CAR-T cells simultaneously targeting BCMA and CD38 and secreting IL-10 and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine, and discloses a CAR-T cell that simultaneously targets BCMA and CD38 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 BCMA-CD38-IL10-CAR or being prepared by an expression vector comprising the nucleic acid molecule. The BCMA-CD38-IL10-CAR-T cell of the present invention effectively avoids the problem of antigen escape, enhances the ability of T cells to increase in value, and alleviates the problem of T cell exhaustion, thereby greatly improving the therapeutic effect of the disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to CAR-T cells that simultaneously target BCMA and CD38 and secrete IL-10, and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is the second most common hematological malignancy in the blood system after non-Hodgkin's lymphoma. Its recurrence and refractory nature have always been a medical problem (Kazandjian, 2016). Although BCMA-based CAR-T therapy has achieved certain success in clinical trials for patients with relapsed or refractory multiple myeloma (R / RMM) and some products have been successfully marketed, there are still significant limitations (Raje et al., 2019, Mishra et al., 2023, Zhang et al., 2023).

[0003] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily. In normal human tissues, BCMA protein and mRNA are almost exclusively found on plasma cells (Cho et al., 2018), and are selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and drug resistance (Tai et al., 2016, Tai et al., 2006, Claudio et al., 2002). The consistent upregulation and uniqueness of BCMA on the surface of MM cells make BCMA an attractive target for MM drug discovery and development (Carpenter et al., 2013).

[0004] Although BCMA-CAR-T cells have a response rate of 73-100% for patients with relapsed or refractory multiple myeloma, in postoperative observations of CAR-T cell therapy targeting BCMA, the proportion of BCMA receptors on the surface of myeloma cells downregulated or disappeared reached 4-33%, leading to the generation of drug resistance and treatment failure (Mei et al., 2021). Studies have shown that after BCMA on MM cells of some patients was downregulated, their CD38 was significantly upregulated (Da Vià et al., 2021).

[0005] CD38 is a type II transmembrane glycoprotein, with a ligand of CD31, and is a member of the Ig gene superfamily. The binding of CD38 and CD31 is closely related to cell migration and receptor-mediated cell adhesion (Malavasi et al., 2008). CD38 is lowly expressed on the surface of lymphoid cells, myeloid cells and other non-hematopoietic tissue cells, but highly expressed on the surface of malignant plasma cells such as myeloma cells. Based on this feature, CD38 has become a popular target for the treatment of multiple myeloma (MM) (Drent et al., 2016).

[0006] Therefore, by adding one or more binding sites to CAR-T cells, that is, targeting two or more antigens or different epitopes of antigens at the same time, it is expected to improve the specificity of treatment, more accurately target tumor cells and reduce off-target toxicity, thereby enhancing the killing toxicity of tumor cells. The BCMA / CD38 CAR-T target combination can solve the problem of disease progression or recurrence after CAR-T cell therapy caused by potential loss of BCMA targets.

[0007] In CAR-T cell therapy, T cell dysfunction caused by T cell exhaustion is one of the main factors hindering the efficacy of CAR-T cell therapy (Hou et al., 2021). Exhausted T cells are characterized by complete or partial loss of proliferation and effector function, upregulation of multiple immunosuppressive receptors, and transcriptional and epigenetic changes (Blank et al., 2019, Wherry and Kurachi, 2015, Franco et al., 2020). Metabolic adaptability is essential for maintaining T cell survival and function. It has been reported that in vivo administration of interleukin 10 (IL-10) Fc fusion protein (IL-10-Fc) can reprogram intratumoral T cell metabolism to mitochondrial pyruvate carrier-dependent oxidative phosphorylation, increase mitochondrial respiratory capacity, and thus enhance the proliferation and effector function of terminally exhausted CD8+ tumor-infiltrating lymphocytes (TILs) (Guo et al., 2021). Studies have shown that CAR-T cells expressing IL-10 can prevent functional impairment associated with T cell exhaustion in the tumor microenvironment, thereby enhancing proliferation and effector function (Zhao et al., 2024). Therefore, loading CAR-T cells with IL-10 is a promising therapeutic strategy to combat T cell exhaustion-related dysfunction and induce effective and long-term anti-tumor immunity. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide a CAR-T cell (BCMA-CD38-IL10-T cell) that simultaneously targets BCMA and CD38 and secretes IL-10, so as to solve the following technical problems: 1) By simultaneously targeting two cell surface antigens, BCMA and CD38, the problem of antigen escape in CAR-T cell therapy is solved; 2) 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] The purpose of the present invention and the solution to its technical problem can be achieved by adopting the following technical solutions.

