CAR-T cell targeting CD38 and secreting IL-15 and preparation method thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a CAR-T cell targeting CD38 and secreting IL-15 and a preparation method of the CAR-T cell, the method comprises the step of infecting the T cell with a recombinant lentivirus, and the recombinant lentivirus contains nucleic acid molecules encoding CD38-IL15-CAR or is prepared through an expression vector containing the nucleic acid molecules. The CD38-IL15-CAR-T cell disclosed by the invention expresses CD38-CAR, can secrete a cell factor IL-15, and can better enhance the survival and proliferation of effector cells such as natural killer cells and CD8 + T cells, so that the killing activity of the CAR-T cell on tumor cells is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a CAR-T cell targeting CD38 and secreting IL-15, and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant tumor caused by abnormal proliferation of plasma cells in the bone marrow. It is characterized by the rapid spread of cancerous plasma cells in the bone marrow, which seriously affects the production of normal blood cells, leading to decreased blood cell indexes, bone damage and kidney damage (Padala et al., 2021). Although existing chemotherapy, radiotherapy, hematopoietic stem cell transplantation and monoclonal antibody treatments have improved the prognosis of MM patients to a certain extent, these traditional treatments are difficult to completely eliminate MM cells and have problems such as high recurrence rate and drug resistance.

[0003] Chimeric Antigen Receptor T-Cell (CAR-T cell) immunotherapy is an emerging immune cell therapy that uses genetic engineering to express chimeric antigen receptors (CARs) on the surface of T cells to identify and eliminate cells expressing specific target antigens, providing new possibilities for the cure of MM.

[0004] B cell maturation antigen (BCMA) is selectively expressed on tumor plasma cells and is involved in the survival and proliferation of MM cells. Therefore, it is considered an ideal target for targeted therapy of multiple myeloma. Anti-BCMA CAR-T cell therapy has a significant effect on multiple myeloma, but its objective response rate fluctuates. Some patients fail to receive treatment or relapse within a short period of time after treatment, which is mainly attributed to antigen escape. Therefore, more effective CAR-T cell therapy is needed.

[0005] Cyclic ADP ribose hydrolase (abbreviated as CD38) is a membrane protein highly expressed in MM cells, but its expression level in normal lymphocytes, myeloid cells and non-hematopoietic tissue cells is low, making it an ideal target for MM treatment (Liao et al., 2023). CD38 is mainly distributed on the cell membrane and has extracellular nicotinamide adenine dinucleotide (NAD) enzyme activity, which can convert NAD +It is hydrolyzed into nicotinamide and (cyclic) adenosine diphosphate (ADP). ADP is catalyzed by CD203a to generate adenosine monophosphate (AMP). AMP is catalyzed by CD73 to generate adenosine (ADO). Adenosine, as an immunoregulatory molecule, can bind to receptors on the cell membrane surface and transmit signals to the interior of the cell, thereby exerting an immunosuppressive function (Takasawa, 2022; Veliz et al., 2024).

[0006] At present, two monoclonal antibody drugs targeting CD38 have been approved for marketing, including Daratumumab, a fully human anti-CD38 IgGκ monoclonal antibody developed by Johnson & Johnson in 2015, and Isatuximab, an IgG1 human-mouse chimeric monoclonal antibody developed by Sanofi in 2020, but their therapeutic effects are limited, and the more promising CD38-CAR-T cell therapy products are still in the research stage. Studies have reported that after CD38-targeted CAR-T cells (CD38-CAR-T cells) are infused into patients, the number of CD38-CAR-T cells will drop 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 research and develop a new CD38-CAR-T cell to promote the sustained survival and proliferation of CD38-CAR-T cells in vivo, thereby improving the therapeutic effect of CD38-CAR-T cells on MM.

