E-CAR-NK cell for treating systemic lupus erythematosus and application thereof

Abstract

The invention discloses an E-CAR-NK cell for treating systemic lupus erythematosus and application of the E-CAR-NK cell, and belongs to the technical field of genetic engineering.A CD19 and BCMA double-target E-CAR is adopted for modifying an NK cell, and the E-CAR-NK cell is obtained; the CD19 and BCMA double-target E-CAR is composed of the following modules: a CD8 signal peptide, a CD19 scFv, a Linker, a BCMA scFv, a CD8 Hinge region, an NKG2D transmembrane region, a CD3 epsilon signal transduction region, a DAP12 costimulatory region, a 2B4 costimulatory region, and a CD3 zeta signal transduction region. Compared with a common CAR-NK cell, the E-CAR-NK cell disclosed by the invention has higher antigen sensitivity and better continuous killing ability, so that the treatment effect of SLE is improved, and the E-CAR-NK cell has lower side effects and higher safety.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to an E-CAR-NK cell for treating systemic lupus erythematosus and its application. Background Art

[0002] Autoimmune diseases are a general term for a class of diseases caused by abnormal functions of the body's immune system, which mistakenly regard normal tissues of the body as foreign threats and launch attacks, resulting in tissue damage, including systemic lupus erythematosus (SLE), scleroderma, pemphigus, multiple sclerosis, and so on. Globally, the incidence of these diseases is estimated to be between 5% and 8%, and they have become the third largest chronic health threat after tumors and cardiovascular diseases.

[0003] Systemic lupus erythematosus (SLE), as a chronic autoimmune disease, can affect multiple organ systems. Its clinical manifestations and treatment effects are heterogeneous, and its pathogenesis is complex, involving the combined effects of multiple factors such as genetics, environment, ecology, and epigenetics, leading to abnormal immune responses and thus triggering autoimmune attacks. At present, SLE cannot be cured, and the main treatment goals are to control symptoms, reduce disease activity, and prevent complications. Commonly used treatment methods include non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, immunosuppressants, and biological agents. Although the above treatment methods can relieve the symptoms of SLE to a certain extent, they are often accompanied by problems such as more side effects and limited efficacy.

[0004] Recently, CAR-T therapy has also made great progress in the treatment of autoimmune diseases such as SLE, scleroderma, and multiple sclerosis, providing new treatment approaches for these difficult-to-cure diseases. However, clinical applications still face many challenges. CAR-T cell therapy is limited in use due to some of its toxic side effects. The most common toxic side effects include cytokine release syndrome (CRS), neurotoxicity, etc. Although the incidence of CRS and neurotoxicity in SLE patients is relatively low, it is still necessary to be vigilant. In addition, due to the existence of human leukocyte antigen (HLA) restrictions, allogeneic CAR-T cells may also cause graft-versus-host disease (GVHD); using autologous T cells to prepare autologous CAR-T cells results in insufficient production in many patients undergoing clinical enrollment evaluation and ultimately unable to proceed. In addition, due to the relatively difficult preparation and high cost of CAR-T cells, it is difficult to popularize.

[0005] Natural killer (NK) cells are an important type of innate immune cells with strong anti-tumor ability, and they do not cause strong cytokine release syndrome (CRS) or graft-versus-host disease (GVHD). Therefore, introducing antigen-specific chimeric antigen receptor (CAR) genes into NK cells to develop CAR-NK cell therapy has advantages. In addition, NK cells can be obtained from healthy donors unrelated to the patient or other sources (such as induced pluripotent stem cells, iPSCs). CAR-NK cell products derived from induced pluripotent stem cells eliminate the collection and patient-specific manufacturing processes and can be immediately used by a large number of patients.

