GPIb alpha CAAR, GPIb alpha CAAR plasmid, GPIb alpha CAAR-Treg, construction method of GPIb alpha CAAR-Treg and application of GPIb alpha CAAR-Treg in treatment of immune thrombocytopenia

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses GPIb alpha CAAR, GPIb alpha CAAR plasmids, GPIb alpha CAAR-Treg, a construction method of the GPIb alpha CAAR-Treg and application of the GPIb alpha CAAR-Treg to treatment of immune thrombocytopenia. The invention relates to GPIb alpha CAAR and a preparation method and application thereof on one hand, relates to GPIb alpha CAAR plasmids and a preparation method and application thereof on the other hand, and further relates to GPIb alpha CAAR-Treg and a preparation method and application thereof. According to the present invention, by constructing the regulatory T cell (Treg) expressing the GPIb alpha chimeric autoantibody receptor (CAAR), the specific recognition and inhibition of the pathogenic B cell secreting the anti-GPIb alpha antibody are achieved, such that the generation of the key pathogenic antibody in the ITP is blocked from the source;

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to GPIbαCAAR, GPIbαCAAR plasmid, GPIbαCAAR-Treg and a construction method thereof and use thereof in treating immune thrombocytopenia. Background Art

[0002] Immune thrombocytopenia (ITP) is a bleeding disorder characterized by a decrease in platelet count. Its core pathological mechanism involves accelerated platelet destruction mediated by antiplatelet autoantibodies secreted by autoreactive B cells and plasma cells, as well as impaired platelet production by megakaryocytes. Pathogenic antibodies targeting the platelet surface glycoproteins GPIIb / IIIa (CD41 / CD61) or GPIb / IX (CD42b / CD42c / CD42a) are detected in approximately 50%-70% of patients. Patients positive for anti-GPIb / IX antibodies exhibit more severe thrombocytopenia and a significantly reduced response to conventional treatments such as corticosteroids, intravenous immunoglobulin, and rituximab. Notably, GPIbα is the most critical subunit of the GPIb / IX complex and the primary target of platelet autoantibodies. GPIbα, a platelet surface protein, binds to the A1 domain of von Willebrand Factor (VWF), activating signaling pathways within platelets, thereby mediating platelet activation and thrombosis. June Li et al. found that anti-GPIbα antibodies are not only key molecules that induce Fc-independent platelet activation, neuraminidase-1 translocation, and desialylation, but also a major mechanism leading to platelet clearance via the aspartyl glycoprotein receptor (ASGPR) pathway on hepatocytes. This finding partially explains why ITP patients with anti-GPIbα antibodies have a poor response to traditional immunosuppressive therapy and are more likely to develop refractory ITP. Therefore, anti-GPIbα antibodies are closely associated with refractory relapse of ITP. For newly diagnosed ITP patients, glucocorticoids and thrombopoietin receptor agonists (TPO-RAs) are currently the most commonly used first- and second-line treatment options, with most patients achieving good initial responses and clinical remission. However, approximately 10–20% of patients may progress from an initial persistent stage to refractory ITP. Refractory ITP requires a personalized, multi-mechanism combined treatment strategy, which is challenging to treat, with limited drug options and a poor overall prognosis. Therefore, early intervention in high-risk patients to prevent the transformation of common ITP to refractory disease will help increase overall remission rates and improve long-term clinical outcomes.

[0003] Although immunotherapy targeting excessive B cell activation has been widely explored in the current field of autoimmune disease treatment, there are still significant limitations in directly regulating pathogenic T cell responses. Studies have shown that effector T cell dysfunction is a key factor leading to refractory and recurrent ITP. Its abnormal activation can trigger a persistent inflammatory response, disrupt immune tolerance homeostasis, and exacerbate platelet destruction. Recent studies have found that regulatory T cells (Treg), as natural immune tolerance regulators, have shown unique therapeutic potential in re-establishing immune balance. However, traditional Treg adoptive cell therapy lacks selective regulation of pathogenic T cell subsets, especially for complex pathological scenarios where B cell-mediated autoantibody production and T cell immune dysregulation coexist. As a result, traditional Treg adoptive cell therapy cannot effectively regulate ITP. There is an urgent need to develop new treatment strategies that can synergistically target multiple pathogenic mechanisms. Summary of the Invention

[0004] The present invention provides GPIbαCAAR, GPIbαCAAR plasmid, GPIbαCAAR-Treg, and a method for constructing the same, as well as a use for treating immune thrombocytopenia, primarily to address the current problem of lack of effective treatments for immune thrombocytopenia in complex pathological scenarios such as the simultaneous presence of B cell-mediated autoantibody production and T cell immune disorders.

