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

Abstract

The present invention discloses an E-CAR-NK cell for treating systemic lupus erythematosus and its application, belonging to the field of genetic engineering technology. NK cells are modified with an E-CAR with dual targets of CD19 and BCMA to obtain E-CAR-NK cells. The CD19 and BCMA dual-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 CD3ε signaling region, a DAP12 costimulatory region, a 2B4 costimulatory region, and a CD3ζ signaling region. Compared with ordinary CAR-NK cells, the E-CAR-NK cells of the present invention have higher antigen sensitivity and better sustained killing ability, thereby improving the therapeutic effect of SLE, and have lower side effects and stronger safety.

Description

Technical Field

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

[0002] Autoimmune diseases are a general term for disorders caused by abnormal immune system function, where the body mistakenly identifies healthy tissues as foreign threats and launches an attack, leading to tissue damage. These diseases include systemic lupus erythematosus (SLE), scleroderma, pemphigus, and multiple sclerosis. Globally, the prevalence of these diseases is estimated to be between 5% and 8%, making them the third most common chronic health threat after cancer and cardiovascular disease.

[0003] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect multiple organ systems. Its clinical manifestations and treatment responses are heterogeneous, and its pathogenesis is complex, involving the combined effects of multiple factors, including genetic, environmental, ecological, and epigenetic factors, which lead to abnormal immune responses and thus autoimmune attacks. Currently, SLE is incurable, and the main treatment goals are to control symptoms, reduce disease activity, and prevent complications. Commonly used treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, immunosuppressants, and biologics. While these treatments can alleviate SLE symptoms to a certain extent, they are often accompanied by significant side effects and limited efficacy.

[0004] Recently, CAR-T therapy has made significant progress in combating autoimmune diseases such as SLE, scleroderma, and multiple sclerosis, providing a new treatment approach for these intractable diseases. However, clinical application still faces numerous challenges. CAR-T cell therapy is limited by its potential side effects, the most common of which include cytokine release syndrome (CRS) and neurotoxicity. Although the incidence of CRS and neurotoxicity in SLE patients is low, they remain a concern. Furthermore, due to human leukocyte antigen (HLA) restriction, allogeneic CAR-T cells can also cause graft-versus-host disease (GVHD). The use of autologous T cells to produce autologous CAR-T cells has resulted in insufficient production for many patients undergoing clinical evaluation, ultimately leading to the discontinuation of treatment. Furthermore, the difficulty and high cost of producing CAR-T cells have hindered widespread adoption.

[0005] Natural killer (NK) cells are an important type of innate immune cell with strong anti-tumor capabilities. They do not cause severe CRS or GVHD. Therefore, the development of CAR-NK cell therapy by introducing antigen-specific CAR genes into NK cells 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). iPS-derived CAR-NK cell products eliminate the need for collection and patient-specific manufacturing processes, making them immediately available to a wide range of patients.

[0006] Currently, most clinical studies in the treatment of SLE target CD19, which can successfully eliminate dysfunctional B cells and rebuild the immune system, thereby effectively controlling SLE. Considering that the severity of systemic lupus erythematosus is associated with increased expression of the BCMA surface antigen on long-lived plasma cells, it may be a potential therapeutic target. Unlike CD19+ cell populations, CD19- long-lived plasma cells are present in the bone marrow, and anti-double-stranded DNA (dsDNA) is produced by CD19+ or CD19- cell populations. If CD19 and BCMA can be targeted simultaneously, can the treatment effect of systemic lupus erythematosus be improved?

[0007] Chinese patent CN115715298A discloses a chimeric antigen receptor CAR or CAR construct targeting BCMA and CD19 and its application. The patent provides a chimeric antigen receptor (CAR) or CAR construct containing antibodies against BCMA and CD19, as well as a nucleic acid molecule encoding the CAR or CAR construct, a modified immune cell, and a method for preparing the immune cell. It is used to prevent and / or treat B cell-related conditions (such as B cell and plasma cell-related malignancies or autoimmune diseases such as systemic lupus erythematosus), can effectively avoid target escape, and prevent the recurrence of multiple myeloma. However, the patent's dual-target cell therapy targeting BCMA and CD19 is less effective in treating SLE and also carries the risk of causing CRS and GVHD.

