Double-target CAR-T cell specifically binding to human CD19 protein and BCMA protein and application

Abstract

The invention discloses a double-target CAR-T cell specifically combined with human CD19 protein and BCMA protein and application, and belongs to the technical field of chimeric antigen receptor T cells, the double-target CAR-T cell specifically combined with human CD19 protein and BCMA protein specifically combined with human CD19 protein and BCMA protein specifically combined with human CD19 protein and BCMA protein specifically combined with human CD19 protein and BCMA protein specifically combined with human CD19 protein and BCMA protein and application thereof. The sequence of the CD19scFv is as shown in SEQ ID NO: 1; the sequence of the BCMA scFv is as shown in SEQ ID NO: 2; the three different linkers are respectively as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. According to the invention, the double-target CAR-T cell is efficiently constructed, and five kinds of CD19-BCMA double-target CAR-T cells with different connection sequences or Linkers are successfully constructed through an optimized experimental process. By optimizing the connection sequence and linker properties, the steric hindrance between two target CAR molecules is reduced, the targeted killing efficiency is improved, double-target CAR-T cells with different construction strategies show significant difference in the aspect of killing target cells, and part of the construction strategies show higher killing efficiency on specific target cells.

Description

Technical Field

[0001] The present invention relates to the technical field of chimeric antigen receptor T cell technology, and particularly relates to a dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein and its application. Background Art

[0002] Neuroimmune diseases with neuromuscular junction (NMJ) transmission disorders mediated by autoantibodies mainly affect the postsynaptic membrane at the neuromuscular junction, resulting in fluctuating weakness and pathological fatigue of systemic or focal skeletal muscles. Symptoms usually worsen after activity and relieve after rest, showing the phenomenon of "lighter in the morning and heavier in the evening". In severe cases, the respiratory muscle group may be involved, resulting in a fatal myasthenic crisis. Acetylcholine receptor (AChR) antibody is the main pathogenic autoantibody in MG (myasthenia gravis). By binding to AChR on the postsynaptic membrane, it activates the complement system, destroys AChR, and leads to NMJ signal transmission disorders, triggering muscle contraction weakness. In addition to AChR antibodies, antibodies against muscle-specific receptor tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4), and ryanodine receptor (RyR) can also be detected in some patients. These autoantibodies cause diseases by interfering with AChR aggregation, affecting AChR function, and NMJ signal transmission.

[0003] Since autoantibodies are mainly produced by autoreactive B cells, inhibiting or depleting B cells is an important strategy for treating MG. Traditional non-specific immunosuppressants (such as azathioprine and mycophenolate mofetil) can reduce the inflammatory response and improve symptoms in most patients, but drug treatment usually needs to be maintained for several years or even lifelong, and may cause immunosuppression and non-targeted side effects. In the past decade or so, monoclonal antibodies against B cells, such as rituximab, have played an important role in improving the disease survival state, greatly improving the disease state of some refractory patients and showing good safety. However, even if peripheral blood B cells are depleted, the symptoms of some patients still cannot be effectively controlled. Studies on other autoimmune diseases have shown that the residual memory B cells in tissues may lead to the failure of rituximab treatment. In addition, monoclonal antibodies still need to be used for a long time, which will also bring cumulative side effects and medical costs. The future treatment goal should focus on "resetting" the immune system, that is, achieving a long-term immune tolerance state through one-time immunosuppression, so as to avoid long-term immunosuppression and disease activity.