[0006] Currently, in the treatment of systemic lupus erythematosus (SLE), most clinical studies target CD19, which can successfully eliminate dysfunctional B cells and reconstruct the immune system, thereby effectively controlling SLE. Considering that the severity of systemic lupus erythematosus is related to the increased expression of the BCMA surface antigen on long-lived plasma cells, it may be a potential therapeutic target. Different from the CD19+ cell population, CD19-long-lived plasma cells are present in the bone marrow, and anti-double-stranded DNA (dsDNA) is produced by the CD19+ or CD19-cell population. Can the therapeutic effect of systemic lupus erythematosus be improved by targeting CD19 and BCMA simultaneously? Chinese Patent CN115715298A discloses a chimeric antigen receptor CAR or CAR construct targeting BCMA and CD19 and its applications. This patent provides a chimeric antigen receptor (CAR) or CAR construct containing antibodies against BCMA and CD19, as well as nucleic acid molecules encoding the CAR or CAR construct, engineered immune cells, and methods for preparing the immune cells. It is used for the prevention and / or treatment of B cell-related conditions (such as B cell- and plasma cell-related malignancies or autoimmune diseases, such as systemic lupus erythematosus, etc.), which can effectively avoid target escape and prevent the recurrence of multiple myeloma. However, the dual-target cell therapy targeting BCMA and CD19 in this patent has poor efficacy in treating SLE and also has the risk of easily causing CRS and GVHD.

[0007] Chinese Patent CN117186229B discloses an anti-human BCMA nanobody with a long CDR3 sequence and CAR-T and its applications. This patent specifically relates to an anti-human BCMA nanobody with a CDR3 sequence of a specific length and CAR-T and its applications. This patent is a single-target CAR-T cell targeting the BCMA target. However, the single-target CAR-T cell therapy cannot avoid the problem of antigen escape, has poor therapeutic effects, and also has the risk of causing CRS and GVHD.

[0008] Although CAR-T cell therapy has shown great potential in the treatment of SLE, its use is limited due to the toxic side effects it causes. Although the incidence of CRS in SLE patients is relatively low, it is still necessary to be vigilant. In addition, there are problems such as allogeneic CAR-T cells may cause graft-versus-host disease (GVHD). Moreover, the preparation of autologous CAR-T cells takes a long time, is difficult and costly, making it difficult to popularize. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the present invention provides an E-CAR-NK cell for treating systemic lupus erythematosus and its application. Compared with ordinary CAR-NK cells, the E-CAR-NK cells of the present invention have higher antigen sensitivity and better continuous killing ability, thereby improving the treatment effect of SLE, and have lower side effects and stronger safety.

[0010] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: An E-CAR-NK cell for treating systemic lupus erythematosus, which uses CD19 and BCMA dual-target E-CAR to modify NK cells to obtain E-CAR-NK cells; The CD19 and BCMA dual-target E-CAR is composed of the following modules: CD8 signal peptide, CD19 scFv, Linker, BCMA scFv, CD8 Hinge region, NKG2D transmembrane region, CD3ε signal transduction region, DAP12 costimulatory region, 2B4 costimulatory region, CD3ζ signal transduction region; The nucleotide artificial sequence of the CD19 scFv is as shown in SEQ ID NO.2 in the sequence listing, the nucleotide artificial sequence of the BCMA scFv is as shown in SEQ ID NO.4 in the sequence listing, and the nucleotide artificial sequence of the CD3ε signal transduction region is as shown in SEQ ID NO.7 in the sequence listing; The nucleotide artificial sequence of the CD8 signal peptide is as shown in SEQ ID NO.1 in the sequence listing, the nucleotide artificial sequence of the Linker is as shown in SEQ ID NO.3 in the sequence listing, the nucleotide artificial sequence of the CD8 Hinge region is as shown in SEQ ID NO.5 in the sequence listing, the nucleotide artificial sequence of the NKG2D transmembrane region is as shown in SEQ ID NO.6 in the sequence listing, the nucleotide artificial sequence of the DAP12 costimulatory region is as shown in SEQ ID NO.8 in the sequence listing, the nucleotide artificial sequence of the 2B4 costimulatory region is as shown in SEQ ID NO.9 in the sequence listing, and the nucleotide artificial sequence of the CD3ζ signal transduction region is as shown in SEQ ID NO.10 in the sequence listing.