[0005] In order to solve the above problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides GPIbαCAAR and a preparation method and use thereof.

[0007] One is a method for constructing GPIbαCAAR, comprising the following steps: integrating the GPIbα extracellular ligand binding domain LBD with a sequence of SEQ ID NO.1 into a second-generation CAR structure comprising a CD8α hinge / transmembrane region, a 4-1BB co-stimulatory domain and an intracellular CD3ζ domain to form a GPIbαCAAR with a sequence of SEQ ID NO.2.

[0008] Secondly, the GPIbαCAAR prepared by the construction method of GPIbαCAAR is mainly evaluated based on its material properties, and it is not required to use a method that is completely consistent with the above method.

[0009] The third is the application of GPIbαCAAR in the preparation of drugs for treating immune thrombocytopenia. GPIbαCAAR can give Treg or Teff cells the ability to recognize specific antigens.

[0010] The second aspect of the present invention provides GPIbαCAAR plasmid and its preparation method and application.

[0011] First, the construction method of GPIbαCAAR plasmid mainly includes the following two parts:

[0012] Construction of GPIbαCAAR: The GPIbα extracellular ligand binding domain (LBD) of SEQ ID NO. 1 was integrated into a second-generation CAR structure comprising a CD8α hinge / transmembrane region, a 4-1BB costimulatory domain, and an intracellular CD3ζ domain to form a GPIbαCAAR of SEQ ID NO. 2.

[0013] Construction of GPIbαCAAR plasmid: GPIbαCAAR with the sequence of SEQ ID NO.2 was inserted into the vector to form GPIbαCAAR plasmid.

[0014] In the above preparation steps, each condition can be selected according to needs, and any one of the conditions can be selected separately: 1) the vector is preferably a lentiviral vector, more preferably, the lentiviral vector is a pLenti-EF1α vector, and more preferably, GPIbαCAAR is inserted into the pLenti-EF1α vector to form a GPIbαCAAR plasmid with a sequence of SEQ ID NO. 3; 2) the integration, insertion, etc. can be performed with reference to the existing technology, and the MOI used for transfection during lentiviral vector transduction is preferably 20.

[0015] Secondly, the GPIbαCAAR plasmid prepared by the construction method is mainly evaluated based on its material properties, and it is not required to use a method that is completely consistent with the above method.

[0016] The third is the application of the GPIbαCAAR plasmid in the preparation of drugs for the treatment of immune thrombocytopenia. The GPIbαCAAR plasmid confers antigen-specific activation advantages to Tregs, enabling them to accurately recognize target antigens and effectively exert local immunosuppression, while retaining the broad-spectrum regulatory function of the natural TCR pathway.

[0017] The third aspect of the present invention provides GPIbαCAAR-Treg and its preparation method and application.

[0018] The first method is to construct GPIbαCAAR-Treg: any of the aforementioned GPIbαCAAR plasmids is transfected into human embryonic kidney cells, and the viral supernatant is collected and concentrated.

[0019] The viral supernatant was co-incubated with activated Treg cells to obtain GPIbαCAAR-Treg cells.

[0020] In the present invention, GPIbαCAAR-Treg cells were prepared. In another study by our team, although GPIbαCAAR-T cells can also play a positive role in immune thrombocytopenia, there are obvious differences in the mode of action of the two.

[0021] In the above preparation steps, each condition can be selected according to needs, and any one of the conditions can be selected separately: 1) the activated Treg cells are human and generally patient-derived Treg cells, further derived from human peripheral blood mononuclear cells; 2) the activated Treg cells are stimulated by CD3 / 28 magnetic beads.

[0022] The second is the GPIbαCAAR-Treg cells prepared by the GPIbαCAAR-Treg construction method.

[0023] Third, the use of GPIbα CAAR-Treg cells in the preparation of drugs for the treatment of immune thrombocytopenia. GPIbα CAAR-Treg cells can significantly inhibit the killing action of antigen-specific T cells in vivo, especially significantly inhibiting the cytotoxicity of CAAR-Teff against hybridoma cells expressing GPIbα. GPIbα CAAR-Treg cells solve the problem of the coexistence of multiple immune disorders in ITP by precisely eliminating pathogenic T cells and then blocking the collaboration of TB cells to reshape immune tolerance. In particular, GPIbα CAAR-Treg cells can have a more significant therapeutic effect on ITP patients who are positive for anti-GPIbα antibodies, effectively preventing ITP from developing into refractory ITP. In other words, GPIbα CAAR-Treg cells can also be used to inhibit the development of ITP into refractory ITP and prevent refractory relapse of ITP. In addition, the GPIbα CAAR-Treg cells of the present invention are less likely to accidentally damage normal cells that do not express anti-GPIbα antibodies, and their targeting and safety are enhanced.

