CAR-T cells expressing dual-targeting CD38 and BCMA serial-parallel hybrid chimeric antigen receptors and secreting IL-15 and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to a CAR-T cell expressing a serial-parallel hybrid chimeric antigen receptor dual-targeting CD38 and BCMA and secreting IL-15, and a preparation method thereof, the method comprising infecting T cells with a recombinant lentivirus, the recombinant lentivirus comprising a nucleic acid molecule encoding a serial-parallel hybrid chimeric antigen receptor dual-targeting CD38 and BCMA and comprising IL-15, or obtained by an expression vector comprising the nucleic acid molecule. The CAR-T cell of the present invention expresses a serial-parallel hybrid CD38-BCMA-IL15-CAR and CD38-CAR, and can secrete the cytokine IL-15, which can better enhance the activation of natural killer cells and CD8 + The survival and proliferation of effector cells such as T cells can be enhanced, thereby enhancing the ability of CAR-T cells to kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a CAR-T cell expressing a dual-targeted CD38 and BCMA serial-parallel hybrid chimeric antigen receptor and secreting IL-15, and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is a plasma cell malignancy caused by the monoclonal proliferation of plasma cells, which leads to the production of monoclonal antibodies and damages end organs such as the bone marrow, further leading to hematopoiesis and bone fragility. Although emerging drug treatments can effectively improve the prognosis of MM patients and prolong their survival, MM is currently incurable, so new treatment strategies are still needed (Mei et al., 2021).

[0003] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) is a new type of immune cell therapy. It uses genetic engineering to express chimeric receptors in T cells, redirect lymphocytes to recognize and eliminate cells expressing specific target antigens, and has a long-lasting and effective clinical response. Currently, there are two CAR-T products targeting CD19 and BCMA for the treatment of B-cell acute leukemia and diffuse B-cell lymphoma, respectively (Chekol et al., 2022, Zhang et al., 2022).

[0004] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor protein superfamily. It is mainly expressed by malignant and normal plasma cells and some mature B cells. It participates in the survival and proliferation of MM cells, making it a major target for multiple myeloma. In a clinical study of BCMA-CAR-T cell therapy, 75% of patients had remission and 50% of patients had complete remission. Several experiments also reported that some patients failed treatment or relapsed within a short period of time after treatment, mainly due to antigen escape. Therefore, more effective CAR-T cell therapy regimens are needed (Yu Min et al., 2024, Parikhand Lonial, 2023).

[0005] Dual-targeted CAR-T cells are one of the methods to solve the problem of antigen escape, such as using CD19 / CD22 to treat leukemia and lymphoma, BCMA / CD19 to treat myeloma, etc. (Xu et al., 2024; Zhou et al., 2024; CN118459604A). CD38 is a single-chain type II transmembrane glycoprotein that is involved in regulating the adhesion of lymphocytes to endothelial cells and is a marker for human leukemia and myeloma cells. CD38 is highly expressed on the surface of MM cells and is considered to be an ideal target for MM immunotherapy (Liao et al., 2023). Anti-CD38 monoclonal antibodies can exert their anti-tumor effects through multiple mechanisms of action and prolong patient survival, and have been approved for clinical treatment; the preparation of anti-CD38-CAR-T cells may have significant therapeutic effects on multiple myeloma (De Novellis et al., 2022).

[0006] Studies have reported that after CAR-T cells are infused into patients, the number of CAR-T cells will drop to the initial level within 10 days, reducing the therapeutic effect, which may be caused by "self-killing" mediated by CAR-T cells (Guo et al., 2020). Therefore, it is necessary to study and develop a new CAR-T cell preparation method to promote the sustained survival and proliferation of CAR-T cells in vivo, thereby improving the therapeutic effect of CAR-T cells on MM.

[0007] It has been reported that IL-15 (Interleukin-15) affects the survival and proliferation of natural killer cells (NK cells) and CD8+T cells, and plays a vital role in the immune system (Waldmann et al., 2020). IL-15 is a member of the 4α-helical bundle cytokine family, with a molecular weight of 14-15kDa, and is an α, β heterodimeric cytokine. IL-15 can bind to the high-affinity receptor IL-15Rα expressed by antigen-presenting cells (APC). It has been reported that injection of IL-15 is helpful for combined immunotherapy of cancer. The IL-15 / IL-15Rα complex binds to the IL-2 / 15Rβ and γc chains on effector cells (including effector NK cells, CD8+T cells, etc.), thereby activating the JAK / STAT pathway (JAK-STATpathway) downstream of the effector cells, thereby promoting the survival and proliferation of effector cells such as NK cells and CD8+T cells. However, the clinical advancement of cytokines as cancer therapeutics is often hampered by the significant toxicity associated with their systemic administration, while local administration often requires multiple injections to achieve the desired therapeutic effect due to the rapid diffusion of the protein.