[0011] Use of the described E-CAR-NK cells in the preparation of a medicament for treating systemic lupus erythematosus.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) After introducing the CD3ε element into the intracellular segment of the CAR molecule in the present invention, this not only enhances the signal function of the E-CAR molecule itself, but also can recruit co-stimulatory molecules such as CD2, making better use of the endogenous co-stimulatory signal, so that the E-CAR-NK cells have higher antigen sensitivity and better continuous killing ability than ordinary CAR-NK cells. In the in vivo efficacy experiment of SLE, compared with ordinary CAR-NK (experimental group E), the E-CAR-NK cells of the present invention (experimental group A) can further reduce the urinary protein, inflammatory cytokines, and the contents of anti-dsDNA antibody and anti-Sm antibody in the serum of SLE mice, improving the treatment effect of SLE; (2) The treatment effect of the dual-target CD19-BCMA-E-CAR-NK cells (experimental group A) of the present invention on SLE is better than that of the single-target CD19-E-CAR-NK cells (experimental group C) and BCMA-E-CAR-NK cells (experimental group D); in addition, compared with the dual-target CD19-BCMA-E-CAR-2-NK cells (experimental group B, where the nucleic acid sequences of CD19 and BCMA are provided by patent CN115715298A), the CD19-BCMA-E-CAR-NK cells (experimental group A) of the present invention can better reduce various indicators of SLE mice (including the expression of urinary protein, cytokines, and the contents of anti-dsDNA antibody and anti-Sm antibody), and have a better treatment effect on systemic lupus erythematosus; (3) Compared with the natural T cell antigen receptor (TCR), the antigen sensitivity of CAR is poor, and its cooperation with other signal molecules is also poor. These disadvantages are because CAR cannot form a mature immune synapse like TCR to integrate each signal pathway. These signal disadvantages will lead to the inability of CAR-T cells to eliminate target cells with low antigen expression, and it is easy to undergo functional exhaustion and difficult to work for a long time. Therefore, optimizing the signal transduction function of CAR is the key to breaking through the current clinical bottleneck of CAR-T cell therapy. After introducing the CD3ε element into the intracellular segment of the CAR molecule in the present invention, the signal function of the E-CAR molecule itself is enhanced, and co-stimulatory molecules such as CD2 can also be recruited, making better use of the endogenous co-stimulatory signal, so that the E-CAR-NK cells have higher antigen sensitivity and better continuous killing ability than ordinary CAR-NK cells, thereby improving the treatment effect of SLE; (4) Compared with the risks of some CRS and GVHD caused by CAR-T cell therapy, the present invention selects different types of immune cells, namely NK cells. Manufacturing therapeutic E-CAR-NK cells will not cause strong CRS, nor will it cause graft-versus-host disease (GVHD), with lower side effects and stronger safety. Moreover, the NK cells in the present invention are induced from induced pluripotent stem cells. Therefore, the E-CAR-NK cell product provided by the present invention omits the collection and manufacturing process for specific patients, can be mass-produced, and can be immediately used by a large number of patients. Description of the Drawings

[0013] Figure 1 It is the design diagram of the fusion gene fragment of the CD19 and BCMA dual-target E-CAR expression vector in Example 1; Figure 2 It is the flow cytometry diagram of the expression rate of cell surface marker CD3-CD16+CD56+ in NK cells in Example 3; Figure 3 It is the flow cytometry diagram of the infection rate of CD19-BCMA-E-CAR recombinant lentivirus on NK cells in Example 3; Figure 4 It is the diagram of the expression levels of IFN-γ and TNF-α inflammatory cytokines in the mouse sera of the control group, CD19-BCMA-E-CAR-NK cell group, CD19-BCMA-E-CAR-2-NK cell group, CD19-E-CAR-NK cell group, BCMA-E-CAR-NK cell group, and CD19-BCMA-CAR-NK cell group in Example 4; Figure 5 It is the diagram of the expression levels of anti-dsDNA antibodies in the mice of the control group, CD19-BCMA-E-CAR-NK cell group, CD19-BCMA-E-CAR-2-NK cell group, CD19-E-CAR-NK cell group, BCMA-E-CAR-NK cell group, and CD19-BCMA-CAR-NK cell group in Example 4; Figure 6 It is the diagram of the expression levels of anti-Sm antibodies in the mice of the control group, CD19-BCMA-E-CAR-NK cell group, CD19-BCMA-E-CAR-2-NK cell group, CD19-E-CAR-NK cell group, BCMA-E-CAR-NK cell group, and CD19-BCMA-CAR-NK cell group. Detailed Embodiments

[0014] In order to have a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed embodiments of the present invention are now described.