[0024] Fourthly, GPIbαCAAR-Treg cells can specifically recognize and bind to anti-GPIbα antibodies and their secreting cells, and thus have important application potential in the treatment of immune thrombocytopenia. Upon recognizing anti-GPIbα antibodies or their corresponding target cells, GPIbαCAAR-Treg cells are significantly activated, thereby enhancing their immunomodulatory function and exerting a targeted immune tolerance effect. For ITP patients who are GPIbα antibody-positive, GPIbαCAAR-Treg cells can effectively suppress abnormal immune responses, thereby achieving more significant therapeutic effects and effectively preventing the disease from progressing to refractory ITP. They also have a significant effect on refractory ITP and can reduce the risk of relapse.

[0025] A fourth aspect of the present invention providesUse of anti-GPIbα antibodies or secretory cells thereof in the preparation of products for stimulating and activating GPIbα CAAR-Treg cells.

[0026] GPIbαCAAR-Treg cells were detected by anti-GPIbα antibody, and the proportion of LAP-GARP positive cells was significantly higher in CAR + The number of GPIbα cells in the GPIbα group increases significantly, and even at a lower ratio of effector cells to target cells (E:T), the activity of effector T cells (Teff) can be effectively suppressed. In specific applications, it can be the use of anti-GPIbα antibodies in the preparation of a biological model with enhanced activation of GPIbαCAAR-Treg cells, in which GPIbαCAAR-Treg cells are significantly activated, and the activation level of GPIbαCAAR-Treg cells in the ordinary model is higher, thereby obtaining a specific biological model. In other words, it can be a method for preparing a biological model (such as a cell model) with high activation of GPIbαCAAR-Treg cells, which mainly includes adding anti-GPIbα antibodies or their secretory cells to significantly activate GPIbαCAAR-Treg cells, thereby making the GPIbαCAAR-Treg cells in the model highly activated, such as the biological model involved in the present invention, by infusing hybridoma cells that secrete anti-GPIbα antibodies, GPIbαCAAR-Treg cells can be highly activated, thereby achieving the purpose of regulating some variables.

[0027] In the present disclosure, by constructing regulatory T cells (Tregs) expressing GPIbα chimeric autoantibody receptors (CAARs), specific recognition and inhibition of pathogenic B cells that secrete anti-GPIbα antibodies are achieved, thereby blocking the production of key pathogenic antibodies in ITP at the source. This method is highly targeted and can avoid side effects caused by nonspecific immunosuppression. In addition, the selection of naive Treg cells as carriers helps to maintain their stable immunoregulatory function during in vitro expansion, while the introduction of 4-1BB and CD3ζ signaling domains enhances the activation and functional persistence of cells under specific antigen stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Design of GPIbαCAAR lentiviral vector plasmid structure;

[0029] Figure 2 The structural composition of GPIbα;

[0030] Figure 3 for Treg sorting and phenotypic monitoring;

[0031] Figure 4 is the expression efficiency of CAAR;

[0032] Figure 5 To verify the specific activation of GPIbαCAAR-Treg;

[0033] Figure 6 To verify the specific inhibitory function of GPIbαCAAR-Treg in vitro;

[0034] Figure 7 This is to verify the in vivo specific inhibitory function of GPIbαCAAR-Treg. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific research projects.

[0036] Experimental content

[0037] 1. Construction and Validation of GPIbα CAAR: The GPIbα extracellular ligand binding domain (LBD) was integrated into the second-generation CAR structure (comprising the CD8α hinge / transmembrane region, the 4-1BB costimulatory domain, and the intracellular CD3ζ domain). GPIbα CAAR was then transiently expressed in HEK 293T cells to test its epitope binding ability.