[0007] It has been reported that interleukin 15 (IL-15) affects natural killer cells (NK cells) and CD8 + The survival and proliferation of T cells play a vital role in the immune system (Waldmann, Dubois, Miljkovic, & Conlon, 2020). IL-15 is a member of the 4α-helical bundle cytokine family with a molecular weight of 14-15kDa and is an α, β heterodimeric cytokine. IL-15 can bind to the high-affinity receptor IL-15Rα expressed by antigen-presenting cells (APC). The IL-15 / IL-15Rα complex binds to effector cells (including effector NK cells, CD8 + T cells, etc.), thereby activating the JAK / STAT pathway downstream of effector cells and promoting the activation of NK cells and CD8 + Survival and proliferation of effector cells such as T cells.

[0008] CD38-CAR-T cells secrete IL-15, which theoretically has the potential to enhance the killing of MM tumor cells by CAR-T cells. However, in view of the problems of antigen escape and unstable therapeutic effect of existing CD38-CAR-T cells, there is no literature report on CD38-CAR-T cells expressing IL-15 for targeted treatment of MM. Summary of the invention

[0009] The present invention aims to solve the problem that CD38-CAR-T cells in the prior art have insufficient survival and proliferation ability in vivo. By making CD38-CAR-T cells co-express IL-15, a new CAR-T cell targeting CD38 and secreting IL-15 (CD38-IL15-CAR-T cell) is prepared to improve the terminal exhaustion of CD8 + The survival and proliferation of CAR-T cells enhance their anti-tumor killing activity.

[0010] Therefore, in the first aspect, the present invention provides a chimeric antigen receptor (CD38-IL15-CAR) targeting CD38 and comprising IL-15, which comprises the following elements: 1) signal peptide 1; 2) anti-CD38 antibody region; 3) IL-15R domain; 4) hinge region; 5) transmembrane region; 6) intracellular signal transduction region; 7) intracellular signal co-stimulatory region; 8) intracellular signal stimulation region; 9) 2A sequence; 10) signal peptide 2; 11) IL-15 sequence.

[0011] The amino acid sequence of the anti-CD38 antibody region is shown in any one of SEQ ID NOs: 2-11.

[0012] In an embodiment of the present invention, signal peptide 1 and signal peptide 2 can be signal peptides of mammalian cell surface proteins, such as human albumin signal peptide, human insulin signal peptide, human CD8 signal peptide or mouse IgG kappa signal peptide. Preferably, the signal peptide is a human albumin signal peptide, and its amino acid sequence is shown in SEQ ID NO: 1.

[0013] In an embodiment of the present invention, the amino acid sequence of the IL-15R domain is shown in SEQ ID NO:12.

[0014] In an embodiment of the present invention, the hinge region is the hinge region of human CD8α, human CD28 or human IL-15R hinge region, preferably the hinge region of human IL-15R, whose amino acid sequence is as shown in SEQ ID NO:13.

[0015] In an embodiment of the present invention, the transmembrane region may be the transmembrane region of human CD8α, human CD28 or human IL-15R. Preferably, the transmembrane region is the transmembrane region of human IL-15R, and its amino acid sequence is as shown in SEQ ID NO:14.

[0016] In an embodiment of the present invention, the amino acid sequence of the intracellular signaling region is shown as SEQ ID NO:15.

[0017] In an embodiment of the present invention, the amino acid sequence of the intracellular co-stimulatory region is shown in SEQ ID NO:16.

[0018] In an embodiment of the present invention, the amino acid sequence of the intracellular stimulatory region is shown in SEQ ID NO:17.

[0019] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is shown in SEQ ID NO:18.

[0020] In an embodiment of the present invention, the amino acid sequence of IL-15 is shown in SEQ ID NO:19.

[0021] In a second aspect, the present invention provides a nucleic acid molecule encoding the CD38-IL15-CAR of the first aspect.

[0022] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0023] In a fourth aspect, the present invention provides a recombinant lentivirus comprising the nucleic acid molecule of the second aspect or prepared by the expression vector of the third aspect.

[0024] 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.

[0025] In an embodiment of the fifth aspect, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0026] 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.

[0027] In a sixth aspect, the present invention provides CAR-T cells (CD38-IL15-CAR-T cells) that target CD38 and secrete IL-15, wherein the CD38-IL15-CAR-T cells express the CD38-IL15-CAR of the first aspect of the present invention.

