Chimeric AQP4 antibody receptor, CAAR-T cell and application

Abstract

The invention discloses a chimeric AQP4 antibody receptor, a CAAR-T cell and application. The chimeric AQP4 antibody receptor comprises an AQP4 antibody binding domain, a costimulatory domain and an activation domain which are sequentially connected from an amino terminal to a carboxyl terminal, wherein the AQP4 antibody binding domain is the full length of AQP4 M1 or the full length of AQP4 M23, the nucleotide sequence of the full length of AQP4 M1 is as shown in SEQ ID NO: 4, and the nucleotide sequence of the full length of AQP4 M23 is as shown in SEQ ID NO: 5. The full-length AQP4 M1 and M23 subtype proteins are expressed on the surface of the T cell through a gene editing technology, the constructed CAAR-T cell can specifically recognize and attack B cells secreting pathogenic antibodies and retain other normal B cell subsets, the effect is remarkable, and the risk caused by long-term use of biological agents is reduced.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy technology, and in particular to a chimeric AQP4 antibody receptor, CAAR-T cells and applications thereof. Background Art

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an antibody-mediated, complement-involved autoimmune disease that primarily targets aquaporin 4 (AQP4) on astrocytes. It predominantly affects young and middle-aged individuals, with a preponderance of women. It is characterized by high relapse rates and severe disability. The etiology of NMOSD is primarily related to autoantibodies against AQP4. The AQP4 antibody-mediated immune inflammatory response primarily targets areas of the central nervous system with high AQP4 expression, primarily the optic nerves and spinal cord, causing recurrent episodes of optic neuritis and / or myelitis. Furthermore, the periependymal region, such as the area postrema of the medulla oblongata, the thalamus, the hypothalamus, the periphery of the third and fourth ventricles, and the corpus callosum, is also a common site of disease. Recurrent attacks of NMOSD can lead to irreversible neurological disability in patients.

[0003] In clinical practice, rituximab (anti-CD20 monoclonal antibody), inebilizumab (anti-CD19 monoclonal antibody), satralizumab and tocilizumab (anti-IL-6 monoclonal antibody), eculizumab (anti-C5 monoclonal antibody), azathioprine, or mycophenolate mofetil are the mainstays of treatment for NMOSD. Although biologics significantly reduce relapse rates, breakthrough relapses still occur in 20%-30% of patients, limiting their efficacy. Furthermore, biologics carry specific risks, and long-term use of immunosuppressants or biologics may increase the risk of opportunistic infections.

[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 chimeric AQP4 antibody receptor, CAAR-T cells and applications. The present application expresses the full-length AQP4 M1 and M23 subtype proteins on the surface of T cells through gene editing technology, so that the AQP4 protein maintains its own protein conformation and connects the co-stimulatory domain and activation domain to obtain a chimeric AQP4 antibody receptor and construct CAAR-T cells. The CAAR-T cells can specifically recognize and attack B cells that secrete pathogenic antibodies while retaining other normal B cell subsets, with significant effects and reducing the risks brought by long-term use of biological agents.

[0006] To achieve the above objectives, this application provides the following technical solutions: According to one aspect of the present application, a chimeric AQP4 antibody receptor is provided, comprising an AQP4 antibody binding domain, a costimulatory domain, and an activation domain sequentially connected from the amino terminus to the carboxyl terminus; wherein the AQP4 antibody binding domain is the full-length AQP4 M1 or the full-length AQP4 M23, the nucleotide sequence of the full-length AQP4 M1 is shown in SEQ ID NO: 4, and the nucleotide sequence of the full-length AQP4 M23 is shown in SEQ ID NO: 5.

[0007] In some embodiments, the costimulatory domain is 4-1BB, and the nucleotide sequence of 4-1BB is shown in SEQ ID NO:6; the activation domain is CD3ξ, and the nucleotide sequence of CD3ξ is shown in SEQ ID NO:7.

[0008] According to another aspect of the present application, a recombinant vector is also provided, comprising the chimeric AQP4 antibody receptor.

[0009] In some embodiments, the recombinant vector is a lentiviral plasmid vector.

[0010] According to another aspect of the present application, a CAAR-T cell is also provided, wherein the CAAR-T cell comprises the recombinant vector.

[0011] In some embodiments, the CAAR-T cells are AQP4 M1 CAAR-T cells or AQP4 M23 CAAR-T cells.

