CAR-T cells expressing parallel chimeric antigen receptors dual-targeting CD38 and BCMA 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 that expresses a parallel chimeric antigen receptor that dual-targets CD38 and BCMA and secretes 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 parallel chimeric antigen receptor that dual-targets CD38 and BCMA and comprises IL-10, or is obtained by an expression vector comprising the nucleic acid molecule. The CAR-T cell of the present invention expresses parallel CD38-CAR and BCMA-CAR, and can secrete the cytokine IL-10, which can better enhance the expression 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 parallel chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-10, and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is the second most common malignant tumor of the blood system, characterized by abnormal proliferation of bone marrow plasma cells, with high morbidity and mortality, with an incidence rate of about 1.6 per 100,000 in my country. Although the development of new drugs such as immunomodulatory drugs (thalidomide and lenalidomide), autologous stem cell transplantation (ASCT), proteasome inhibitors (PI) and CD38 monoclonal antibodies has improved the survival rate of patients, the prognosis of MM remains grim, and there is an urgent need to develop safe and long-acting drugs (Munshi et al., 2021).

[0003] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) is a new type of immunotherapy that shows great potential in the long-term disease control of some hematological malignancies. This therapy uses genetic engineering to construct a chimeric antigen receptor expression vector to transfect T cells, so that the T cells express chimeric receptors that can recognize target cells on their surface, specifically recognize target cell surface antigens, and achieve specific killing of target cells.

[0004] B cell maturation antigen (BCMA) is selectively expressed on tumor plasma cells, participates in the survival and proliferation of MM cells, and is considered an ideal target for MM targeted therapy. Anti-BCMA C-T cell therapy has a significant effect on MM, and its objective response rate fluctuates greatly, ranging from 30% to 100% (Gazeau et al., 2021). Several experiments also reported that some patients failed treatment or relapsed within a short period of time after treatment, mainly due to antigen escape (Samur et al., 2021, Da et al., 2021). Therefore, more effective CAR-T cell therapy is needed.

[0005] Dual-targeted CAR-T cells are one of the ways to solve the problem of antigen escape, such as using CD19 / CD22 to treat leukemia and lymphoma, and BCMA / CD19 to treat myeloma. CD38 is a single-chain type II transmembrane glycoprotein that participates in cell migration and signal transduction of lymphocytes and endothelial cells by binding to CD31 (Deaglio et al., 1998). CD38 is widely expressed on plasma cells and is more highly expressed on the surface of tumor plasma cells, making it an ideal therapeutic target for MM (Li et al., 2021). Anti-CD38 monoclonal antibodies exert their anti-tumor effects through multiple mechanisms of action and prolong patient survival. Several CD38 antibodies have been approved for clinical treatment (Dimopoulos et al., 2021, Lokhorst et al., 2015, Dimopoulos et al., 2021). On this basis, the affinity of anti-CD38 monoclonal antibodies was modified, and the prepared anti-CD38-CAR-T cells had significant therapeutic effects on multiple myeloma, so CD38 is also a new target for targeted therapy of multiple myeloma. However, some studies have reported that after CD38-targeted CAR-T cells were infused into patients, the number of CAR-T cells dropped to the initial level within 10 days, reducing its therapeutic effect, which may be caused by "self-killing" mediated by CD38-CAR-T cells (Yelei Guo et al., 2020). Therefore, it is necessary to study and develop a new method for preparing CD38-CAR-T cells to promote the sustained survival and proliferation of CAR-T cells in vivo, thereby improving the therapeutic effect of CAR-T cells on MM.

[0006] The cytokine interleukin 10 (IL-10) is an immunomodulatory cytokine that has the effect of counteracting T cell dysfunction in the tumor microenvironment. It has been reported that after exogenous injection of an Fc fusion protein of IL-10 with an extended half-life, the Fc fusion protein of IL-10 with an extended half-life promotes CD8+T cell oxidative phosphorylation (OXPHOS) in a mitochondrial pyruvate carrier (MPC)-dependent manner, that is, directly and effectively enhances the proliferation and tumor killing activity of terminally exhausted CD8+ tumor-infiltrating lymphocytes through the pyruvate-MPC-OXPHOS metabolic pathway (Yugang Guo et al., 2021). However, for clinical treatment, IL-10 still needs to be administered subcutaneously every day, and its sustained immune activity is low and its convenience is poor.