[0004] Chimeric antigen receptor T cell therapy (CAR-T therapy) is a precise targeted therapy. This therapy uses gene modification technology to transfer genetic material with specific antigen recognition domains and T cell activation signals into T cells, enabling T cells to bind to specific antigens on the surface of target cells and be activated. It exerts direct killing effects by releasing perforin, granzyme B, etc., and also recruits endogenous immune cells in the human body to kill target cells by releasing cytokines, thereby achieving the therapeutic purpose. In addition, CAR-T can form memory T cells, thus obtaining long-term killing of specific target cells. This therapy has achieved remarkable efficacy in the treatment of certain types of hematological malignancies (such as acute lymphoblastic leukemia and certain types of lymphoma), and CAR-T therapy targeting B cells has shown the ability to deeply clear B cells in autoimmune diseases such as systemic lupus erythematosus. Currently, the CAR-T cells targeting B cells with more treatment evidence mainly include:

[0005] 1. CD19-CAR-T cells. CD19 is a leukocyte differentiation antigen expressed on B cells and belongs to the Ig superfamily (immunoglobulin superfamily). It begins to be expressed in the early stage of B cell development and is expressed in all B cell lines except plasma cells. CD19-CAR-T is mainly used in the treatment of B cell malignancies, such as acute lymphoblastic leukemia and certain types of non-Hodgkin lymphoma, in the field of oncology. It is also the earliest type of CAR-T cells used in autoimmune diseases. In 2021, it was first used to treat a 20-year-old female with severe refractory systemic lupus erythematosus. The results showed that it is feasible to produce CAR-T cells from patients with autoimmune diseases. At the same time, patients have good tolerance to CAR-T cell infusion and no serious toxic effects are caused. This treatment method enables rapid clearance of B cells in the body, accompanied by rapid expansion of CAR-T cells in peripheral blood. The patient's symptoms were relieved after 3 months, including the disappearance of proteinuria, and seroconversion of disease-related antibodies against double-stranded DNA was observed. In addition, all immunosuppressive agents (including glucocorticoids) were discontinued, and there was no sign of recurrence of the old disease within 18 months.

[0006] 2. BCMA-CAR-T cells, also known as TNFRSF17 or CD269, are transmembrane glycoprotein members of the tumor necrosis factor receptor (TNFR) superfamily, distributed on the surface of B cells and plasma cells, and are an important target on the surface of multiple myeloma cells in the field of oncology. For autoimmune diseases, since BCMA is highly expressed on plasma cells, it may theoretically clear plasma cells that cannot be cleared by CD19-targeted therapy, and these plasma cells are an important source of autoantibodies.

[0007] Both CAR-T cell technologies have relatively mature applications. CD19 CAR-T can effectively target most B cells, but it cannot eliminate terminally differentiated plasma cells; BCMACAR-T can effectively eliminate plasma cells but cannot target naive B cells, and both have certain limitations.

[0008] Based on this, the present invention designs a dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein and its application to solve the above problems. Summary of the Invention

[0009] In view of the above-mentioned disadvantages of the prior art, the present invention provides a dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein and its application.

[0010] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0011] A dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein, comprising: 5 different CD19 and BCMA dual-target CAR-T cells;

[0012] The CD19 scFv sequence is as shown in SEQ ID NO:1;

[0013] The BCMA scFv sequence is as shown in SEQ ID NO:2;

[0014] Three different linkers are respectively as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5;

[0015] The Flag-DuCAR1 sequence is as shown in SEQ ID NO:6;

[0016] The Flag-DuCAR2 sequence is as shown in SEQ ID NO:7;

[0017] The Flag-DuCAR3 sequence is as shown in SEQ ID NO:8;

[0018] The Flag-DuCAR4 sequence is as shown in SEQ ID NO:9;

[0019] The Flag-DuCAR5 sequence is as shown in SEQ ID NO:10.

[0020] An application of the dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein, wherein the CAR-T cell is applied to autoimmune diseases or malignant tumors caused by immune cells expressing CD-19 and BCMA targets.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The present invention efficiently constructs dual-target CAR-T cells. Through an optimized experimental process, five different CD19-BCMA dual-target CAR-T cells with different connection orders or Linkers are successfully constructed. By optimizing the connection order and linker, a connection method with the smallest steric hindrance is screened out. This construction strategy can fully exert the binding ability of the two CAR molecules in the dual-target CAR-T cells, further optimize the functional performance of CAR-T cells, including enhancing their affinity for target antigens and signal transduction efficiency. This optimized construction method not only improves the preparation success rate of CAR-T cells but also provides a solid technical foundation for subsequent clinical applications, contributing to the wide application of CAR-T cell therapy in various autoimmune diseases.

