Plasmid vector, chimeric MOG antibody receptor T cell and application of chimeric MOG antibody receptor T cell

Abstract

The invention discloses a plasmid vector, a chimeric MOG antibody receptor T cell and application of the chimeric MOG antibody receptor T cell. The plasmid vector comprises a chimeric autoantibody receptor, and the chimeric autoantibody receptor is sequentially composed of a signal peptide, an extracellular fragment of an MOG antigen, a transmembrane region CD8alpha, a costimulatory domain 4-1BB and an activation domain CD3xi from an amino terminal to a carboxyl terminal; the base sequence of the signal peptide is as shown in SEQ ID NO: 1; the base sequence of the extracellular fragment of the MOG antigen is as shown in SEQ ID NO: 2; the base sequence of the transmembrane region CD8 alpha is as shown in SEQ ID NO: 3; the base sequence of the costimulatory domain 4-1BB is as shown in SEQ ID NO: 4; the base sequence of the activation domain CD3xi is as shown in SEQ ID NO: 5; the chimeric MOG antibody receptor T cell provided by the invention can specifically kill pathogenic B cells which generate the MOG antibody with high selectivity, so that precise treatment on MOGAD is realized.

Description

Technical Field

[0001] The present invention relates to the field of biology and medicine technology, and in particular to a plasmid vector, a chimeric MOG antibody receptor T cell and applications thereof. Background Art

[0002] Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is an autoimmune disease of the central nervous system, characterized by recurrent episodes of optic neuritis, myelitis, and brainstem encephalitis, sometimes leading to severe neurological impairment. It is currently believed that MOGAD is caused by a combination of genetic susceptibility and environmental factors, leading to the immune system misidentifying MOG as a foreign antigen and producing specific antibodies against it.

[0003] In related technologies, MOGAD patients are still prone to relapse after conventional treatment and monoclonal antibody treatment, and there is no targeted drug for the precise treatment of MOGAD.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] The technical task of the present application is to address the above deficiencies and provide a plasmid vector of a chimeric MOG antibody receptor T cell and its application. By designing a plasmid vector of a chimeric MOG antibody receptor T cell and a chimeric MOG antibody receptor T cell, the present application can specifically and highly selectively kill pathogenic B cells that produce MOG antibodies, thereby achieving precise treatment of MOGAD.

[0006] To achieve the above objectives, this application provides the following technical solutions: According to one aspect of the present application, a plasmid vector is provided, comprising: a chimeric autoantibody receptor, wherein the chimeric autoantibody receptor is composed of a signal peptide, an extracellular segment of the MOG antigen, a transmembrane region CD8α, a costimulatory domain 4-1BB, and an activation domain CD3ξ from the amino terminus to the carboxyl terminus; the base sequence of the signal peptide is shown in SEQ ID NO: 1; the base sequence of the extracellular segment of the MOG antigen is shown in SEQ ID NO: 2; the base sequence of the transmembrane region CD8α is shown in SEQ ID NO: 3; the base sequence of the costimulatory domain 4-1BB is shown in SEQ ID NO: 4; and the base sequence of the activation domain CD3ξ is shown in SEQ ID NO: 5.

[0007] According to another aspect of the present application, a chimeric MOG antibody receptor T cell is also provided. The chimeric MOG antibody receptor T cell is obtained by transfecting the plasmid vector and the packaging plasmid into 293T cells, wherein the packaging plasmids are psPAX2 and pMD2.G, the transfection reagent is Lipofectamine 2000, and the mass ratio of the transfection reagent, the plasmid vector, the psPAX2 and the pMD2.G is 16:4:3:1.

[0008] In some embodiments, the chimeric MOG antibody receptor T cells can kill B cells expressing specific MOG antibodies.

[0009] In some embodiments, the MOG antibody is the 8-18C5 antibody.

[0010] In some embodiments, the B cells are Nalm6 B cells.

[0011] According to another aspect of the present application, there is also provided a use of a plasmid vector or a chimeric MOG antibody receptor T cell in the preparation of a drug for treating MOGAD.

[0012] In some embodiments, the drug can specifically kill pathogenic B cells expressing MOG antibodies.

[0013] In some embodiments, the MOG antibody is the 8-18C5 antibody.

