A chimeric AQP4 antibody receptor, CAAR-T cells and their applications

Abstract

This application discloses a chimeric AQP4 antibody receptor, CAAR-T cells, and their applications, comprising an AQP4 antibody-binding domain, a co-stimulatory domain, and an activation domain sequentially linked from the amino terminus to the carboxyl terminus; wherein the AQP4 antibody-binding domain is either 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. This application utilizes gene editing technology to express the full-length AQP4 M1 and M23 isotype proteins on the surface of T cells. The constructed CAAR-T cells can specifically recognize and attack B cells secreting pathogenic antibodies while preserving other normal B cell subsets, demonstrating significant efficacy and reducing the risks associated with long-term use of biological agents.

Description

Technical Field

[0001] This invention relates to the field of cell therapy technology, and more specifically, to a chimeric AQP4 antibody receptor, CAAR-T cells, and their applications. Background Technology

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an antibody-mediated, complement-involved autoimmune disease that primarily targets aquaporin 4 (AQP4) on astrocytes. It commonly affects young and middle-aged adults, with a predominantly female population, and is characterized by high relapse rates and high disability rates. The etiology of NMOSD is mainly related to autoantibodies against AQP4. AQP4 antibody-mediated immune inflammatory responses primarily attack areas of high AQP4 expression in the central nervous system, mainly the optic nerve and spinal cord, causing recurrent optic neuritis and / or myelitis. Other common sites include the periependymal region such as the last medulla oblongata, thalamus, hypothalamus, the periventricular region of the third and fourth ventricles, and the corpus callosum. The recurrent episodes of NMOSD cause irreversible neurological disability in patients.

[0003] In related technologies, rituximab (anti-CD20 monoclonal antibody), inebilizumab (anti-CD19 monoclonal antibody), satralizumab and tocilizumab (anti-IL-6 monoclonal antibodies), eculizumab (anti-C5 monoclonal antibody), azathioprine, or mycophenolate mofetil are the main drugs used clinically to treat NMOSD. Although biologics significantly reduce the relapse rate, breakthrough relapse still occurs in 20%-30% of patients, indicating limited efficacy. Furthermore, biologics may carry specific risks; 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 background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a chimeric AQP4 antibody receptor, CAAR-T cells, and their applications. This application expresses the full-length AQP4 M1 and M23 isoform proteins on the surface of T cells using gene editing technology, thereby maintaining the original protein conformation of the AQP4 protein and connecting the co-stimulatory domain and activation domain to obtain a chimeric AQP4 antibody receptor. CAAR-T cells are then constructed. These CAAR-T cells can specifically recognize and attack B cells that secrete pathogenic antibodies while preserving other normal B cell subsets, resulting in significant effects and reducing the risks associated with long-term use of biological agents.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] According to one aspect of this application, a chimeric AQP4 antibody receptor is provided, comprising an AQP4 antibody-binding domain, a co-stimulatory domain, and an activation domain sequentially connected from the amino terminus to the carboxyl terminus; wherein the AQP4 antibody-binding domain is full-length AQP4 M1 or full-length AQP4 M23, the nucleotide sequence of full-length AQP4 M1 is shown in SEQ ID NO:4, and the nucleotide sequence of full-length AQP4 M23 is shown in SEQ ID NO:5.

[0008] In some embodiments, the co-stimulatory domain is 4-1BB, the nucleotide sequence of which is shown in SEQ ID NO:6; the activation domain is CD3ξ, the nucleotide sequence of which is shown in SEQ ID NO:7.

[0009] According to another aspect of this application, a recombinant vector comprising the chimeric AQP4 antibody receptor is also provided.

[0010] In some embodiments, the recombinant vector is a lentiviral plasmid vector.

[0011] According to another aspect of this application, a CAAR-T cell is also provided, the CAAR-T cell comprising the recombinant vector described above.

[0012] In some embodiments, the CAAR-T cells are AQP4 M1 CAAR-T cells or AQP4 M23 CAAR-T cells.

[0013] In some embodiments, the CAAR-T cells can bind to plasma cell antibodies in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorders. 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:18. As shown in NO:20, the heavy chain variable region nucleotide sequence of the Clone22 antibody is shown in SEQ ID NO:21, the light chain variable region nucleotide sequence of the Clone22 antibody is shown in SEQ ID NO:22, the heavy chain variable region nucleotide sequence of the Clone25 antibody is shown in SEQ ID NO:23, and the light chain variable region nucleotide sequence of the Clone25 antibody is shown in SEQ ID NO:24.

