CAR-T cells expressing dual-targeting CD38 and BCMA serial-parallel hybrid chimeric antigen receptors and secreting IL-10 and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to a CAR-T cell expressing a dual-targeted CD38 and BCMA serial-parallel hybrid chimeric antigen receptor and secreting IL-10, 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 dual-targeted CD38 and BCMA serial-parallel hybrid chimeric antigen receptor comprising IL-10, 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-CAR and CD38-CAR, and can secrete the cytokine IL-10, promote the proliferation and effector function of CAR-T cells, and enhance anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a series-parallel hybrid CAR-T cell that dual-targets CD38 and BCMA and secretes IL-10, and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant disease of abnormal proliferation of clonal plasma cells. It is the second most common malignant tumor of the blood system, with high morbidity and mortality. The incidence rate in my country is about 1.6 per 100,000. Although the development of new drugs such as autologous stem cell transplantation, immunomodulatory imide drugs (thalidomide and lenalidomide), and proteasome inhibitors (PI) in the past decade has significantly improved the survival rate of patients, the 5-year overall survival can reach 40% to 87%. However, most patients will eventually develop refractory diseases and relapse, so safe and long-acting drugs are still needed (Roex et al., 2020, Mishra et al., 2023).

[0003] Chimeric Antigen Receptor (CAR) T cell therapy is a breakthrough innovative strategy for the treatment of hematological malignancies, and has shown significant efficacy in clinical practice. B Cell Maturation Antigen (BCMA) is selectively expressed on tumor plasma cells and is considered an ideal target for targeted therapy of multiple myeloma. Currently, several BCMA CAR-T cells have been approved for clinical use, and their experimental data show that BCMA CAR-T cells have significant efficacy in multiple myeloma, with objective response rates ranging from 30% to 97% (Zhang et al., 2022). However, some patients fail treatment or relapse within a short period of time after treatment, which may be due to clonal evolution and loss of antigens in tumor cells, immunosuppression in the bone marrow microenvironment, or CAR-T cell exhaustion (Zhang et al., 2023).

[0004] One of the limitations of existing therapies is the high relapse rate. Although new drugs have improved survival rates, most MM patients will eventually relapse and develop refractory disease. The second limitation is antigen escape. After BCMA CAR-T treatment, tumor cells may escape immune surveillance by downregulating or losing BCMA. The third limitation is immunosuppression and CAR-T cell exhaustion. Immunosuppression and CAR-T cell exhaustion in the bone marrow microenvironment limit the durability of the treatment effect.

[0005] One way to solve antigen escape is to use dual-targeted CAR-T cells. CD38 is a single-chain type II transmembrane glycoprotein that is highly expressed on the surface of myeloma cells and is also an ideal therapeutic target for multiple myeloma (Liao et al., 2023). Daratumumab and Isatuximab are two anti-CD38 monoclonal antibodies that can induce rapid apoptosis of tumor cells through multiple mechanisms of action, prolong patient survival, and have no serious inhibitory effect on myeloid cell growth. They have been approved for clinical treatment (DeNovellis et al., 2022, van de Donk et al., 2018). At the same time, CD38-targeted CAR-T cells have shown anti-MM activity in preclinical studies (Li et al., 2023).

[0006] The cytokine interleukin-10 (IL10) is an immunomodulatory cytokine that can reduce inflammatory responses (Moore et al., 2001). The combination of IL-10 and CAR-T cells can enable CAR-T cells to maintain intact mitochondrial structure and function in the tumor microenvironment and counteract T cell dysfunction in the tumor microenvironment (Heuser-Loy, 2024). In addition, the literature reports that IL-10 is negatively correlated with the development of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs), which destroys tumor immunosuppression and helps reduce tumor development, growth and metastasis (Tanikawa et al., 2012). However, the combination therapy of IL-10 requires peritumoral administration to reduce systemic adverse reactions, and the treatment convenience is poor. It is a challenge to develop a clinical application that can effectively deliver IL-10 to the site of action with minimal systemic side effects.

[0007] It can be seen from this that the existing single-target BCMA CAR-T cells have problems of drug resistance and poor persistence of CAR-T cells in the body during the treatment of MM. It is necessary to develop new CAR-T cells that dual-target CD38 and BCMA and can secrete IL-10. Summary of the invention

[0008] The inventors of the present invention designed a CAR that dual-targets CD38 and BCMA to enhance the targeted killing ability of CAR-T cells and alleviate the exhaustion of CAR-T cells. On this basis, they further designed a series-parallel hybrid CAR that dual-targets CD38 and BCMA and contains IL-10, which contains an anti-BCMA single-chain variable fragment (scFv) and a humanized anti-CD38 scFv and a secretory IL-10 peptide. The series-parallel hybrid recombinant vector expressing dual-targeting CD38 and BCMA and secreting IL10 was prepared, and CAR-T cells were further prepared. The main technical problems solved include: First, by dual-targeting CD38 and BCMA two cell surface antigens, the problem of antigen escape in CAR-T therapy is solved; second, by co-expressing IL-10, the survival and proliferation of terminally exhausted CAR-T cells are improved, thereby enhancing the anti-tumor killing activity.