[0014] Compared with the existing technology, the advantages and positive effects of the present application are: by designing a plasmid vector of a chimeric MOG antibody receptor T cell and a chimeric MOG antibody receptor T cell, the present application can specifically and highly selectively kill pathogenic B cells that produce MOG antibodies, thereby achieving precise treatment of MOGAD. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 A schematic diagram of the design of the MOG CAAR-T cell plasmid sequence in Example 1 of the present application is shown.

[0017] Figure 2 A schematic diagram of the plasmid sequence design of the 8-18C5 Nalm6 cell line in Example 1 of the present application is shown.

[0018] Figure 3 The schematic diagram of the design of the EGFP plasmid sequence for the extracellular segment of MOG antigen in Example 1 of the present application is shown.

[0019] Figure 4 The results of the 8-18C5 Nalm6 cell line test in Example 2 of the present application are shown.

[0020] Figure 5 The results of the binding test between 8-18C5 and the extracellular segment of MOG antigen in Example 2 of the present application are shown.

[0021] Figure 6 The results of T cell infection efficiency in Example 3 of the present application are shown.

[0022] Figure 7 The CAAR-T cell killing results in Example 4 of the present application are shown.

[0023] Figure 8 The results of the soluble protein killing experiment in Example 4 of the present application are shown.

[0024] Figure 9 The results of flow cytometry detection of B cells and B cell subsets in Example 6 of the present application are shown. DETAILED DESCRIPTION

[0025] In order to more clearly understand the above-mentioned objects, features and advantages of the present application, the present application is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other without conflict.

[0026] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1: Construction of CAAR-T, 8-18C5 and MOG antigen fused EGFP lentiviral vector.

[0028] 1) Construct a CAAR-T cell plasmid that overexpresses the extracellular segment of MOG antigen.

[0029] The human MOG antigen sequence was downloaded from the Uniprot website. The extracellular domain of the MOG antigen was selected (protein number: G16653-1, amino acid sequence: G30-G154). The CD19 antibody binding sequence FMC63 from patent WO2019 / 159193A1 (country: India, publication date: 2019-08-22) was also used to construct the CD19 CAR-T cell plasmid as a positive control. The MOG CAAR-T and CD19 CAR-T cell plasmids use the same signal peptide, CD8α, 4-1BB, and CD3ξ to eliminate differences in killing effects caused by the costimulatory domain 4-1BB and activation domain CD3ξ, thereby enhancing the comparability of the killing effects of MOG CAAR-T cells with those of CD19 CAR-T cells.

[0030] The signal peptide is followed by the extracellular segment sequence of MOG antigen, the transmembrane region CD8α, the costimulatory domain 4-1BB and the activation domain CD3ξ, which is the target sequence of MOG CAAR-T. The sequence design is shown in ( Figure 1 shown).

[0031] The signal peptide is followed by the variable regions of the light and heavy chains (scFV single-chain antibody sequence) of the CD19 antibody FMC63, the transmembrane region CD8α, the co-stimulatory domain 4-1BB, and the activation domain CD3ξ, which is the target sequence of CD19 CAR-T.

[0032] The signal peptide base sequence is shown in SEQ ID NO: 1; The extracellular segment sequence of MOG antigen is shown in SEQ ID NO: 2; The CD19 CAR-T scFV sequence (the light and heavy chain variable region sequences of the CD19 antibody FMC63) is shown in SEQ ID NO: 13; The transmembrane region CD8α sequence is shown in SEQ ID NO: 3; The costimulatory domain 4-1BB sequence is shown in SEQ ID NO: 4; The activation domain CD3ξ sequence is shown in SEQ ID NO:5; After sequence design, it was synthesized by Jinweizhi Biotechnology Co., Ltd. and ligated with the pCDHGFP plasmid vector double-digested with XbaI and BamHI to obtain the MOG CAAR-T lentiviral plasmid vector and the CD19 CAR-T lentiviral plasmid vector (positive control).

[0033] This plasmid vector enables the MOG antigen expressed on the surface of T cells to bind to target cells expressing its specific antibody 8-18C5, thereby reducing the distance between MOG CAAR-T cells and target cells and further exercising the killing effect of MOG CAAR-T cells on 8-18C5 target cells.

[0034] 2) Construction of lentiviral plasmid vector overexpressing 8-18C5 The heavy chain variable region VH of 8-18C5 antibody was connected to the human heavy chain constant region IgG1, and the light chain variable region VL was connected to the kappa chain (CLκ) of the human light chain constant region. The sequence design is shown in ( Figure 2 shown).