[0010] In a first aspect, the present invention provides a chimeric antigen receptor (BCMA-CD38-IL10-CAR) that simultaneously targets BCMA and CD38 and comprises IL-10, comprising the following elements:

[0011] 1) signal peptide region, 2) anti-CD38 antibody region; 3) linker 1; 4) anti-BCMA heavy chain region; 5) linker 2; 6) anti-BCMA light chain region; 7) hinge region; 8) transmembrane region; 9) intracellular signal co-stimulatory region; 10) intracellular signal stimulatory region, 11) 2A sequence; and 12) human IL-10 sequence,

[0012] The amino acid sequence of the anti-CD38 antibody region is as shown in any one of SEQ ID NOs: 4-13,

[0013] The positions of the anti-BCMA heavy chain region and the anti-BCMA light chain region can be interchanged.

[0014] And wherein, the positions of the anti-CD38 antibody region and the BCMA antibody region containing the BCMA heavy chain region, the BCMA light chain region and the linker 2 can be interchanged.

[0015] In an embodiment of the present invention, the signal peptide region can be a signal peptide of a 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 and 3, respectively.

[0016] 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.

[0017] In an embodiment of the present invention, the amino acid sequence of the anti-BCMA light chain region is shown in SEQ ID NO:15.

[0018] In an embodiment of the present invention, the amino acid sequence of linker 1 between the anti-CD38 antibody region and the anti-BCMA heavy chain region is shown in SEQ ID NO:16.

[0019] In an embodiment of the present invention, the amino acid sequence of linker 2 between the anti-BCMA heavy chain region and the anti-BCMA light chain region is shown in SEQ ID NO:17.

[0020] 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:18.

[0021] 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:19.

[0022] In an embodiment of the present invention, the amino acid sequence of the intracellular co-stimulatory region is shown in SEQ ID NO:20.

[0023] In an embodiment of the present invention, the amino acid sequence of the intracellular stimulatory region is shown in SEQ ID NO:21.

[0024] In an embodiment of the present invention, the amino acid sequence of 2A is shown in SEQ ID NO:22.

[0025] In an embodiment of the present invention, the amino acid sequence of human IL-10 is shown in SEQ ID NO:23.

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

[0027] In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule encoding a chimeric antigen receptor (BCMA-CD38-IL10-CAR) that simultaneously targets BCMA and CD38 and comprises IL-10.

[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 mammalian cells 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 a sixth aspect, the present invention provides CAR-T cells (BCMA-CD38-IL10-CAR-T cells) that simultaneously target BCMA and CD38 and secrete IL-10, which cells express the chimeric antigen receptor (BCMA-CD38-IL10-CAR) of the first aspect of the present invention that simultaneously targets BCMA and CD38 and contains IL-10.

[0033] In a seventh aspect, the present invention provides a method for preparing the BCMA-CD38-IL10-CAR-T cells of the sixth aspect, comprising infecting T cells with the recombinant lentivirus of the fourth aspect.

[0034] In a specific embodiment of the seventh aspect of the present invention, the method for preparing the BCMA-CD38-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 BCMA-CD38-IL10-CAR;

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

[0037] (3) Isolate peripheral blood mononuclear cells (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 BCMA-CD38-IL10-CAR-T cells expressing BCMA-CD38-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 BCMA-CD38-IL10-CAR-T cells.

[0044] In an eighth aspect, the present invention provides a disease treatment drug, which comprises the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the recombinant lentivirus of the fourth aspect of the present invention, or the BCMA-CD38-IL10-CAR-T cell of the sixth aspect of the present invention.

[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 BCMA-CD38-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 BCMA and / or CD38 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, BCMA-CD38-IL10-CAR represents a chimeric antigen receptor simultaneously targeting BCMA and CD38 and comprising IL-10, CAR-T cells represent chimeric antigen receptor T cells, BCMA-CAR-T cells represent chimeric antigen receptor T cells targeting BCMA, and BCMA-CD38-IL10-CAR-T cells represent T cells that simultaneously target BCMA and CD38 and secrete IL-10.