[0014] According to another aspect of this application, the use of the chimeric AQP4 antibody receptor, the recombinant vector, and the CAAR-T cells in the preparation of a medicament for treating neuromyelitis optica spectrum disorders is also provided.

[0015] In some embodiments, the drug is capable of specifically killing cells expressing AQP4 antibodies.

[0016] Compared with existing technologies, the advantages and positive effects of this application are as follows: This application expresses the full-length AQP4 M1 and M23 isoform proteins on the surface of T cells using gene editing technology, allowing the AQP4 protein to maintain its original protein conformation and connect its co-stimulatory and activation domains to obtain a chimeric AQP4 antibody receptor. This allows for the construction of CAAR-T cells, which can specifically recognize and attack B cells secreting pathogenic antibodies while preserving other normal B cell subsets. This approach is highly effective and reduces the risks associated with long-term use of biological agents. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A schematic diagram of the AQP4 protein structure is shown.

[0019] Figure 2 The image shows a heatmap of VJ gene rearrangement in the Smartseq3 experiment in an embodiment of this application.

[0020] Figure 3 The present invention illustrates the frequency of occurrence of gene V, gene J, and relative frequency of CDR3 in the Smartseq3 experiment in the embodiments of this application.

[0021] Figure 4 The results of cell immunofluorescence detection in the embodiments of this application are shown.

[0022] Figure 5 This diagram illustrates the construction of the AQP4 plasmid vector in an embodiment of this application.

[0023] Figure 6 This diagram illustrates the construction of the Clone2 / Clone8 / rAb53 plasmid in an embodiment of this application.

[0024] Figure 7 This diagram illustrates the construction of an AQP4 M23 subunit fused EGFP plasmid vector in an embodiment of this application.

[0025] Figure 8 The killing efficiency of each group on rAb53 Nalm6 B cells in the embodiments of this application is shown.

[0026] Figure 9 The killing efficiency of each group on Clone2 and Clone8 cell lines in the embodiments of this application is shown.

[0027] Figure 10 The results of soluble protein killing experiments in the embodiments of this application are shown.

[0028] Figure 11 The concentrations of IFNγ and Granzyme B cytokines in each group are shown in the embodiments of this application. Detailed Implementation

[0029] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0030] NMOSD: Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune disease of the central nervous system in which the patient's immune system attacks 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 target for damage.

[0031] AQP4: Aquaporin 4, Figure 1 The diagram shows the structure of the AQP4 protein. AQP4 has two isoforms: AQP4-M1 and AQP4-M23. The M23 subunit has 22 fewer amino acids than the M1 subunit; these first 22 amino acids are all located intracellularly. M23 has three discontinuous extracellular domains: pep3 (loop A), pep5 (loop C), and pep7 (loop E). Loop A has 11 extracellular amino acid sequences, loop C has 18 amino acid sequences, and loop E has 25 amino acid sequences, with 11 of these amino acids located intracellularly.

[0032] 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's body in vitro, enabling them to express pathogenic sequences of antigen proteins and connect transmembrane regions and intracellular structural domains. These modified T cells are then reinfused into the patient, allowing them to specifically recognize and attack B cells that secrete pathogenic antibodies while preserving other normal B cell subsets, thereby achieving the goal of treating the disease.

[0033] Smartseq3 technology: Single-cell transcriptome sequencing technology with significant advantages such as high resolution and wide coverage, enabling comprehensive and detailed analysis of gene expression in individual cells.

[0034] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0035] I. Obtain the full-length sequence of plasma cell antibodies from the cerebrospinal fluid of NMOSD patients.

[0036] Cerebrospinal fluid (CSF) samples were obtained from inpatients in the Department of Neurology at Tianjin Medical University General Hospital, all of whom had signed informed consent forms. The full-length sequence of plasma cell antibodies from the CSF of NMOSD patients was obtained using SmartSeq3 technology and synthesized. The obtained CSF samples were stained, and plasma cells were then sorted by flow cytometry and isolated into single-cell samples in 96-well plates. After adding lysis buffer, the full-length sequence of the B cell receptor (BCR) on the surface of each single cell was predicted through reverse transcription and template conversion, PCR pre-amplification, Tn5 transposase fragmentation, and sequencing library construction. Details are as follows:

[0037] (1) Cerebrospinal fluid sample processing: Centrifuge 10 ml of NMOSD patient cerebrospinal fluid at 1800 rpm for 6 minutes. Carefully aspirate the supernatant, leaving only 100 μl of cerebrospinal fluid. Gently pipette and mix well. Add four flow cytometry antibodies: APC-CY7 CD19, BV421 CD27, FITC CD38, and Percp5.5 7AAD. - 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 it is incubated on ice in the dark for 30 minutes.