[0012] In some embodiments, the CAAR-T cells are capable of binding to plasma cell antibodies in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorder, wherein the plasma cell antibody is one of Clone2 antibody, Clone8 antibody, Clone17 antibody, Clone19 antibody, Clone22 antibody, and Clone25 antibody, the heavy chain variable region nucleotide sequence of the Clone2 antibody is shown in SEQ ID NO:9, the light chain variable region nucleotide sequence of the Clone2 antibody is shown in SEQ ID NO:13, the heavy chain variable region nucleotide sequence of the Clone8 antibody is shown in SEQ ID NO:10, the light chain variable region nucleotide sequence of the Clone8 antibody is shown in SEQ ID NO:14, the heavy chain variable region nucleotide sequence of the Clone17 antibody is shown in SEQ ID NO:17, the light chain variable region nucleotide sequence of the Clone17 antibody is shown in SEQ ID NO:18, the heavy chain variable region nucleotide sequence of the Clone19 antibody is shown in SEQ ID NO:19, and the light chain variable region nucleotide sequence of the Clone19 antibody is shown in SEQ ID NO: NO:20, the nucleotide sequence of the heavy chain variable region of the Clone22 antibody is shown in SEQ ID NO:21, the nucleotide sequence of the light chain variable region of the Clone22 antibody is shown in SEQ ID NO:22, the nucleotide sequence of the heavy chain variable region of the Clone25 antibody is shown in SEQ ID NO:23, and the nucleotide sequence of the light chain variable region of the Clone25 antibody is shown in SEQ ID NO:24.

[0013] According to another aspect of the present application, there is also provided the use of the chimeric AQP4 antibody receptor, the recombinant vector and the CAAR-T cell in the preparation of a drug for treating neuromyelitis optica spectrum disorders.

[0014] In some embodiments, the drug is capable of specifically killing cells expressing the AQP4 antibody.

[0015] Compared with the existing technology, the advantages and positive effects of this application are: this application expresses the full length of AQP4 M1 and M23 subtype proteins on the surface of T cells through gene editing technology, so that the AQP4 protein maintains its own protein conformation, and connects the costimulatory domain and the activation domain to obtain a chimeric AQP4 antibody receptor and construct CAAR-T cells. The CAAR-T cells can specifically recognize and attack B cells that secrete pathogenic antibodies while retaining other normal B cell subsets, which has a significant effect and reduces the risks brought by the long-term use of biological agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 Shown is a schematic diagram of the AQP4 protein structure.

[0018] Figure 2 A heat map of VJ gene rearrangement in the Smartseq3 experiment in the examples of this application is shown.

[0019] Figure 3 The V gene occurrence frequency, J gene occurrence frequency and CDR3 relative frequency of the Smartseq3 experiment in the examples of this application are shown.

[0020] Figure 4 The results of cell immunofluorescence detection in the examples of this application are shown.

[0021] Figure 5 Shown is a schematic diagram of the construction of the AQP4 plasmid vector in the examples of this application.

[0022] Figure 6 A schematic diagram of the construction of the Clone2 / Clone8 / rAb53 plasmid in the examples of this application is shown.

[0023] Figure 7 Schematic diagram of the construction of the AQP4 M23 subunit fused EGFP plasmid vector in the examples of this application is shown.

[0024] Figure 8 The figures show the killing efficiency of rAb53 Nalm6 B cells by each group in the examples of this application.

[0025] Figure 9 The killing efficiency of each group in the examples of this application on Clone2 and Clone8 cell lines is shown.

[0026] Figure 10 The results of the soluble protein killing experiment in the examples of this application are shown.

[0027] Figure 11 The concentrations of IFNγ and Granzyme B cell factor in each group in the examples of this application are shown. DETAILED DESCRIPTION

[0028] 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.

[0029] NMOSD: Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune disease of the central nervous system in which the patient's immune system invades the optic nerve and spinal cord, leading to vision loss and paralysis. It is an antibody-mediated, complement-involved autoimmune disease with aquaporin 4 (AQP4) on astrocytes as the main immune damage target.

[0030] AQP4: Aquaporin 4, Figure 1 A schematic diagram of the AQP4 protein structure is shown. AQP4 has two isoforms: AQP4-M1 and AQP4-M23. M23 lacks the first 22 amino acids of the M1 subunit, all of which are located intracellularly. M23 has three discrete extracellular domains: pep3 (loop A), pep5 (loop C), and pep7 (loop E). Loop A consists of 11 extracellular amino acids, loop C consists of 18 amino acids, and loop E consists of 25 amino acids, 11 of which are located intracellularly.

[0031] CAAR-T: Chimeric autoantibody receptor T cell therapy. Its core principle lies in the ingenious use of genetic engineering technology to precisely modify T cells extracted from the patient in vitro, so that they express the pathogenic sequence of the antigen protein and connect the transmembrane region and intracellular domain. These modified T cells are then returned to the patient's body, enabling them to specifically recognize and attack B cells that secrete pathogenic antibodies while retaining other normal B cell subsets, thereby achieving the purpose of treating the disease.

[0032] Smartseq3 technology: Single-cell transcriptome sequencing technology, with significant advantages such as high resolution and wide coverage, can perform comprehensive and detailed analysis of gene expression in individual cells.

[0033] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0034] 1. Obtain the full-length sequences of plasma cell antibodies in the cerebrospinal fluid of NMOSD patients.