[0007] It can be seen that the existing single-target BCMA CAR-T cells have drug resistance problems in the treatment of MM, the essence of which is the reduced expression of BCMA on the tumor surface, resulting in antigen escape. Therefore, it is necessary to design a parallel enhanced 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 antibody and a secretory IL-10 fragment, so as to prepare a recombinant vector expressing the receptor, and further prepare CAR-T cells to solve the above problems. Summary of the invention

[0008] The existing single-target BCMA CAR-T cells have drug resistance problems in the treatment of MM, the essence of which is the reduced expression of BCMA on the tumor surface, which leads to antigen escape; on the other hand, the survival time of CAR-T cells in the body after infusion is short, and they do not proliferate or proliferate less, causing T cell exhaustion. In response to the above two key issues, the present invention designs and verifies a series of parallel CAR structures that can dual-target CD38 and BCMA, effectively avoiding the problem of antigen escape; IL-10 is also included in the CAR structure, which effectively enhances the proliferation ability of T cells and alleviates the problem of T cell exhaustion, thereby greatly improving the treatment effect of the disease.

[0009] Therefore, in a first aspect, the invention provides a parallel chimeric antigen receptor (pCD38-BCMA-IL10-CAR) that dually targets CD38 and BCMA and contains IL-10, comprising the following elements:

[0010] 1) signal peptide 1; 2) anti-CD38 antibody region; 3) dimerization sequence; 4) hinge region; 5) transmembrane region; 6) intracellular signal co-stimulatory region; 7) intracellular signal stimulatory region; 8) 2A sequence; 9) signal peptide 2; 10) anti-BCMA heavy chain region; 11) linker; 12) anti-BCMA light chain region; 13) dimerization sequence, 14) 2A sequence and 15) human IL-10 sequence,

[0011] The amino acid sequence of the anti-CD38 antibody region is as 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, the linker 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 an anti-BCMA heavy chain region, a linker and an anti-BCMA light chain region form a parallel 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 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.

[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 CD8α or human CD28. Preferably, the hinge region is the hinge region of human CD8α, whose amino acid sequence is shown in SEQ ID NO:17.

[0018] In an embodiment of the present invention, the transmembrane region may 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 in SEQ ID NO:18.

[0019] In an embodiment of the present invention, the amino acid sequence of the intracellular signaling co-stimulatory region is shown as SEQ ID NO:19.

[0020] In an embodiment of the present invention, the amino acid sequence of the intracellular signal stimulation region is shown in SEQ ID NO:20.

[0021] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is shown in SEQ ID NO:21.

[0022] In an embodiment of the present invention, the amino acid sequence of the dimerization sequence is shown in SEQ ID NO:16.

[0023] In an embodiment of the present invention, the amino acid sequence of the linker is shown in SEQ ID NO:22.

[0024] In an embodiment of the present invention, the amino acid sequence of human IL-10 is shown in SEQ ID NO:23.

[0025] In a second aspect, the present invention provides a nucleic acid molecule encoding a parallel chimeric antigen receptor (pCD38-BCMA-IL10-CAR) that dually targets CD38 and BCMA and contains IL-10.

[0026] In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule encoding a parallel chimeric antigen receptor (pCD38-BCMA-IL10-CAR) targeting CD38 and BCMA and comprising IL-10.

[0027] 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.

[0028] 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.

[0029] In an embodiment of the fifth aspect, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0030] 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.

[0031] In a sixth aspect, the present invention provides a CAR-T cell (pCD38-BCMA-IL10-CAR-T cell) that expresses a parallel chimeric antigen receptor that dual-targets CD38 and BCMA and secretes IL-10, which expresses the chimeric antigen receptor (pCD38-BCMA-IL10-CAR) that dual-targets CD38 and BCMA and contains IL-10 of the first aspect of the present invention.

[0032] In a seventh aspect, the present invention provides a method for preparing the pCD38-BCMA-IL10-CAR-T cells of the sixth aspect, comprising infecting T cells with the recombinant lentivirus of the fifth aspect.

[0033] In an embodiment of the seventh aspect of the present invention, the method for preparing pCD38-BCMA-IL10-CAR-T cells comprises the following steps:

[0034] (1) constructing a recombinant lentiviral expression vector carrying a nucleotide sequence encoding pCD38-BCMA-IL10-CAR;

[0035] (2) transfecting host cells with the recombinant lentiviral expression vector and the helper plasmid to prepare a recombinant lentivirus that can infect T cells;

[0036] (3) Isolate PBMCs from peripheral blood provided by the donor and use magnetic beads to separate and activate T cells;

[0037] (4) Infecting T cells with the recombinant lentivirus obtained in step (2) to produce T cells expressing pCD38-BCMA-IL10-CAR.