[0008] It can be seen that the existing single-target BCMA CAR-T cells have the problem of drug resistance in the treatment of MM. The essence of this problem is the reduced expression of BCMA on the tumor surface, which leads to antigen escape. At the same time, the persistence of CAR-T cells in the body is poor, which affects the efficacy. Therefore, it is necessary to develop new CAR-T cells to effectively eliminate target cells expressing CD38 and BCMA. Summary of the invention

[0009] In view of the problems existing in the treatment of MM with existing single-target BCMA CAR-T cells, the present invention designs and verifies a series of series-parallel hybrid CAR structures that can dual-target CD38 and BCMA and contain IL-15, which not only avoids the problem of antigen escape but also improves the proliferation ability of CAR-T cells, thereby greatly improving the treatment effect of the disease.

[0010] Therefore, in a first aspect, the invention provides a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL15-CAR) dual-targeting CD38 and BCMA, comprising the following elements:

[0011] 1) signal peptide 1; 2) anti-CD38 antibody A; 3) linker 2; 4) anti-BCMA heavy chain region; 5) linker 1; 6) anti-BCMA light chain region; 7) IL-15R extracellular region; 8) hinge region; 9) transmembrane region; 10) IL-15R intracellular signal transduction region; 11) intracellular signal co-stimulatory region; 12) intracellular signal stimulatory region; 13) 2A sequence; 14) signal peptide 2; 15) anti-CD38 antibody B; 16) linker 3; and 17) IL-15 sequence,

[0012] The amino acid sequence of the anti-CD38 antibody A or anti-CD38 antibody B is shown in any one of SEQ ID NOs: 4-13.

[0013] And wherein, the positions of the anti-BCMA heavy chain region and the anti-BCMA light chain region can be interchanged, and the positions of the anti-CD38 antibody region and the anti-BCMA antibody region containing the anti-BCMA heavy chain region, linker 1 and the anti-BCMA light chain region can be interchanged.

[0014] In an embodiment of the present invention, the anti-CD38 antibody region and the anti-BCMA antibody region comprising the anti-BCMA heavy chain region, linker 1 and the anti-BCMA light chain region form a series-parallel hybrid double-chain structure.

[0015] In an embodiment of the present invention, the amino acid sequence of the anti-BCMA heavy chain region is shown in SEQ ID NO: 14, and the amino acid sequence of the anti-BCMA heavy chain region is shown in SEQ ID NO: 15.

[0016] In an embodiment of the present invention, signal peptide 1 and signal peptide 2 can be signal peptides of mammalian cell surface proteins. Preferably, the signal peptide is a human albumin signal peptide, a human insulin signal peptide or a mouse IgG kappa signal peptide, and its amino acid sequence is shown in SEQ ID NO: 1, 2 or 3, respectively.

[0017] In an embodiment of the present invention, the amino acid sequence of the dimerization sequence is shown in SEQ ID NO:16.

[0018] In an embodiment of the present invention, the hinge region may be the hinge region of human CD8 or human IL-15R. Preferably, the hinge region is the hinge region of human IL-15R, and its amino acid sequence is shown in SEQ ID NO:18.

[0019] In an embodiment of the present invention, the transmembrane region may be the transmembrane region of human CD8 or human IL-15R. Preferably, the transmembrane region is the transmembrane region of human IL-15R, and its amino acid sequence is shown in SEQ ID NO:19.

[0020] In an embodiment of the present invention, the amino acid sequence of the intracellular signaling co-stimulatory region is shown as SEQ ID NO:21.

[0021] In an embodiment of the present invention, the amino acid sequence of the intracellular signal stimulation region is shown in SEQ ID NO:22.

[0022] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is shown in SEQ ID NO:23.

[0023] In an embodiment of the present invention, the amino acid sequence of the extracellular region of IL-15R is shown in SEQ ID NO: 17;

[0024] In an embodiment of the present invention, the amino acid sequence of the IL-15R intracellular signaling region is shown as SEQ ID NO:20; the amino acid sequence of the IL-15 sequence is shown as SEQ ID NO:24.

[0025] In an embodiment of the present invention, the amino acid sequences of linkers 1, 2, and 3 are shown in SEQ ID NO:16.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL15-CAR) that dually targets CD38 and BCMA and contains IL-15.

[0027] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0028] In a fourth aspect, the present invention provides a recombinant lentivirus, which comprises the nucleic acid molecule of the second aspect or is prepared by the expression vector of the third aspect.

[0029] In a fifth aspect, the present invention provides a method for preparing a recombinant lentivirus, wherein the expression vector of the third aspect and a helper plasmid are co-transfected into a mammalian host cell to obtain a recombinant lentivirus.

[0030] In an embodiment of the fifth aspect, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0031] In the embodiments of the fifth aspect, the mammalian host cells may be, but are not limited to, HEK293 cells, human PER.C6 cells, human Hela cells, mouse CHO cells, etc., preferably HEK293 cells.

[0032] In the sixth aspect, the present invention provides a CAR-T cell (hCD38-BCMA-IL15-CAR-T cell) that expresses a tandem-parallel hybrid chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-15, which expresses a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL15-CAR) that dual-targets CD38 and BCMA and contains IL-15.