[0028] In a seventh aspect, the present invention provides a method for preparing the CD38-IL15-CAR-T cells of the sixth aspect, comprising infecting T cells with the recombinant lentivirus of the fourth aspect.

[0029] In a specific embodiment of the seventh aspect of the present invention, the method for preparing CD38-IL15-CAR-T cells comprises the following steps:

[0030] S1) constructing a recombinant lentiviral expression vector carrying a nucleotide sequence encoding CD38-IL15-CAR,

[0031] S2) transfecting host cells with the above-mentioned recombinant lentiviral expression vector and helper plasmid to prepare a recombinant lentivirus capable of infecting T cells;

[0032] S3) isolating peripheral blood mononuclear cells (PBMC) from peripheral blood provided by the donor, and separating and activating T cells using magnetic beads;

[0033] S4) Infect T cells with the recombinant lentivirus obtained in step S2) to produce CD38-IL15-CAR-T cells expressing CD38-IL15-CAR.

[0034] In further embodiments, the helper plasmid may be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0035] In a further embodiment, the method may further comprise the following steps after step S4):

[0036] S5) culturing the cells obtained in step S4) in vitro;

[0037] S6) amplifying the cells obtained in step S5) in large quantities; and

[0038] S7) Collect CD38-IL15-CAR-T cells.

[0039] 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 CD38-IL15-CAR-T cell of the sixth aspect of the present invention.

[0040] 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 CD38-IL15-CAR-T cell in the preparation of a drug for treating a disease.

[0041] In an embodiment of the present invention, the disease refers to a disease characterized by high expression of CD38 molecules on the cell surface. In a specific embodiment, the disease can be multiple myeloma, lymphoma, leukemia, systemic lupus erythematosus, Sjögren's syndrome, etc.

[0042] In an embodiment of the present invention, CAR represents a chimeric antigen receptor, CD38-CAR represents a chimeric antigen receptor targeting CD38, CD38-IL15-CAR represents a chimeric antigen receptor targeting CD38 and comprising IL-15, CAR-T cells represent chimeric antigen receptor T cells, CD38-CAR-T cells represent chimeric antigen receptor T cells targeting CD38, and CD38-IL15-CAR-T cells represent chimeric antigen receptor T cells targeting CD38 and secreting IL-15.

[0043] The CD38-IL15-CAR-T cells of the present invention specifically recognize and bind to the CD38 glycoprotein highly expressed on the surface of the MM cell membrane through the expressed CD38-CAR. At the same time, the CD38-IL15-CAR-T cells of the present invention secrete cytokine IL-15, which can promote the terminal exhaustion of CD8 + The survival and proliferation of T cells enhance the anti-tumor killing activity compared with CD38-CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the structure of CD38-IL15-CAR, wherein (A) represents the primary sequence structure diagram of CD38-IL15-CAR, and (B) represents the schematic diagram of the three-dimensional structure after CD38-IL15-CAR expression. The structure of CD38-CAR lacks the 2A, IL-15 and IL-15R domain sequences compared to CD38-IL15-CAR, and no further illustration is required.

[0045] Figure 2 The figure shows the CD38-CAR positivity rate detection of CD38-IL15-CAR-T cells on the 7th and 9th days after virus transfection in three parallel experiments.

[0046] Figure 3 This is a comparison chart of the proliferation trends of CD38-IL15-CAR-T cells, CD38-CAR-T cells, and unmodified T cells. The proliferation rate of unmodified T cells was taken as 1, and the proliferation of CD38-CAR-T cells and CD38-IL15-CAR-T cells was counted.