[0035] Wherein, the heavy chain variable region VH sequence is shown in SEQ ID NO: 7; The heavy chain constant region IgG1 sequence is shown in SEQ ID NO: 8; The 8-18C5 signal peptide sequence is shown in SEQ ID NO: 6; The light chain variable region VL sequence is shown in SEQ ID NO: 9; The light chain constant region CLκ sequence is shown in SEQ ID NO: 10; After sequence design, the lentiviral vector was synthesized by Jinweizhi Biotechnology Co., Ltd. and ligated into the pLV plasmid vector digested with XbaI and BamHI to obtain a lentiviral plasmid vector overexpressing the MOG antibody 8-18C5.

[0036] The primary cause of MOGAD disease is the rapid proliferation and differentiation of B cells into plasma cells, which express and secrete antibodies targeting the MOG antigen. The 8-18C5 antibody targets the membrane-associated region of MOG, specifically a discontinuous epitope within the extracellular immunoglobulin V-like domain of MOG. Therefore, we synthesized the 8-18C5 lentiviral plasmid vector, packaged in 293T cells, and then infected the Nalm6 B cell line. The 8-18C5 Nalm6 cell line is used to mimic the pathogenic antibody-secreting B cell population in MOGAD patients. In vitro, MOG CAAR-T cells specifically target the 8-18C5 Nalm6 target cell line. When co-incubated with B cells from MOGAD patients, MOG CAAR-T cells theoretically kill all cells that specifically bind to the MOG antigen—that is, the pathogenic B cell subset expressing MOG-specific antibodies—while sparing other B cell subsets.

[0037] 3) Construct a lentiviral plasmid vector fused with MOG antigen and EGFP.

[0038] The human MOG antigen sequence was downloaded from the Uniprot website. The extracellular segment and transmembrane region of the MOG antigen (protein number: G16653-1, amino acid sequence: M1-F175) were selected. The transmembrane region was fused with the fluorescent protein EGFP. The sequence design is shown in ( Figure 3 ).

[0039] The base sequence of the extracellular segment and transmembrane region of MOG antigen (M1-F175) is shown in SEQ ID NO: 11; The EGFP base sequence is shown in SEQ NO ID: 12.

[0040] The target sequence was amplified by PCR and ligated with the pCDH plasmid vector double-digested with BamHI and EcoRI by homologous recombination. After correct Sanger sequencing, the MOG antigen extracellular segment overexpression plasmid fused with EGFP protein was obtained.

[0041] Example 2: Lentivirus packaging and acquisition of stably transfected cell lines.

[0042] 293T cells were used as the tool cell line, and lentivirus expressing CD19 CAR-T, MOG CAAR-T, and 8-18C5 was packaged using the CaCl2 method. After the target gene plasmid, envelope plasmid, and capsid plasmid were transfected into 293T cells, the lentivirus was formed and secreted into the culture supernatant solution, which is the lentiviral suspension. This lentiviral suspension has viral infection characteristics. Different target gene plasmids can be used during the packaging process to package lentiviral suspensions expressing different genes. The lentiviral concentrate is used to concentrate the lentiviral suspension to increase the viral titer and enhance the cell infection efficiency. The Nalm6 cell line is a B cell tool cell line. The primary pathogenicity of MOGAD is due to the production of pathogenic antibodies against the MOG antigen by antibody-secreting cells within the patient's B cell subset. Therefore, we expressed the 8-18C5 pathogenic antibody sequence in the Nalm6 B cell line to verify that the MOG antigen extracellular domain sequence in MOG CAAR-T successfully binds to the pathogenic antibody sequence in MOGAD and further promotes downstream killing. To this end, a viral suspension of 8-18C5 overexpressing the target gene was collected and infected with Nalm6 cells in the presence of 6 μg / ml hexadimethrine bromide (Polybrene). After one week, stably transfected cells overexpressing the 8-18C5 BCR were sorted. Continued subculture resulted in a 100% transfected Nalm6 cell line stably expressing the fusion protein. Similarly, a viral suspension containing the extracellular segment of the MOG antigen fused to EGFP overexpressing the target gene was collected and infected with a 293T cell line in the presence of 6 μg / ml hexadimethrine bromide (Polybrene). After 48 hours, the 293T cells were selected with 4 μg / ml puromycin to obtain a 293T positive cell line overexpressing the extracellular segment of the MOG antigen fused to EGFP. Subculture the cells at a maintained puromycin concentration of 2 μg / ml to obtain a 100% transfected 293T cell line stably expressing the extracellular segment of the MOG antigen fused to EGFP.