[0038] (2) Flow cytometry sorting: First, adjust the fluorescence voltage and compensation of the 4 channels, and start the sorting program of the flow cytometer. According to the gating settings, separate the target cell population from the sample. The target cell population for sorting is 7AAD. - CD19 + CD27 high CD38 high Cell subpopulations. Using 96-well plates with cell lysis buffer pre-added to the bottom, sort individual cells. After sorting, carefully remove the plates and seal them with clean sealing film to avoid cell contamination or loss.

[0039] (3) Obtaining plasma cell BCR sequence: The Smartseq3 experiment was completed by Beijing Bio-Tech Co., Ltd.

[0040] (4) Sequence Analysis: Recombinant antibodies consist of two parts: a light chain and a heavy chain. Both the light and heavy chains have variable regions encoded by a single exon, but this exon is composed of multiple gene fragments. These gene fragments can be categorized into four types: V (Variable), D (Diversity), J (Joining), and C (Constant). Only V, D, and J gene fragments participate in variable region recombination. The C gene encodes the constant region of the antibody, which is linked to the recombined VJ or VDJ fragment after transcription. The light chain lacks the D gene fragment, thus VJ recombination occurs in the light chain, and VDJ recombination occurs in the heavy chain. Therefore, after obtaining Smartseq3 data, we can obtain the V and J genes of different antibodies and their frequencies. The antigen-binding region of an antibody has three variable regions: CDR1, CDR2, and CDR3. CDR1 and CDR2 are located in the middle of the V gene fragment, 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, the clonal amplification and immune diversity of the final obtained sequences were determined by analyzing the frequency of VJ gene rearrangement, CDR3 region repetition, V gene occurrence, and J gene occurrence. Figure 2 The figure shows a heatmap of VJ gene rearrangement pairings in the Smartseq3 experiment in this application embodiment. 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 (i.e., the more frequent the occurrence of occurrences is, the stronger the antibody diversity in the cerebrospinal fluid cells of the patient. The higher the frequency of different VJ gene rearrangements in the figure, the higher the clonal amplification rate of that VJ gene rearrangement. Figure 3 The Smartseq3 experiment in this application illustrates the frequencies of the V and J genes, as well as the relative frequency of CDR3, indicating which types of V 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; a higher CDR3 frequency indicates a higher clonal amplification rate of recombinant antibodies containing that CDR3. Relative V Usage (V gene frequency), Relative J Usage (J gene frequency), and Relative Frequency (CDR3 relative frequency) were determined by rearranging a large number of antibody sequences, resulting in 37 possible antibody arrangements. Therefore, these 37 antibody sequences were synthesized and exist as recombinant secreted proteins.

[0041] (5) Synthesis of crude antibody protein: The crude protein supernatant of 37 pairs was synthesized by Yiqiao Shenzhou Biotechnology Co., Ltd.

[0042] II. Cell immunofluorescence assay to detect the binding efficiency of 37 soluble proteins to the M23 subtype of AQP4 protein.

[0043] According to gene rearrangement ( Figure 2-3 Based on the sequence of the plasmid, we synthesized a total of 37 pairs of crude protein supernatant antibodies, and then performed cell immunofluorescence detection on the synthesized 37 pairs of crude protein supernatant. Figure 4 The results of cell immunofluorescence detection in the embodiments of this application are shown. Results ( Figure 4 The results showed that six pairs of antibodies could bind to 293T cells expressing the M23 subtype of AQP4 protein, proving that these six pairs of antibodies are specific antibodies against AQP4 protein.

[0044] Specifically as follows:

[0045] (1) Constructing a lentiviral plasmid vector fused with the AQP4 M23 subunit and EGFP

[0046] Download the human AQP4 antigen M23 subunit sequence (protein ID: P55087-2, amino acid sequence M1-V301) from the Uniprot website, fuse the protein terminal with fluorescent protein EGFP, and see the sequence design (see [link to Uniprot website]). Figure 7 ),

[0047] The full-length base sequence of AQP4 M23 is shown in SEQ ID NO: 5.

[0048] The EGFP base sequence is shown in SEQ ID NO: 25.