[0009] Therefore, in a first aspect, the invention provides a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL10-CAR) that dual-targets CD38 and BCMA and contains IL-10, comprising the following elements:

[0010] 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; 17) IL-15 sequence; 18) 2A sequence; and 19) human IL-10 sequence,

[0011] 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;

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In an embodiment of the present invention, signal peptide 1 and signal peptide 2 can be signal peptides of any mammalian cell surface protein. 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.

[0016] In an embodiment of the present invention, the amino acid sequence of the dimerization sequence is shown in SEQ ID NO:16.

[0017] 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.

[0018] 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.

[0019] In an embodiment of the present invention, the amino acid sequence of the intracellular signaling co-stimulatory region is shown in SEQ ID NO:21.

[0020] In an embodiment of the present invention, the amino acid sequence of the intracellular signal stimulation region is shown in SEQ ID NO:22.

[0021] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is shown in SEQ ID NO:23.

[0022] 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.

[0023] 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.

[0024] In an embodiment of the present invention, the amino acid sequences of linkers 1, 2, and 3 are shown in SEQ ID NO:16.

[0025] In an embodiment of the present invention, the amino acid sequence of human IL-10 sequence is shown as SEQ ID NO:25.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL10-CAR) that dually targets CD38 and BCMA and contains IL-10.

[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, comprising co-transfecting a mammalian host cell with the expression vector of the third aspect and a helper plasmid 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-IL10-CAR-T cell) that expresses a tandem-parallel hybrid chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-10, which expresses a tandem-parallel hybrid chimeric antigen receptor (hCD38-BCMA-IL10-CAR) that dual-targets CD38 and BCMA and contains IL-10.

[0033] In a seventh aspect, the present invention provides a method for preparing the hCD38-BCMA-IL10-CAR-T cells of the sixth aspect, comprising transfecting 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 the hCD38-BCMA-IL10-CAR-T cells of the sixth aspect comprises the following steps:

[0035] (1) constructing a recombinant lentiviral expression vector carrying a nucleotide sequence encoding hCD38-BCMA-IL10-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-IL10-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-IL10-CAR.

[0044] In an eighth aspect, the present invention provides a disease treatment drug, comprising a nucleic acid molecule encoding hCD38-BCMA-IL10-CAR of the present invention, an expression vector comprising a nucleic acid molecule encoding hCD38-BCMA-IL10-CAR, the recombinant lentivirus of the fourth aspect, or the hCD38-BCMA-IL10-CAR-T cell of the sixth aspect of the present invention.

[0045] In a ninth aspect, the present invention provides use of the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant lentivirus or the above-mentioned hCD38-BCMA-IL10-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-IL10-CAR represents a tandem-parallel hybrid chimeric antigen receptor that dual-targets CD38 and BCMA and contains IL-10, BCMA-CAR-T cells represent chimeric antigen receptor T cells targeting BCMA, and hCD38-BCMA-IL10-CAR-T cells represent T cells that express a tandem-parallel hybrid chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-10.

[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 uses CD38 and BCMA as targets to obtain antibody gene sequences that specifically recognize CD38 and BCMA antigens, which are unique and distinctive. The hCD38-BCMA-IL10-CAR-T cells constructed and prepared on this basis are also unique and distinctive in recognizing CD38-positive and / or BCMA-positive tumor cells. The hCD38-BCMA-IL10-CAR-T cells of the present invention simultaneously target two surface markers of multiple myeloma, CD38 and BCMA, and can effectively avoid the problem of antigen escape and prevent tumor recurrence caused by the loss of a single target. The hCD38-BCMA-IL10-CAR-T cells of the present invention secrete IL-10 on the basis of targeting CD38 and BCMA, thereby improving cell activity and proliferation ability, alleviating T cell exhaustion, enhancing the killing activity of CAR-T cells, effectively reducing the tumor recurrence rate after treatment, and prolonging the patient's survival period. The killing activity of the hCD38-BCMA-IL10-CAR-T cells of the present invention on multiple myeloma cells MM.1S-GFP is significantly higher than that of ordinary T cells and single-target BCMA-CAR cells. Using a low dose of effector cells, the myeloma cell line MM.1S-GFP cells can be effectively killed, and the hCD38-BCMA-IL10-CAR-T cells have excellent myeloma targeted killing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of the structure of hCD38-BCMA-IL10-CAR, where (A) represents the primary sequence structure diagram; (B) represents the three-dimensional structure diagram after CAR protein expression, where the a / b / c / d three-dimensional structures shown in (B) correspond to the a / b / c / d primary sequence structures shown in (A). 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 This is a comparison chart of the proliferation trends of hCD38-BCMA-IL10-CAR-T cells of the present invention and unmodified T cells. The horizontal axis is the number of days, and the vertical axis is the number of target cells.