[0048] Compared with the prior art, the present invention has obvious beneficial effects. By means of the above technical solution, the present invention has at least the following beneficial technical effects:

[0049] 1. The present invention uses BCMA and CD38 as targets to obtain monoclonal antibody gene sequences that specifically recognize BCMA and CD38, which are unique and distinctive. The BCMA-CD38-IL10-CAR-T cells constructed and prepared on this basis are also unique and distinctive in recognizing CD38-positive and BCMA-positive tumor cells.

[0050] 2. BCMA-CD38-IL10-CAR-T cells simultaneously target two surface markers of multiple myeloma, BCMA and CD38, which can effectively avoid the problem of antigen escape and prevent tumor recurrence caused by the loss of a single target.

[0051] 3. The killing activity of BCMA-CD38-IL10-CAR-T cells against multiple myeloma cells MM.1S-GFP was significantly higher than that of unmodified T cells and single-target BCMA-CAR-T cells. Using a low dose of effector cells (0.2 times to 0.4 times the number of target cells), the myeloma cell line MM.1S-GFP can be effectively killed, and it has excellent myeloma targeted killing activity.

[0052] 4. The CAR-T cells of the present invention also express IL-10, which significantly enhances the in vitro proliferation ability of CAR-T cells, is conducive to the large-scale preparation of CAR-T cells, and brings good news to cancer patients; at the same time, IL-10 also enhances the persistence of CAR-T in the body, which is conducive to the continuous clearance of tumors and reduces recurrence.

[0053] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagrams of the structures of BCMA-CD38-IL10-CAR and BCMA-CAR provided by the present invention, wherein (A) and (C) respectively represent the primary sequence diagram of BCMA-CD38-IL10-CAR and the three-dimensional structure diagram after CAR protein expression, and (B) represents the primary sequence diagram of BCMA-CAR.

[0055] Figure 2 These are the CAR positivity test results of BCMA-CD38-IL10-CAR-T cells and BCMA-CAR-T cells provided by the present invention on the 5th and 7th days after virus transfection.

[0056] Figure 3 This is the in vitro proliferation of BCMA-CD38-IL10-CAR-T cells, BCMA-CAR-T cells and T cells provided by the present invention.

[0057] Figure 4 This is a comparison of the 24-hour in vitro killing results of BCMA-CD38-IL10-CAR-T cells, BCMA-CAR-T cells and T cells provided by the present invention on MM.1S-GFP target cells. The horizontal axis is the effect-target ratio, and the vertical axis is the number of remaining target cells.

[0058] Figure 5This is a comparison of the 24-hour in vitro killing results of BCMA-CD38-IL10-CAR-T cells, BCMA-CAR-T cells and T cells provided by the present invention on MM.1S-GFP target cells. The horizontal axis is the effect-target ratio, and the vertical axis is the killing rate. DETAILED DESCRIPTION

[0059] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions fall within the scope of the claims of the present invention.

[0060] 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.

[0061] 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: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or the conditions recommended by the manufacturer's instructions.

[0062] Materials and Methods

[0063] 1. Design and construction of plasmids encoding BCMA-CD38-IL10-CAR and BCMA-CAR

[0064] The plasmid encoding BCMA-CD38-IL10-CAR was synthesized by Shenzhen BGI Co., Ltd., and the plasmid contained nucleotide sequences encoding the following elements in order from 5' to 3' direction of the CAR structure: signal peptide region (SEQ ID NO: 1, 2 or 3), anti-CD38 antibody region (SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13), linker 1 (SEQ ID NO: 16), anti-BCMA heavy chain region (SEQ ID NO: 14), linker 2 (SEQ ID NO: 17), anti-BCMA light chain region (SEQ ID NO: 15), hinge region (SEQ ID NO: 18), transmembrane region (SEQ ID NO: 19), intracellular signal co-stimulatory region (SEQ ID NO: 20), intracellular signal stimulatory region (SEQ ID NO: 21), 2A sequence (SEQ ID NO: 22) and human IL-10 sequence (SEQ ID NO: 23). The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newpro Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid encoding BCMA-CD38-IL10-CAR, which is called pMSGV-sBCMA-CD38-IL10 and is a recombinant lentiviral expression vector in viral packaging.

[0065] 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 anti-CD38 antibody region can be interchanged with the anti-BCMA antibody region (including the heavy chain, light chain and linker), such as Figure 1 As shown in (A) and (C).