[0049] The target sequence was amplified by PCR and ligated to the pCDH plasmid vector digested with BamHI and EcoRI via homologous recombination. After Sanger sequencing confirmed the sequence was correct, an overexpression plasmid of the MOG antigen extracellular fragment expressing EGFP protein was obtained.

[0050] (2) Using 293T cells as the tool cell line, lentivirus expressing AQP4 M23 subtype fusion with 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. One week later, positive and stable transfected cells were sorted. The cells were then passaged to obtain a 100% transfected 293T cell line stably expressing AQP4 M23 subtype fusion with EGFP. For details of the implementation method, see "IV. Lentiviral Packaging and Obtaining Stable Transfected Cell Lines".

[0051] (3) Cell seeding: The 293T cell line with the AQP4 M23 antigen extracellular segment gene stabilized was mixed with the wild-type 293T cell line at a ratio of 7:3 and passaged in a 24-well plate with cell spreaders. When the cells reached 80% confluence, subsequent tests were performed.

[0052] (4) Cell immunofluorescence: The old culture medium was aspirated, and the cells were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. The cells were washed three 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 Sinocare Biotechnology Co., Ltd. The soluble protein was diluted at ratios of 1:10, 1:32 and 1:100, and 50 μl was added to each well. The cells were incubated overnight at 4 degrees Celsius in the dark. The cells were washed three times with PBS for 5 minutes each time, and biotin-SP-conjugated high-affinity anti-human IgG goat antibody (1:700, diluted with PBS, total volume 50 μl) was added. The cells were incubated at room temperature for 30 minutes. The cells were washed three times with PBS for 5 minutes each time. Alexfluor 647-conjugated streptavidin (1:700, diluted with PBS, total volume 50 μl) was added. The cells were washed three times with PBS for 5 minutes each time. Mounting tablets with mounting medium.

[0053] (5) Fluorescence microscopy analysis: Using cells that have not been incubated with antibodies as a control, the presence or absence of red fluorescence under a 20x objective lens was used to indicate whether the soluble protein binds to the extracellular domain of AQP4 protein M23. The results ( Figure 4 The results showed that the extracellular domain of the AQP4 antigen M23 subunit can bind to 6 pairs of soluble antibody sequences.

[0054] The nucleotide sequences of these 6 pairs of antibodies are as follows:

[0055] The Clone2 heavy chain variable region sequence is shown in SEQ ID NO:9;

[0056] The variable region sequence of the Clone2 light chain is shown in SEQ ID NO:13;

[0057] The Clone8 heavy chain variable region sequence is shown in SEQ ID NO:10;

[0058] The Clone8 light chain variable region sequence is shown in SEQ ID NO:14;

[0059] The Clone17 heavy chain variable region sequence is shown in SEQ ID NO:17;

[0060] The variable region sequence of the Clone17 light chain is shown in SEQ ID NO:18;

[0061] The Clone19 heavy chain variable region sequence is shown in SEQ ID NO:19;

[0062] The variable region sequence of the Clone19 light chain is shown in SEQ ID NO:20;

[0063] The heavy chain variable region sequence of Clone22 is shown in SEQ ID NO:21;

[0064] The variable region sequence of the Clone22 light chain is shown in SEQ ID NO:22;

[0065] The Clone25 heavy chain variable region sequence is shown in SEQ ID NO:23;

[0066] The variable region sequence of the Clone25 light chain is shown in SEQ ID NO:24.

[0067] III. Construction of plasmid vectors for AQP4 CAAR-T, Clone2 / Clone8 / rAb53 Nalm6 B cell lines.

[0068] 1. Construction of AQP4 CAAR-T lentiviral plasmid vector

[0069] Human AQP4 antigen sequences were downloaded from the Uniprot website, and the AQP4 pep3-pep7 sequence (protein ID: P55087-1, amino acid sequence: N58-T208), the full-length M1 sequence (protein ID: P55087-1, amino acid sequence: M1-V323), and the full-length M23 sequence (P55087-2, amino acid sequence: M1-V301) were selected as antigen recognition regions for CAAR-T cells. The co-stimulatory domain 4-1BB and activation domain CD3ξ, identical to those in CD19 CAR-T cells from WO2019 / 159193A1 (country of origin: India, publication date: 2019-08-22), were also selected.