[0052] Figure 3 It is a CAR positivity detection graph of the three cells of the present invention, namely, hCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells and unmodified T cells, on the 7th day and the 21st day after virus transfection.

[0053] Figure 4 This is a comparison of the killing ability of hCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells and unmodified T cells of the present invention on MM.1S-GFP target cells. The number of MM.1S-GFP cells remaining in the killing system was counted, with the horizontal axis being the effect-target ratio and the vertical axis being the number of target cells.

[0054] Figure 5 This is a comparison chart of the killing results of the three types of cells, hCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells and unmodified T cells of the present invention. 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, and the vertical axis is the killing rate.

[0055] Figure 6 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

[0056] 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. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used can all be obtained through commercial channels.

[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-IL10-CAR

[0061] The plasmid encoding hCD38-BCMA-IL10-CAR was synthesized by Nanjing Biode Biotechnology Co., Ltd., and the plasmid contained nucleotide sequences encoding the following elements in order from the 5' to the 3' direction of the CAR structure: signal peptide (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 region (SEQ ID NO: 20), intracellular signal co-stimulatory region (SEQ ID NO: 21), intracellular signal stimulatory region (SEQ ID NO: 22), 2A sequence (SEQ ID NO: 23), signal peptide 2 (SEQ 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), IL-15 sequence (SEQ ID NO: 24), 2A sequence (SEQ ID NO: 23) and IL-10 sequence (SEQ ID NO: 25), 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-IL10-CAR. The plasmid was called pMSGV-hCD38-BCMA-IL10, 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 containing 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 containing 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-IL10-CAR of the present invention, a plasmid expressing BCMA-CAR was constructed at the same time. The plasmid encoding BCMA-CAR was synthesized by Nanjing Baode Biotechnology Co., Ltd. The plasmid sequentially contains nucleotide sequences encoding the following elements from the 5' to 3' direction of the CAR structure: signal peptide 1 (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 is 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-IL10 or pMSGV-BCMA is exactly the same. The following uses pMSGV-hCD38-BCMA-IL10 as an example to illustrate the process of preparing recombinant lentivirus.

[0066] The main plasmid pMSGV-hCD38-BCMA-IL10 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-IL10, 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 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 the virus can be 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-IL10-CAR-T cells.

[0082] To verify the advantages of the killing effect of the hCD38-BCMA-IL10-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 obtained in method 1 according to the specific operation process described in section "2-5" above.

[0083] 7. Determination of hCD38-BCMA-IL10-CAR-T cell positive rate and proliferation activity

[0084] 7.1 hCD38-BCMA-IL10-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 7 and 21, respectively, and the positive rates of CD38-CAR and BCMA-CAR in CAR-T cells were detected by flow cytometry.

[0085] 7.2 On the day of recombinant T cell preparation, 1×10 5 hCD38-BCMA-IL10-CAR-T cells and T cells without virus introduction were placed in 6-well plates for culture. The day of hCD38-BCMA-IL10-CAR-T cell preparation was day 0. Cell counts were performed every 3 days to measure cell proliferation activity.

[0086] 8. Screening of anti-CD38 antibodies

[0087] According to the specific operation procedures described in Methods 3-7, CD38-CAR-T cells were prepared using the pMSGV-CD38 plasmid obtained in Method 1.

[0088] 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:

[0089] 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.

[0090] 8.2 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.

[0091] 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.

[0092] 8.4 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.

[0093] 8.5 All samples were detected by flow cytometry FITC detection channel to detect the positive rate of GFP in MM.1S cells.

[0094] 8.6 Calculate the killing rate of CD38-CAR-T cells and T cells against MM.1S-GFP cells; the killing activity is expressed as killing rate.

[0095] The formula for the killing rate is: killing rate (%) = (1-(A*B) / C) × 100%;

[0096] 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.