[0043] 1) Cell Culture: Nalm6 and 293T cell lines were routinely cultured in our laboratory. Nalm6 cell line was purchased from Wuhan Pronose Biotechnology Co., Ltd. under the catalog number CL-0701; 293T cell line was purchased from Wuhan Pronose Biotechnology Co., Ltd. under the catalog number CL-0005. Nalm6 cells were cultured in 1640 medium supplemented with 10% inactivated fetal bovine serum, while 293T cells were cultured in high-glucose DMEM supplemented with 10% inactivated fetal bovine serum. The cells were incubated at 37°C in a 5% CO2 incubator and cultured according to conventional methods. Cells in the logarithmic growth phase were used for experiments.

[0044] 2) Cell Preparation: Resuspend 293T cells in high-glucose DMEM supplemented with 10% FBS in a 10 cm dish and culture in a 37°C incubator with 5% CO2. Allow cells to adhere and reach a confluence of approximately 80%-90%. Transfection can then be performed. 2 hours before transfection, replace the old medium with 6-8 ml of fresh complete medium.

[0045] 3) Plasmid Transfection: Mix the core plasmid and packaging plasmids (psPAX2 and pMD2.G) thoroughly. Add 500 μl of low-serum medium to a sterile 1.5 ml EP tube, followed by 20 μg of the mixed plasmids (core plasmid:psPAX2:pMD2.G = 4:3:1). In another sterile 1.5 ml EP tube, add 500 μl of low-serum medium and 40 μl of Lipofectamine 2000 (cationic liposome transfection reagent) (Lipofectamine 2000:plasmid = 2:1). Mix thoroughly and let stand at room temperature for 5 minutes. Slowly add Lipofectamine 2000 to the plasmid tube, mix thoroughly, and let stand at room temperature for 15-20 minutes. Add 1 ml of the mixture dropwise to the 293T cell culture medium. Gently shake the plate to mix thoroughly, and incubate in a 37°C incubator with 5% CO2.

[0046] It should be noted that different core plasmids were used to package different lentiviral suspensions depending on the experimental purpose. When constructing the Nalm6-overexpressing 8-18C5 cell line, the 8-18C5 lentiviral core plasmid was used to package the lentiviral suspension; when constructing MOG CAAR-T, the MOG CAAR-T core plasmid was used to package the lentiviral suspension; when constructing CD19 CAR-T, the CD19CAR-T core plasmid was used to package the lentiviral suspension; when constructing a 293T cell line expressing the extracellular segment of the MOG antigen fused to EGFP, the MOG antigen extracellular segment fused to EGFP core plasmid was used to package the lentiviral suspension. When packaging the lentiviral suspension, the ratio of core plasmid to packaging plasmid was the same, and the lentiviral packaging steps were also the same.

[0047] 4) Virus Collection: After 4-6 hours, aspirate the old medium and add 12-15 ml of fresh complete medium pre-warmed at 37°C. Continue incubating the cells in the incubator. After approximately 72 hours, collect the supernatant into a 15 ml centrifuge tube. Filter the supernatant through a 0.45 μm filter and add 4× Lentiviral Concentrate (purchased from Takara, Cat. No. 631231). This concentrate can reduce the volume of the obtained viral suspension by 100-1000-fold, thereby increasing viral titer and enhancing infection efficiency. Incubate on a shaker at 4°C overnight. Centrifuge at 3500 g for 30 minutes. Resuspend the supernatant in PBS at 1 / 100 volume, aliquot, and store at -80°C until use.

[0048] 5) Virus titer determination (dilution counting method): Titer unit: TU / ml, refers to the number of biologically active virus particles contained in each milliliter. "TU" is the abbreviation of "transducing units", which means the number of viral genomes that can infect and enter the target cell. Different dilutions of the virus are used to infect the same number of 293T cells. On the fifth day, the proportion of cells with positive fluorescence expression is detected by flow cytometry to calculate the virus titer. The details are as follows: Day 1 Cell preparation: Digest and count 293T cells that are growing well and dilute to 1×10 4 / ml, add 100 μl / well to a 96-well plate, prepare 10 wells for each virus, and culture in a 37°C, 5% CO2 incubator.