[0033] In a seventh aspect, the present invention provides a method for preparing the hCD38-BCMA-IL15-CAR-T cells of the sixth aspect, comprising infecting T cells with the recombinant lentivirus of the fifth aspect.

[0034] In an embodiment of the seventh aspect of the present invention, the method for preparing hCD38-BCMA-IL15-CAR-T cells comprises the following steps:

[0035] (1) constructing a recombinant lentiviral expression vector carrying a nucleotide sequence encoding hCD38-BCMA-IL15-CAR;

[0036] (2) transfecting host cells with the recombinant lentiviral expression vector and the helper plasmid to prepare a recombinant lentivirus that can infect T cells;

[0037] (3) Isolate PBMCs from peripheral blood provided by the donor and use magnetic beads to separate and activate T cells;

[0038] (4) Infecting T cells with the recombinant lentivirus obtained in step (2) to produce T cells expressing hCD38-BCMA-IL15-CAR.

[0039] In further embodiments, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0040] In a further embodiment, the method may further comprise the following steps after step (4):

[0041] (5) culturing the cells obtained in step (4) in vitro;

[0042] (6) amplifying the cells obtained in step (5) in large quantities; and

[0043] (7) Collect T cells expressing hCD38-BCMA-IL15-CAR.

[0044] In an eighth aspect, the present invention provides a disease treatment drug, comprising a nucleic acid molecule encoding hCD38-BCMA-IL15-CAR of the present invention, an expression vector comprising a nucleic acid molecule encoding hCD38-BCMA-IL15-CAR, the recombinant lentivirus of the fourth aspect, or the hCD38-BCMA-IL15-CAR-T cell of the sixth aspect.

[0045] In a ninth aspect, the present invention provides the use of the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant lentivirus or the above-mentioned hCD38-BCMA-IL15-CAR-T cell in the preparation of a drug for treating a disease.

[0046] In an embodiment of the present invention, the disease refers to a disease characterized by high expression of CD38 molecules and / or BCMA molecules on the cell surface. In a specific embodiment, the disease can be multiple myeloma, lymphoma, leukemia, systemic lupus erythematosus, Sjögren's syndrome, etc.

[0047] In an embodiment of the present invention, CAR represents a chimeric antigen receptor, BCMA-CAR represents a chimeric antigen receptor targeting BCMA, CAR-T cells represent chimeric antigen receptor T cells, hCD38-BCMA-IL15-CAR represents a tandem-parallel hybrid chimeric antigen receptor that dually targets CD38 and BCMA and contains IL-15, BCMA-CAR-T cells represent chimeric antigen receptor T cells targeting BCMA, and hCD38-BCMA-IL15-CAR-T cells represent T cells that express a tandem-parallel hybrid chimeric antigen receptor that dually targets CD38 and BCMA and secretes IL15-.

[0048] In the context of the present invention, a tandem-parallel hybrid chimeric antigen receptor refers to a chimeric antigen receptor containing two parallel protein chains, one of which contains two antibodies and the other contains one antibody. Therefore, a "tandem-parallel hybrid chimeric antigen receptor dually targeting CD38 and BCMA" refers to a chimeric antigen receptor containing two parallel protein chains that target CD38 and BCMA respectively, one of which contains two antibodies, anti-CD38 and anti-BCMA, connected in tandem, and the other chain contains only an anti-CD38 antibody.

[0049] The present invention targets CD38 and BCMA, two highly expressed markers of multiple myeloma, and targets multiple myeloma cells, which can effectively prevent tumor recurrence caused by loss of a single target, and prolong the patient's survival period. The hCD38-BCMA-IL15-CAR-T cells of the present invention express CD38-CAR on the basis of expressing targeted CD38-BCMA-IL15-CAR, that is, there are two CARs, CD38-BCMA-IL15-CAR and CD38-CAR, on the cell surface, which enhances the targeted killing activity of CAR-T cells, and uses low-dose effector cells to effectively kill myeloma cell line MM.1S-GFP cells; in addition, the hCD38-BCMA-IL15-CAR-T cells of the present invention, in addition to expressing CD38-BCMA-IL15-CAR and CD38-CAR, also secrete cytokine IL-15, which enhances the proliferation activity of CAR-T and is conducive to the removal of tumor cells in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of the structure of the hCD38-BCMA-IL15-CAR series-parallel hybrid CAR in the present invention, wherein (A) represents the primary sequence structure diagram; (B) represents the three-dimensional structure schematic diagram after CAR protein expression, wherein a / b / c / d shown in (B) has 4 three-dimensional structures corresponding to a / b / c / d shown in (A) has 4 primary sequence structure diagrams. Hinge-IL15Rα is the hinge region, TM-IL15R is the transmembrane region, IL-15Rα is the IL-15R extracellular region, IL15Rα is the IL-15R intracellular signal transduction region, 4-1BB is the intracellular signal costimulatory region, and CD3z is the intracellular signal stimulation region.

[0051] Figure 2 These are the positive rate detection results of BCMA and CD38 in hCD38-BCMA-IL15-CAR-T cells and single-target BCMA-CAR-T cells provided by the present invention on the 5th and 7th days after transfection.