[0097] 9. Determination of the activity of hCD38-BCMA-IL10-CAR-T cells in killing multiple myeloma cells in vitro

[0098] 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-IL10-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-IL10-CAR-T cells were effector cells, and BCMA-CAR-T cells were used as positive control group cells. The specific experimental steps are as follows:

[0099] 9.1MM.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.

[0100] 9.2 When hCD38-BCMA-IL10-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.

[0101] 9.3 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.

[0102] 9.4 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.

[0103] 9.5 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.

[0104] 9.6 All samples were detected by flow cytometry FITC detection channel to detect the positive rate of GFP in MM.1S cells.

[0105] 9.7 Calculate the killing rate of hCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells, and T cells against MM.1S-GFP cells; the killing activity is expressed as the killing rate.

[0106] The formula for the killing rate is: killing rate (%) = (1-(A*B) / C) × 100%;

[0107] 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.

[0108] Results and Discussion

[0109] BCMA-CAR-T cells and 16 types of hCD38-BCMA-IL10-CAR-T cells were constructed according to the above method.

[0110] Specifically, hCD38-BCMA-IL10-CAR-T cells were identified according to the positions of the signal peptide region, the heavy 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 types of hCD38-BCMA-IL10-CAR-T cells can be constructed. The inventors of the present invention have previously screened signal peptides, and SEQ ID NO: 1 is selected for signal peptide 1 and SEQ ID NO: 2 is selected for signal peptide 2. On this basis, a total of 400 types of hCD38-BCMA-IL10-CAR-T cells can be constructed by combining anti-CD38 antibodies in pairs. In the early stage of the present invention, anti-CD38 antibodies were screened by in vitro killing experiments of CD38-CAR-T cells (such as Figure 6 As shown in the figure, the two antibody sequences with the best killing effect are screened out, and the anti-CD38 antibody is therefore selected as SEQ ID NO: 5 and / or SEQ ID NO: 7. To this end, when the anti-CD38 antibody is preferably SEQ ID NO: 5 and / or SEQ ID NO: 7, according to Figure 1There are 4 structures a / b / c / d shown in (A), and a total of 16 pMSGV-hCD38-BCMA-IL10 plasmids were constructed. Through these 16 plasmids, according to the specific operation process described in parts 3-7 of the method, the Figure 1 There are 4 structures a / b / c / d shown in (B), and a total of 16 types of hCD38-BCMA-IL10-CAR-T cells. Table 1 lists these 16 types of cells.

[0111] Table 1. 16 types of hCD38-BCMA-IL10-CAR-T cells constructed by the present invention

[0112]

[0113]

[0114] *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.

[0115] The experimental results showed that the BCMA-CAR positivity of most of the 16 hCD38-BCMA-IL10-CAR-T cells was higher than 80%, and higher than that of single-target BCMA-CAR-T cells; the CD38-CAR positivity was above 50%. On the 21st day of preparing hCD38-BCMA-IL10-CAR-T cells, the BCMA-CAR positivity of some hCD38-BCMA-IL10-CAR-T cells was still maintained at 90%, and the CD38-CAR positivity was maintained at more than 40%; and the number of cell proliferation was always greater than that of empty T cells, which effectively alleviated the exhaustion of T cells. Figure 2 , 3 It was shown that the number of hCD38-BCMA-IL10-CAR-T cells proliferated by anti-CD38 antibody A (SEQ ID NO: 5) and anti-CD38 antibody B (SEQID NO: 7) according to structure a was always higher than that of empty T cells; on the 5th day of preparing CAR-T cells, the positive rate of CD38-CAR was as high as 74%, and the positive rate of BCMA-CAR was as high as 94%. On the 21st day, the positive rate of CD38-CAR remained at 47%, and the positive rate of BCMA-CAR remained at 90%.

[0116] In the in vitro killing experiment of hCD38-BCMA-IL10-CAR-T cells on target cells, these 16 types of hCD38-BCMA-IL10-CAR-T cells also showed similar killing effects. hCD38-BCMA-IL10-CAR-T cells have a significant killing effect on MM.1s-GFP target cells. When the effect-target ratio is 0.2, the killing rate is above 80%. For example, hCD38-BCMA-IL10-CAR-T cells formed according to structure a by anti-CD38 antibody A (SEQ ID NO: 5) and anti-CD38 antibody B (SEQ ID NO: 7) are compared with empty T cells, such as Figure 4 , 5 As shown, within 12 hours, when the effector-target ratio was only 0.1, hCD38-BCMA-IL10-CAR-T cells could efficiently mediate T cell killing of tumor cells, with a killing rate of up to 82%; when the effector-target ratio was increased to 0.4, hCD38-BCMA-IL10-CAR-T cells could kill MM.1S-GFP cells by up to 98.4%.