[0047] Figure 4 This is a comparison chart of the killing results of CD38-IL15-CAR-T cells, CD38-CAR-T cells and unmodified T cells. The horizontal axis is the effector-target ratio, that is, the ratio of the number of T cells or CAR-T cells to the target cell MM.1S-GFP, and the vertical axis is the killing rate. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] Materials and methods

[0051] 1. Design and construction of plasmids encoding CD38-CAR and CD38-IL15-CAR

[0052] The plasmid encoding CD38-IL15-CAR was synthesized by Nanjing Biode Biotechnology Co., Ltd., and the plasmid contained nucleic acid sequences encoding the following elements in sequence from the 5' to the 3' direction of the CAR structure: signal peptide 1 (SEQ ID NO: 1), anti-CD38 antibody region (SEQ ID No: 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11), IL-15R domain (SEQ ID NO: 12), hinge region (SEQ ID NO: 13), transmembrane region (SEQ ID NO: 14), intracellular signal transduction region (SEQ ID NO: 15), intracellular signal co-stimulation region (SEQ ID NO: 16), intracellular signal stimulation region (SEQ ID NO: 17), 2A sequence (SEQ ID NO: 18), signal peptide 2 (SEQ ID NO: 1) and IL-15 sequence (SEQ ID NO: 19). The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newpro Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid encoding CD38-IL15-CAR, which is called pMSGV-CD38-IL15 and is a recombinant lentiviral expression vector in lentiviral packaging. The structure of CD38-IL15-CAR is as follows Figure 1 shown.

[0053] In addition, a plasmid encoding CD38-CAR was constructed for comparison. Compared with the plasmid encoding CD38-IL15-CAR, the constructed CD38-CAR plasmid lacks the 2A sequence (SEQ ID NO: 18), IL-15R domain (SEQ ID NO: 12) and IL-15 sequence (SEQ ID NO: 19), and the other sequences remain the same. The plasmid encoding CD38-CAR constructed by this method is called pMSGV-CD38.

[0054] 2. Preparation of recombinant lentivirus expressing CD38-CAR and CD38-IL15-CAR

[0055] The main plasmid pMSGV-CD38 or pMSGV-CD38-IL15 and three auxiliary plasmids pMDLG-pRRE-Kana (Changsha Aibiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0374), pMD2G-Kana (Changsha Aibiwei Biotechnology Co., Ltd., Catalog No.: HG-VMA0648), 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), and CD38-CAR and CD38-IL15-CAR recombinant lentiviruses that can infect T cells can be prepared. The specific operation process is as follows:

[0056] 2.1 293T cells were cultured with DMEM (Situofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30243.01) + 10% FBS (Situofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30256.01) in a 37°C, 5% CO2 cell culture incubator until the confluence reached about 80%.

[0057] 2.2 Take 144 μL of 1 mg / mL PEI (Merck Biotechnology Co., Ltd., Catalog No.: 937762) and add it to 1 mL OPTI-MEM (Thermo Fisher Scientific Biotechnology Co., Ltd., Catalog No.: 11058021), mix well, and let stand at room temperature for 20 min.

[0058] 2.3 Add 16 μg of main plasmid pMSGV-CD38 or pMSGV-CD38-IL15, 12 μg of pMDLG-pRRE-Kana, 4 μg of pMD2G-Kana and 4 μg of pRSV-REV-Kana auxiliary plasmid into 1 mL OPTI-MEM and mix well.

[0059] 2.4 Add the PEI and 1 mL OPTI-MEM mixed solution prepared in step 2.2 to the solution prepared in step 2.3, mix well, and let stand at room temperature for 20 min.

[0060] 2.5 Take 8 mL of DMEM + 10% FBS complete medium and add it to the solution in step 2.4 and mix well.

[0061] 2.6 Aspirate the medium in the 293T cell culture flask and add the medium prepared in step 2.5.

[0062] 2. After 748 hours, the cell culture medium supernatant was filtered using a vacuum filter with a pore size of 0.45 μm (Corning (Shanghai) Co., Ltd., Catalog No.: 430770) to obtain a supernatant containing the recombinant lentivirus. The supernatant containing the recombinant lentivirus was placed in a 250 mL collection bottle sterilized by high pressure and placed in a refrigerator at 2-8°C for 2 hours.

[0063] 2.8 Centrifuge the supernatant in step 2.7 at 2000×g and 4°C for 2h. After centrifugation, discard the supernatant, use 250μL of Lymphocyte Serum-Free Medium (Corning Life Sciences (Wujiang) Co., Ltd., Cat. No.: 88-581-CM) per bottle to resuspend the recombinant lentivirus at the bottom, aliquot the virus, and sample 25μL in a 1.5mL centrifuge tube and place it in a 4°C refrigerator for the virus infection titer test in the following method 3. The aliquoted virus is frozen in a -80°C refrigerator.