[0008] Chinese patent CN117186229B discloses anti-human BCMA nanobodies with long CDR3 sequences, CAR-Ts, and their applications. The patent specifically relates to an anti-human BCMA nanobody with a CDR3 sequence of a specific length, CAR-Ts, and their applications. The patent is for single-target CAR-T cells targeting BCMA, but single-target CAR-T cell therapy cannot avoid the problem of antigen escape, resulting in poor therapeutic efficacy and the risk of CRS and GVHD.

[0009] Although CAR-T cell therapy has shown great potential in treating SLE, its use is limited by its toxic side effects. While the incidence of CRS in SLE patients is low, caution is still warranted. Furthermore, there are concerns about the potential for allogeneic CAR-T cells to cause graft-versus-host disease (GVHD). Furthermore, the lengthy preparation time, difficulty, and high cost of autologous CAR-T cells hinder widespread adoption. Summary of the Invention

[0010] In response to the shortcomings of the existing technology, the present invention provides an E-CAR-NK cell and its application for treating systemic lupus erythematosus. Compared with ordinary CAR-NK cells, the E-CAR-NK cell of the present invention has higher antigen sensitivity and better sustained killing ability, thereby improving the therapeutic effect of SLE, and has lower side effects and stronger safety.

[0011] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0012] 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;

[0013] 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 signaling region, DAP12 costimulatory region, 2B4 costimulatory region, and CD3ζ signaling region;

[0014] The artificial nucleotide sequence of the CD19 scFv is shown in SEQ ID NO. 2 in the sequence listing, the artificial nucleotide sequence of the BCMA scFv is shown in SEQ ID NO. 4 in the sequence listing, and the artificial nucleotide sequence of the CD3ε signaling region is shown in SEQ ID NO. 7 in the sequence listing;

[0015] The artificial nucleotide sequence of the CD8 signal peptide is shown in SEQ ID NO.1 in the sequence listing, the artificial nucleotide sequence of the Linker is shown in SEQ ID NO.3 in the sequence listing, the artificial nucleotide sequence of the CD8 Hinge region is shown in SEQ ID NO.5 in the sequence listing, the artificial nucleotide sequence of the NKG2D transmembrane region is shown in SEQ ID NO.6 in the sequence listing, the artificial nucleotide sequence of the DAP12 co-stimulatory region is shown in SEQ ID NO.8 in the sequence listing, the artificial nucleotide sequence of the 2B4 co-stimulatory region is shown in SEQ ID NO.9 in the sequence listing, and the artificial nucleotide sequence of the CD3ζ signaling region is shown in SEQ ID NO.10 in the sequence listing.

[0016] The use of the E-CAR-NK cells in the preparation of a drug for treating systemic lupus erythematosus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention introduces the CD3ε element into the intracellular segment of the CAR molecule, which not only enhances the signaling function of the E-CAR molecule itself, but also recruits co-stimulatory molecules such as CD2, making better use of endogenous co-stimulatory signals, so that E-CAR-NK cells have higher antigen sensitivity and better sustained killing ability than ordinary CAR-NK cells. In the SLE in vivo efficacy experiment, compared with ordinary CAR-NK (experimental group E), the E-CAR-NK cells of the present invention (experimental group A) can further reduce the urine protein, inflammatory cytokines, and anti-dsDNA antibodies and anti-Sm antibodies in the serum of SLE mice, thereby improving the therapeutic effect of SLE;

[0019] (2) The dual-target CD19-BCMA-E-CAR-NK cells (experimental group A) of the present invention have a better therapeutic effect on SLE than single-target CD19-E-CAR-NK cells (experimental group C) and BCMA-E-CAR-NK cells (experimental group D); in addition, compared with dual-target CD19-BCMA-E-CAR-2-NK cells (experimental group B, in which 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 urine protein, cytokines, and anti-dsDNA antibodies and anti-Sm antibodies), and have a better effect in treating systemic lupus erythematosus;