[0035] The patient's cerebrospinal fluid samples were obtained from inpatients in the Department of Neurology at Tianjin Medical University General Hospital, and the patients have signed informed consent. The full-length sequence of plasma cell antibodies in the cerebrospinal fluid of NMOSD patients was obtained and synthesized using SmartSeq3 technology. The cerebrospinal fluid obtained from NMOSD patients was stained, and after staining, the plasma cells in the cerebrospinal fluid of NMOSD patients were sorted out using a flow cytometer and sorted into a 96-well plate as single cells. After adding lysis buffer, the full-length sequence of the B cell receptor (BCR) on the surface of the single cell was predicted through reverse transcription and template conversion, PCR pre-amplification, Tn5 transposase fragmentation, and sequencing library construction. The details are as follows: (1) Cerebrospinal fluid sample processing: 10 ml of NMOSD patient cerebrospinal fluid was centrifuged at 1800 rpm for 6 minutes. The supernatant was carefully aspirated, leaving only 100 μl of cerebrospinal fluid, which was gently pipetted and mixed. Four flow cytometry antibodies, APC-CY7 CD19, BV421 CD27, FITC CD38, and Percp5.5 7AAD, were added. - The cell population is the living cell population, CD19 + The cell subset is a B cell subset, CD19 + CD27 + CD38 + The cell subpopulation is the target cell population, namely the plasma cell subpopulation, and incubate on ice in the dark for 30 minutes.

[0036] (2) Flow cytometry sorting: First, adjust the 4-channel fluorescence voltage and compensation, and start the sorting program of the flow cytometer. According to the gate setting, the target cell population is separated from the sample. The sorted target cell population is 7AAD. - CD19 + CD27 high CD38 high Use a 96-well plate and add cell lysis buffer to the bottom of the plate in advance to sort single cells. After sorting, carefully remove the plate and seal it with a clean sealing film to avoid cell contamination or loss.

[0037] (3) Plasma cell BCR sequence acquisition: Smartseq3 experiment was completed by Beijing BioIntelligence Technology Co., Ltd.

[0038] (4) Sequence analysis: Recombinant antibodies are composed of two parts: light chain and heavy chain. Whether it is the light chain or the heavy chain of the antibody, its variable region is encoded by one exon, but this exon is recombined from multiple gene segments. Gene segments can be divided into four types: V (Variable), D (Diversity), J (Joining), and C (Constant). Only three gene segments, V, D, and J, participate in variable region recombination. The C gene encodes the constant region of the antibody and is connected to the VJ or VDJ segment after transcription. The light chain has no D gene segment, so the light chain undergoes VJ recombination and the heavy chain undergoes VDJ recombination. Therefore, after we obtain Smartseq3 data, we can obtain the V genes and J genes of different antibodies and their frequency of occurrence. The antigen binding region of the antibody has three variable regions: CDR1, CDR2, and CDR3. CDR1 and CDR2 are located in the middle of the V gene segment, while the CDR3 region is generated by V(D)J rearrangement. Therefore, the diversity of CDR3 is much greater than that of CDR1 and CDR2. After excluding unqualified sequences from the obtained sequences, the clonal amplification and immune diversity of the final obtained sequences were determined by analyzing VJ gene rearrangement, the frequency of CDR3 region duplication, the frequency of V gene appearance, and the frequency of J gene appearance. Figure 2 A heat map of the VJ gene rearrangement pairing in the Smartseq3 experiment in an embodiment of the present application is shown. The vertical axis represents the V gene detected by Smartseq3, and the horizontal axis represents the J gene detected by Smartseq3. The more VJ gene rearrangements appear in the figure (i.e., the frequency of occurrence is greater than 0), the greater the antibody diversity in the patient's cerebrospinal fluid cells. The higher the frequency of occurrence of different VJ gene rearrangement processes in the figure, the higher the clonal amplification rate of the VJ gene rearrangement method. Figure 3 The V gene frequency, J gene frequency, and CDR3 relative frequency of the Smartseq3 experiment in the examples of this application are shown, respectively indicating which types of V genes and J genes plasma cells in the cerebrospinal fluid of NMOSD patients are more likely to synthesize. CDR3 is more diverse than CDR1 and CDR2. The higher the frequency of CDR3, the higher the clonal amplification of recombinant antibodies with this CDR3. Relative V Usage (V gene frequency), Relative J Usage (J gene frequency), Relative Frequency (CDR3 relative frequency) After gene rearrangement of a large number of antibody sequences, a total of 37 antibody arrangements were discovered. Therefore, we synthesized these 37 antibody sequences and present them in the form of recombinant secreted proteins.

[0039] (5) Antibody crude protein synthesis: 37 pairs of crude protein supernatants were synthesized by Sino Biological Technology Co., Ltd.