[0015] Example 1 Construction of CD19 and BCMA Dual-Target E-CAR Expression Vector In the present invention, the CD3ε element in the T cell antigen receptor (TCR) is introduced into the intracellular segment of the CAR molecule (abbreviated as E-CAR), and the nucleic acid sequence of CD3ε is optimized to enhance the signal function of the E-CAR molecule itself, making it have higher antigen sensitivity and persistence in vivo.

[0016] The connection schematic diagram of each module of CD19 and BCMA dual-target E-CAR is as Figure 1 shown. The modules and nucleotide artificial sequences of CD19 and BCMA dual-target E-CAR are as follows: (1) CD8 signal peptide (its nucleotide artificial sequence is shown as SEQ ID NO.1 in the sequence listing); (2) CD19 scFv (its nucleotide artificial sequence is shown as SEQ ID NO.2 in the sequence listing); (3) Linker (its nucleotide artificial sequence is shown as SEQ ID NO.3 in the sequence listing); (4) BCMA scFv (its nucleotide artificial sequence is shown as SEQ ID NO.4 in the sequence listing); (5) CD8 Hinge region (its nucleotide artificial sequence is shown as SEQ ID NO.5 in the sequence listing); (6) NKG2D transmembrane region (its nucleotide artificial sequence is shown as SEQ ID NO.6 in the sequence listing); (7) CD3ε signal transduction region (its nucleotide artificial sequence is shown as SEQ ID NO.7 in the sequence listing); (8) DAP12 costimulatory region (its nucleotide artificial sequence is shown as SEQ ID NO.8 in the sequence listing); (9) 2B4 costimulatory region (its nucleotide artificial sequence is shown as SEQ ID NO.9 in the sequence listing); (10) CD3ζ signal transduction region (its nucleotide artificial sequence is shown as SEQ ID NO.10 in the sequence listing); Construction of recombinant plasmid: According to Figure 1 Connect the above sequences in turn, entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize its entire expression cassette and insert it onto the standard vector pUC57 to obtain pUC-CD19-BCMA-CAR. Transform it into E.coli (DH5α), and after correct sequencing, extract the recombinant plasmid from the positive clone and name it CD19-BCMA-E-CAR.

[0017] Replace the hCD19 scFv (the artificial nucleotide sequence of which is shown as SEQ ID NO.11 in the sequence listing) and hBCMA scFv (the artificial nucleotide sequence of which is shown as SEQ ID NO.12 in the sequence listing) described in TanCAR 08 in the chimeric antigen receptor CAR or CAR construct targeting BCMA and CD19 and its application in Chinese Patent CN115715298A with the CD19 scFv (the artificial nucleotide sequence of which is shown as SEQ ID NO.2 in the sequence listing) and BCMA scFv (the artificial nucleotide sequence of which is shown as SEQ ID NO.4 in the sequence listing) in this technical solution respectively; specifically, connect the artificial nucleotide sequences of SEQ ID NO.1, SEQ ID NO.11, SEQ ID NO.3, SEQ ID NO.12, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 in sequence, and construct a recombinant plasmid according to the above method, named CD19-BCMA-E-CAR-2.

[0018] Omit SEQ ID NO.3 and SEQ ID NO.4, connect the artificial nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 in sequence, and construct a recombinant plasmid according to the above method, named CD19-E-CAR.

[0019] Omit SEQ ID NO.2 and SEQ ID NO.3, connect the artificial nucleotide sequences of SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 in sequence, and construct a recombinant plasmid according to the above method, named BCMA-E-CAR.

[0020] Omit SEQ ID NO.7, connect the artificial nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 in sequence, and construct a recombinant plasmid according to the above method, named CD19-BCMA-CAR.

[0021] The concentrations of the above recombinant plasmids were all 1.0 μg / μL.

[0022] Example 2 Lentivirus packaging and titer detection (1) Inoculate the lentivirus packaging cell line 293T cells into 10 mL of DMEM medium containing 10 vol% FBS, and culture them in a culture dish at 37 °C and 5% CO2. When the cell attachment rate reaches 70%, prepare for transfection.