[0064] 3. Lentiviral infection titer detection

[0065] The operating methods for CD38-CAR and CD38-IL15-CAR lentiviral infection titer detection are the same.

[0066] The specific steps are as follows:

[0067] 3.1 Take Jurkat cells (American Biological Standard Collection Center (ATCC), catalog number: TIB-152) after 2 generations of resuscitation culture, and centrifuge at 300×g at room temperature for 10 minutes.

[0068] 3.2 Discard the culture supernatant and resuspend the cells in RPMI 1640 medium (Situofan Biotechnology (Hangzhou) Co., Ltd., Catalog No.: SH30809-01) to a density of 2×10 6 cells / mL, and add the transfection reagent Protamine (Sigma-Aldrich, Catalog No.: 53597-25-4, prepared stock concentration: 10 mg / mL) to the cell suspension at a ratio of 2 μL / mL and mix well.

[0069] 3.3 Take a 48-well plate and inoculate 100 μL of Jurkat cells (2×10 5 Three rows were inoculated with a suspension of 10 cells / well, and three sets of repeated experiments were performed. In the wells adjacent to the outer circle of inoculated cells, 1 mL of PBS was added.

[0070] 3.4 Take a 96-well plate and add 133 μL, 120 μL, 120 μL, 120 μL, 120 μL, 120 μL, 120 μL of serum-free RPMI 1640 medium to the first well of the first row of the 96-well plate. Take 7 μL of the virus solution collected and sampled in method 2, add it to the first well, mix well, and obtain virus dilution 1. Take 120 μL of virus dilution 1, add it to the second well, mix well, and obtain virus dilution 2. Take 120 μL of virus dilution 2, add it to the third well, mix well, and obtain virus dilution 3. Take 120 μL of virus dilution 3, add it to the fourth well, mix well, and obtain virus dilution 4. Take 120 μL of virus dilution 4, add it to the fifth well, mix well, and obtain virus dilution 5. Take 120 μL of virus dilution 5, add it to the sixth well, mix well, and obtain virus dilution 6. Thus, the first group of gradient dilutions containing different amounts of virus were obtained.

[0071] 3.5 According to the operation method in step 3.4 above, dilute the virus solution in wells 1 to 6 in the second row and wells 1 to 6 in the third row of the 96-well plate, respectively, to obtain the second and third groups of virus gradient dilution solutions.

[0072] 3.6 Take 100 μL of virus dilutions 2, 3, 4, 5 and 6 from the first set of virus gradient dilutions and add them to wells 3 to 7 of the second row of the 48-well plate inoculated with Jurkat cells in step 3.3. Add 100 μL of serum-free RPMI 1640 medium to well 2 as control 1. Mix wells thoroughly.

[0073] 3.7 According to the operation method of step 3.6 above, add the second and third groups of virus gradient dilutions to the third and fourth rows of wells 3 to 7 corresponding to the 48-well plate inoculated with Jurkat cells in step 3.3. Add 100 μL of serum-free RPMI 1640 medium to the second well of the third and fourth rows as control 2 and control 3. Mix each well thoroughly and culture in a cell culture incubator at 37°C and 5% CO2.

[0074] 3. After 86 hours, add 800 μL of RPMI 1640 + 10% FBS complete culture medium and culture in a cell culture incubator at 37° C. and 5% CO 2 .

[0075] 3. After 924 hours, carefully aspirate 500 μL of culture medium from the surface of each well (Jurkat cells have adhered to the bottom of the well plate), and add 500 μL / well of RPMI 1640 + 10% FBS complete culture medium. Continue to culture in a cell culture incubator at 37°C and 5% CO2.

[0076] 3. After 1048 hours, take the cells from each well of the three experimental groups and put them into 1.5 mL centrifuge tubes. The cells from the three control wells were divided into two parts, centrifuged at 300×g, room temperature for 30 minutes, and discarded the supernatant.