[0052] Figure 3This is a comparison of the killing ability of hCD38-BCMA-IL15-CAR-T cells, BCMA-CAR-T cells and unmodified T cells on MM.1S-GFP target cells. The number of MM.1S-GFP cells remaining in the killing system was counted, with the horizontal axis representing the effect-target ratio and the vertical axis representing the number of target cells.

[0053] Figure 4 This is a comparison of the killing results of hCD38-BCMA-IL15-CAR-T cells, BCMA-CAR-T cells and unmodified T cells. The horizontal axis is the effector-target ratio, that is, the ratio of the number of T cells or CAR-T cells to the target cell MM.1S-GFP, and the vertical axis is the killing rate.

[0054] Figure 5 This is the screening result of 10 kinds of anti-CD38 antibodies, that is, the comparison of the killing results of 10 kinds of CD38-CAR-T cells and unmodified T cells. The horizontal axis is the effect-target ratio, that is, the ratio of the number of T cells or CAR-T cells to the target cell MM.1S-GFP, and the vertical axis is the killing rate. Among them, 1-the anti-CD38 antibody in CD38-CAR-T cells is SEQ ID NO:4; 2-the anti-CD38 antibody in CD38-CAR-T cells is SEQ ID NO:5; and so on. DETAILED DESCRIPTION

[0055] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or the conditions recommended by the manufacturer's instructions.

[0056] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0057] Materials and methods

[0058] 1. Design and construction of plasmid encoding CD38-CAR

[0059] In order to screen suitable anti-CD38 antibodies, a plasmid encoding CD38-CAR was designed and constructed. The plasmid was synthesized by Nanjing Baode Biotechnology Co., Ltd. The plasmid contained nucleotide sequences encoding the following elements in order from the 5' to 3' direction of the CAR structure: signal peptide 1 (SEQ ID NO: 1, 2 or 3), anti-CD38 antibody (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), hinge region (SEQ ID NO: 18), transmembrane region (SEQ ID NO: 19), intracellular signal costimulatory region (SEQ ID NO: 21), intracellular signal stimulation region (SEQ ID NO: 22). The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newpu Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid encoding CD38-CAR, which was called pMSGV-CD38, and is a recombinant lentiviral expression vector in virus packaging.

[0060] 2. Design and Construction of Plasmid Encoding hCD38-BCMA-IL15-CAR

[0061] The plasmid encoding hCD38-BCMA-IL15-CAR was synthesized by Nanjing Biode Biotechnology Co., Ltd. The plasmid contains nucleotide sequences encoding the following elements in order from the 5' to the 3' direction of the CAR structure: signal peptide 1 (SEQ ID NO: 1, 2 or 3), anti-CD38 antibody A (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), linker 2 (SEQ ID NO: 16), anti-BCMA heavy chain region (SEQ ID NO: 14), linker 1 (SEQ ID NO: 16), anti-BCMA light chain region (SEQ ID NO: 15), IL-15R extracellular region (SEQ ID NO: 17), hinge region (SEQ ID NO: 18), transmembrane region (SEQ ID NO: 19), IL-15R intracellular signal transduction region (SEQ ID NO: 20), intracellular signal costimulatory region (SEQ ID NO: 21), intracellular signal stimulation region (SEQ ID NO: 22), 2A sequence (SEQ ID No: 23), signal peptide 2 (SEQ ID NO: 24), ID NO: 1, 2 or 3), anti-CD38 antibody B (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), linker 3 (SEQ ID NO: 16) and IL-15 sequence (SEQ ID NO: 24), the structure is as follows Figure 1The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newpro Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid encoding hCD38-BCMA-IL15-CAR. The plasmid was called pMSGV-hCD38-BCMA-IL15, which is a recombinant lentiviral expression vector in virus packaging.

[0062] During the plasmid construction process, the positions of the anti-BCMA heavy chain region and the anti-BCMA light chain region can be interchanged, and the positions of the anti-CD38 antibody region and the anti-BCMA antibody region comprising the anti-BCMA heavy chain region, linker 1 and the anti-BCMA light chain region can be interchanged; and the anti-CD38 antibody region and the anti-BCMA antibody region comprising the anti-BCMA heavy chain region, linker 1 and the anti-BCMA light chain region form a series-parallel mixed double-chain structure, such as Figure 1 As shown in (A).

[0063] To verify the advantages of the hCD38-BCMA-IL15-CAR of the present invention, a plasmid encoding BCMA-CAR was constructed at the same time. The plasmid expressing BCMA-CAR was synthesized by Nanjing Baode Biotechnology Co., Ltd., and the plasmid sequentially contained nucleotide sequences encoding the following elements from the 5' to 3' direction of the CAR structure: signal peptide 2 (SEQ ID NO: 1, 2 or 3), anti-BCMA heavy chain region (SEQID NO: 14), linker 1 (SEQ ID NO: 22), anti-BCMA light chain region (SEQ ID NO: 15), hinge region (SEQ ID NO: 17), transmembrane region (SEQ ID NO: 18), intracellular signal costimulatory region (SEQ ID NO: 19) and intracellular signal stimulation region (SEQID NO: 20). The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES to obtain a plasmid encoding BCMA-CAR, which was called pMSGV-BCMA, and was a recombinant lentiviral expression vector in viral packaging.