[0040] 2. Cell immunofluorescence was used to detect the binding efficiency of 37 soluble proteins with the M23 subtype of AQP4 protein.

[0041] According to gene rearrangement ( Figure 2-3 ) sequence, we synthesized 37 pairs of antibody crude protein supernatants with plasmids, and performed cell immunofluorescence detection on the synthesized 37 pairs of crude protein supernatants. Figure 4 The results of cell immunofluorescence detection in the examples of this application are shown. Figure 4 ) showed that 6 pairs of antibodies could bind to 293T cells expressing the M23 subtype of AQP4 protein, proving that these 6 pairs of antibodies were specific antibodies for AQP4 protein.

[0042] The details are as follows: (1) Construction of a lentiviral plasmid vector containing the AQP4 M23 subunit fused with EGFP The human AQP4 antigen M23 subunit sequence (protein number: P55087-2 amino acid sequence M1-V301) was downloaded from the Uniprot website. The protein terminal was fused with the fluorescent protein EGFP. The sequence design is shown in ( Figure 7 ), The full-length base sequence of AQP4 M23 is shown in SEQ ID NO: 5. The base sequence of EGFP is shown in SEQ ID NO: 25.

[0043] 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.

[0044] (2) Using 293T cells as a tool cell line, a lentivirus expressing the AQP4 M23 subtype fused to EGFP was packaged using the CaCl2 method. The viral suspension was collected and infected with 293T cells in the presence of 6 μg / ml Polybrene. After one week, positive stably transfected cells were sorted. Subculture was continued to obtain a 100% transfected 293T cell line stably expressing the AQP4 M23 subtype fused to EGFP. For specific implementation methods, see "IV. Lentivirus Packaging and Obtaining Stably Transfected Cell Lines."

[0045] (3) Cell inoculation: Mix the 293T cell line stably transfected with the extracellular segment of the AQP4 M23 antigen gene and the wild-type 293T cell line in a ratio of 7:3, and passage them into a 24-well plate covered with cell slides. When the cells reach 80% confluence, perform subsequent testing.

[0046] (4) Cell immunofluorescence: The old culture medium was aspirated, the cells were washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 15 minutes. The cells were washed 3 times with PBS for 5 minutes each time, blocked with 5% BSA in PBS for 1 hour, and soluble crude protein diluted with PBS was added. The soluble protein was synthesized by Sino-Bio Technologies Co., Ltd. and diluted at a ratio of 1:10, 1:32, and 1:100. 50 μl was added to each well and incubated overnight at 4 degrees in the dark. The cells were washed 3 times with PBS for 5 minutes each time, and biotin-SP-conjugated affinipure goat anti-human IgG (1:700, diluted in PBS, total volume 50 μl) was added and incubated at room temperature for 30 minutes. The cells were washed 3 times with PBS for 5 minutes each time. Alexfluor 647-conjugated streptavidin (1:700, diluted in PBS, total volume 50 μl) was added. The cells were washed 3 times with PBS for 5 minutes each time. Mount the sections with mounting medium.

[0047] (5) Fluorescence microscopy analysis: Using cells not incubated with antibodies as a control, the expression of red fluorescence was used to indicate whether the soluble protein bound to the extracellular segment of AQP4 protein M23 under a 20x objective lens. The results were ( Figure 4 ) showed that the extracellular segment of the AQP4 antigen M23 subunit can bind to 6 pairs of soluble antibody sequences.

[0048] The nucleotide sequences of these six pairs of antibodies are: The heavy chain variable region sequence of Clone2 is shown in SEQ ID NO:9; The light chain variable region sequence of Clone2 is shown in SEQ ID NO: 13; The heavy chain variable region sequence of Clone8 is shown in SEQ ID NO: 10; The light chain variable region sequence of Clone8 is shown in SEQ ID NO: 14; The heavy chain variable region sequence of Clone17 is shown in SEQ ID NO: 17; The light chain variable region sequence of Clone17 is shown in SEQ ID NO: 18; The heavy chain variable region sequence of Clone19 is shown in SEQ ID NO: 19; The light chain variable region sequence of Clone19 is shown in SEQ ID NO:20; The heavy chain variable region sequence of Clone22 is shown in SEQ ID NO:21; The light chain variable region sequence of Clone22 is shown in SEQ ID NO:22; The heavy chain variable region sequence of Clone25 is shown in SEQ ID NO: 23; The sequence of the light chain variable region of Clone25 is shown in SEQ ID NO: 24.

[0049] 3. Construction of AQP4 CAAR-T, Clone2 / Clone8 / rAb53 Nalm6 B cell line plasmid vectors.