[0023] 2. The present invention improves the targeted killing efficiency. Dual-target CAR-T cells with different construction strategies show significant differences in killing target cells, and some construction strategies exhibit higher killing efficiency on specific target cells. By simultaneously targeting two antigens, CD19 and BCMA, the dual-target CAR-T cells increase the killing breadth of CAR-T cells, effectively reducing the possibility of target cells escaping through a single antigen, thereby achieving deep immune reset. This dual-target design not only enhances the recognition ability of CAR-T cells for target cells but also improves their killing efficiency, providing a new strategy for the treatment of relapsed or refractory tumors or autoimmune diseases. In addition, the optimized CAR structure design further improves the signal transduction efficiency of CAR-T cells, enabling them to release cytotoxic granules more efficiently when contacting target cells, thus achieving a more precise and efficient anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0025] Figure 1 is the map of the shuttle plasmid pCDH;

[0026] Figure 2 is the construction schematic diagram of five different dual-target CAR-Ts;

[0027] Figure 3 is the doubling curve of dual-target CAR-T cells;

[0028] Figure 4 The detection results of the positive rates of several different CAR-T cells;

[0029] Figure 5 The killing efficiency graph of dual-target CAR-T cells against Daudi cells in vitro;

[0030] Figure 6 The IFN-γ secretion of dual-target CAR-T cells;

[0031] Figure 7 The quantitative scoring form for the severity of myasthenia gravis;

[0032] Figure 8 The daily activity scoring form for myasthenia gravis;

[0033] Figure 9 The comprehensive scoring form for myasthenia gravis;

[0034] Figure 10 The 15-item questionnaire for the quality of life of myasthenia gravis;

[0035] Figure 11 The trend graph of CART cells and B cells;

[0036] Figure 12 The scoring trend graph. Detailed implementation mode

[0037] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0038] Example 1: Construction of a CD19-BCMA dual-target CAR vector;

[0039] In this example, 5 different dual-target CAR plasmids were constructed. Among them, the DNA sequences of CD19 scFv (FM63) and BCMA scFv are shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequences of Linker-1, 2, and 3 were artificially synthesized;

[0040] The fragments of CD19 scFv, BCMA scFv, CD19-VH, CD19-VL, BCMA-VH, and BCMA-VL were amplified respectively. According to Figure 2As shown, different fragments are combined together by overlap PCR. After amplifying the full-length fragment, Flag tags and signal peptides are added to the primers, and then cloned into the pCDH-EF1-CART-nonGFP vector( Figure 1 ); The Flag tag is removed from the dual-target CAR-T vector used in clinical practice.

[0041] The primers and sequencing services used in this experiment were provided by Shanghai Branch of Beijing Tsingke Biotechnology Co., Ltd.

[0042] Example 2: Preparation of CD19-BCMA dual-target CAR-T;

[0043] Step 1: Preparation of recombinant virus;

[0044] The calcium phosphate method is used to transfect 293T cells;

[0045] Specifically: Select healthy 293T cells for passage. After 23 hours, when the cells grow to cover 60% of the bottom of the culture dish, perform transfection. Mix the recombinant vector, auxiliary plasmids REV, RRE, and VSVG in a ratio of 2.5:1:1.25:1, then add CaCl2. Add 2×HBSS (Hanks balanced salt solution) to the prepared solution, pipette until milky white, immediately drop it into the culture dish and shake well. Place it in the incubator for 9 hours and then change the medium. After 48 hours of transfection, collect the supernatant, centrifuge and filter, and then purify it with a concentration column.