[0038] a. GPIbαCAAR plasmid construction

[0039] (1) Vector backbone design: second-generation CAR basic plasmid (containing CD8α hinge / transmembrane region, 4-1BB costimulatory domain, CD3ζ intracellular domain, pLenti-EF1α vector);

[0040] (2) Target sequence: human GPIbα extracellular ligand-binding domain (LBD, NCBI reference sequence NP_000164.1, amino acid residues 1-290);

[0041] LBD sequence (SEQ ID NO.1):

[0042] MPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTD;

[0043] GPIbα CAAR sequence (SEQ ID NO.2):

[0044] MALPVTALLLPLALLLHAARPMPLLLLLLLLPSPLHPHPICEVSKVASHLEVNCDKRNLTALPPDLPKDTTILHLSENLLYTFSLATLMPYTRLTQLNLDRCELTKLQVDGTLPVLGTLDLSHNQLQSLPLLGQTLPALTVLDVSFNRLTSLPLGALRGLGELQELYLKGNELKTLPPGLLTPTPKLEKLSLANNNLTELPAGLLNGLENLDTLLLQENSLYTIPKGFFGSHLLPFAFLHGNPWLCNCEILYFRRWLQDNAENVYVWKQGVDVKAMTSNVASVQCDNSDKFPVYKYPGKGCPTLGDEGDTDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

[0045] GPIbα CAAR plasmid sequence (SEQ ID NO.3):

[0046]

[0047] b. HEK 293T cells transiently expressing GPIbαCAAR:

[0048] (1) One day before transfection, seed an appropriate amount of HEK293T cells into a six-well plate (cell density is about 70% at the time of transfection the next day);

[0049] (2) Before transfection, replace the cell DMEM complete medium with serum-free Opti-MEM medium; prepare the transfection complex by adding 3 μg of plasmid to 250 μl of Opti-MEM solution in a sterile centrifuge tube and gently pipetting to mix. In another sterile centrifuge tube, add 12 μg of polyethyleneimine (PEI) to 250 μl of Opti-MEM solution and gently pipetting to mix. Incubate at room temperature for 5 minutes.

[0050] (3) Gently mix the DNA solution and PEI solution using a pipette and let it stand at room temperature for 20 minutes; add 500 μl of the PEI-DNA mixture to one well of a six-well plate and gently shake to mix; place in an incubator for 4-6 hours, and then replace with DMEM complete medium;

[0051] (4) After 48 hours, cells were harvested and the expression efficiency of the target gene was detected.

[0052] c. Epitope detection of GPIbαCAAR:

[0053] (1) HEK293T cells transfected with GPIbαCAAR were trypsinized, resuspended and counted, and 10% resuspended cells were added to each flow cytometry tube in 200 μl flow cytometry buffer. 6 cells;

[0054] (2) Add 2 μl of anti-human platelet GPIbα (CD42b) flow cytometry antibody (clone number: HIP1; FITC) to each tube and incubate at room temperature in the dark for 15 minutes;

[0055] (3) Streaming machine testing

[0056] 2. Treg Isolation and CAAR-Treg Construction

[0057] a. Treg isolation and culture

[0058] (1) Primary CD4+CD25hiCD127lo / -CD45RA+ cells were enriched from human peripheral blood mononuclear cells (PBMCs) using flow cytometry (FACS) Treg cells, ensure cell purity >95%;

[0059] (2) Culture the cells in Xvivo-15 medium containing 10% fetal bovine serum, 1% non-essential amino acids, 200 mM glutamine, 0.01 mM β2-mercaptoethanol, and 1000 IU / mL IL-2. Stimulate with CD3 / 28 magnetic beads, and perform a half-medium change every 2-3 days.

[0060] b. Lentiviral vector transduction

[0061] (1) Lentivirus production and concentration: GPIbαCAAR plasmid was transfected into HEK293T cells together with packaging plasmids psPAX2 and pMD2G. The supernatants at 48 and 72 hours were filtered through a 40 nm filter and ultracentrifuged at 25,000 rpm for 2.5 hours at 4°C. The resulting pellets were resuspended and stored at -80°C until use.

[0062] (2) Lentivirus transfection and CAAR-Treg culture: CD3 / 28 activated for 48 hours Treg cells were resuspended at a density of 1x10 6 cells / ml, set the MOI for transfection to 20, take the corresponding amount of lentivirus and polybrane, add them to the culture flask and place it in a CO2 incubator for further culture. After 12-16 hours of infection, remove the viral supernatant and continue to culture. Observe and count the cells every day to maintain the cell density at 1x10 6 cells / ml; after 3 days of expansion, appropriate cells were collected for analysis and infection efficiency was assessed by flow cytometry. The cells were then cultured in Xvivo-15 medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 200mM glutamine, 0.01mM β2-mercaptoethanol, and 1000IU / mL IL-2 for 10-11 days before in vitro and in vivo functional experiments. The expression of FOXP3, CTLA-4, and Helios was also assessed by flow cytometry to monitor the suppressive phenotype of Tregs in vitro.