[0070] The secretory peptide base sequence is shown in SEQ ID NO:1;

[0071] The CD8α transmembrane region base sequence is shown in SEQ ID NO: 2;

[0072] The AQP4 pep3-pep7 base sequence is shown in SEQ ID NO: 3;

[0073] The full-length base sequence of AQP4 M1 is shown in SEQ ID NO:4;

[0074] The full-length base sequence of AQP4 M23 is shown in SEQ ID NO: 5;

[0075] The 4-1BB base sequence is shown in SEQ ID NO: 6;

[0076] The CD3ξ base sequence is shown in SEQ ID NO: 7.

[0077] After sequence design, the pCDHGFP plasmid vector was synthesized by Genewiz Biotechnology Co., Ltd. and ligated with the pCDHGFP plasmid vector digested with XbaI and BamHI to obtain the AQP4 CAAR-T lentiviral plasmid vector. Figure 5 This diagram illustrates the construction of the AQP4 plasmid vector in an embodiment of this application. Figure 5 a is the AQP4 pep3-pep7 CAAR-T plasmid vector. Figure 5 b is the AQP4 M1 CAAR-T plasmid vector. Figure 5 c represents the AQP4 M23 CAAR-T plasmid vector. It's important to note that AQP4 has two subunits (M1 and M23). M1 has 323 amino acids, and M23 has 301 amino acids. The difference lies in the protein initiation site; M1 has 21 more amino acids than M23 (i.e., AQP4 pep1). Figure 5 The sum of the blue and brown parts in b is the total length of AQP4 M1. M1 and M23 differ in their spatial conformation. Figure 1 M23 is more likely to aggregate to form an OAP structure, while M1 is less likely to form an OAP structure.

[0078] 2. Construct a Nalm6 BCR cell line overexpressing Clnoe2 / Clone8 / rAb53.

[0079] The validated Clone2 / Clone8 / rAb53 sequence was used to link the heavy chain variable region VH to the heavy chain constant region IgG1, and the light chain variable region VL to the κ chain of the human light chain constant region. rAb53 is a recombinant p53 adenovirus (rAd-p53) and a positive antibody for AQP4, which can specifically bind to AQP4. This was used to verify whether CAAR-T cells could successfully kill the virus. See sequence design below. Figure 6 .

[0080] in,

[0081] The base sequence of the signal peptide is shown in SEQ ID NO:8;

[0082] The base sequence of the variable region (VH) of the heavy chain of Clone2 is shown in SEQ ID NO: 9;

[0083] The base sequence of the variable region (VH) of the 8-chain Clone is shown in SEQ ID NO: 10;

[0084] The base sequence of the variable region (VH) of the heavy chain of rAb53 is shown in SEQ ID NO: 11;

[0085] The heavy chain constant region IgG1 base sequence is shown in SEQ ID NO: 12;

[0086] The base sequence of the variable region (VL) of the light chain of Clone2 is shown in SEQ ID NO: 13;

[0087] The base sequence of the variable region (VL) of the Clone8 light chain is shown in SEQ ID NO: 14;

[0088] The base sequence of the variable region (VL) of the rAb53 light chain is shown in SEQ ID NO: 15;

[0089] The base sequence of the light chain constant region κ (CLκ) is shown in SEQ ID NO: 16.

[0090] After sequence design, the pCDH plasmid vector, which was synthesized by Genewiz Biotechnology Co., Ltd., was ligated into a pCDH plasmid vector that was double-digested with XbaI and BamHI to obtain a lentiviral plasmid vector overexpressing Clone2 / Clone8. The plasmid sequence was derived from plasma cells in the cerebrospinal fluid of NMOSD patients. Therefore, the construction of the Clone2 / Clone8 Nalm6 cell line and the in vitro killing experiment are of great significance for the diagnosis and treatment of NMOSD.

[0091] IV. Lentiviral packaging and obtaining stable transfected cell lines.

[0092] Using 293T cells as the tool cell line, lentiviruses expressing the target gene were packaged using the CaCl2 method. Viral suspensions were collected and used to infect Nalm6 cells in the presence of 6 μg / ml polybrene. After one week, positive and stably transfected cells were sorted. Further passage culture was performed to obtain a 100% transfected Nalm6 cell line stably expressing the fusion protein.

[0093] 1) Cell culture: Nalm6 and 293T cell lines were cultured routinely in our laboratory. Nalm6 cells were placed in 1640 medium containing 10% inactivated fetal bovine serum, and 293T cells were placed in high-glucose DMEM medium containing 10% inactivated fetal bovine serum. They were placed in a 37°C, 5% CO2 incubator and cultured according to routine methods. Cells in the logarithmic growth phase were used for experiments.