[0049] On the second day, add virus: Perform 8-fold serial dilutions in EP tubes, for a total of eight dilutions. Measure the viral titers of the MOG CAAR-T lentivirus, CD19 CAR-T lentivirus, 8-18C5 BCR-overexpressing lentivirus, and MOG antigen extracellular domain fused to EGFP lentivirus. Dilution protocol: Prepare eight 1.5ml EP tubes for each virus, add 100µl of culture medium to each tube, add 50µl of the viral stock solution to the first tube, mix thoroughly, then pipette 50µl into the second tube and mix thoroughly. Repeat this process for eight dilutions. Add 50µl of the diluted viral solution to each well of cells in a 96-well plate, fill the well with culture medium to 200µl, and label the well.

[0050] On the fifth day, the results were observed and the titer was calculated: the infection efficiency was detected by flow cytometry, and the number of fluorescent cell clones with an infection efficiency of 10% to 20% was selected. The titer (TU / ml) = 1000 × infection efficiency × dilution factor × 20.

[0051] Viral infection and stable cell line screening: Cell lines in logarithmic growth phase were infected with viral concentrate (containing 6 μg / ml Polybrene) at an MOI of 5. The multiplicity of infection (MOI) refers to the ratio of the number of viruses capable of infecting cells to the total number of cells in a culture. After 48 hours, 2 μg / ml puromycin was added, and fresh screening medium was replaced approximately every two days. The 8-18C5 Nalm6 cell line and a 293T cell line containing the extracellular domain of MOG fused to EGFP fluorescent protein were constructed using this method. After approximately two weeks of culture, 8-18C5 BCR expression was assessed by flow cytometry.

[0052] Figure 4 The results of the 8-18C5 Nalm6 cell line test in Example 2 of the present application are shown. Figure 4 As shown, BCR structure (B cell receptor structure) can be detected in the 8-18C5Nalm6 cell line (Nalm6 8-18C5 group), while BCR structure is not detected in the Nalm6 wild-type cell line (control group, Nalm6 B cells were not treated, Nalm6 Control), indicating that 8-18C5BCR was successfully overexpressed in the Nalm6 B cell tool cell line. Figure 5 The results of the binding test between 8-18C5 and the extracellular segment of MOG antigen in Example 2 of the present application are shown. Figure 5 As shown, the extracellular segment of 293T MOG antigen was fused with EGFP. The cell line could normally express EGFP fluorescence and successfully bind to 8-18C5 soluble protein.

[0053] Example 3: Extraction of human peripheral blood mononuclear cells (PBMC) and construction of MOG CAAR-T cells 1) PBMC extraction: Human peripheral blood was obtained from inpatients in the Department of Neurology, Tianjin Medical University General Hospital, and all patients signed informed consent. Take 3 ml of fresh anticoagulated blood and mix it with 3 ml of PBS. Take another 15 ml centrifuge tube and add 6 ml of human peripheral blood lymphocyte separation solution. Human peripheral blood lymphocyte separation solution was purchased from TBD with the product number LTS1077 for extracting PBMC cells. Carefully spread the diluted blood sample on the separation solution, and centrifuge at 2000 rpm with the speed of 1 increase and 1 decrease set for 20 minutes. After centrifugation, the centrifuge tube is divided into 4 layers from top to bottom. The white ring layer is PBMC. Add 3 times the volume of washing solution and mix it. Centrifuge at 2000 rpm for 5 minutes. Add 1 ml of red blood cell lysis solution and mix it by pipetting. Let it stand at room temperature for 10 minutes. Add 3 times the volume of washing solution and mix it. Centrifuge at 2000 rpm for 5 minutes. PBMC cells can be obtained and the cells are used for magnetic bead sorting to obtain CD3 + T cells.

[0054] 2) CD3 + T cell sorting: The PBMCs were counted and the number of 7 Resuspend the cells in 40ul MACS Buffer and add 10ul CD3 + Antibodies used for T cell sorting were mixed and incubated at 4°C in the dark for 5 minutes. 7 Add 30ul MACS Buffer and 20ul CD3 + The magnetic beads used for T cell sorting were mixed and incubated in the dark at 4°C for 10 minutes. 3 ml of MACS Buffer was added and the cells were centrifuged at 300 g for 10 minutes. 3 ml of MACS Buffer was added to resuspend the cells.