[0077] 3.11 Add 1 mL of PBS to each tube to resuspend the cells, centrifuge at 300 × g at room temperature for 3 min, discard the excess supernatant, and keep only 20-30 μL of cell suspension in each tube for incubation in the following experimental steps.

[0078] 3.12 One of the samples of the three groups of control cells was recorded as control-blank and was not incubated with FITC-CD38 antigen (Beijing Sino Biological Technology Co., Ltd., Catalog No.: 10818-H08H-F). 2 μL of FITC-CD38 antigen was added to each tube of the remaining cell samples, mixed thoroughly, and incubated at room temperature for 30 minutes.

[0079] 3.13 Add 1 mL of PBS to the cell sample incubated with CD38 antigen to resuspend the cells, centrifuge at 300×g at room temperature for 3 min, and discard the supernatant.

[0080] 3.14 Add 200 μL PBS to each tube of cell sample and resuspend it. Detect the positive rate of CD38-CAR by flow cytometry.

[0081] 4. T cell isolation and activation

[0082] 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 700×g for 20 minutes, and aspirate the plasma for later use.

[0083] 4.2 During the centrifugation period, follow the Dynabeads TM RetroNectin coating solution was prepared as described in the instruction manual of Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific, Cat. No. 11452D), added to a 6-well plate at 1.5 mL / well, and allowed to stand at room temperature for 5 h.

[0084] 4.3 Add PBS to the blood cells in step 4.1 at a ratio of 1:1 and mix well, then add lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., catalog number: LTS1077) at a ratio of 1:2. Centrifuge at 700×g, room temperature for 20 minutes.

[0085] 4.4 Pipette the white film layer above the separation solution into a new centrifuge tube, add PBS to wash the white film layer, centrifuge at 500×g, room temperature for 10 minutes, discard the supernatant, and PBMC (Peripheral Blood Mononuclear Cell) is obtained.

[0086] 4.5 T lymphocytes were isolated from PBMCs according to the instructions of CD3 Microbeads (Human) (Miltenyi Biotec, catalog number: 130-050-101) magnetic beads, and the cells were counted.

[0087] 4.6 The T cells obtained in step 4.5 were centrifuged at 4°C, 500×g for 10 min, and the supernatant was discarded. X-VIVO containing 40 IU / mL human interleukin 2 (Interleukin 2, IL-2) (Suzhou Jinan 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), and the cell concentration was adjusted to 1×10 6 cells / mL.

[0088] 4.7 Discard the RetroNectin coating solution in the 6-well plate from step 4.2 and plate the T cells resuspended in step 4.6 at a concentration of 2 × 10 6 Cells / well were inoculated in a 6-well cell culture plate, with a total of 9 wells inoculated for the preparation of CD38-CAR-T cells and CD38-IL15-CAR-T cells in method 5. The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24-48 hours for activation to obtain unmodified T cells.

[0089] 5. Preparation of CD38-CAR-T cells and CD38-IL15-CAR-T cells

[0090] The operation methods of CD38-CAR-T cells and CD38-IL15-CAR-T cells are the same. The specific operation steps are as follows:

[0091] 5.1 Culture for 24-48 hours and observe the state of activated T cells in method 4. T cells grow adherently and have a fusion degree of 20-50%. Virus infection of T cells is possible.

[0092] 5.2 Carefully discard 1 mL of culture medium from the top of each well and add X-VIVO containing the transfection reagent Protamine (20 μg / mL) TM 15 Add 1 mL of serum-free medium to each well and mix by gently pipetting.

[0093] 5.3 Calculate the amount of CD38-CAR or CD38-IL15-CAR virus added to each well according to MOI=3 (the amount of virus added to each well = MOI×2×10 6 / virus titer). The CD38-CAR and CD38-IL15-CAR virus-infected T cell experiments were set up in triplicate, and one untransformed T cell that was not infected with the virus was set up in each group of replicates as a control.

[0094] 5.4 After mixing, centrifuge at 1000×g at room temperature for 30 min, and then culture in a cell culture incubator at 37°C and 5% CO2.