[0094] 2) Cell preparation: 293T cells were resuspended in high-glucose DMEM medium containing 10% FBS in 10cm culture dishes and placed in a 37°C incubator containing 5% CO2. After the cells adhered to the wall and the density reached about 80%-90%, they were ready for transfection. The medium was changed 2 hours before transfection (6-8ml of fresh complete medium replaced the old medium).

[0095] 3) Plasmid transfection: Mix the core plasmid and packaging plasmid. Take a sterile 1.5ml EP tube, add 500ul Opti-MEM, and add 20ug of mixed plasmid (core plasmid: psPAX2: pMD2.G = 4:3:1). Take another sterile 1.5ml EP tube, add 500ul Opti-MEM, and add 40ul Lipofectamine 2000 (Lipofectamine 2000: plasmid = 2:1). Mix thoroughly and incubate at room temperature for 5 minutes. Slowly add Lipofectamine 2000 to the plasmid tube, mix thoroughly, and incubate at room temperature for 15-20 minutes. Add 1ml of the mixture dropwise to the culture medium of 293T cells, gently shake the petri dish to mix, and incubate in a 37°C incubator with 5% CO2.

[0096] 4) Virus collection: After 4-6 hours, remove the old culture medium, add 12-15 ml of fresh, preheated 37°C complete culture medium, and continue to incubate the cells in an incubator. After about 72 hours, collect the supernatant into a 15 ml centrifuge tube, filter the supernatant through a 0.45 μm filter, add 4× lentivirus concentrate, incubate overnight on a shaker at 4°C, centrifuge at 3500 g for 30 minutes, resuspend the supernatant with 1 / 100 volume of PBS, aliquot and store at -80°C for later use.

[0097] 5) Viral titer assay (dilution counting method): Titer unit: TU / ml, which refers to the number of biologically active viral particles contained in each milliliter. "TU" is an abbreviation for "transducing units", which represents the number of viral genomes that can infect and enter target cells.

[0098] Day 1 Cell Preparation: Digest and count healthy 293T cells, then dilute to 1×10⁻⁶. 4 Add 100 μL / well to a 96-well plate, preparing 10 wells for each virus. Incubate at 37°C in a 5% CO2 incubator.

[0099] On the second day, add the virus: Perform 8-fold serial dilutions in EP tubes, creating 8 consecutive dilutions. The dilution method is as follows: Prepare 8 1.5ml EP tubes for each virus. Add 100ul of culture medium to each tube. Add 50ul of the original virus solution to the first tube, mix well, and then add 50ul to the second tube and mix well. Continue in this manner to create 8 dilutions. Add 50ul of the diluted virus solution to each well of a 96-well container, and bring the culture medium to a final volume of 200ul. Label the container accordingly.

[0100] On the fifth day, observe the results and calculate the titer: Flow cytometry was used to detect the infection efficiency, and the number of fluorescent cell clones with an infection efficiency of 10%-20% was selected. The titer (TU / ml) was calculated as 1000 × infection efficiency × dilution factor × 20.

[0101] 6) Viral Infection and Stable Cell Line Screening: Cell lines in the logarithmic growth phase were infected with a concentrated virus solution (containing 6 μg / ml Polybrene) with an MOI of 5. The multiplicity of infection (MOI) is the ratio of the number of viruses capable of infecting cells to the total number of cells in a system. After 48 hours, 2 μg / ml puromycin was added. The selection medium was changed to fresh medium approximately every 2 days. Positive cell lines were obtained after about 2 weeks of culture. When infected with the AQP4 BCR Clone2 / Clone8 / rAb53 virus, Nalm6 positive cell lines expressing AQP4 BCR were obtained for subsequent cell killing experiments and to verify the function of the Nalm6 cell line. When infected with the AQP4 M23 subtype fusion EGFP virus, 293T cell lines expressing AQP4 M23 subtype fusion EGFP were successfully obtained, and subsequent immunofluorescence experiments were performed to demonstrate the function of the cell lines. The AQP4 BCRNalm6 cell line and the 293T cell line fused with EGFP fluorescent protein of the AQP4 M23 subtype were both constructed according to this method.

[0102] V. Extraction of human peripheral blood mononuclear cells (PBMCs) and construction of AQP4 CAAR-T cells

[0103] (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 forms. 3 ml of fresh anticoagulated blood was mixed with 3 ml of PBS sample diluent. 6 ml of human peripheral blood lymphocyte separation medium (purchased from TBD, catalog number LTS1077) was added to a separate 15 ml centrifuge tube for PBMC extraction. The cells were then sorted using magnetic beads to obtain CD4+ cells. 3+ T cells.