[0050] 1. Construction of AQP4 CAAR-T lentiviral plasmid vector The human AQP4 antigen sequence was downloaded from the Uniprot official website, and the AQP4 pep3-pep7 sequence (protein number: P55087-1 amino acid sequence: N58-T208), M1 full-length sequence (protein number P55087-1 amino acid sequence M1-V323) and M23 full-length sequence (P55087-2 amino acid sequence M1-V301) were selected as the antigen recognition region of CAAR-T cells, and the same costimulatory domain 4-1BB and activation domain CD3ξ as those in CD19 CAR-T in WO2019 / 159193A1 (country: India, publication date: 2019-08-22) were selected. The secretory peptide base sequence is shown in SEQ ID NO: 1; The base sequence of CD8α transmembrane region is shown in SEQ ID NO: 2; The base sequence of AQP4 pep3-pep7 is shown in SEQ ID NO: 3; The full-length base sequence of AQP4 M1 is shown in SEQ ID NO: 4; The full-length base sequence of AQP4 M23 is shown in SEQ ID NO: 5; The base sequence of 4-1BB is shown in SEQ ID NO: 6; The base sequence of CD3ξ is shown in SEQ ID NO:7.

[0051] After the 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 AQP4 CAAR-T lentiviral plasmid vector. Figure 5 Schematic diagram of the construction of the AQP4 plasmid vector in the embodiment of the present application is shown, wherein: Figure 5 a is the AQP4 pep3-pep7 CAAR-T plasmid vector, Figure 5 b is the AQP4 M1 CAAR-T plasmid vector, Figure 5c is the AQP4 M23 CAAR-T plasmid vector. It should be noted that AQP4 has two subunits (M1 and M23), M1 has 323 amino acids, and M23 has 301 amino acids. The difference between the two is that at the beginning of the protein, M1 has 21 more amino acids than M23 (i.e. AQP4 pep1), that is, Figure 5 The sum of the blue and brown parts in b is the full length of AQP4 M1. M1 and M23 have different spatial conformations ( Figure 1 ), M23 will aggregate more to form OAP structure, and M1 is less likely to form OAP structure.

[0052] 2. Construction of Clnoe2 / Clone8 / rAb53 Nalm6 BCR-overexpressing cell lines The heavy chain variable region VH of the verified Clone2 / Clone8 / rAb53 sequence was connected to the heavy chain constant region IgG1, and the light chain variable region VL was connected to the human light chain constant region κ chain. rAb53 is a recombinant p53 adenovirus (rAd-p53). rAb53 is a positive antibody for AQP4 and can specifically bind to AQP4. It is used to verify whether CAAR-T cells can successfully kill. Sequence design is shown in Figure 6 .

[0053] in, The signal peptide base sequence is shown in SEQ ID NO: 8; The base sequence of the Clone2 heavy chain variable region (VH) is shown in SEQ ID NO: 9; The base sequence of the heavy chain variable region (VH) of Clone 8 is shown in SEQ ID NO: 10; The base sequence of the rAb53 heavy chain variable region (VH) is shown in SEQ ID NO: 11; The base sequence of the heavy chain constant region IgG1 is shown in SEQ ID NO: 12; The base sequence of the light chain variable region (VL) of Clone2 is shown in SEQ ID NO: 13; The base sequence of the Clone8 light chain variable region (VL) is shown in SEQ ID NO: 14; The base sequence of the rAb53 light chain variable region (VL) is shown in SEQ ID NO: 15; The base sequence of the light chain constant region κ (CLκ) is shown in SEQ ID NO: 16.

[0054] After the sequence design, it was synthesized by Jinweizhi Biotechnology Co., Ltd. and ligated to the pCDH plasmid vector double-digested with XbaI and BamHI to obtain a lentiviral plasmid vector overexpressing Clone2 / Clone8. This plasmid sequence was derived from plasma cells in the cerebrospinal fluid of NMOSD patients. Therefore, we constructed the Clone2 / Clone8 Nalm6 cell line and conducted in vitro killing experiments, which are of great significance for the diagnosis and treatment of NMOSD disease.

[0055] 4. Lentivirus packaging and acquisition of stably transfected cell lines.

[0056] Using 293T cells as a tool cell line, a lentivirus expressing the target gene was packaged using the CaCl2 method. The viral suspension was collected and used to infect Nalm6 cells in the presence of 6 μg / ml Polybrene. After one week, positive stably transfected cells were sorted. Continued subculture resulted in a 100% transfected Nalm6 cell line stably expressing the fusion protein.

[0057] 1) Cell Culture: Nalm6 and 293T cell lines were routinely cultured in our laboratory. Nalm6 cells were cultured in 1640 medium supplemented with 10% inactivated fetal bovine serum, and 293T cells were cultured in high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum. The cells were placed in a 37°C, 5% CO2 incubator and cultured according to conventional methods. Cells in the logarithmic growth phase were used for experiments.