[0066] In order to compare with BCMA-CD38-IL10-CAR, the present invention also constructed a plasmid encoding BCMA-CAR, whose coding region sequentially comprises nucleotide sequences encoding the following elements: a signal peptide region (SEQ ID NO: 1), an anti-BCMA heavy chain region (SEQ ID NO: 14), a linker 2 (SEQ ID NO: 17), an anti-BCMA light chain region (SEQ ID NO: 15), a hinge region (SEQ ID NO: 18), a transmembrane region (SEQ ID NO: 19), an intracellular signal co-stimulatory region (SEQ ID NO: 20) and an intracellular signal stimulatory region (SEQ ID NO: 21), the structure is as follows Figure 1 The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES to obtain a plasmid encoding BCMA-CAR, which is called pMSGV-BCMA and is a recombinant lentiviral expression vector in virus packaging.

[0067] 2. Preparation of recombinant lentivirus

[0068] The main plasmid pMSGV-sBCMA-CD38-IL10 or pMSGV-BCMA is co-transfected with three auxiliary plasmids into mammalian cells to prepare recombinant lentivirus that can infect T cells. The specific steps are as follows:

[0069] 2.1. Prepare a culture area of ​​525 cm 2 Cell culture flask, seeded with 6×10 7 WayneLVPro HEK293 cells (SuspensionAdapted) (Zhongshan Kangsheng Biotechnology Co., Ltd., Catalog No. A23109, hereinafter referred to as 293T cells) were added with complete culture medium containing DMEM (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30243.01) and 10% FBS (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30256.01), and cultured in a 37°C, 5% CO2 cell culture incubator overnight.

[0070] 2.2. Take 576 μL of 1 mg / mL PEI (Merck Biotechnology Co., Ltd., Catalog No.: 937762), add it to 4 mL OPTI-MEM (Thermo Fisher Scientific Biotechnology Co., Ltd., Catalog No.: 11058021), mix by pipetting, and let stand at room temperature for 20 min.

[0071] 2.3. Add 54 μg of the main plasmid pMSGV-sBCMA-CD38-IL10 or pMSGV-BCMA and three auxiliary plasmids, 16 μg pMD2G-Kana (Changsha Abiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0648), 16 μg pRSV-REV-Kana (Changsha Abiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0370) and 48 μg pMDLG-pRRE-Kana (Changsha Abiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0374) into 4 mL OPTI-MEM and mix well.

[0072] 2.4. Add the PEI and OPTI-MEM mixed solution in step 2.2 to the solution in step 2.3, mix well, and let stand at room temperature for 20 minutes.

[0073] 2.5. Add 32 mL of DMEM + 10% FBS complete medium to the solution in step 2.4 and mix well.

[0074] 2.6. Aspirate the culture medium in the 293T cell culture flask and add the culture medium prepared in step 2.5.

[0075] 2.7. After culturing 293T cells in a 37°C, 5% CO2 incubator for 6 h, the culture medium (DMEM+10% FBS+0.5% sodium butyrate (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Cat. No.: 303410)) was replaced and culture was continued for 48 h.

[0076] 2.8. Centrifuge the above culture medium, take the supernatant, filter it through 0.22 μm and place it at -20℃ for 30 min.

[0077] 2.9. Centrifuge the supernatant in step 2.8 at 1800g at 4°C for 2h, discard the supernatant, resuspend the virus particles in 500μL KBM581 (Corning (Shanghai) Management Co., Ltd., Catalog No.: 88-581-CM) serum-free culture medium, and store at -80°C.

[0078] 3. Recombinant Lentivirus Titer Detection

[0079] 3.1. Take Jurkat cells (purchased from ATCC: MD130) after resuscitation culture for 2 generations and centrifuge at 400g for 10 minutes.

[0080] 3.2. Resuspend the cells to 2×10 in RPMI1640 medium (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30809-01). 6 cells / mL, 48-well plate, 2×10 5 cells / well.

[0081] 3.3. Take 100 μL of the recombinant lentivirus prepared in step 2 diluted 200 times with RPMI1640 and add it to the plated cells and mix thoroughly.

[0082] 3.4.6 hours later, add 800 μL of RPMI1640 + 10% FBS complete culture medium and culture in a 37°C, 5% CO2 cell culture incubator.

[0083] 3.5. Perform flow cytometry detection after 48 hours.

[0084] 4. T cell isolation and activation

[0085] 4.1. Take 15 mL of peripheral blood from a healthy donor under sterile conditions, let it stand at room temperature for 30 minutes, centrifuge it at 400 g for 30 minutes, and aspirate the upper plasma for later use.