[0038] In further embodiments, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0039] In a further embodiment, the method may further comprise the following steps after step (4):

[0040] (5) culturing the cells obtained in step (4) in vitro;

[0041] (6) amplifying the cells obtained in step (5) in large quantities; and

[0042] (7) Collect pCD38-BCMA-IL10-CAR-T cells.

[0043] In an eighth aspect, the present invention provides a disease treatment drug, comprising a nucleic acid molecule encoding pCD38-BCMA-IL10-CAR of the present invention, an expression vector comprising a nucleic acid molecule encoding pCD38-BCMA-IL10-CAR, the recombinant lentivirus of the fourth aspect, or the pCD38-BCMA-IL10-CAR-T cell of the sixth aspect of the present invention.

[0044] 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 pCD38-BCMA-IL10-CAR-T cell in the preparation of a drug for treating a disease.

[0045] 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.

[0046] In an embodiment of the present invention, CAR represents a chimeric antigen receptor, BCMA-CAR represents a chimeric antigen receptor targeting BCMA, pCD38-BCMA-IL10-CAR represents a parallel chimeric antigen receptor that dually targets CD38 and BCMA and contains IL10, CAR-T cells represent chimeric antigen receptor T cells, BCMA-CAR-T cells represent chimeric antigen receptor T cells targeting BCMA, and pCD38-BCMA-IL10-CAR-T cells represent T cells that express a parallel chimeric antigen receptor that dually targets CD38 and BCMA and secretes IL-10.

[0047] In the context of the present invention, a parallel chimeric antigen receptor refers to a chimeric antigen receptor containing two parallel protein chains, and thus a "parallel 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.

[0048] The present invention recognizes and binds to CD38 and BCMA molecules highly expressed on the surface of multiple myeloma MM.1S cells by expressing pCD38-BCMA-CAR, thereby activating T cells, secreting IL-10 cytokines and perforin, and other mechanisms mediating MM cell lysis and death. At the same time, T cells in the present invention dual-target CD38 and BCMA to solve the problem of antigen escape in CAR-T therapy; in addition, IL-10 is simultaneously expressed and secreted in the CAR structure, which effectively enhances the proliferation ability of T cells, alleviates the problem of T cell exhaustion, and enhances anti-tumor killing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the structure of pCD38-BCMA-IL10-CAR, wherein (A) represents the primary sequence structure diagram, and (B) represents the three-dimensional structure diagram after CAR protein expression.

[0050] Figure 2 This is a comparison chart of the proliferation trend of pCD38-BCMA-IL10-CAR-T cells and unmodified T cells. The horizontal axis is the number of days and the vertical axis is the number of target cells.

[0051] Figure 3 This is a graph showing the CAR positivity rates of pCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells and unmodified T cells on the 7th and 21st days after viral transfection.

[0052] Figure 4 This is a comparison of the killing ability of pCD38-BCMA-IL10-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 5 This is a comparison of the killing results of pCD38-BCMA-IL10-CAR-T cells, BCMA-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. DETAILED DESCRIPTION

[0054] 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.

[0055] 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.

[0056] Materials and methods

[0057] 1. Design and construction of plasmid encoding pCD38-BCMA-IL10-CAR

[0058] The plasmid encoding pCD38-BCMA-IL10-CAR was synthesized by Nanjing Biode Biotechnology Co., Ltd., which 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 (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), dimerization sequence (SEQ ID NO: 16), hinge region (SEQ ID NO: 17), transmembrane region (SEQ ID NO: 18), intracellular signal co-stimulatory region (SEQ ID NO: 19), intracellular signal stimulation region (SEQ ID NO: 20), 2A sequence (SEQ ID NO: 21), signal peptide 2 (SEQ ID NO: 1, 2 or 3), anti-BCMA heavy chain region (SEQ ID NO: 14), linker (SEQ ID NO: 22), anti-BCMA light chain region (SEQ ID NO: 15), dimerization sequence (SEQ ID NO: 16), 2A sequence (SEQ ID NO: 23), NO:21) and human IL-10 sequence (SEQ ID NO:23), the structure is as Figure 1 The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newpro Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid encoding pCD38-BCMA-IL10-CAR. The plasmid was called pMSGV-pCD38-BCMA-IL10, which is a recombinant lentiviral expression vector in virus packaging.