[0063] 3. Verification of Specific Activation of GPIbα CAAR-Treg

[0064] a. Stimulus condition design

[0065] (1) Antigen-specific stimulation group: 5 μg / ml anti-GPIbα (CD42b) antibody (clone number HIP1) was added to simulate the binding of CAAR receptors to GPIbα-targeting autoantibodies; at the same time, a hybridoma cell co-culture group secreting anti-GPIbα antibodies (E:T ratio = 1:1) was set up to simulate the in vivo B cell-mediated antigen presentation microenvironment;

[0066] (2) Nonspecific activation group: Nonspecific T cell receptor (TCR) signal activation was performed using 5 μg / ml CD3 / CD28 antibody (solid phase coated).

[0067] b. Inhibitory marker detection:

[0068] After stimulation, cells were collected and the expression of inhibitory molecules was analyzed by multicolor flow cytometry: ① surface markers: anti-LAP (latency associated peptide), anti-GARP (glycoprotein A repeat predominant); ② gating strategy: CD4 + CD25 + CAAR + The target cell population was identified by anti-CD42b binding, and the expression intensity (MFI) of each molecule and the proportion of positive cells were analyzed.

[0069] 4. Verification of GPIbαCAAR-Treg specific inhibitory function in vitro

[0070] a. Construction of co-culture system

[0071] (1) Effector cells: CD4+CD25- Teff cells were sorted from the peripheral blood of healthy donors, labeled with CFSE, and preactivated with anti-CD3 / CD28 antibodies (5 μg / ml) for 24 hours;

[0072] (2) Target cells: Specific stimulation group: magnetic beads coated with anti-GPIbα (CD42b) antibody; nonspecific stimulation group: Teffs were activated only with anti-CD3 / CD28 antibody (5 μg / ml);

[0073] (3) Suppressor cells: GPIbαCAAR-Treg (experimental group) or non-transduced wild-type Treg (NTD-Treg, negative control).

[0074] b. Experimental groups and conditions

[0075] (1) Effector-target ratio gradient: setting the effector-target ratio of Teff to CAAR-Treg (E:T = 0:1, 1:4, 1:2, 2:1, 4:1);

[0076] (2) Culture time: After 96 h of co-culture, the inhibitory effect was detected by flow cytometry using the change in the MFI value of FITC.

[0077] 5. Verification of the specific inhibitory function of GPIbαCAAR-Treg in vivo

[0078] a. Animal Model Construction

[0079] (1) Mouse strain: NOD / SCID / IL-2Rγnull (NSG) immunodeficient mice;

[0080] (2) Tumor burden model:

[0081] Luciferase (Luc)-labeled GPIbα hybridoma cells (5×10^5 / mouse) were injected into the tail vein to simulate the malignant proliferation of autoantibody-secreting B cells;

[0082] Tumor dissemination was dynamically monitored by in vivo imaging (IVIS) (baseline value was set at day 0).

[0083] b. Treatment groups

[0084] G1: hybridoma cells + NTD-Treg (empty vector Treg cell control);

[0085] G2: hybridoma cells + CAAR-Treg (experimental group);

[0086] G3: hybridoma cells + CAAR-Teff (CAR-T cells targeting GPIbα, positive killing control);

[0087] G4: hybridoma cells + CAAR-Teff + CAAR-Treg (combination treatment group ①);

[0088] G5: Hybridoma cells + CAAR-Teff + NTD-Treg (combination therapy group ②):

[0089] ① Tumor burden monitoring: IVIS imaging twice a week to quantitatively measure total flux (photons / s) to assess tumor growth;

[0090] End point time: The mice were sacrificed 28 days after infusion, and the spleen and bone marrow were collected for flow cytometry analysis.

[0091] ②Key indicators

[0092] Tumor inhibition rate: Calculate the percentage decrease in relative photon flux among each group;

[0093] Specific killing regulation: Compare the differences in tumor burden among the groups to verify the negative regulation of CAAR-Treg on CAAR-T overactivation.