[0117] 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.

[0118] References

[0119] [1]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".

[0120] [2]HEUSER-LOY, C. (2024), "Interleukin-10 gives exhausted chimericantigen receptor(CAR)T cells a second breath", Cancer Commun(Lond), Vol.44No.7, pp.787-790.

[0121] [3]LI,H.,LI,J.,WU,J.,SHI,Z.,GAO,Y.,SONG,W.,LI,J.,LI,Z.&ZHANG,M.(2023),"Asecond-generation CD38-CAR-T cell for the treatment ofmultiplemyeloma",Cancer Med,Vol.12 No.9,pp.10804-10815.

[0122] [4]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-SpecificCARIntegrated into CD38 Locus Driven by Different Promoters Causes DistinctAntitumorActivities ofT andNK Cells",Adv Sci(Weinh),Vol.10 No.27,pp.e2207394.

[0123] [5]MISHRA,A.K.,GUPTA,A.,DAGAR,G.,DAS,D.,CHAKRABORTY,A.,HAQUE,S.,PRASAD,C.P.,SINGH,A.,BHAT,A.A.,MACHA,M.A.,BENALI,M.,SAINI,K.S.,PREVIS,R.A.,SAINI,D.,SAHA,D.,DUTTA,P.,BHATNAGAR,A.R.,DARSWAL,M.,SHANKAR,A.&SINGH,M.(2023),"CAR-T-Cell Therapy in Multiple Myeloma:B-Cell Maturation Antigen(BCMA)and Beyond",Vaccines(Basel),Vol.11 No.11,pp.

[0124] [6]MOORE,KW,de WAAL,MR,COFFMAN,RL&O'GARRA,A.(2001),"Interleukin-10 andthe interleukin-10 receptor",Annu Rev Immunol,Vol.19683-765.

[0125] [7]ROEX,G.,TIMMERS,M.,WOUTERS,K.,CAMPILLO-DAVO,D.,FLUMENS,D.,SCHROYENS,W.,CHU,Y.,BERNEMAN,ZN,LION,E.,LUO,F.&ANGUILLE,S.(2020),"Safety and efficiency of the CAR-T clinical clinic in BC." in multiple myeloma”,JHematol Oncol,Vol.13 No.1,pp.164.

[0126] [8]van de DONK,NW,van der HOLT,B.,MINNEMA,MC,VELLENGA,E.,CROOCKEWIT,S.,KERSTEN,MJ,von DEM,BP,YPMA,P.,SCHAAFSMA,R.,the WEERDT,O.,KLEIN,SK,DELFORGE,M.,LEVIN,MD,BOS,GM,JIE,KG,SINNIGE,H.,COENEN,JL,deWAAL,EG,ZWEEGMAN,S.,SONNEVELD,P.&LOKHORST,HM(2018),"Thalidomide and stem cell transplantation recently before autologous diagnosedmultiple myeloma(HOVON-50):long-term results from the phase 3,randomizedcontrolled trial",LancetHaematol,Vol.5 No.10,pp.e479-e492.

[0127] [9]ZHANG,X.,ZHANG,H.,LAN,H.,WU,J.&XIAO,Y.(2023),"CAR-T cell therapyinmultiplemyeloma:Currentlimitations andpotential strategies",Front Immunol,Vol.141101495.

[0128]

[10] ZHANG,X.,ZHU,L.,ZHANG,H.,CHEN,S.&XIAO,Y.(2022),"CAR-T CellTherapyin Hematological Malignancies:Current Opportunities and Challenges",Front Immunol,Vol.13927153.

[0129]

[11] TANIKAWA,T.,WILKE,C.M.,KRYCZEK,I.,CHEN,G.Y.,KAO,J., G.&ZOU,W.(2012),"Interleukin-10 ablationpromotes tumor development,growth,andmetastasis.",Cancerresearch,Vol.72 No.2,pp.420-9.

Claims

1. A series-parallel hybrid chimeric antigen receptor that dually targets CD38 and BCMA and contains IL-10, 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; 17) IL-15 sequence; 18) 2A sequence; and 19) Human IL-10 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-10 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 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 a CAR-T cell expressing a dual-targeting CD38 and BCMA serial-parallel hybrid chimeric antigen receptor and secreting IL-10, 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-10, 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 expressing a series-parallel hybrid chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-10 as described in 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 of claim 2, the expression vector of claim 3, the recombinant lentivirus of claim 4, or the CAR-T cell expressing a dual-targeting CD38 and BCMA series-parallel hybrid chimeric antigen receptor and secreting IL-10 as claimed in claim 7 in the preparation of a drug for treating a disease.

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