[0046] Step 2: PBMC (peripheral blood mononuclear cell) resuscitation;

[0047] Take out 4 tubes of PBMC from the liquid nitrogen tank for the preparation of dual-target CAR-T cells, resuscitate them in a 37-degree water bath. Add the completely dissolved cell solution to a centrifuge tube containing 10 mL of X-vivo15 (serum-free medium for immune cells), centrifuge at 1800 r for 10 minutes. After discarding the supernatant, resuspend the cells with 8 mL of X-vivo15, and spread the cells on a 24-well plate, 2 mL per well, for a total of 4 wells. Let the cells stand overnight.

[0048] Step 3: PBMC activation;

[0049] Collect the cells resuscitated in Step 2 into a 15 mL centrifuge tube, pipette them evenly and then count the cells. Take 2*10 5Flow cytometry antibody staining was used to identify the proportion of CD3 T cells, and the amount of beads (magnetic beads) was calculated (beads: T cells = 3:1). The cells were centrifuged at 1800 r for 10 minutes. Meanwhile, 66 μL of beads was added to a cryotube containing 1 mL of X-vivo medium, and the buffer in the beads was washed away. The cryotube was placed on a magnetic stand to adsorb the beads, and the supernatant was completely discarded. 900 μL of X-vivo medium was taken to resuspend the cell pellet and then added to the cryotube containing only beads (the cells were co-incubated with the beads at a density of 1×10 7 cells / mL). The cells and beads were blown evenly, the cryotube was sealed with a sealing film and then placed on a shaker at room temperature and shaken for 45 minutes to allow the beads to fully bind to CD3 T cells; finally, the cryotube was placed on the magnetic stand for 2 minutes, the supernatant was discarded, and the CD3 T cells bound to the beads were attracted by the magnetic beads. The cells were resuspended with 1 mL of X-vivo medium, and the cells were cultured at a density of 2×10 6 cells / mL. The cells were placed in a 24-well plate, with a total of 2 wells, 2 mL per well, and cultured at 37 °C with 5% CO₂ for 24 hours.

[0050] Step 4: Virus infection;

[0051] The cells activated for 24 hours in Step 3 were collected into a centrifuge tube, blown evenly and then counted. 2×10 5 cells were taken for flow cytometry to identify the activation level of T cells. Then the PBMC cells were centrifuged and resuspended and counted. The cells were resuspended to 1×10 6 cells / mL with X-vivo medium (containing 200 IU of interleukin). 0.5 mL of the cell suspension was added to each well of a 24-well plate. The amount of virus used was calculated according to the virus titer and the multiplicity of infection MOI. A control well without virus (mock) was set up, and the required DuCAR1 (dual target CAR-T), DuCAR2, DuCAR3, DuCAR4 and DuCAR5 viruses were added to each well accordingly. In addition, 2 μL of the transfection reagent polybrene (8 μg / mL) was added to each well and mixed evenly in turn; finally, the well plate was wrapped with plastic wrap and centrifuged at 2000 g, with an acceleration of 3 minutes to the target speed and a deceleration of 1 minute after centrifugation ended (the time for the centrifuge to accelerate to the target speed was set to 3 minutes, and the time for the centrifuge to decelerate to a stop after centrifugation ended was set to 1 minute), at 32 °C (the centrifugation operation was carried out at 32 °C), and the centrifugation time was 90 minutes. After centrifugation ended, the volume was made up to the final volume of 2 mL, and static culture was carried out. After 48 hours of virus infection, the virus was removed, and the medium was completely replaced with X-vivo15 medium.

[0052] Step 5: Cell passage and statistical analysis of proliferation fold;

[0053] The culture density of DuCAR-T cell passage was maintained at 5×10 5 cells / mL. On day 3, day 5, day 6, day 8, day 10, and day 12 respectively, CAR-T cells were collected and counted. After counting, fresh medium was supplemented to adjust the cell density to 5×10 5 cells / mL and the cells were cultured continuously. The CAR-T cell doubling curve was plotted as shown in Figure 3 The figure shows that the doubling of all 5 types of dual-target CAR-T cells was normal, and they could be amplified about 400-fold on Day 12. Among them, the amplification trend of DuCAR2 was better.