[0023] (2) Take a sterile 5 mL centrifuge tube and prepare the reaction system according to the following components: Serum-free DMEM: 3 mL; CD19-BCMA-E-CAR recombinant plasmid: 10 μg; GM easyTM Lentiviral Mix (lentivirus packaging auxiliary plasmid mixture): 10 μL (10 μg); HG Transgene TM Reagent (high-efficiency transfection reagent): 60 μL. After mixing, let it stand at room temperature for 20 min, and then evenly add it dropwise to the culture dish containing 293T cells obtained in step (1), and place it in a 37 °C and 5% CO2 incubator for transfection culture.

[0024] (3) After 12 h of transfection culture, carefully aspirate the cell culture medium and discard it into a waste liquid cup containing disinfectant. Then add 15 mL of DMEM medium containing 10 vol% serum to the culture dish, and continue to culture for 48 h. Then aspirate the cell supernatant into a 50 mL centrifuge tube, centrifuge at 4 °C and 500 g for 5 min. After filtering the supernatant with a 0.45 μm filter, transfer it to a new centrifuge tube to obtain the virus solution.

[0025] (4) Concentrate the above virus solution to obtain CD19-BCMA-E-CAR recombinant lentivirus. After detecting the titer, store it in a -80 °C low-temperature refrigerator for standby.

[0026] The titer of CD19-BCMA-E-CAR recombinant lentivirus is 5.81×10 7 pfu / mL.

[0027] Package the recombinant plasmids CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR into lentiviruses according to the above method respectively, and obtain CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR recombinant lentiviruses respectively. The detected titers are 6.22×10 7 pfu / mL, 5.96×10 7 pfu / mL, 6.32×10 7 pfu / mL, and 6.56×10 7pfu / mL; Adjust the titers of the above recombinant lentiviruses to 5×10 7 pfu / mL.

[0028] Example 3 Preparation of CAR-NK Cells 1. Inducing NK cells from human induced pluripotent stem cells (hiPSCs) According to the methods of Example 1, Example 2 and Example 3 in Chinese Patent CN115896019B, a method for inducing differentiation of induced pluripotent stem cells into NK cells, first induce human induced pluripotent stem cells (hiPSCs) into CD34+ hematopoietic stem cells; then further induce the differentiation of CD34+ hematopoietic stem cells into NK cells.

[0029] 2. Expansion of NK cells When expanding NK cells, the cell culture flask is coated with anti-CD3 monoclonal antibody (OKT3). Before use, wash it twice with NK cell expansion medium (purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.), and inoculate the cells into the culture flask at a density of 2×10 4 cells / mL, add NK cell expansion medium containing factors, and culture it in an incubator at 37°C, low oxygen (5% O2), and 5% CO2 for 2 weeks. Replace half of the NK cell expansion medium containing factors every three days. Collect the expanded NK cells, and use flow cytometry to detect the expression rate of CD3-CD16+CD56+ in NK cells (CD3-FITC, CD16 / CD56-PE antibodies are purchased from BECKMAN company, A07735). As Figure 2 shown, the expression rate of CD3-CD16+CD56+ in NK cells is 81.4%.

[0030] 3. Lentiviral infection of NK cells Take out the CD19-BCMA-E-CAR recombinant lentivirus prepared in Example 2 from -80°C, thaw it and resuspend the NK cells to obtain a cell suspension. Add the cell suspension to a 6-well plate, 1 mL per well, so that the ratio of the number of virus particles to the number of NK cells is 50:1. After culturing in an incubator at 37°C and 5% CO2 for 6 h, add 1 mL of NK cell medium to each well and continue culturing. After a total infection time of 1 week, the obtained CD19-BCMA-E-CAR-NK cells are detected, and the expression of chimeric antigen receptor is detected by flow cytometry.

[0031] Figure 3 The expression of the CD19-BCMA-E-CAR vector constructed by the present invention on the surface of NK cells detected by flow cytometry shows that the transfection efficiency of the CD19-BCMA-E-CAR-NK cells constructed by the present invention is 38.6%.

[0032] CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR recombinant lentiviruses were used to infect NK cells according to the above methods, respectively, to obtain CD19-BCMA-E-CAR-2-NK cells, CD19-E-CAR-NK cells, BCMA-E-CAR-NK cells, and CD19-BCMA-CAR-NK cells. The transfection efficiencies were 39.5%, 39.2%, 38.6%, and 40.5%, respectively. The specific results are shown in Table 1.