[0104] 2) CD3 + T cell sorting: The PBMC count to be obtained for the construction of M1 CAAR-T, M23 CAAR-T cells and CD19 CAR-T cells, per 10 7 Cells were resuspended in 40 μL of magnetic bead sorting buffer, and then 10 μL of CD was added. 3+ The antibodies used for T cell sorting were mixed and incubated at 4 degrees Celsius in the dark for 5 minutes. Every 10 7 Cells were added to 30 μL of magnetic bead sorting buffer and 20 μL of CD. 3+After mixing the magnetic beads used for T cell sorting, incubate at 4 degrees Celsius in the dark for 10 minutes, add 3 ml of magnetic bead sorting buffer, centrifuge at 300g for 10 minutes, and resuspend the cells in 3 ml of magnetic bead sorting buffer.

[0105] Prepare an LS sorting column in advance. Rinse the column with 3 ml of magnetic bead sorting buffer, add cell suspension, and wash the column twice, adding 3 ml of magnetic bead sorting buffer each time. Collect the liquid flowing out of the sorting column and centrifuge. Resuspend the column in 1 ml of 1640 medium containing 10% FBS. After resuspending, count the cells and adjust the density to 1×10⁻⁶. 6 / ml, cultured into well plates, and add the corresponding amount of CD3 / CD28 activator per ml of cells.

[0106] 3) Construction of CD19 CAR-T cells and AQP4 CAAR-T cells

[0107] T cells were infected with the corresponding virus concentrate at MOI=5 24 hours after CD3 / CD28 activation. Fresh complete culture medium was added every two days. The T cell infection efficiency was detected by flow cytometry after 7-9 days, and luciferase killing assay was performed.

[0108] VI. Firefly luciferase killing assay to detect CAAR-T cell killing efficiency

[0109] The firefly luciferase killing assay is a mature scientific method for indicating the degree of cell killing. Luciferase is pre-transformed into the Nalm6 B cell line that pre-expresses BCR. After the killing assay, firefly luciferase substrate is added, and the chemiluminescence value is detected. The higher the number of surviving target cells and the higher the chemiluminescence value, the more likely the CAAR-T cells have not killed the target cells. Conversely, the lower the number of surviving target cells and the lower the chemiluminescence value, the more likely the CAAR-T cells have successfully killed the target cells.

[0110] 1) Nalm6 cell plating: Adjust the density of the Nalm6 B cell line to 1×10⁻⁶ cells / cells. 4 / ml, cells were seeded in a white opaque microplate for chemiluminescence detection. 100ul of cells were added to each well. A portion of the cells without T cells served as a negative control, and another portion of the cells were centrifuged and resuspended in sterile water as a positive control.

[0111] 2) NTD-T cells (negative control T cells, no treatment), CD19 CAR-T cells, AQP4 pep3-pep7 CAAR-T cells, M1 CAAR-T cells, and M23 CAAR-T cells were added according to different E:T ratios (effect-target ratios of 0.5:1, 1:1, 2:1, and 5:1, respectively), and the culture volume of each well was increased to 200 μL. The co-cultured cells were placed in a 37°C 5% CO2 incubator for incubation.

[0112] 3) The killing efficiency was determined by measuring the chemiluminescence values ​​using a multi-functional microplate reader at 5 hours and 24 hours of co-culture. Killing efficiency = (Negative well value - Target well value) / (Negative well value - Positive well value).

[0113] Figure 8 The killing efficiency of each group on rAb53 Nalm6 B cells in the embodiments of this application is shown. Figure 9 The killing efficiency of each group against Clone2 and Clone8 cell lines in the embodiments of this application is shown. Figure 8-9 As shown, 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, with killing efficiency similar to that of CD19 CAR-T cells. This indicates that when constructing AQP4 CAAR-T cells, it is essential to ensure that the AQP4 protein forms the correct conformational epitope, rather than simply a linear epitope. Furthermore, we used M1 CAAR-T and M23 CAAR-T cells to kill Clone2 and Clone8 cell lines. The results showed that M1 CAAR-T and M23 CAAR-T cells successfully killed the Clone2 and Clone8 Nalm6 cell lines, with a killing efficiency similar to that of CD19 CAAR-T cells, demonstrating the killing effect of M1 CAAR-T and M23 CAAR-T cells. Moreover, M1 CAAR-T and M23 CAAR-T cells could not successfully kill the Nalm6 cell line (Nalm6 Control) that does not express AQP4 antibodies, indicating that the killing effect of M1 CAAR-T and M23 CAAR-T cells is specific and cannot achieve a killing effect on the Nalm6 cell line that does not express AQP4 antibodies.