[0064] 3. Preparation of recombinant lentivirus

[0065] The method for preparing recombinant lentivirus using pMSGV-CD38, pMSGV-hCD38-BCMA-IL15 or pMSGV-BCMA is exactly the same. The following uses pMSGV-hCD38-BCMA-IL15 as an example to illustrate the process of preparing recombinant lentivirus.

[0066] The main plasmid pMSGV-hCD38-BCMA-IL15 and three auxiliary plasmids pMDLG-pRRE-Kana (Changsha Aibiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0374), pMD2G-Kana (Changsha Aibiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0648) and pRSV-REV-Kana (Changsha Aibiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0370) were co-transfected into WayneLVPro HEK293 cell line (SuspensionAdapted) (Zhongshan Kangsheng Biotechnology Co., Ltd., Catalog No. A23109, hereinafter referred to as 293T cells) to prepare recombinant lentivirus that can infect T cells. The specific operation process is as follows:

[0067] 3.1 Prepare a culture area of ​​525 cm 2 Cell culture flasks were seeded with 6 × 10 7 293T cells were added with 120 mL of complete medium containing DMEM high glucose medium (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30243.01) and 10% FBS (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30256.01), and placed in a 37°C, 5% CO2 incubator for overnight culture.

[0068] 3.2 Add 500 μg of recombinant lentiviral expression vector pMSGV-hCD38-BCMA-IL15, 250 μg of pMDLG-pRRE-Kana, 150 μg of pMD2G-Kana and 150 μg of pRSV-REV-Kana to 10 mL of opti-MEM (Thermo Fisher Scientific, Catalog No. 11058021), pipette up and down to mix thoroughly, then add 2 mL of 100 μM PEI (Merck Biotechnology, Catalog No. 937762), immediately pipette up and down to mix evenly, and let stand at room temperature for 20 minutes.

[0069] 3.3 Pour out the culture medium in the cell culture flask in step 2.1, add the above DNA / PEI complex to the cell culture flask, add 120mL complete culture medium (10% FBS + DMEM), gently shake the culture flask, and mix thoroughly. Place the culture dish in a 37°C, 5% CO2 incubator. After 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0070] 3.4 After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.22μm filter cup, incubate it at 4℃ for 2 hours, transfer it to a centrifuge bottle, balance it, and centrifuge it at 20000g at 4℃ for 2 hours. After the centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 120μL PBS buffer to resuspend the precipitate, and you can get the recombinant lentivirus, which is stored at -80℃.

[0071] 4. Lentiviral titer detection

[0072] Jurkat cells (ATCC, catalog number: MD130) after 2 generations of resuscitation culture were centrifuged at 300 g for 10 minutes and resuspended in RPMI1640 medium (Situofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30809-01) to 2×10 6 Cells / mL were inoculated into 48-well plates, 0.1 mL was inoculated into each well, 100 μL of RPMI1640 medium was taken to dilute 10 μL of the virus obtained in "2. Preparation of recombinant lentivirus", 10 μL of the diluted virus was added to the plated cells, and mixed thoroughly. After 6 hours, 800 μL of RPMI 1640 + 10% FBS complete culture medium was added and cultured in a cell culture incubator at 37°C and 5% CO2, and flow cytometry was performed after 48 hours.

[0073] 5. T cell isolation and activation

[0074] 5.1 Take 15 mL of peripheral blood from a healthy donor under sterile conditions and let it stand at room temperature for 30 hours; centrifuge at 400 g for 30 minutes and aspirate the plasma for later use.

[0075] 5.2 Dilute the anticoagulated blood with PBS at a ratio of 1:1 and mix well, then add lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., product number: LTS1077) at a ratio of 1:2. Centrifuge at 300g for 10 minutes.

[0076] 5.3 After centrifugation, aspirate the buffy coat layer above the separation solution into a new centrifuge tube, add PBS to wash the buffy coat layer, centrifuge at 300g for 10 minutes, and discard the supernatant to obtain PBMC (peripheral blood mononuclear cells).

[0077] 5.4 Use CD3 Microbeads (Human) (Miltenyi Biotec, catalog number: 130-050-101) magnetic beads instructions to sort and separate T lymphocytes from PBMCs and perform cell counting.

[0078] 5.5 Take 9×10 6 CD3 +T cells were centrifuged at 300 g for 10 min at 4°C, the supernatant was discarded, and X-VIVO containing 40 IU / mL human IL-2 (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CD66) was added. TM 15 serum-free culture medium (Lonza Investment Co., Ltd., China, catalog number: DL-102), adjust the cell concentration to 1×10 6 cells / mL. According to Dynabeads TM Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific, Cat. No. 11452D) was used to activate T cells. T cells were cultured at a concentration of 1 × 10 6 cells / mL, 1 mL was inoculated into each well of a 24-well cell culture plate; and the plates were cultured in a cell culture incubator for 48 hours to obtain activated T cells.