[0054] Step 6: Identification of DuCAR-T positive rate and phenotype;

[0055] Take the DuCAR-T cells on Day 11 in Step 5 respectively, and perform cell counting under a microscope. Take 2×10 5 cells and add them to the flow antibodies for detecting the positive rate of CAR (chimeric antigen receptor) respectively. Incubate in the dark at 4 °C for 15 min, add 1 mL of PBS (phosphate buffered saline) to wash the cells, centrifuge at 4500 r for 3 minutes. After discarding the supernatant, add 0.2 mL of PBS to resuspend the cells and perform detection on the machine. The flow antibodies used were APC anti-DYKDDDDK tag antibody (Biolegend-637307), PE BCMA-mFc protein (produced in the laboratory), and PE CD19-mFc protein (produced in the laboratory). The positive rate results on Day 11 were as shown in Figure 4 For each of the 5 types of dual-target CAR-T cells constructed, detection was performed with different secondary antibodies, and the positive rate did not change significantly, proving that the CAR molecule had been expressed and correctly folded, and there would be slight differences between different construction strategies.

[0056] Example 3: Detection of the killing efficiency and cytokine release of CD19-BCMA dual-target CAR-T;

[0057] Step 1: Identification of killing by the method of labeling target cells with CAM (fluorescein diacetate);

[0058] The CAR-T cells and target cells were counted respectively, and the target cells were labeled with CAM. This experimental system only contained CAR-T cells and target cells. The effector-to-target ratios were set as 1:1, 3:1, and 10:1. The positive control was the sample well with CAM-labeled target cells added with 1% Triton. Among them, the CAM-labeled target cells were the negative control wells (background wells). The cells were mixed according to the effector-to-target ratio and added to a 96-well plate, and co-cultured for 6 hours. The supernatant was collected and centrifuged, and the supernatant was transferred to a 96-well plate and detected on the EnSpire multifunctional microplate reader. The killing efficiency was calculated according to the detected values, as shown in Figure 5As shown, daudi cells that co-express CD19 and BCMA were selected as target cells. Dual-target CAR-T cells constructed with different connection sequences or different linkers had significant differences in killing ability. Among them, DuCAR1 and DuCAR3 showed no killing effect at a ratio of 1:1. The killing effects of DuCAR1, DuCAR3, DuCAR4, and DuCAR5 were significantly different from those of the single-target CAR-T control cells at ratios of 3:1 and 10:1. DuCAR2 maintained a killing effect similar to that of the single-target control CAR-T cells at different effector-to-target ratios.

[0059] Killing efficiency = (CAR-T - background value) / (positive control - background value) × 100%.

[0060] Step 3: Detection of cytokine IFN-γ (interferon-γ) secretion;

[0061] Mock, DuCAR1, DuCAR2, DuCAR3, DuCAR4, and DuCAR5 and target cells in good growth state were centrifuged separately. Finally, they were resuspended and counted with X-vivo15. CAR-T (CAR+) and target cells were mixed at a ratio of 1:2 and added to a 96-well plate for culture for 24 h. The supernatant was collected to detect the release of cytokine IFN-γ. The capture antibody used in cytokine detection was IFNG antibody (recombinant anti-interferon γ rabbit monoclonal antibody) (SinoBiological; catalog number: 11725-R121), and the detection antibody was IFN-gamma antibody-HRP (horseradish peroxidase-labeled anti-interferon γ rabbit polyclonal antibody) (SinoBiological; catalog number: 11725-RP02-H). As Figure 6 shown, after co-incubation with different target cells, the IFN-γ secretion abilities of dual-target CAR-T cells obtained by several different construction strategies were different. After co-incubation with daudi cells, several dual-target CAR-T cells all had significant IFN-γ, and among them, the IFN-γ secretion level of DuCAR2 was relatively high.

[0062] Clinical trial:

[0063] On the premise of not undergoing lymphocyte depletion, the above-mentioned CAR-T cells can be directly reinfused for application.