[0033] Table 1 CAR transfection efficiency

[0034] Example 4 In vivo efficacy experiment of CD19 and BCMA dual-target E-CAR Animal model: NZB / WF1 mice were selected, which is a commonly used systemic lupus erythematosus (SLE) model that can naturally develop clinical and pathological features similar to human SLE.

[0035] Experimental setting: 8-week-old female NZB / W F1 mice were randomly divided into six groups, with 5 mice in each group. Each mouse was given 2×10 6 cells prepared in Example 3 by tail vein injection. After 8 weeks of treatment, the mice were anesthetized with 1% sodium pentobarbital, and after successful anesthesia, the eyeballs of the mice were removed to collect blood.

[0036] The specific grouping is as follows: Control group: Infused with an equal volume of normal saline; Experimental group A: Infused with CD19-BCMA-E-CAR-NK cells; Experimental group B: Infused with CD19-BCMA-E-CAR-2-NK cells; Experimental group C: Infused with CD19-E-CAR-NK cells; Experimental group D: Infused with BCMA-E-CAR-NK cells; Experimental group E: Infused with CD19-BCMA-CAR-NK cells; Observation indicators: (1) Record the weight changes of the mice weekly and observe the diet status of the mice; As shown in Table 2, during the treatment of SLE mice, no adverse reactions such as weight loss and loss of appetite were observed; and there was no significant change in the body weight of the mice in the control group and the experimental groups.

[0037] Table 2 Body weight changes of mice (g)

[0038] (2)Urinary protein detection: Urine samples of mice were collected at the same time period every 2 weeks for urinary protein detection, and the urinary protein content was measured by a urine analyzer.

[0039] The results are shown in Table 3. Compared with the control group, the urinary protein content of the experimental group mice showed an obvious downward trend, indicating that the infusion of E-CAR-NK (CAR-NK) cells could reduce the expression level of urinary protein in SLE mice and had a certain therapeutic effect on SLE. Among them, the urinary protein content of the mice in experimental group A decreased the fastest and was also the lowest in vivo, indicating that the dual-target CD19-BCMA-E-CAR-NK cells provided by the present invention had the best therapeutic effect on SLE.

[0040] The urinary protein content of the mice in experimental group A was lower than that in experimental group E, indicating that after the introduction of the CD3ε element into the CAR molecule, the signal function of the E-CAR molecule itself was enhanced, further reducing the urinary protein content in the mice and improving the therapeutic effect on SLE; the urinary protein content of the mice in experimental group A was also lower than that in experimental groups C and D, indicating that the dual-target CD19-BCMA-E-CAR-NK cells had a better therapeutic effect on SLE than the single-target CD19-E-CAR-NK cells (experimental group C) and BCMA-E-CAR-NK cells (experimental group D).

[0041] Table 3 Mean change table of urinary protein content in mice of each group (mg / mL)

[0042] Note: "0 week" indicates the urinary protein content in the SLE mice of each group before treatment. (3)Cytokine analysis: After 8 weeks, the eyeballs of the mice were removed to collect blood, and after the blood was collected, serum was separated and extracted, and then the content changes of IFN-γ and TNF-α inflammatory cytokines in the mouse serum were measured by ELISA; Separation and extraction of mouse serum: After obtaining blood from the mice by eye blood collection method, the blood was collected into labeled EP tubes, and then placed in a centrifuge with a temperature adjusted to 4°C and centrifuged at a speed of 1000g for 10 minutes. After centrifugation, the supernatant in the EP tube was carefully taken out and stored in a newly labeled EP tube, and stored in a -80°C refrigerator for future use.

[0043] The results are shown in Table 4 and Figure 4As shown, compared with the control group, the expression levels of inflammatory cytokines (IFN-γ, TNF-α) in the experimental group of mice were significantly decreased, indicating that the infusion of E-CAR-NK (CAR-NK) cells could reduce the inflammatory response in SLE mice and had a certain therapeutic effect on SLE. Among them, the expression levels of inflammatory cytokines (IFN-γ, TNF-α) in mice in experimental group A were the lowest, indicating that the dual-target CD19-BCMA-E-CAR-NK cells (experimental group A) provided by the present invention had the best therapeutic effect on SLE.