[0055] Prepare the LS column in advance, add 3 ml of MACS Buffer to rinse the column, add the cell suspension, and wash the column twice, adding 3 ml of MACS Buffer each time. Collect the liquid flowing out of the column and centrifuge it. Resuspend it in 1 ml of 1640 medium containing 10% FBS, count the cells after resuspension, and adjust the density to 1×10 6 / ml, cultured in well plates, and added corresponding amount of CD3 / CD28 activator per ml of cells.

[0056] 3) Construction of CD19 CAR-T cells and MOG CAAR-T cells After 24 hours of CD3 / CD28 activation, the corresponding virus concentrate was added to infect T cells at an MOI of 5. Fresh complete culture medium was replaced every 2 days. The T cell infection efficiency was detected by flow cytometry 7-9 days later, and a luciferase killing experiment was performed. Figure 6 The results of T cell infection efficiency in Example 3 of the present application are shown, wherein NTD-T cells do not have a killing effect and are used as a negative control, CD19 CAR-T cells can kill cells expressing CD19 including Nalm6 cell lines or B cells in patients, and are used as a positive control to detect the killing effect of MOG CAAR-T, and MOG-CAART is MOG CAAR-T cells.

[0057] Example 4: Firefly luciferase killing assay to detect the killing efficiency of CAAR-T cells.

[0058] The firefly luciferase killing assay is a mature scientific method to indicate the degree of cell killing. Luciferase is pre-transferred into the 8-18C5 Nalm6 target cell line. After the killing assay, firefly luciferase substrate is added and the chemiluminescence value is detected. If a large number of surviving target cells and a high chemiluminescence value indicate that CAAR-T cells have not killed the target cells, while a small number of surviving target cells and a low chemiluminescence value indicate that CAAR-T cells have successfully killed the target cells.

[0059] 1) Plating of 8-18C5 Nalm6 cell line: Adjust the target cell density to 1×10 4 / ml, cells were seeded in a white opaque ELISA plate for chemiluminescence detection, 100ul of cells were added to each well, a portion of cells was not added with T cells as a negative control, and another portion of cells was centrifuged and resuspended in sterile water as a positive control 2) Add NTD-T cells, CD19 CAR-T cells, and MOG CAAR-T cells at varying E:T ratios (0.5:1, 1:1, 2:1, 5:1, and 8:1, respectively). NTD-T cells have no cytotoxic effect as a negative control, while CD19 CAR-T cells can kill cells expressing CD19, including the Nalm6 cell line or patient B cells, and serve as a positive control to test the cytotoxicity of MOG CAAR-T cells. The culture volume in each well was filled to 200 μL, and the co-cultured cells were incubated at 37°C in a 5% CO2 incubator.

[0060] 3) The killing efficiency was determined by measuring the chemiluminescence value using a multifunctional microplate reader at 5 and 24 hours of co-culture. Figure 7 The results of CAAR-T cell killing in Example 4 of the present application are shown. NTD-T cells were used as a negative control, and CD19CAR-T cells were used as a positive control to detect the killing efficiency of MOG CAAR-T cells. Killing efficiency = (negative well value - target well value) / (negative well value - positive well value). Figure 7 As shown in the figure, the test results indicate that MOG CAAR-T cells can successfully kill 8-18C5 Nalm6 target cells. At 5 hours and 24 hours, MOG CAAR-T cells kill the 8-18C5 Nalm6 cell line at a similar efficiency as CD19 CAR-T cells, suggesting that MOG CAAR-T cells can exert the same killing effect as CD19 CAR-T cells. In addition, MOG CAAR-T cells cannot successfully kill the Nalm6 control cell line (i.e., a cell line that only expresses CD19 and does not express MOG-specific antibodies), indicating that MOG CAAR-T cells can only bind to and kill cell lines that express MOG antibody sequences.