[0058] 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 containing 5% CO2. Wait until the cells adhere to the wall and reach a confluence density 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. 3) Plasmid transfection: Mix the core plasmid and packaging plasmid, take a sterile 1.5ml EP tube, add 500ul Opti-MEM, add 20ug of mixed plasmid (core plasmid: psPAX2: pMD2.G = 4:3:1), take another sterile 1.5ml EP tube, add 500ul Opti-MEM, add 40ul Lipofectamine 2000 (Lipofectamine 2000: plasmid = 2:1), mix thoroughly, let it stand at room temperature for 5 minutes, slowly add Lipofectamine 2000 to the plasmid tube, mix thoroughly, let it stand at room temperature for 15-20 minutes. Add 1ml of the mixture dropwise to the culture medium of 293T cells, gently shake the plate to mix, and then incubate in a 37-degree incubator with 5% CO2. 4) Virus Collection: After 4-6 hours, aspirate the old culture medium and add 12-15 ml of fresh complete culture medium preheated at 37°C. Continue incubating the cells in an 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. Incubate on a shaker at 4°C overnight. Centrifuge at 3500 g for 30 minutes. Resuspend the supernatant at 1 / 100 volume in PBS, aliquot, and store at -80°C until use.

[0059] 5) Viral titer determination (dilution counting method): Titer unit: TU / ml, refers to the number of biologically active viral particles per milliliter. "TU" stands for "transducing units," which indicates the number of viral genomes capable of infecting and entering target cells.

[0060] 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.

[0061] On the second day, add the virus: Perform an 8-fold serial dilution in EP tubes, for eight consecutive dilutions. The dilution method is as follows: Prepare eight 1.5ml EP tubes for each virus. Add 100µl of culture medium to each tube. Add 50µl of the virus 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 virus solution to each well of the cells in a 96-well plate. Fill the well with culture medium to 200µl and label the well.

[0062] 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%-20% was selected. The titer (TU / ml) = 1000 × infection efficiency × dilution factor × 20.

[0063] 6) Viral infection and stable cell line screening: Infect cells in the logarithmic growth phase with virus 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, add 2 μg / ml puromycin. Replace the selection medium approximately every two days. After approximately two weeks of culture, positive cell lines will be obtained. When the infected virus is AQP4 BCR Clone2 / Clone8 / rAb53, a Nalm6-positive cell line expressing the AQP4 BCR can be obtained for subsequent cell killing experiments and verification of the function of the Nalm6 cell line. When the infected virus is AQP4 M23 subtype fused to EGFP, a 293T cell line expressing the AQP4 M23 subtype fused to EGFP can be successfully obtained and subsequent immunofluorescence experiments can be performed to verify the function of the cell line. The AQP4 BCRNalm6 cell line and the 293T cell line containing the AQP4 M23 isoform fused with EGFP fluorescent protein were constructed according to this method.

[0064] 5. Extraction of Human Peripheral Blood Mononuclear Cells (PBMCs) and Construction of AQP4 CAAR-T Cells (1) PBMC extraction: Human peripheral blood was obtained from inpatients in the Department of Neurology, Tianjin Medical University General Hospital. All patients signed informed consent. 3 ml of fresh anticoagulated blood was mixed with 3 ml of PBS sample diluent. 6 ml of human peripheral blood lymphocyte separation solution was added to a 15 ml centrifuge tube. Human peripheral blood lymphocyte separation solution was purchased from TBD, product number LTS1077, and was used to extract PBMC cells. The cells were used for magnetic bead sorting to obtain CD 3+ T cells.

[0065] 2) CD3 + T cell sorting: Count the PBMCs obtained for constructing M1 CAAR-T, M23 CAAR-T cells and CD19 CAR-T cells, and count the cells every 10 7 Resuspend the cells in 40ul magnetic bead separation buffer and add 10ul CD 3+ Antibodies used for T cell sorting were mixed and incubated at 4°C in the dark for 5 minutes. 7 Add 30ul magnetic bead separation buffer and 20ul CD 3+ Mix the magnetic beads used for T cell sorting and incubate in the dark at 4°C for 10 minutes. Add 3 ml of magnetic bead sorting buffer, centrifuge at 300 g for 10 minutes, and add 3 ml of magnetic bead sorting buffer to resuspend the cells.

[0066] Prepare the LS sorting column in advance, add 3 ml of magnetic bead sorting buffer to rinse the sorting column, add the cell suspension, and wash the sorting column twice, adding 3 ml of magnetic bead sorting buffer each time. Collect the liquid flowing out of the sorting column and centrifuge it. Resuspend it in 1 ml of 1640 medium containing 10% FBS, count it 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.

[0067] 3) Construction of CD19 CAR-T cells and AQP4 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 two days. The T cell infection efficiency was detected by flow cytometry 7-9 days later, and a luciferase killing experiment was performed.