[0044] Table 4 Mean content of cytokines in mice in each group (pg / mL)

[0045] (4)Detection of anti-double-stranded DNA (anti-dsDNA) antibody and anti-Smith (anti-Sm) antibody: After 8 weeks of treatment, the eyeballs of the mice were removed to collect blood. After collecting the blood, serum was separated and extracted, and then the expression levels of anti-dsDNA antibody and anti-Sm antibody in the mouse serum were detected by ELISA.

[0046] Anti-dsDNA antibody: Mouse anti-double-stranded DNA antibody ELISA kit (Enzyme-linked Biology; ml063514).

[0047] Anti-Sm antibody: Mouse anti-Smith antibody (Sm) ELISA kit (Shanghai Lianzu Biology; LZ-E032313).

[0048] Separation and extraction of mouse serum: After obtaining blood from the mice by eye bleeding, the blood was collected into labeled EP tubes, and then placed in a centrifuge adjusted to a temperature of 4°C and centrifuged at a speed of 1000g for 10 minutes. After centrifugation, the supernatant in the EP tube was carefully taken out and stored in a newly labeled EP tube, and stored in a -80°C refrigerator for future use.

[0049] The results are shown in Table 5 and Figure 5 、 Figure 6 As shown, compared with the control group, the expressions of anti-dsDNA antibody ( Figure 5 ), anti-Sm antibody ( Figure 6 ) in the serum of the experimental group of mice were significantly decreased, and there were significant differences. Among them, the expression levels of anti-dsDNA antibody and anti-Sm antibody in the serum of mice in experimental group A were the lowest, indicating that the dual-target CD19-BCMA-E-CAR-NK cells (experimental group A) provided by the present invention could significantly reduce the contents of anti-dsDNA antibody and anti-Sm antibody in SLE mice and had the best therapeutic effect on SLE.

[0050] Table 5 Expression of anti-dsDNA antibody and anti-Sm antibody in sera of mice in each group (OD 450nm mean value)

[0051] In summary, compared with the control group, all indicators (urinary protein, cytokines, anti-dsDNA antibody, and anti-Sm antibody) in the experimental group were significantly improved, indicating that the infusion of E-CAR-NK (CAR-NK) cells can improve the symptoms of systemic lupus erythematosus; however, all indicators in experimental group A were better than those in other experimental groups, indicating that the dual-target CD19-BCMA-E-CAR-NK cells provided by the present invention have the best therapeutic effect on systemic lupus erythematosus.

Claims

1. An E-CAR-NK cell for treating systemic lupus erythematosus, characterized in that, NK cells are modified with a dual-target E-CAR targeting CD19 and BCMA to obtain E-CAR-NK cells; The dual-target E-CAR targeting CD19 and BCMA consists of the following modules: CD8 signal peptide, CD19 scFv, Linker, BCMA scFv, CD8 Hinge region, NKG2D transmembrane region, CD3ε signaling region, DAP12 co-stimulatory region, 2B4 co-stimulatory region, CD3ζ signaling region; The nucleotide artificial sequence of the CD19 scFv is as shown in SEQ ID NO.2 in the sequence listing, the nucleotide artificial sequence of the BCMA scFv is as shown in SEQ ID NO.4 in the sequence listing, and the nucleotide artificial sequence of the CD3ε signaling region is as shown in SEQ ID NO.7 in the sequence listing.

2. The E-CAR-NK cells for treating systemic lupus erythematosus according to claim 1, wherein The nucleotide artificial sequence of the CD8 signal peptide is as shown in SEQ ID NO.1 in the sequence listing, the nucleotide artificial sequence of the Linker is as shown in SEQ ID NO.3 in the sequence listing, the nucleotide artificial sequence of the CD8 Hinge region is as shown in SEQ ID NO.5 in the sequence listing, the nucleotide artificial sequence of the NKG2D transmembrane region is as shown in SEQ ID NO.6 in the sequence listing, the nucleotide artificial sequence of the DAP12 co-stimulatory region is as shown in SEQ ID NO.8 in the sequence listing, the nucleotide artificial sequence of the 2B4 co-stimulatory region is as shown in SEQ ID NO.9 in the sequence listing, and the nucleotide artificial sequence of the CD3ζ signaling region is as shown in SEQ ID NO.10 in the sequence listing.

3. Use of the E-CAR-NK cells according to any one of claims 1-2 in the preparation of a medicament for treating systemic lupus erythematosus.

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