[0086] 4.2. Dilute the lower layer of blood cells with PBS at a ratio of 1:1 and mix well. Carefully add human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biological Products Co., Ltd., catalog number: LTS1077) to the upper layer at a ratio of 1:2 and centrifuge at 300g for 10 minutes.

[0087] 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 Cell).

[0088] 4.4. Isolate T lymphocytes by magnetic bead sorting using CD3 Microbeads (Human) (Miltenyi Biotec, Cat. No. 130-050-101) and count the cells.

[0089] 4.5. Take the CD3 obtained by sorting + T cells were centrifuged at 300 g for 10 min at 4°C.

[0090] 4.6. Discard the supernatant and add X-VIVO containing 40 IU / mL human IL-2 (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CD66) TM 15 serum-free culture medium (Lonza (China) Investment Co., Ltd., catalog number: DL-102), and the cell concentration was adjusted to 1×10 6 cells / mL.

[0091] 4.7. 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 and inoculate them in 24-well cell culture plates at a concentration of 1 × 10 6 cells / mL, 1 mL / well; culture in a cell culture incubator for 48 hours to obtain activated unmodified T cells.

[0092] 5. Preparation of CAR-T cells

[0093] 5.1. Take a 24-well plate, isolate and activate T cells for 48 hours, add 30 μL of 1 mg / mL protamine (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Catalog No.: P4020) to each well, and mix by pipetting.

[0094] 5.2. According to MOI=3, calculate the required amount of virus. The calculation formula is as follows: Required amount of virus (mL) = (MOI*number of cells) / virus titer. According to the calculation results, add BCMA-CD38-IL10-CAR and BCMA-CAR lentiviruses into a 24-well plate respectively and mix thoroughly.

[0095] 5.3. Seal the four sides of the 24-well plate with sealing film, centrifuge at 1000g for 30 minutes, and culture in a cell culture incubator at 37°C and 5% CO2.

[0096] 5.4. All samples were stained with PE-BCMA (Beijing Sino Biological Science Co., Ltd., Catalog No.: 10620-H02H) and FITC-CD38 (Beijing Sino Biological Science Co., Ltd., Catalog No.: 10818-H08H-F) antigens and FITC-labeled IL-10 antibody (Thermo Fisher Scientific, Catalog No.: 25-7108-42), and CAR expression was detected by flow cytometry.

[0097] 6. In vitro proliferation experiment of CAR-T cells

[0098] Virus infection was started on Day 0, and the cell number was 1.0×10 5 The total infection volume was 1 mL. The infection was completed on Day 3 and the cells were counted for the first time. TM 15 T cells were cultured in serum-free medium, and then the medium was changed and cells were counted every three days. Unmodified T cells were used as controls, and the cell proliferation of BCMA-CD38-IL10-CAR-T cells and BCMA-CAR-T cells was counted.

[0099] 7. In vitro killing experiment of CAR-T cells on target cells

[0100] MM.1S cells (Cell Bank of the Chinese Academy of Sciences, Catalog No.: SCSP-5017) were transformed into MM.1S-GFP cells as target cells. BCMA-CD38-IL10-CAR-T cells, BCMA-CAR-T cells and unmodified T cells were used as effector cells for killing activity determination. The specific steps are as follows:

[0101] 7.1. Adjust the target cells to the logarithmic growth phase and subculture them twice before conducting the experiment.

[0102] 7.2. Adjust the target cell density to 1×10 6 2 mL per well was inoculated in a 6-well plate.

[0103] 7.3. Set the ratio of effector cells to target cells to 0.2 and 0.4 respectively, and adjust the concentration of effector cells to 1×10 6 cells / mL and added into the target cell wells for co-culture.

[0104] 7.4. Adjust the concentration of unmodified T cells to 1×10 6According to the amount of effector cells added in the experimental group, unmodified T cells were added to the target cell wells as controls, and the target cell wells without effector cells were used as blank controls.

[0105] 7.5. After the above 6-well plate was placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, the culture medium volume and cell concentration in each well were calculated.

[0106] 7.6 All samples were subjected to flow cytometry, and the positive rate of target cells was detected through the FITC channel.