[0079] 6. Preparation of Recombinant T Cells

[0080] 6.1 Calculate the required amount of virus according to the multiplicity of infection MOI = 3. The calculation formula is as follows: Required amount of virus (mL) = (MOI*number of cells) / virus titer.

[0081] 6.2 Add the recombinant lentivirus to the prepared T cells according to the required amount calculated in 5.1, pipet evenly, and culture in a 37°C, 5% CO2 cell culture incubator to obtain hCD38-BCMA-IL15-CAR-T cells.

[0082] 6.3 hCD38-BCMA-IL15-CAR-T cells were incubated with FITC-labeled CD38 (SinoBiological, Catalog No.: 10818-H08H-F) and anti-BCMAscFv antibody (SinoBiological, Catalog No.: 10620-H02H) on days 5 and 7, respectively, and the positive rates of CD38-CAR and BCMA-CAR in CAR-T cells were detected by flow cytometry.

[0083] To verify the advantages of the killing effect of the hCD38-BCMA-IL15-CAR-T cells of the present invention, we also prepared single-targeted BCMA-CAR-T cells. The BCMA-CAR-T cells were obtained using the pMSGV-BCMA plasmid prepared in method 2 according to the specific operation process described in section "3-6" above.

[0084] 7. Screening of anti-CD38 antibodies

[0085] According to the specific operation procedures described in Methods 3-6, CD38-CAR-T cells were prepared using the pMSGV-CD38 plasmid obtained in Method 1.

[0086] Since the multiple myeloma cell line MM.1S highly expresses CD38 molecules, this experiment transformed this cell line as a target cell for the in vitro killing experiment of CD38-CAR-T cells; MM.1S cells (Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-5017) were introduced with GFP protein and transformed into MM.1S-GFP cells as target cells; T cells without virus introduction were used as control group cells; the prepared CD38-CAR-T cells were used as experimental group cells. The specific experimental steps are as follows:

[0087] 7.1MM.1S-GFP cells were seeded into six-well culture plates, and duplicate wells were set for killing experiments. A concentration of 1×10 6 2mL of MM.1S cells with 1 cell / mL.

[0088] 7.2 When CD38-CAR-T cells are used as effector cells, the concentration of effector cells is adjusted to 1×10 6 cells / mL, and effector cells were added to the target cell culture wells at effector-target ratios of 0.2 and 0.4.

[0089] 7.3 When T cells without virus were used as control cells, the concentration was adjusted to 1×10 6 According to the amount of effector cells added in the experimental group, blank T cells were added to the target cell culture wells as the control group; the target cell wells without effector cells were used as the negative control group.

[0090] 7.4 After the above culture plate is placed in a CO2 incubator at 37°C for 12 hours, the culture plate is removed and the volume of culture medium and the number of viable cells in the culture wells are calculated.

[0091] 7.5 All samples were detected by flow cytometry FITC detection channel to detect the positive rate of GFP in MM.1S cells.

[0092] 7.6 Calculate the killing rate of CD38-CAR-T cells and T cells against MM.1S-GFP cells; the killing activity is expressed as the killing rate.

[0093] The formula for the killing rate is: killing rate (%) = (1-(A*B) / C) × 100%;

[0094] Among them, A represents the positive rate of MM.1S-GFP cells in the experimental group or control group; B represents the number of living cells in the experimental group or control group; C represents the number of living cells in the negative control group.

[0095] 8. Determination of the activity of hCD38-BCMA-IL15-CAR-T cells in killing multiple myeloma cells in vitro

[0096] Since the multiple myeloma cell line MM.1S highly expresses BCMA and CD38 molecules, this experiment transformed this cell line as a target cell for the in vitro killing experiment of hCD38-BCMA-IL15-CAR-T cells; MM.1S cells (Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-5017) were introduced with GFP protein and transformed into MM.1S-GFP cells as target cells; T cells without virus introduction were used as control group cells; prepared hCD38-BCMA-IL15-CAR-T cells were experimental group cells, and BCMA-CAR-T cells were positive control group cells. The specific experimental steps are as follows:

[0097] 8.1MM.1S-GFP cells were seeded into six-well culture plates, and duplicate wells were set for killing experiments. A concentration of 1×10 6 2mL of MM.1S cells with 1 cell / mL.

[0098] 8.2 When hCD38-BCMA-IL15-CAR-T cells are used as effector cells, the concentration of effector cells is adjusted to 1×10 6 cells / mL, and effector cells were added to the target cell culture wells at effector-target ratios of 0.2 and 0.4.

[0099] 8.3 When T cells without virus were used as control cells, the concentration was adjusted to 1×10 6 According to the amount of effector cells added in the experimental group, blank T cells were added to the target cell culture wells as the control group; the target cell wells without effector cells were used as the negative control group.