[0020] (3) Compared with the natural T cell antigen receptor (TCR), CAR has poor antigen sensitivity and poor synergy with other signaling molecules. These disadvantages are because CAR cannot form mature immune synapses to integrate various signaling pathways like TCR. These signal disadvantages will cause CAR-T cells to be unable to eliminate target cells with low antigen expression, and are prone to 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 the present invention introduces the CD3ε element into the intracellular segment of the CAR molecule, the signal function of the E-CAR molecule itself is enhanced, and co-stimulatory molecules such as CD2 can be recruited to better utilize endogenous co-stimulatory signals, so that E-CAR-NK cells have higher antigen sensitivity and better sustained killing ability than ordinary CAR-NK cells, thereby improving the treatment effect of SLE;

[0021] (4) Compared with the risks of CRS and GVHD caused by CAR-T cell therapy, the present invention selects a different type of immune cell, namely NK cells, to produce therapeutic E-CAR-NK cells, which will not cause strong CRS or graft-versus-host disease (GVHD), and has lower side effects and stronger safety. Moreover, the NK cells in the present invention are induced by induced pluripotent stem cells, so the E-CAR-NK cell product provided by the present invention eliminates the collection and manufacturing process for specific patients, and can be mass-produced and immediately available to a large number of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the design diagram of the CD19 and BCMA dual-target E-CAR expression vector fusion gene fragment in Example 1;

[0023] Figure 2 Flow cytometry graph showing the expression rates of cell surface markers CD3-CD16+CD56+ in NK cells in Example 3;

[0024] Figure 3 This is a flow cytometric graph of the infection rate of the CD19-BCMA-E-CAR recombinant lentivirus on NK cells in Example 3;

[0025] Figure 4 This is a graph showing the expression levels of IFN-γ and TNF-α inflammatory cytokines in the serum of mice in 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;

[0026] Figure 5This is a graph showing the anti-dsDNA antibody expression levels of mice in 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;

[0027] Figure 6 This is a graph showing the anti-Sm antibody expression levels of mice in 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 DESCRIPTION

[0028] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0029] Example 1 Construction of CD19 and BCMA dual-target E-CAR expression vector

[0030] The present invention introduces the CD3ε element (abbreviated as E-CAR) in the T cell antigen receptor (TCR) into the intracellular segment of the CAR molecule and optimizes the nucleic acid sequence of CD3ε, thereby enhancing the signaling function of the E-CAR molecule itself, giving it higher antigen sensitivity and persistence in the body.

[0031] The connection diagram of each module of CD19 and BCMA dual-target E-CAR is as follows Figure 1 As shown, the modules and artificial nucleotide sequences of the CD19 and BCMA dual-target E-CAR are as follows:

[0032] (1) CD8 signal peptide (its artificial nucleotide sequence is shown in SEQ ID NO.1 in the sequence listing);

[0033] (2) CD19 scFv (its artificial nucleotide sequence is shown as SEQ ID NO. 2 in the sequence listing);

[0034] (3) Linker (its artificial nucleotide sequence is shown as SEQ ID NO.3 in the sequence listing);

[0035] (4) BCMA scFv (its artificial nucleotide sequence is shown as SEQ ID NO. 4 in the sequence listing);

[0036] (5) CD8 Hinge region (its artificial nucleotide sequence is shown in SEQ ID NO. 5 in the sequence listing);

[0037] (6) NKG2D transmembrane region (its artificial nucleotide sequence is shown as SEQ ID NO.6 in the sequence listing);

[0038] (7) CD3ε signal transduction region (its artificial nucleotide sequence is shown as SEQ ID NO.7 in the sequence listing);

[0039] (8) DAP12 costimulatory region (its artificial nucleotide sequence is shown as SEQ ID NO. 8 in the sequence listing);

[0040] (9) 2B4 co-stimulatory region (its artificial nucleotide sequence is shown as SEQ ID NO. 9 in the sequence listing);

[0041] (10) CD3ζ signal transduction region (its artificial nucleotide sequence is shown as SEQ ID NO.10 in the sequence listing);

[0042] Construction of recombinant plasmid:

[0043] according to Figure 1 The above sequences were connected sequentially, and the entire expression cassette was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and inserted into the standard vector pUC57 to obtain pUC-CD19-BCMA-CAR. After transformation into E. coli (DH5α), the recombinant plasmid was extracted from the positive clone after correct sequencing and named CD19-BCMA-E-CAR.