[0059] 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, the linker 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, the linker and the anti-BCMA light chain region form a parallel double-chain structure, such as Figure 1 As shown in (A).

[0060] To verify the advantages of the pCD38-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 Baaode 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 (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, a recombinant lentiviral expression vector in viral packaging.

[0061] 2. Preparation of recombinant lentivirus

[0062] The method for preparing recombinant lentivirus using pMSGV-pCD38-BCMA-IL10 or pMSGV-BCMA is exactly the same. The following uses pMSGV-pCD38-BCMA-IL10 as an example to illustrate the process of preparing recombinant lentivirus.

[0063] The main plasmid pMSGV-pCD38-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:

[0064] 2.1 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.

[0065] 2.2 Add 500 μg of recombinant lentiviral expression vector pMSGV-pCD38-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.

[0066] 2.3 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.

[0067] 2.4 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℃.

[0068] 3. Lentiviral titer detection

[0069] 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 6Cells / 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.

[0070] 4. T cell isolation and activation

[0071] 4.1 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.

[0072] 4.2 Dilute the anticoagulated blood with PBS at a ratio of 1:1 and mix well, then add lymphocyte separation solution (Tianjin Haoyang Biological, catalog number: LTS1077) at a ratio of 1:2. Centrifuge at 300g for 10 minutes.

[0073] 4.3 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).

[0074] 4.4 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.

[0075] 4.5 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 6cells / 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.

[0076] 5. Preparation of Recombinant T Cells

[0077] 5.1 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.

[0078] 5.2 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 pCD38-BCMA-IL10-CAR-T cells.

[0079] To verify the advantages of the killing effect of the pCD38-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 methods 2-5.

[0080] 6. Determination of pCD38-BCMA-IL10-CAR-T cell positive rate and proliferation activity

[0081] 6.1 pCD38-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.

[0082] 6.2 On the day of recombinant T cell preparation, 1×10 5 pCD38-BCMA-IL10-CAR-T cells and T cells without virus were cultured in 6-well plates. The day of pCD38-BCMA-IL10-CAR-T cell preparation was day 0. Cell counts were performed every 3 days to measure cell proliferation activity.

[0083] 7. Determination of the activity of pCD38-BCMA-IL10-CAR-T cells in killing multiple myeloma cells in vitro

[0084] 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 pCD38-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; the prepared pCD38-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:

[0085] 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.

[0086] 7.2 When pCD38-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.

[0087] 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.

[0088] 7.4 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.

[0089] 7.5 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 live cells in the culture wells are calculated.

[0090] 7.6 All samples were detected by flow cytometry FITC detection channel to detect the positivity rate of GFP in MM.1S cells.

[0091] 7.7 Calculate the killing rate of pCD38-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.

[0092] The formula for the kill rate is: Kill rate (%) = (1-(A*B) / C) × 100%;

[0093] 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.

[0094] Results and Discussion

[0095] According to the above method, BCMA-CAR-T cells and 40 pCD38-BCMA-IL10-CAR-T cells were constructed. These 40 pCD38-BCMA-IL10-CAR-T cells were divided into different groups according to the positions of the heavy chain and light chain regions of anti-BCMA, the anti-CD38 antibody region and the anti-BCMA antibody region (including heavy chain, light chain and linker) in CAR (e.g. Figure 1 Table 1 lists these 40 cells, where the amino acid sequence of signal peptide 1 is SEQ ID NO: 1, and the amino acid sequence of signal peptide 2 is SEQ ID NO: 2.

[0096] Table 1. 40 types of pCD38-BCMA-IL10-CAR-T cells constructed by the present invention

[0097]

[0098]

[0099] *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.

[0100] The experimental results showed that the BCMA-CAR positive rates of these 40 pCD38-BCMA-IL10-CAR-T cells were all above 90%, which was higher than the BCMA-CAR positive rate of single-target BCMA-CAR-T cells; the CD38-CAR positive rates were all above 30%. On the 21st day of preparing pCD38-BCMA-IL10-CAR-T cells, the BCMA-CAR positive rate could still be maintained at 85%, and the CD38-CAR positive rate could still be maintained at more than 33%; and the number of cell proliferation was always greater than that of empty T cells, which effectively alleviated the exhaustion of T cells. Figure 2The pCD38-BCMA-IL10-CAR-T cells formed according to structure a of the anti-CD38 antibody region (SEQ ID NO: 5) are shown. On the 5th day of preparing the pCD38-BCMA-IL10-CAR-T cells, the positive rates of CD38-CAR were higher than 50%, and the positive rates of BCMA-CAR were 90%.