[0095] 5.5 Samples were taken on Day 7 and Day 9 after transfection, and the expression of CD38-CAR in untransformed T cells, CD38-CAR-T cells, and CD38-IL15-CAR-T cells was detected by flow cytometry.

[0096] 6. In vitro proliferation experiment of CD38-IL15-CAR-T cells

[0097] 6.1 The CD38-IL15-CAR-T cells transfected in method 5 were used in this experiment, and the untransformed T cells (obtained in step 4.7 of method 4) and CD38-CAR-T cells (obtained in method 5) were used as controls. The time of virus transfection was recorded as Day 0, and the infection was terminated on Day 3. The cells were counted for the first time, and then X-VIVO was used to TM 15% serum-free medium to recover T cells.

[0098] 6.2 T cells were counted before each culture medium change. The proliferation of CD38-CAR-T cells and CD38-IL15-CAR-T cells was counted using unmodified T cells as the standard.

[0099] 7. T cell killing experiment in vitro

[0100] 7.1 GFP protein was introduced into MM.1S cells (Cell Bank of Chinese Academy of Sciences, Catalog No.: SCSP-5017) to transform them into MM.1S-GFP cells as target cells. Unmodified T cells (obtained in step 4.7 of method 4), CD38-CAR-T cells (obtained in method 5) and CD38-IL15-CAR-T cells (obtained in method 5) were used as effector cells and controls for killing activity determination.

[0101] 7.2 Before the killing experiment, samples of MM.1S-GFP cells, unmodified T cells, CD38-CAR-T cells and CD38-IL15-CAR-T cells were taken for flow cytometry detection, the GFP positivity rate of MM.1S-GFP cells was measured, and the CD3-CAR positivity rate of unmodified T cells, CD38-CAR-T cells and CD38-IL15-CAR-T cells was measured.

[0102] 7.3 Using X-VIVO TM 15 Serum-free medium was used to adjust the concentrations of MM.1S-GFP cells, CD38-IL15-CAR-T cells, CD38-CAR-T cells, and unmodified T cells to 1×10 6 / mL.

[0103] 7.4 Two groups of experiments were set up with effector cells and MM.1S-GFP target cells with effector-target ratios (E:T) of 0.2:1 and 0.5:1, respectively. Each group of experiments was conducted in 24-well plates with no T cells added (as a control), unmodified T cells, CD38-CAR-T cells, and CD38-IL15-CAR-T cells, and each group of experiments was repeated 3 times.

[0104] 7.5MM.1S-GFP target cells are 5×10 5 / well (500 μL), no T cells were added to the experimental wells without T cells, and for the experimental wells without modified T cells, CD38-CAR-T cells, CD38-IL15-CAR-T cells, etc., the required amount of T cells was calculated according to the effector-target ratio (E:T=0.2:1, E:T=0.5:1) set in step 7.4 of this method 7 and the CD38-CAR positive rate detected in step 7.2 of this method 7 (required volume of T cells = E / T×5×10 5 / (1×10 6 ×T cell CD38-CAR positive rate)) and added to 24-well culture plates to fill each well with X-VIVO TM 15 Serum-free medium to 1000 μL, mix well, and place in a 5% CO2, 37°C incubator for culture.

[0105] 7.6After 24 hours, measure the volume of the remaining culture medium in each well, mix well and count the cells in each well. At the same time, take samples for flow cytometry to detect the positive rate of MM.1S-GFP target cells, and calculate the killing rate of MM.1S-GFP target cells killed by T cells in two groups of experiments and three repeated experiments, and finally calculate the average killing rate of the three repeated experiments.

[0106] Results and Discussion

[0107] According to the above method, a total of 10 chimeric antigen T cells targeting CD38 and secreting IL-15, namely CD38-IL15-CAR-T cells, and 10 chimeric antigen T cells targeting CD38, namely CD38-CAR-T cells, were constructed according to the different anti-CD38 antibody region sequences.

[0108] The experimental results showed that the positive rates of CD38-CAR of these 10 types of CD38-CAR-T cells and 10 types of CD38-IL15-CAR-T cells were all higher than 90%. Figure 2 The positive rate of CD38-CAR in CD38-CAR-T cells and CD38-IL15-CAR-T cells composed of anti-CD38 antibody 1 (SEQ ID NO: 2) is shown.

