARO, M., CONNARN, J.N., KAISER, S., PATEL, P., HUANG, L., CAMPBELL, T.B., HEGE, K. & SAN-MIGUEL, J. (2021), "Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma", N Engl J Med, Vol. 384 No. 8, pp. 705-716.

[0087] PADALA, S.A., BARSOUK, A., BARSOUK, A., RAWLA, P., VAKITI, A., KOLHE, R., KOTA, V. & AJEBO, G.H. (2021), "Epidemiology, Staging, and Management of Multiple Myeloma", Med Sci (Basel), Vol. 9 No. 1, pp.

[0088] RAJE, N., BERDEJA, J., LIN, Y., SIEGEL, D., JAGANNATH, S., MADDURI, D., LIEDTKE, M., ROSENBLATT, J., MAUS, M. V., TURKA, A., LAM, L. P., MORGAN, R. A., FRIEDMAN, K., MASSARO, M., WANG, J., RUSSOTTI, G., YANG, Z., CAMPBELL, T., HEGE, K., PETROCCA, F., QUIGLEY, M. T., MUNSHI, N. & KOCHENDERFER, J. N. (2019), "Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma", N Engl J Med, Vol. 380 No. 18, pp. 1726-1737. STERNER, R. C. & STERNER, R. M. (2021), "CAR-T cell therapy: current limitations and potential strategies", Blood Cancer J, Vol. 11 No. 4, pp. 69.

Claims

1. A CAR targeting CD38, characterized in that The chimeric antigen receptor comprises the following regions: 1) Signal peptide region; 2) anti-CD38 antibody region; 3) Hinge region; 4) transmembrane region; 5) intracellular co-stimulatory region; and 6) Intracellular stimulation zone, Wherein, the amino acid sequence of the anti-CD38 antibody region is shown in any one of SEQ ID NOs: 4-13.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CD38-targeting chimeric antigen receptor of claim 1.

3. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 2.

4. A recombinant lentivirus, characterized in that The recombinant lentivirus comprises the nucleic acid molecule of claim 2 or is prepared by the expression vector of claim 3.

5. The method for preparing the recombinant lentivirus according to claim 4, characterized in that: The method comprises the following steps: co-transfecting mammalian host cells with the vector according to claim 3 and an auxiliary plasmid.

6. A method for preparing CAR-T cells targeting CD38, characterized in that: The method comprises the following steps: infecting T cells with the recombinant lentivirus according to claim 4.

7. CAR-T cells targeting CD38, prepared by the method of claim 6.

8. A disease treatment drug, comprising the nucleic acid molecule of claim 2, the expression vector of claim 3, the recombinant lentivirus of claim 4, or the CD38-targeting CAR-T cell of claim 7.

9. The disease treating drug according to claim 8, characterized in that: The diseases are multiple myeloma, lymphoma, leukemia, systemic lupus erythematosus and Sjögren's syndrome.

10. Use of the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the recombinant lentivirus according to claim 4, or the CD38-targeted CAR-T cell according to claim 7 in the preparation of a drug for treating a disease.

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