[0101] In the in vitro killing experiment of pCD38-BCMA-IL10-CAR-T cells on target cells, these 40 CAR-T cells also showed similar killing effects. For example, pCD38-BCMA-IL10-CAR-T cells (see Table 1) had a significant killing effect on MM.1s-GFP target cells, and the pCD38-BCMA-IL10-CAR-T cells formed according to structure a of the anti-CD38 antibody region (SEQ ID NO: 5) were effector cells; Figure 4 , 5 As shown, three types of cells: pCD38-BCMA-IL10-CAR-T cells, BCMA-CAR-T cells and T cells without virus introduction all have a killing effect on the target cell MM.1S-GFP. Regardless of whether the effector-target ratio is 0.2 or 0.4, the killing effect of pCD38-BCMA-IL10-CAR-T on MM.1S-GFP is stronger than that of BCMA-CAR-T and empty T cells: within 12 hours, when the effector-target ratio is 0.2, pCD38-BCMA-IL10-CAR-T cells can effectively kill tumor cells with a killing rate of 86%; when the effector-target ratio is increased to 0.4, the killing rate of pCD38-BCMA-IL10-CAR-T cells on MM.1S-GFP cells is as high as 97%.

[0102] 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.

[0103] References

[0104] [1]DA,VM,DIETRICH,O.,TRUGER,M.,ARAMPATZI,P.,DUELL,J.,HEIDEMEIER,A.,ZHOU,X.,DANHOF,S.,KRAUS,S.,CHATTERJEE,M.,MEGGENDORFER,M.,TWARDZIOK,SER,ME,GOPP,ME,HUMS. DECEK,M.,PROMMERSBERGER,S.,HEGE,K.,KAISER,S.,FUHR,V.,WEINHOLD,N.,ROSENWALD,A.,ERHARD,F.,HAFERLACH,C.,EINSELE,H.,KORTUM,KM,SALIBA,AE&RASCHE,L. BCMA gene deletioninresponseto anti-BCMACAR Tcells inapatient withmultiplemyeloma”,NatMed,Vol.27No.4,pp.616-619.

[0105] [2]DEAGLIO,S.,MORRA,M.,MALLONE,R.,AUSIELLO,CM,PRAGER,E.,GARBARINO,G.,DIANZANI,U.,STOCKINGER,H.&MALAVASI,F.(1998),"Human CD38(ADP-ribosyl cyclase) is an acceptor of ICD-1, superfamily member",JImmunol,Vol.160No.1,pp.395-402.

[0106] [3]DIMOPOULOS,MA,LELEU,X.,MOREAU,P.,RICHARDSON,PG,LIBERATI,AM,HARRISON,SJ,MILES,PH,OCIO,EM,ASSADOURIAN,S.,CAMPANA,F.,MALINGE,L.,SEMIOND,D.,VAN DE VELDE,H.&YONG,K.(2021),"Isatuximab plus pomalidomide anddexamethasone in relapsed / refractory multiple myeloma patients with renal impairment:ICARIA-MMsubgroup analysis",Leukemia,Vol.35No.2,pp.562-572.

[0107] [4]DIMOPOULOS,M.,BRINGHEN,S.,ANTILLA,P.,CAPRA,M.,CAVO,M.,COLE,C.,GASPARETTO,C.,HUNGRIA,V.,JENNER,M.,VO ROBYEV, V., RUIZ, EY, YIN, JY, SALEEM, R., HELLET, M., MACE, S., PAIVA, B. & VIJ, R. (2021),"Isatuximabasmonotherapyand combinedwith dexamethasone in patients with relapsed / refractory multiple myeloma",Blood,Vol.137No.9,pp.1154-1165.

[0108] [5]GAZEAU,N.,BEAUVAIS,D.,YAKOUB-AGHA,I.,MITRA,S.,CAMPBELL,TB,FACON,T.&MANIER,S.(2021),"Effective anti-BCMA retreatment in multiple myeloma",BloodAdv,Vol.5 No.15,pp.3016-3020.