[0061] 4) Soluble protein killing experiment: The cell density of 8-18C5 Nalm6 cells was adjusted to 1×10 4 / ml. Cells were seeded in white opaque ELISA plates for chemiluminescence detection. 100 μl of cells were added to each well. A portion of cells was not added with T cells as a negative control, and another portion of cells was centrifuged and resuspended in sterile water as a positive control. MOG CAAR-T cells were added to both groups at an E:T ratio of 10:1. 8-18C5 soluble protein was synthesized by GenScript Biotech Co., Ltd. The sequence of the 8-18C5 soluble protein plasmid is similar to that of the 8-18C5 overexpression plasmid in that the light chain variable region, heavy chain variable region, and light chain constant region are identical. The difference between the two plasmids is that the heavy chain constant region of the 8-18C5 soluble protein lacks the transmembrane region and the intracellular topological domain. The heavy chain sequence of the 8-18C5 soluble protein is set forth in SEQ ID NO: 14. The soluble protein density was adjusted to 100ug / ml and added to the co-culture system. The culture system was replenished to 200ul and incubated in a 37-degree 5% CO2 incubator for 24 hours. The killing efficiency was detected by a multifunctional microplate reader. Figure 8 The results of the soluble protein killing experiment in Example 4 of the present application are shown as follows: Figure 8 As shown, the presence of soluble protein did not affect the killing efficiency of MOG CAAR-T cells.

[0062] Example 5: Cell Immunofluorescence Detection of the Binding Efficiency of Soluble Proteins to the Extracellular Segment of MOG Antigen 1) Cell inoculation: Mix a 293T cell line stably transduced with the extracellular domain of the MOG antigen and a wild-type 293T cell line (untreated) at a ratio of 7:3. Resuspend in culture medium and passage the cells onto a 24-well plate covered with a cell slide. Perform subsequent assays when the cells reach 80% confluency.

[0063] 2) Cell immunofluorescence: Aspirate the old culture medium, wash the cells 3 times with PBS, fix them with 4% paraformaldehyde for 15 minutes at room temperature, wash them with PBS 3 times for 5 minutes each time, block them with 5% BSA in PBS for 1 hour, add soluble protein 8-18C5 diluted in PBS * Soluble protein was synthesized by GenScript Biotechnology Co., Ltd. at a final concentration of 5 μg / ml and incubated overnight at 4°C in the dark. The sections were washed three times with PBS for 5 minutes each. Biotin-conjugated human IgG antibody (Biotin-SP-conjugate daffinipure goat anti-human IgG, diluted 1:700 in PBS, total volume 50 μl) was added and incubated at room temperature for 30 minutes. The sections were washed three times with PBS for 5 minutes each. Alex fluor 647-conjugated streptavidin secondary antibody (1:700 in PBS, total volume 50 μl) was added, and the sections were washed three times with PBS for 5 minutes each. The sections were then mounted with mounting medium.

[0064] 3) Fluorescence microscopy analysis: Using cells not incubated with antibodies as a control, the expression of red fluorescence was used to indicate whether soluble proteins bound to the extracellular segment of MOG protein under a 20x objective lens ( Figure 5 ), the results showed that the extracellular segment of MOG antigen can bind to soluble 8-18C5.

[0065] Example 6: Experiment on the killing of PBMCs by CAAR-T cells in MOGAD patients Blood from MOGAD patients and from patients for constructing NTD-T cells, MOG CAAR-T, and CD19 CAR-T cells were obtained from inpatients in the Department of Neurology at Tianjin Medical University General Hospital. All patients signed informed consent forms. PBMCs from MOGAD patients were extracted according to the experimental procedures in Example 3, and NTD-T cells, CD19 CAR-T cells, and MOG CAAR-T cells were constructed. After co-culturing NTD-T cells, CD19 CAR-T cells, and MOG CAAR-T cells with MOGAD patient PBMCs, the proportions of B cells and B cell subsets were detected by flow cytometry.

[0066] 1) PBMC plating: PBMCs were extracted from the blood of MOGAD patients and counted, and the density was adjusted to 1×10 6 / ml, 500,000 PBMC cells were added to each well, and NTD-T cells, CD19 CAR-T cells and MOG CAAR-T cells were added in a 1:1 ratio, and incubated in a 37-degree constant temperature incubator for 24 hours.

[0067] 2) Flow cytometry detection of B cell subset proportions: The remaining cells were collected and stained by flow cytometry. After adding B cell subset-related flow cytometry antibodies such as CD19, CD27, IgD, and CD38, the cells were incubated in the dark at 4°C for 30 minutes. The cells were washed twice with PBS and the changes in each B cell subset were detected by flow cytometry.