[0100] 8.4 When BCMA-CAR-T cells were used as positive control cells, the cell concentration was adjusted to 1×10 6 cells / mL, and effector cells were added to the target cell culture wells at effector-target ratios of 0.2 and 0.4.

[0101] 8.5 After the above culture plate is placed in a CO2 incubator at 37°C for 12 hours, the culture plate is removed and the volume of culture medium and the number of viable cells in the culture wells are calculated.

[0102] 8.6 All samples were detected by flow cytometry FITC detection channel to detect the GFP positivity rate in MM.1S cells.

[0103] 8.7 Calculate the killing rate of hCD38-BCMA-IL15-CAR-T cells, BCMA-CAR-T cells, and T cells against MM.1S-GFP cells; the killing activity is expressed as the killing rate.

[0104] The formula for the killing rate is: killing rate (%) = (1-(A*B) / C) × 100%;

[0105] Among them, A represents the positive rate of MM.1S-GFP cells in the experimental group or control group or positive control group; B represents the number of living cells in the experimental group or control group; C represents the number of living cells in the negative control group.

[0106] Results and Discussion

[0107] BCMA-CAR-T cells and 16 types of hCD38-BCMA-IL15-CAR-T cells were constructed according to the above method.

[0108] Specifically, hCD38-BCMA-IL15-CAR-T cells were identified according to the different positions of the signal peptide region, the heavy chain and light chain regions of anti-BCMA, the anti-CD38 antibody A, the anti-CD38 antibody B, and the anti-BCMA antibody region (including the anti-BCMA heavy chain region, the anti-BCMA light chain region, and the linker 1) in the CAR (e.g. Figure 1 There are 4 structures of a / b / c / d shown in (B), and the following table is referred to as a / b / c / d), and a total of 3600 hCD38-BCMA-IL15-CAR-T cells can be constructed. In the early stage of the present invention, the signal peptide was screened, and signal peptide 1 is preferably SEQ ID NO: 1, and signal peptide 2 is preferably SEQ ID NO: 2. On this basis, a total of 400 hCD38-BCMA-IL15-CAR-T cells can be constructed by combining anti-CD38 antibodies in pairs. The inventors of the present invention previously screened anti-CD38 antibodies through an in vitro killing experiment of CD38-CAR-T cells (such as Figure 5 As shown in the figure, two antibody sequences with the best killing effect are screened out, and SEQ ID NO: 5 and / or SEQ ID NO: 7 are selected as the anti-CD38 antibody. To this end, when the anti-CD38 antibody is selected as SEQ ID NO: 5 and / or SEQ ID NO: 7, according to Figure 1 There are 4 structures a / b / c / d shown in (A), and a total of 16 pMSGV-hCD38-BCMA-IL10 plasmids were constructed. These 16 plasmids were used according to the specific operation process described in parts 3-7 of the method to construct the Figure 1 There are 4 structures a / b / c / d shown in (B), and a total of 16 types of hCD38-BCMA-IL15-CAR-T cells. Table 1 lists these 16 types of cells.

[0109] Table 1. 16 types of hCD38-BCMA-IL15-CAR-T cells constructed by the present invention

[0110]

[0111] *The first column indicates the killing rate of MM.1s-GFP target cells on the second day when the effector-target ratio is 0.2, and the second column indicates the killing rate of MM.1s-GFP target cells on the second day when the effector-target ratio is 0.4.

[0112] The experimental results showed that the BCMA-CAR positivity rates of most of the 16 types of hCD38-BCMA-IL15-CAR-T cells were between 80% and 95%, which were higher than those of single-target BCMA-CAR-T cells; the CD38-CAR positivity rates were all above 40%. Figure 2 It was shown that the positive rate of CD38-CAR in hCD38-BCMAIL15-CAR-T cells formed by anti-CD38 antibody A (SEQ ID NO: 5) and anti-CD38 antibody B (SEQ ID NO: 7) according to structure a was as high as 73%, and the positive rate of BCMA-CAR was as high as 94%.

[0113] In the in vitro killing experiment of hCD38-BCMA-IL15-CAR-T cells on target cells, these 16 types of hCD38-BCMA-IL15-CAR-T cells also showed similar killing effects. When the effect-target ratio was 0.2, the killing rate was basically above 80%; when the effect-target ratio was 0.4, the killing rate was basically up to 95%. Figure 3 , 4 As shown, hCD38-BCMA-IL15-CAR-T cells formed by anti-CD38 antibody A (SEQ ID NO: 5) and anti-CD38 antibody B (SEQ ID NO: 7) according to structure a can efficiently mediate T cell killing of tumor cells when the effector-target ratio is 0.2, and the killing rate can reach 86% compared with empty T cells; when the effector-target ratio is increased to 0.4, the killing of MM.1S-GFP cells by hCD38-BCMA-IL15-CAR-T cells is maintained above 90%, and can reach up to 97.7%.

[0114] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

[0115] References

[0116] [1]CHEKOL,A.E.,YIBELTAL,S.M.,TADELE,A.F.&ASMAMAW,D.T.(2022),"Ciltacabtagene autoleucel:The second anti-BCMA CAR T-cell therapeuticarmamentarium of relapsed or refractory multiple myeloma",Front Immunol,Vol.13991092.