[0044] The hCD19 scFv (its artificial nucleotide sequence is shown as SEQ ID NO.11 in the sequence listing) and the hBCMA scFv (its artificial nucleotide sequence is shown as SEQ ID NO.12 in the sequence listing) described in Chinese patent CN115715298A, a chimeric antigen receptor CAR or CAR construct targeting BCMA and CD19 and its application, and TanCAR 08 were used to replace the CD19 scFv (its artificial nucleotide sequence is shown as SEQ ID NO.2 in the sequence listing) and the BCMA scFv (its artificial nucleotide sequence is shown as SEQ ID NO.4 in the sequence listing) in the present technical solution, respectively; specifically, 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 were sequentially connected, and a recombinant plasmid was constructed according to the above method, which was named CD19-BCMA-E-CAR-2.

[0045] SEQ ID NO.3 and SEQ ID NO.4 were omitted, and 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 were connected in sequence. The recombinant plasmid was constructed according to the above method and named CD19-E-CAR.

[0046] SEQ ID NO. 2 and SEQ ID NO. 3 were omitted, and 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 were connected in sequence. The recombinant plasmid was constructed according to the above method and named BCMA-E-CAR.

[0047] SEQ ID NO.7 was omitted, and 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 were connected in sequence. The recombinant plasmid was constructed according to the above method and named CD19-BCMA-CAR.

[0048] The concentration of the above recombinant plasmids was 1.0 μg / μL.

[0049] Example 2 Lentivirus packaging and titer detection

[0050] (1) Inoculate 293T cells, a lentiviral packaging cell line, into 10 mL of DMEM medium containing 10 vol% FBS and culture in a culture dish at 37°C and 5% CO2. Prepare for transfection when the attachment rate reaches 70%.

[0051] (2) Take a sterile 5mL centrifuge tube and prepare the reaction system according to the following components: serum-free DMEM: 3mL; CD19-BCMA-E-CAR recombinant plasmid: 10μg; GM easyTM Lentiviral Mix (lentiviral packaging auxiliary plasmid mixture): 10μL (10μg); HG TransgeneTM Reagent (high-efficiency transfection reagent): 60μL. After mixing, let it stand at room temperature for 20 minutes, and evenly add it dropwise to the culture dish containing 293T cells obtained in step (1), and place it in a 37℃, 5% CO2 incubator for transfection culture.

[0052] (3) After 12 hours of transfection culture, carefully aspirate the cell culture medium and discard it in a waste liquid cup containing disinfectant. Then add 15 mL of DMEM culture medium containing 10 vol% serum to the culture dish. After continuing to culture for 48 hours, aspirate the cell supernatant into a 50 mL centrifuge tube, centrifuge at 4°C, 500g for 5 minutes, filter the supernatant with a 0.45 μm filter, and transfer it to a new centrifuge tube to obtain the virus liquid.

[0053] (4) The above virus liquid was concentrated to obtain CD19-BCMA-E-CAR recombinant lentivirus. After the titer was detected, it was stored in a -80°C low-temperature refrigerator for future use.

[0054] The titer of CD19-BCMA-E-CAR recombinant lentivirus was 5.81×10 7 pfu / mL.

[0055] The recombinant plasmids CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR were packaged into lentivirus according to the above method, and CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR recombinant lentiviruses were obtained, with the detection titers of 6.22×10 7 pfu / mL, 5.96×10 7 pfu / mL, 6.32×10 7 pfu / mL and 6.56×10 7 pfu / mL; the titers of the above recombinant lentiviruses were adjusted to 5×10 7 pfu / mL.

[0056] Example 3 Preparation of CAR-NK cells

[0057] 1. Induction of NK cells from induced pluripotent stem cells (hiPSCs)

[0058] According to the methods of Examples 1, 2, and 3 of Chinese patent CN115896019B, a method for inducing induced pluripotent stem cells to differentiate into NK cells, induced pluripotent stem cells (hiPSCs) are first induced into CD34+ hematopoietic stem cells; and then the CD34+ hematopoietic stem cells are further induced to differentiate into NK cells.