[0068] Figure 9 The results of flow cytometry detection of B cells and B cell subsets in Example 6 of the present application are shown, wherein 9A is a schematic diagram of the results after culturing NTD-T cells, CD19 CAR-T cells and MOG CAAR-T cells with MOG AD patients. The results show that compared with the NTD-T cell culture group, CD19 CAR-T cells kill most of the CD19-positive B cell populations, while MOG CAAR-T cells retain most of the B cell subsets; 9B is a diagram of memory B cells (CD19) after culturing NTD-T cells, CD19 CAR-T cells and MOG CAAR-T cells with MOG AD patients. + CD27 + IgD -) data analysis chart; 9C is a schematic diagram of the B cell subsets after culturing NTD-T cells, CD19CAR-T cells and MOG CAAR-T cells with MOG AD patients. Memory B cells and double-negative B cells (DN B cells) are currently believed to have pathogenic effects on neuroimmune diseases. The results show that compared with NTD-T cells, CD19 CAR-T cells kill most types of B cells, leaving most cells in the initial B cells (CD19 + IgD + CD27 - ) state, MOG CAAR-T cells mainly killed memory B cells and some double-negative B cells, and had no effect on non-converted memory B cells (CD19 + CD27 + IgD + ) cell subsets; 9D is the double negative B cells (CD19) cultured from NTD-T cells, CD19 CAR-T cells and MOG CAAR-T cells with MOG AD patients. + CD27 - IgD - ) Data analysis chart. Figure 9 As shown, compared to the NTD-T cell co-culture group, the CD19 CAR-T cell killing group killed the majority of B cells. However, compared to the CD19 CAR-T cell co-culture group, the MOG CAAR-T cell co-culture group preserved most B cell subsets, killing only plasmablasts, plasma cells, memory B cells, and some double-negative B cells. This preserved subsets of cells, including non-switched memory B cells and naive B cells, thereby preserving the function of other B cells to the greatest extent possible. Furthermore, the antibody sequences in MOGAD patients are diverse, and the MOG CAAR-T cell killing results revealed that MOG CAAR-T cells can recognize and subsequently kill B cells expressing MOG antigen-specific antibodies.

[0069] Through the above specific embodiments, those skilled in the art can easily implement the present application. However, it should be understood that the present application is not limited to the above specific embodiments. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to implement different technical solutions.

Claims

1. A plasmid vector, characterized in that include: A chimeric autoantibody receptor, wherein the chimeric autoantibody receptor is composed of a signal peptide, an extracellular segment of the MOG antigen, a transmembrane region CD8α, a costimulatory domain 4-1BB, and an activation domain CD3ξ from the amino terminus to the carboxyl terminus; The base sequence of the signal peptide is shown in SEQ ID NO: 1; The base sequence of the extracellular segment of the MOG antigen is shown in SEQ ID NO: 2; The base sequence of the transmembrane region CD8α is shown in SEQ ID NO: 3; The base sequence of the costimulatory domain 4-1BB is shown in SEQ ID NO: 4; The base sequence of the activation domain CD3ξ is shown in SEQ ID NO:

5.

2. A chimeric MOG antibody receptor T cell, characterized in that: The chimeric MOG antibody receptor T cells are obtained by transfecting the plasmid vector and the packaging plasmid described in claim 1 into 293T cells, wherein the packaging plasmids are psPAX2 and pMD2.G, the transfection reagent is Lipofectamine 2000, and the mass ratio of the transfection reagent, the plasmid vector, the psPAX2 and the pMD2.G is 16:4:3:

1.

3. The chimeric MOG antibody receptor T cell according to claim 2, characterized in that: The chimeric MOG antibody receptor T cells can kill B cells expressing specific MOG antibodies.

4. The chimeric MOG antibody receptor T cell according to claim 3, characterized in that: The MOG antibody is 8-18C5 antibody.

5. The chimeric MOG antibody receptor T cell according to claim 4, characterized in that: The B cells are Nalm6 B cells.

6. Use of the plasmid vector according to claim 1 or the chimeric MOG antibody receptor T cell according to any one of claims 2 to 5 in the preparation of a drug for treating MOGAD.

7. The use according to claim 6, characterized in that The drug can specifically kill pathogenic B cells expressing MOG antibodies.

8. The use according to claim 7, characterized in that The MOG antibody is 8-18C5 antibody.

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