[0117] [2]De NOVELLIS,D.,FONTANA,R.,GIUDICE,V.,SERIO,B.&SELLERI,C.(2022),"Innovative Anti-CD38 and Anti-BCMA Targeted Therapies in Multiple Myeloma:Mechanisms of Action and Resistance".

[0118] [3]LIAO,C.,WANG,Y.,HUANG,Y.,DUAN,Y.,LIANG,Y.,CHEN,J.,JIANG,J.,SHANG,K.,ZHOU,C.,GU,Y.,LIU,N.,ZENG,X.,GAO,X.,TANG,Y.&SUN,J.(2023),"CD38-SpecificCAR Integrated into CD38 Locus Driven by Different Promoters Causes DistinctAntitumor Activities of T and NK Cells",Adv Sci(Weinh),Vol.10 No.27,pp.e2207394.

[0119] [4]MEI,H.,LI,C.,JIANG,H.,ZHAO,X.,HUANG,Z.,JIN,D.,GUO,T.,KOU,H.,LIU,L.,TANG,L.,YIN,P.,WANG,Z.,AI,L.,KE,S.,XIA,Y.,DENG,J.,CHEN,L.,CAI,L.,SUN,C.,XIA,L.,HUA,G.&HU,Y.(2021),"A bispecific CAR-T cell therapy targeting BCMA andCD38 in relapsed orrefractory multiplemyeloma",J Hematol Oncol,Vol.14No.1,pp.161.

[0120] [5]PARIKH,R.H.&LONIAL,S.(2023),"Chimeric antigen receptor T-celltherapy in multiple myeloma:A comprehensive review of current data andimplications for clinical practice",CA CancerJ Clin,Vol.73No.3,pp.275-285.

[0121] [6]WALDMANN,T.A.,DUBOIS,S.,MILJKOVIC,M.D.&CONLON,K.C.(2020),"IL-15inthe Combination Immunotherapy of Cancer.",Frontiers in immunology,Vol.11868.

[0122] [7]XU,H.,GUAN,C.,XU,P.,ZHOU,D.,XU,Y.,CHEN,B.&BAI,H.(2024),"Clinicalefficacy and safety of combined anti-BCMA and anti-CD19 CAR-T cell therapyfor relapsed / refractory multiple myeloma:a systematic review and meta-analysis",Front Oncol,Vol.141355643.

[0123] [8] ZHANG, X., ZHU, L., ZHANG, H., CHEN, S. &

[0124] [9]ZHOU, Oncol,Vol.13No.1,pp.100.

[0125]

[10] Yu Min, Kong Fancong, Zhou Yulan, Qi Ling, and Li Fei (2024), "Long-term efficacy and influencing factors of BCMACAR-T in the treatment of patients with relapsed / refractory multiple myeloma", Chinese Journal of Clinical Oncology, Vol. 51 No. 7, pp. 342-347.

Claims

1. A series-parallel hybrid chimeric antigen receptor that dually targets CD38 and BCMA and contains IL-15, characterized in that: The serial-parallel hybrid chimeric antigen receptor comprises the following elements in sequence: 1) Signal peptide 1; 2) Anti-CD38 antibody A; 3) Linker 2; 4) anti-BCMA heavy chain region; 5) Linker 1; 6) Anti-BCMA light chain region; 7) IL-15R extracellular region; 8) Hinge region; 9) transmembrane region; 10) IL-15R intracellular signal transduction region; 11) Intracellular signaling co-stimulatory region; 12) Intracellular signal stimulation area; 13) 2A sequence; 14) Signal peptide 2; 15) anti-CD38 antibody B; 16) Linker 3; and 17) IL-15 sequence, Wherein, the amino acid sequence of the anti-CD38 antibody A or the anti-CD38 antibody B is as shown in any one of SEQ ID NOs: 4-13, Wherein, the positions of the anti-BCMA heavy chain region and the anti-BCMA light chain region can be interchanged, and the positions of the anti-CD38 antibody region and the anti-BCMA antibody region containing the anti-BCMA heavy chain region, the linker 1 and the anti-BCMA light chain region can be interchanged; And wherein, the anti-CD38 antibody region and the anti-BCMA antibody region containing the anti-BCMA heavy chain region, the linker 1 and the anti-BCMA light chain region form a series-parallel mixed double-chain structure.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the serial-parallel hybrid chimeric antigen receptor that dually targets CD38 and BCMA and contains IL-15 as described in 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 from 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 a CAR-T cell expressing a dual-targeting CD38 and BCMA serial-parallel hybrid chimeric antigen receptor and secreting IL-15, characterized in that: The method comprises infecting T cells with the recombinant lentivirus of claim 4.

7. A CAR-T cell expressing a dual-targeting CD38 and BCMA tandem hybrid chimeric antigen receptor and secreting IL-15, characterized in that: The cells are 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 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 CAR-T cell according to claim 7 in the preparation of a drug for treating a disease.

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