[0114] 4) Soluble protein killing experiment: The density of the Nalm6B cell line was adjusted to 1×10⁻⁶ cells / year. 4Cells were seeded in white opaque microplates for chemiluminescence detection. 100 μL of cells were added to each well. A portion of the cells without T cells served as a negative control, while another portion 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. Soluble Clone2 / Clone8 proteins were synthesized by Genscript Biotech Co., Ltd. The soluble protein density was adjusted to 100 μg / ml and added to the co-culture system. The culture volume was brought to a final volume of 200 μL, and the plates were incubated at 37°C with 5% CO2 for 24 hours. The killing efficiency was then measured using a multi-mode microplate reader. Figure 10 The results of soluble protein killing experiments in the embodiments of this application are shown, such as... Figure 10 As shown, 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 some extent. A possible reason is that some CAAR-T cells competitively bind to soluble proteins when they are functioning, thereby reducing the number of CAAR-T cells that bind to pathogenic target cells and further affecting the killing efficiency. However, M1 CAAR-T and M23 CAAR-T cells still have a killing effect.

[0115] VII. ELISA was used to detect the levels of interferon-γ (IFNγ) and granzyme B cytokines in the supernatant of M1 CAAR-T cells and M23 CAAR-T cells after killing.

[0116] 1) Cell plating: Adjust the Nalm6 target cell density to 1×10⁶ cells / cells. 5 / ml, cells were seeded in 96-well plates, 100ul of cells were added to each well, and NTD-T cells and CAAR-T cells were added at an E:T ratio of 1:1 and 10:1, respectively. Co-incubation was terminated at 5 hours and 24 hours, respectively. Cells were centrifuged and the supernatant was collected.

[0117] 2) IFNγ and Granzyme B ELISA detection: The detection kits were all purchased from Wuhan Aibote Biotechnology Co., Ltd., IFNγ detection kit catalog number RK00015, Granzyme B detection kit catalog number RK00089.

[0118] Figure 11 The concentrations of IFNγ and Granzyme B cytokines in each group in the embodiments of this application are shown, such as Figure 11As 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, suggesting that CAAR-T cells were activated and exerted a killing effect.

[0119] In summary, this application utilized Smartseq3 experimental technology to conduct an in-depth investigation of plasma cells in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorder (NMOSD). Through detailed sequencing analysis of plasma cell samples, the full-length sequences of antibodies produced by these plasma cells were successfully predicted. After obtaining the full-length antibody sequences, we used bioinformatics analysis methods to screen and compare the antibody sequences, and combined this with immunological experimental techniques to verify the binding ability of candidate antibodies to target proteins. After rigorous screening and testing, six pairs of antibody sequences were finally identified that specifically bind to the M23 subtype of aquaporin 4 (AQP4). To further verify the biological function of these antibody sequences, we selected two pairs from these six antibody sequences and successfully constructed them into the Nalm6 B cell line. The Nalm6 B cell line is a commonly used cell model with stable genetic characteristics and good growth status, providing a reliable experimental platform for subsequent experiments. By introducing selected antibody sequences into the Nalm6 B cell line, we obtained a Nalm6 cell line expressing specific BCRs (Clone2 and Clone8). Next, we conducted in vitro killing experiments to evaluate the ability of CAAR-T cells to kill Nalm6 cell lines expressing both Clone2 and Clone8 BCRs. During the experiments, we co-cultured CAAR-T cells with Nalm6 cell lines expressing both Clone2 and Clone8 BCRs and closely observed cell growth and death. The results showed that CAAR-T cells successfully killed Nalm6 cell lines expressing both Clone2 and Clone8 BCRs.

[0120] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A CAAR-T cell, characterized in that, The CAAR-T cells contain a recombinant vector, which includes a chimeric AQP4 antibody receptor, wherein the chimeric AQP4 antibody receptor comprises an AQP4 antibody binding domain, a co-stimulatory domain, and an activation domain, from the amino terminus to the carboxyl terminus. The AQP4 antibody binding domain is either 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. The co-stimulatory 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; The recombinant vector is a lentiviral plasmid vector.

2. The use of the CAAR-T cells according to claim 1 in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

Images