[0107] Results and Discussion

[0108] According to the above method, BCMA-CAR-T cells and 40 types of BCMA-CD38-IL10-CAR-T cells were constructed. According to the different positions of the anti-BCMA heavy chain region and anti-BCMA light chain region, the anti-CD38 antibody region and the anti-BCMA antibody region (including the anti-BCMA heavy chain region, the anti-BCMA light chain region and the linker) in CAR, the BCMA-CAR-T cells were selected according to the above method. Figure 1 The four structures of BCMA-CD38-IL10-CAR a / b / c / d shown in (A), referred to as a / b / c / d in Table 1 below), and the difference in the sequence of the anti-CD38 antibody region, a total of 40 BCMA-CD38-IL10-CAR-T cells were constructed. Table 1 lists these 40 cells.

[0109] Table 1. 40 types of BCMA-CD38-IL10-CAR-T cells constructed by the present invention

[0110]

[0111]

[0112] *. The first column indicates the killing rate of MM.1s-GFP target cells after 24 hours when the effector-target ratio is 0.2, and the second column indicates the killing rate of MM.1s-GFP target cells after 24 hours when the effector-target ratio is 0.4.

[0113] The experimental results showed that the BCMA and IL-10 positivity rates of these 40 BCMA-CD38-IL10-CAR-T cells were all higher than 80%, and the average CD38 positivity rate was 62.4% (see Table 1). Figure 2 It shows that the BCMA-CD38-IL10-CAR-T cells with cell number 27 (i.e., the anti-CD38 antibody region sequence is SEQ ID NO: 10, and the CAR structure is c) have a BCMA and IL-10 positivity rate higher than 90% on the 5th and 7th days after viral transfection; the BCMA positivity rate of BCMA-CAR-T cells is also higher than 90%.

[0114] In the in vitro proliferation experiment of CAR-T cells, the in vitro proliferation of dual-target BCMA-CD38-IL10-CAR-T cells (i.e., the anti-CD38 antibody region sequence is SEQ ID NO: 10, and the CAR structure is c) and single-target BCMA-CAR-T cells were tested with unmodified T cells as controls. The results are as follows: Figure 3 As shown: IL-10 does promote the proliferation of CAR-T cells. The proliferation rate of BCMA-CD38-IL10-CAR-T cells is significantly faster than that of BCMA-CAR-T cells, and even stronger than that of unmodified T cells. On Day 12, BCMA-CD38-IL10-CAR-T cells still maintain a good proliferation state.

[0115] In the in vitro killing experiment of CAR-T cells on MM.1S-GFP target cells, these 40 types of BCMA-CD38-IL10-CAR-T cells also showed similar killing effects (see Table 1). Figure 4 and Figure 5 The results showed that at two effector-target ratios, the killing effect of BCMA-CD38-IL10-CAR-T cells with cell number 27 on MM.1s-GFP target cells was stronger than that of T cells expressing BCMA-CAR alone, and when the effector-target ratio was 0.4, the tumor killing ability of BCMA-CD38-IL10-CAR-T cells was as high as 90.2%.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the concept, spirit and scope of the present invention; and these modifications or replacements fall within the scope of protection claimed in the claims of the present invention.

[0117] References

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Claims

1. A chimeric antigen receptor that simultaneously targets BCMA and CD38 and contains IL-10, characterized in that: The chimeric antigen receptor comprises the following elements: 1) Signal peptide region; 2) anti-CD38 antibody region; 3) Linker 1; 4) anti-BCMA heavy chain region; 5) Connector 2; 6) Anti-BCMA light chain region; 8) Hinge region; 9) transmembrane region; 9) Intracellular signaling co-stimulatory region; 10) Intracellular signal stimulation area; 11) 2A sequence; and 12) 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, The positions of the anti-BCMA heavy chain region and the anti-BCMA light chain region can be interchanged, And wherein, the positions of the anti-CD38 antibody region and the BCMA antibody region containing the BCMA heavy chain region, the BCMA light chain region and linker 2 can be interchanged.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor that simultaneously targets BCMA and CD38 and contains IL-10 as claimed 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 expression vector of claim 3 and a helper plasmid.

6. A method for preparing CAR-T cells that simultaneously target BCMA and CD38 and secrete IL-10, characterized in that: The method comprises infecting T cells with the recombinant lentivirus of claim 4.

7. CAR-T cells that simultaneously target BCMA and CD38 and secrete IL-10, characterized in that: The cells are prepared by the method of claim 6.

8. A drug for treating a disease, characterized in that: The drug comprises 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 disease is multiple myeloma, lymphoma, leukemia, systemic lupus erythematosus or 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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