[0109] [6]GUO,Y.,FENG,K.,TONG,C.,JIA,H.,LIU,Y.,WANG,Y.,TI,D.,YANG,Q.,WU,Z.&HAN,W.(2020)Efficiency and side effects ofanti-CD38 CAR T cells in an adultpatient with relapsedB-ALL after failure ofbi-specific CD19 / CD22 CAR T celltreatment...

[0110] [7]GUO,Y.,XIE,Y.,GAO,M.,ZHAO,Y.,FRANCO,F.,WENES,M.,SIDDIQUI,I.,BEVILACQUA,A.,WANG,H.,YANG,H.,FENG,B.,XIE,X.,SABATEL,C.M.,TSCHUMI,B.,CHAIBOONCHOE,A.,WANG,Y.,LI,W.,XIAO,W.,HELD,W.,ROMERO,P.,HO,P.&TANG,L.(2021),"Metabolic reprogramming of terminally exhausted CD8(+)T cells by IL-10enhancesanti-tumor immunity.",Nature immunology,Vol.22 No.6,pp.746-756.

[0111] [8]LI,X.,FENG,Y.,SHANG,F.,YU,Z.,WANG,T.,ZHANG,J.,SONG,Z.,WANG,P.,SHI,B.&WANG,J.(2021),"Characterization of the Therapeutic Effects of NovelChimeric AntigenReceptor T Cells Targeting CD38 on Multiple Myeloma",FrontOncol,Vol.11703087.

[0112] [9]LOKHORST,HM,PLESNER,T.,LAUBACH,JP,NAHI,H.,GIMSING,P.,HANSSON,M.,MINNEMA,MC,LASSEN,U.,KREJCIK,J.,PALUMBO,A.,van de DONK,NW,AHMADI,T.,KHAN,I.,UHLAR,CM,WANG,J.,SASSER,AK,LOSIC,N.,LISBY,S.,BASSE,L.,BRUN,N.&RICHARDSON,PG(2015),"Targeting CD38 with Daratumumab Monoplex Therapy in Multiplexed Multiplex. No.13,pp.1207-19.

[0113]

[10] MUNSHI,NC,ANDERSON,LJ,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.,GO LDSCHMIDT,H.,WEISEL,K.,RAMBALDI,A.,REECE,D.,PETROCCA,F.,MASSARO,M.,CONNARN,JN,KAISER,S.,PATEL,P.,HUANG,L.,CAMPBELL,TB,HEGE,K.&SAN-MIGUEL,J. Vicleucel in Relapsed andRefractoryMultiple Myeloma”,NEnglJMed,Vol.384 No.8,pp.705-716.

[0114]

[11] SAMUR,M.K.,FULCINITI,M.,AKTAS,S.A.,BAZARBACHI,A.H.,TAI,Y.T.,PRABHALA,R.,ALONSO,A.,SPERLING,A.S.,CAMPBELL,T.,PETROCCA,F.,HEGE,K.,KAISER,S.,LOISEAU,H.A.,ANDERSON,K.C.&MUNSHI,N.C.(2021),"Biallelic loss ofBCMA as aresistance mechanism to CAR T cell therapy in a patient with multiplemyeloma",NatCommun,Vol.12 No.1,pp.868.

Claims

1. A parallel chimeric antigen receptor that dually targets CD38 and BCMA and contains IL-10, characterized in that: The parallel chimeric antigen receptor comprises the following elements in sequence: 1) Signal peptide 1; 2) Anti-CD38 single-chain antibody; 3) dimerization sequence; 4) Hinge region; 5) transmembrane region; 6) Intracellular signaling co-stimulatory region; 7) Intracellular signal stimulation area; 8) 2A sequence; 9) Signal peptide 2; 10) Anti-BCMA heavy chain region; 11) Linker; 12) Anti-BCMA light chain region; 13) dimerization sequence; 14) 2A sequence; and 15) Human IL-10 sequence, Wherein, the amino acid sequence of the anti-CD38 antibody region 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 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 and the anti-BCMA light chain region form a parallel double-chain structure.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the parallel 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 co-transfecting a mammalian host cell with the vector of claim 3 and a helper plasmid.

6. A method for preparing a CAR-T cell expressing a parallel chimeric antigen receptor that dual-targets CD38 and BCMA and secretes 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 parallel chimeric antigen receptor that dual-targets CD38 and BCMA and secretes 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 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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