[0068] 6. Firefly luciferase killing assay to detect CAAR-T cell killing efficiency The firefly luciferase killing assay is a mature scientific method to indicate the degree of cell killing. Luciferase is pre-transferred into the Nalm6 B cell line that pre-expresses the BCR. After the killing assay, the firefly luciferase substrate is added and the chemiluminescence value is detected. The greater the number of surviving target cells, the higher the chemiluminescence value, indicating that the CAAR-T cells have not killed the target cells. The fewer the number of surviving target cells, the lower the chemiluminescence value, indicating that the CAAR-T cells have successfully killed the target cells.

[0069] 1) Nalm6 cell plating: Adjust the density of Nalm6 B cell line 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.

[0070] 2) Add NTD-T cells (negative control T cells, untreated), CD19 CAR-T cells, AQP4 pep3-pep7 CAAR-T cells, M1 CAAR-T cells, and M23 CAAR-T cells according to different E:T ratios (effector-target ratios of 0.5:1, 1:1, 2:1, and 5:1, respectively), and fill the culture volume of each well to 200 μL. Place the co-cultured cells in a 37°C 5% CO2 incubator for incubation.

[0071] 3) Analyze the chemiluminescence value using a multi-function microplate reader at 5 and 24 hours of co-culture to determine the killing efficiency. Killing efficiency = (value of negative well - value of target well) / (value of negative well - value of positive well).

[0072] Figure 8 The figures show the killing efficiency of rAb53 Nalm6 B cells by each group in the examples of this application. Figure 9 The killing efficiency of each group in the examples of this application on Clone2 and Clone8 cell lines is shown. Figure 8-9 As shown in the results, AQP4 pep3-pep7 CAAR-T cells could not successfully kill the rAb53 Nalm6 B cell line, while M1 CAAR-T and M23 CAAR-T cells could successfully kill the rAb53 Nalm6 B cell line, and the killing efficiency was similar to that of CD19 CAR-T cells. This indicates that when constructing AQP4 CAAR-T cells, it should be ensured that the AQP4 protein forms the correct conformational epitope rather than a simple linear epitope. Furthermore, we used M1 CAAR-T and M23 CAAR-T cells to kill Clone2 and Clone8 cell lines. The killing results showed that M1 CAAR-T and M23 CAAR-T can successfully kill the Nalm6 cell lines of Clone2 and Clone8, and the killing efficiency is similar to that of CD19 CAAR-T, proving that M1 CAAR-T and M23 CAAR-T have killing effects. In addition, M1 CAAR-T cells and M23 CAAR-T cells cannot successfully kill the Nalm6 cell line that does not express AQP4 antibodies, namely Nalm6 Control. This shows that M1 CAAR-T and M23 CAAR-T cells have specific killing effects and cannot achieve killing effects on the Nalm6 cell line that does not express AQP4 antibodies.

[0073] 4) Soluble protein killing assay: The density of Nalm6 B cell line was adjusted to 1×10 4 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. NTD-T cells, M1 CAAR-T cells, and M23 CAAR-T cells were added at two different E:T ratios of 1:1 and 1:10. Clone2 / Clone8 soluble proteins were synthesized by GenScript Biotechnology Co., Ltd. and adjusted to 100 μg / ml. The soluble proteins were added to the co-culture system, and the volume was made up to 200 μl. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours, and the killing efficiency was measured using a multi-function microplate reader. Figure 10 The results of the soluble protein killing experiment in the examples of this application are shown as follows: Figure 10As shown in the results, the presence of soluble proteins partially affects the killing efficiency of AQP4 CAAR-T cells and reduces the killing efficiency of M1 CAAR-T and M23 CAAR-T cells to a certain extent. The possible reason is that some CAAR-T cells competitively bind to soluble proteins when they function, thereby reducing the number of CAAR-T cells binding to pathogenic target cells, further affecting the killing efficiency, but M1 CAAR-T and M23 CAAR-T still have a killing effect.

[0074] 7. ELISA was used to detect the levels of interferon γ (IFNγ) and granzyme B cytokines in the cell supernatant after M1 CAAR-T cells and M23 CAAR-T cells were killed.

[0075] 1) Cell plating: Adjust the density of Nalm6 target cells to 1×10 5 / ml, cells were seeded in 96-well plates, 100ul of cells were added to each well, NTD-T cells and CAAR-T cells were added at an E:T ratio of 1:1 and 10:1, and co-incubation was terminated after 5 hours and 24 hours, respectively. The cells were centrifuged and the supernatant was obtained.

[0076] 2) IFNγ and Granzyme B ELISA assays: The assay kits were purchased from Wuhan Abotek Biotechnology Co., Ltd. (catalog number RK00015 for the IFNγ assay kit and catalog number RK00089 for the Granzyme B assay kit).

[0077] Figure 11 The concentrations of IFNγ and Granzyme B cell factors in each group in the examples of this application are shown as follows: Figure 11 As shown, the concentrations of IFNγ and Granzyme B increased after M1CAAR-T cells and M23 CAAR-T cells were co-incubated with Clone2 / Clone8 target cells, indicating that CAAR-T cells were activated and exerted a killing effect.