[0109] In the in vitro proliferation experiment, IL-15 can indeed promote the proliferation of CD38-IL15-CAR-T cells. Figure 3 As shown, on Day 25, the CD38-IL15-CAR-T cells composed of anti-CD38 antibody 1 (SEQ ID NO: 2) still maintained a good proliferation state, which was significantly better than CD38-CAR-T cells.

[0110] In the in vitro killing experiment of CAR-T cells on target cells, CD38-CAR-T cells and CD38-IL15-CAR-T cells also showed similar killing effects. Figure 4 As shown, the killing rate of CD38-CAR-T cells and CD38-IL15-CAR-T composed of anti-CD38 antibody 1 (SEQ ID NO: 2) exceeded 80% when the effector-target ratio was 0.2:1, and exceeded 95% when the effector-target ratio was 0.5:1.

[0111] The above shows that the CD38-IL15-CAR-T cells of the present invention maintain a proliferation state superior to that of CD38-CAR-T cells while maintaining a similar killing effect as CD38-CAR-T cells.

[0112] 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.

[0113] References

[0114] [1]Guo,Y.,Feng,K.,Tong,C.,Jia,H.,Liu,Y.,Wang,Y.,Han,W.(2020).Efficiencyand side effects of anti-CD38-CAR T cells in an adult patient with relapsedB-ALL after failure of bi-specific CD19 / CD22 CAR T cell treatment.Cellular&Molecular Immunology,17(4),430-432.doi:10.1038 / s41423-019-0355-5

[0115] [2]Liao,C.,Wang,Y.,Huang,Y.,Duan,Y.,Liang,Y.,Chen,J.,Sun,J.(2023).CD38-Specific CAR Integratedinto CD38 Locus Driven by Different PromotersCauses Distinct Antitumor Activities of T and NK Cells.Advanced Science,10(27).doi:10.1002 / advs.202207394

[0116] [3]Padala,S.A.,Barsouk,A.,Barsouk,A.,Rawla,P.,Vakiti,A.,Kolhe,R.,Ajebo,G.H.(2021).Epidemiology,Staging,and Management of MultipleMyeloma.Medical Sciences,9(1).doi:10.3390 / medsci9010003

[0117] [4]Takasawa,S.(2022).CD38–Cyclic ADP-Ribose Signal SysteminPhysiology,Biochemistry,and Pathophysiology.International Journal ofMolecular Sciences,23(8).doi:10.3390 / ijms23084306

[0118] [5]Veliz,K.,Shen,F.,Shestova,O.,Shestov,M.,Shestov,A.,Sleiman,S.,.Gill,S.(2024).Deletion of CD38 enhances CD19 chimeric antigen receptor Tcell function.Molecular Therapy:Oncology,32(2).doi:10.1016 / j.omton.2024.200819

[0119] [6]Waldmann,T.A.,Dubois,S.,Miljkovic,M.D.,&Conlon,K.C.(2020).IL-15inthe Combination Immunotherapy of Cancer.Frontiers in Immunology,11.doi:10.3389 / fimmu.2020.00868。

Claims

1. A chimeric antigen receptor CAR targeting CD38 and comprising IL-15, characterized in that: The chimeric antigen receptor comprises the following elements in order: 1) Signal peptide 1; 2) anti-CD38 antibody region; 3) IL-15R domain; 4) Hinge region; 5) transmembrane region; 6) Intracellular signal transduction region; 7) Intracellular signaling co-stimulatory region; 8) Intracellular signal stimulation area; 9) 2A sequence; 10) signal peptide 2; and 11) IL-15 sequence, Wherein, the amino acid sequence of the anti-CD38 antibody region is shown in any one of SEQ ID NOs: 2-11.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CAR targeting CD38 and comprising IL-15 according to 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. A method for preparing a recombinant lentivirus, 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 target CD38 and secrete IL-15, characterized in that: The method comprises infecting T cells with the recombinant lentivirus of claim 4.

7. CAR-T cells targeting CD38 and secreting IL-15, 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 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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