[0059] 2. Expansion of NK cells

[0060] When NK cells were expanded, the cell culture flask was coated with anti-CD3 monoclonal antibody (OKT3). Before use, the cells were washed twice with NK cell expansion medium (purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.) and plated at 2×10 4The cells were inoculated into culture flasks at a density of 100 cells / mL, and NK cell expansion medium containing factors was added. The cells were cultured in a 37°C, hypoxic (5% O2), 5% CO2 incubator for 2 weeks. Half of the NK cell expansion medium containing factors was replaced every three days. The expanded NK cells were collected and the expression rate of CD3-CD16+CD56+ in NK cells was detected by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodies were purchased from BECKMAN, A07735). Figure 2 As shown, the expression rate of CD3-CD16+CD56+ in NK cells was 81.4%.

[0061] 3. Lentiviral infection of NK cells

[0062] The CD19-BCMA-E-CAR recombinant lentivirus prepared in Example 2 was taken out from -80 ° C, thawed, and resuspended in NK cells to obtain a cell suspension. The cell suspension was added 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 was 50:1. After culturing in a 37 ° C, 5% CO2 incubator for 6 hours, 1 mL of NK cell culture medium was added to each well and continued to be cultured. After a total infection time of 1 week, the obtained CD19-BCMA-E-CAR-NK cells were tested, and the expression of the chimeric antigen receptor was detected by flow cytometry.

[0063] Figure 3 The expression of the CD19-BCMA-E-CAR vector constructed by the present invention on the surface of NK cells was detected by flow cytometry. The results showed that the transfection efficiency of the CD19-BCMA-E-CAR-NK cells constructed by the present invention was 38.6%.

[0064] NK cells were infected with CD19-BCMA-E-CAR-2, CD19-E-CAR, BCMA-E-CAR, and CD19-BCMA-CAR recombinant lentiviruses according to the above method, resulting in 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 results are shown in Table 1.

[0065] Table 1 CAR transfection efficiency

[0066]

[0067] Example 4 In vivo efficacy experiment of CD19 and BCMA dual-target E-CAR

[0068] Animal model: NZB / WF1 mice were used, a commonly used systemic lupus erythematosus (SLE) model that naturally develops clinical and pathological features similar to those of human SLE.

[0069] Experimental setting: 8-week-old NZB / W F1 female mice were randomly divided into six groups, with 5 mice in each group. Each mouse was given 2×10 6 After 8 weeks of treatment, the mice were anesthetized with 1% sodium pentobarbital. After successful anesthesia, the eyeballs of the mice were removed and blood was collected.

[0070] The specific groups are as follows:

[0071] Control group: infused with an equal volume of normal saline;

[0072] Experimental group A: infusion of CD19-BCMA-E-CAR-NK cells;

[0073] Experimental group B: infusion of CD19-BCMA-E-CAR-2-NK cells;

[0074] Experimental group C: infusion of CD19-E-CAR-NK cells;

[0075] Experimental group D: infusion of BCMA-E-CAR-NK cells;

[0076] Experimental group E: infusion of CD19-BCMA-CAR-NK cells;

[0077] Observation indicators:

[0078] (1) Record the weight changes of mice and observe their dietary status every week;

[0079] As shown in Table 2, no adverse reactions such as weight loss and loss of appetite were observed in SLE mice during the treatment process; and there was no significant change in the weight of mice in the control group and the experimental group.

[0080] Table 2 Body weight changes of mice (g)

[0081]

[0082] (2) Urine protein detection: Urine samples of mice were collected at the same time period every 2 weeks for urine protein detection, and the urine protein content was measured using a urine analyzer.

[0083] The results, shown in Table 3, showed a significant decrease in urine protein levels in all experimental groups compared to the control group. This indicates that infusion of E-CAR-NK (CAR-NK) cells can reduce urine protein levels in SLE mice and demonstrate a therapeutic effect. The urine protein levels in mice in experimental group A decreased most rapidly and were also the lowest, demonstrating that the dual-target CD19-BCMA-E-CAR-NK cells provided by the present invention have the greatest therapeutic effect on SLE.

[0084] The urine protein content of mice in experimental group A was lower than that in experimental group E, indicating that the introduction of the CD3ε element into the CAR molecule enhanced the signaling function of the E-CAR molecule itself, further reduced the urine protein content in the mice, and improved the therapeutic effect on SLE; the urine protein content of 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 have a better therapeutic effect on SLE than single-target CD19-E-CAR-NK cells (experimental group C) and BCMA-E-CAR-NK cells (experimental group D).