[0078] In summary, in this application, we utilized Smartseq3 experimental technology to conduct an in-depth study of plasma cells in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorder (NMOSD). Through meticulous sequencing analysis of plasma cell samples, we successfully predicted the full-length sequences of antibodies produced by these plasma cells. After obtaining the full-length antibody sequences, we used bioinformatics analysis methods to screen and align the antibody sequences. Furthermore, we combined immunological experimental techniques to verify the binding ability of the candidate antibodies to the target protein. After multiple rounds of screening and rigorous testing, we ultimately identified six pairs of antibody sequences that specifically bind to the M23 isoform of aquaporin-4 (AQP4). To further validate the biological function of these antibody sequences, we selected two pairs from these six and successfully engineered them into the Nalm6 B cell line. The Nalm6 B cell line is a commonly used cell model with stable genetic characteristics and well-developed growth conditions, providing a reliable experimental platform for subsequent experiments. By introducing the selected antibody sequences into the Nalm6 B cell line, we generated Nalm6 cell lines expressing specific BCRs (i.e., Clone2 and Clone8). Next, we conducted in vitro cytotoxicity assays to evaluate the ability of CAAR-T cells to kill Nalm6 cell lines expressing both Clone2 and Clone8 BCRs. In this experiment, CAAR-T cells were co-cultured with Nalm6 cell lines expressing both Clone2 and Clone8 BCRs and cell growth and death were closely monitored. The results showed that CAAR-T cells were able to successfully kill Nalm6 cell lines expressing both Clone2 and Clone8 BCRs.

[0079] 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 chimeric AQP4 antibody receptor, characterized in that It includes an AQP4 antibody binding domain, a costimulatory domain, and an activation domain connected sequentially from the amino terminus to the carboxyl terminus; wherein, The AQP4 antibody binding domain is the full-length AQP4 M1 or the full-length AQP4 M23. The nucleotide sequence of the full-length AQP4 M1 is shown in SEQ ID NO: 4, and the nucleotide sequence of the full-length AQP4 M23 is shown in SEQ ID NO:

5.

2. A chimeric AQP4 antibody receptor according to claim 1, characterized in that: The costimulatory domain is 4-1BB, and the nucleotide sequence of 4-1BB is shown in SEQ ID NO: 6; the activation domain is CD3ξ, and the nucleotide sequence of CD3ξ is shown in SEQ ID NO:

7.

3. A recombinant vector, characterized in that The chimeric AQP4 antibody receptor according to claim 2 is included.

4. A recombinant vector according to claim 3, characterized in that The recombinant vector is a lentiviral plasmid vector.

5. A CAAR-T cell, characterized in that The CAAR-T cells contain the recombinant vector according to claim 4.

6. The CAAR-T cell according to claim 5, characterized in that The CAAR-T cells are AQP4 M1 CAAR-T cells or AQP4 M23 CAAR-T cells.

7. The CAAR-T cell according to claim 6, characterized in that The CAAR-T cells are capable of binding to plasma cell antibodies in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorder, wherein the plasma cell antibody is one of Clone2 antibody, Clone8 antibody, Clone17 antibody, Clone19 antibody, Clone22 antibody, and Clone25 antibody, the heavy chain variable region nucleotide sequence of the Clone2 antibody is shown in SEQ ID NO:9, the light chain variable region nucleotide sequence of the Clone2 antibody is shown in SEQ ID NO:13, the heavy chain variable region nucleotide sequence of the Clone8 antibody is shown in SEQ ID NO:10, the light chain variable region nucleotide sequence of the Clone8 antibody is shown in SEQ ID NO:14, the heavy chain variable region nucleotide sequence of the Clone17 antibody is shown in SEQ ID NO:17, the light chain variable region nucleotide sequence of the Clone17 antibody is shown in SEQ ID NO:18, the heavy chain variable region nucleotide sequence of the Clone19 antibody is shown in SEQ ID NO:19, and the light chain variable region nucleotide sequence of the Clone19 antibody is shown in SEQ ID NO: NO:20, the nucleotide sequence of the heavy chain variable region of the Clone22 antibody is shown in SEQ ID NO:21, the nucleotide sequence of the light chain variable region of the Clone22 antibody is shown in SEQ ID NO:22, the nucleotide sequence of the heavy chain variable region of the Clone25 antibody is shown in SEQ ID NO:23, and the nucleotide sequence of the light chain variable region of the Clone25 antibody is shown in SEQ ID NO:

24.

8. Use of the chimeric AQP4 antibody receptor according to any one of claims 1-2, the recombinant vector according to any one of claims 3-4, or the CAAR-T cell according to any one of claims 5-7 in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

9. The use according to claim 8, characterized in that The drug can specifically kill cells expressing AQP4 antibodies.

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