[0064] Application of dual-target CAR-T cells that specifically bind to human CD19 protein and BCMA protein. The CAR-T cells are applied to autoimmune diseases or malignant tumors caused by immune cells expressing CD-19 and BCMA targets.

[0065] Application of CD19-BCMA-targeted CAR-T cell therapy in three patients with highly recurrent and refractory myasthenia gravis. All patients were positive for acetylcholine receptor antibody (AChR-IgG). The aim of the study was to evaluate the safety and efficacy of CAR-T cell therapy in autoimmune diseases and its potential mechanism of action.

[0066] Three patients received CD19-BCMA-targeted CAR-T cell therapy, and their clinical outcomes were monitored for up to 140 days, with an average follow-up of 87 days. Serial blood samples after infusion were stained for CAR-T cells by longitudinal flow cytometry analysis (FACS). These analyses were aimed at monitoring the in vivo expansion of CAR-T cells and determining the peak expansion time after infusion.

[0067] All patients showed good safety and sustained clinical improvement within 60 to 140 days after CAR-T cell infusion. At baseline, the activities of daily living (ADL) scores of the three patients were 8, 7, and 15 respectively. Within one month after infusion, two patients had symptom improvement, and the other patient had symptom improvement within two months. Notably, the ADL scores of all patients dropped to zero. In addition, all patients were able to reduce the use of corticosteroids, and one patient completely discontinued corticosteroid use for more than three months. The CAR-T cell levels in peripheral blood peaked on average at day 10 after infusion. The cytokine release syndrome (CRS) was generally mild and occurred within two weeks, and no severe CRS cases were reported.

[0068] CD19-BCMA-targeted CAR-T cell therapy is safe and effective in patients with highly recurrent and refractory myasthenia gravis. All three patients showed significant clinical improvement, with ADL scores dropping to zero and successfully reducing or discontinuing the use of corticosteroids. The therapy was well tolerated, with only mild CRS observed, and the CAR-T cell levels peaked on average at day 10 after infusion.

[0069] Table 1: Baseline characteristics and treatment details of 3 MG (myasthenia gravis) patients

[0070]

[0071]

[0072] Plot the scores in Table 1 (Myasthenia Gravis Activities of Daily Living Score, Myasthenia Gravis Quantitative Score, Myasthenia Gravis 15-Item Quality of Life Questionnaire, Myasthenia Gravis Composite Score) as a line graph (as Figures 11 - 12 shown).

[0073] In summary, the present invention efficiently constructs dual-target CAR-T cells: through an optimized experimental process, the best-performing one among the CD19-BCMA dual-target CAR-T cells with five different construction strategies is successfully screened out, which is applied to myasthenia gravis for the first time and achieves good therapeutic effects, providing diverse options for CAR-T cell therapy;

[0074] The present invention improves the targeted killing efficiency: the experimental results show that there are significant differences in the killing of target cells among the dual-target CAR-T cells with different construction strategies, and some construction strategies exhibit higher killing efficiency on specific target cells.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein, characterized in that, Including: Five different CD19 and BCMA dual-target CAR-T cells; The CD19 scFv sequence is shown as SEQ ID NO:1; The BCMA scFv sequence is shown as SEQ ID NO:2; The three different linkers are shown as SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 respectively; The Flag-DuCAR1 sequence is shown as SEQ ID NO:6; The Flag-DuCAR2 sequence is shown as SEQ ID NO:7; The Flag-DuCAR3 sequence is shown as SEQ ID NO:8; The Flag-DuCAR4 sequence is shown as SEQ ID NO:9; The Flag-DuCAR5 sequence is shown as SEQ ID NO:

10.

2. Use of a dual-target CAR-T cell that specifically binds to human CD19 protein and BCMA protein as described in claim 1, characterized in that, The CAR-T cells are applied to autoimmune diseases or malignancies caused by immune cells expressing CD19 and BCMA targets.

Images