[0085] Table 3 Changes in mean urine protein content of mice in each group (mg / mL)

[0086]

[0087] Note: “0 week” refers to the urine protein content of SLE mice in each group before treatment

[0088] (3) Cytokine analysis: After 8 weeks, the eyeballs of the mice were removed and blood was collected. After the blood was collected, the serum was separated and extracted, and the changes in the levels of IFN-γ and TNF-α inflammatory cytokines in the mouse serum were measured by ELISA;

[0089] Isolation and extraction of mouse serum: After obtaining blood from the mouse eyeball, the blood was collected into a labeled EP tube and then placed in a centrifuge adjusted to 4°C and centrifuged at 1000g for 10 minutes. After centrifugation, the supernatant in the EP tube was carefully removed and stored in a newly labeled EP tube. It was stored in a -80°C refrigerator for use.

[0090] The results are shown in Table 4 and Figure 4 As shown in the data, compared with the control group, the expression levels of inflammatory cytokines (IFN-γ, TNF-α) in the experimental group mice were significantly reduced, indicating that the infusion of E-CAR-NK (CAR-NK) cells can reduce the inflammatory response in SLE mice and have 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 have the best therapeutic effect on SLE.

[0091] Table 4 Mean cytokine levels in mice of each group (pg / mL)

[0092]

[0093] (4) Anti-double-stranded DNA (anti-dsDNA) antibody and anti-Smith (anti-Sm) antibody detection:

[0094] After 8 weeks of treatment, the eyeballs of the mice were removed and blood was collected. After the blood was collected, the serum was separated and extracted, and the expression of anti-dsDNA antibodies and anti-Sm antibodies in the mouse serum was detected by ELISA.

[0095] Anti-dsDNA antibody: Mouse anti-double-stranded DNA antibody ELISA kit (ELISA; ml063514).

[0096] Anti-Smith antibody: Mouse anti-Smith antibody (Sm) ELISA kit (Shanghai Lianzu Biotechnology; LZ-E032313).

[0097] Isolation and extraction of mouse serum: After obtaining blood from the mouse eyeball, the blood was collected into a labeled EP tube and then placed in a centrifuge adjusted to 4°C and centrifuged at 1000g for 10 minutes. After centrifugation, the supernatant in the EP tube was carefully removed and stored in a newly labeled EP tube. It was stored in a -80°C refrigerator for use.

[0098] The results are shown in Table 5. Figure 5 、 Figure 6 As shown, compared with the control group, the anti-dsDNA antibodies in the serum of the experimental group mice ( Figure 5 )、anti-Sm antibody( Figure 6 ) expression was significantly reduced in both groups, with significant differences. The expression of anti-dsDNA and anti-Sm antibodies in the serum of mice in experimental group A was the lowest, indicating that the dual-target CD19-BCMA-E-CAR-NK cells (experimental group A) provided by the present invention can significantly reduce the levels of anti-dsDNA and anti-Sm antibodies in SLE mice and has the best therapeutic effect on SLE.

[0099] Table 5 Expression of anti-dsDNA antibodies and anti-Sm antibodies in the serum of mice in each group (OD 450nm mean)

[0100]

[0101] In summary, compared with the control group, all indicators (urinary protein, cytokines, anti-dsDNA antibodies, and anti-Sm antibodies) 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 of experimental group A were better than those of 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 were modified with CD19 and BCMA dual-target E-CAR to generate E-CAR-NK cells; The CD19 and BCMA dual-target E-CAR is composed of the following modules connected in sequence: CD8 signal peptide, CD19 scFv, Linker, BCMA scFv, CD8 Hinge region, NKG2D transmembrane region, CD3ε signaling region, DAP12 costimulatory region, 2B4 costimulatory region, CD3ζ signaling region; The artificial nucleotide sequence of the CD19 scFv is shown in SEQ ID NO. 2 in the sequence listing, the artificial nucleotide sequence of the BCMA scFv is shown in SEQ ID NO. 4 in the sequence listing, and the artificial nucleotide sequence of the CD3ε signaling region is shown in SEQ ID NO. 7 in the sequence listing.

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

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