[0094] Experimental results

[0095] 1. Such as Figure 1 As shown in Figure 2, the GPIbαCAAR lentiviral vector plasmid structure design The CAAR expression plasmid is constructed based on the pLenti-EF1α lentiviral vector. Its core components include a second-generation CAR expression cassette driven by the strong EF1α promoter. The CAR structure is composed of a tandem sequence of the human GPIbα extracellular ligand-binding domain (LBD, aa 1-290), the CD8α hinge / transmembrane region, the 4-1BB co-stimulatory domain, and the CD3ζ intracellular activation domain. The plasmid also retains the 5'LTR, Ψ packaging signal, and 3' self-inactivating LTR (SIN-LTR) required for lentiviral packaging, ultimately obtaining the GPIbαCAAR target plasmid that can be used for lentiviral packaging, achieving efficient transduction and stable expression of Treg cells.

[0096] 2. Such as Figure 2 As shown in the figure, the chimeric antibody receptor GPIbα-CAAR is composed of the GPIbα antigen, the CD8 transmembrane domain, the common signaling domain of 4-1BB (CD137) and the signaling domain of the human CD3 complex (chain). The GPIbα extracellular ligand binding domain (LBD) is selected as the antigen epitope embedded in CAAR.

[0097] 3. Such as Figure 3 As shown in Results of Treg sorting and phenotype monitoring. Figure 3 As shown in A, CD4+CD25hiCD127lo / -CD45RA+ cells were sorted by flow cytometry as Tregs were cultured in vitro for 28 days. Figure 3 As shown in Figure B, its inhibitory phenotype remains stable, and the expression rates of its inhibitory markers (FOXP3, CTLA-4, and Helios) remain at a high level.

[0098] 4. Such as Figure 4 As shown in , the expression efficiency of CAAR. Figure 4 Center A shows the detection strategy of GPIbαCAAR-Treg CAAR. Figure 4 Middle B shows the expression rate of CAAR at different MOIs, among which the expression rate of CAAR is the highest at MOI=20. This indicates that MOI=20 is the optimal transfection Optimal conditions for Treg cells.

[0099] 5. Such as Figure 5The results show that GPIbα CAAR-Treg activation was specifically validated. For GPIbα CAAR-Treg, LAP and GARP expression were significantly upregulated in both the anti-GPIbα (CD42b) antibody stimulation group and the hybridoma co-culture group, with the same trend as in the CD3 / CD28 polyclonal activation group. However, NTD-Treg only experienced upregulation of LAP and GARP expression upon CD3 / CD28 stimulation, suggesting that CAAR-Treg can activate suppressive function through antigen-dependent signaling pathways. Furthermore, the LAP / GARP co-expression rate was significantly increased in the anti-GPIbα (CD42b) antibody stimulation group compared to the hybridoma co-culture group, suggesting that direct antibody stimulation is more effective than target cell contact.

[0100] 6. Such as Figure 6 The in vitro specific inhibitory function of GPIbαCAAR-Treg is verified as shown. Under GPIbα (CD42b) antibody stimulation, GPIbαCAAR-Tregs showed strong inhibition of Teff cell activity at a low E:T ratio, while non-transduced (NTD) Tregs only showed inhibitory effects at a high E:T ratio. Similarly, under TCR stimulation, both cell types showed equal inhibitory effects, highlighting the antigen-specific inhibitory effect conferred by GPIbαCAAR-Tregs. GPIbαCAAR confers antigen-specific activation advantages on Tregs, enabling them to accurately recognize target antigens and efficiently exert local immunosuppression, while retaining the broad-spectrum regulatory function of the natural TCR pathway. This feature is expected to break through the risk of non-specific systemic immunosuppression of traditional Tregs therapy, provide a new strategy for the treatment of antibody-mediated ITP, and achieve a balance between targeted inhibition of lesions and normal immune homeostasis through a "dual-signal synergy" mechanism, which has important clinical translation potential.

[0101] 7. Such as Figure 7The results show the in vivo validation of the specific suppressive function of GPIbαCAAR-Treg. While GPIbαCAAR-Treg exhibited some inhibitory effect on hybridomas compared to NTD-Treg, this was significantly weaker than the specific killing of target cells by CAAR-Teff (which mimics antigen-specific T cells in vivo). The NTD-Treg group had the highest tumor burden and exhibited virtually no therapeutic effect, suggesting that GPIbαCAAR endows Treg or Teff cells with the ability to recognize specific antigens. Furthermore, the tumor burden in the CAAR-Treg + CAAR-Teff group was significantly greater than that in the NTD-Treg + CAAR-Teff group, indicating that after CAAR-Treg recognizes and binds to target cells, its suppressive function is activated, significantly suppressing the surrounding CAAR-Teff cells with specific killing functions, leading to an increase in tumor burden. This was not achieved in the NTD-Treg combination group. These results demonstrate that GPIbαCAAR-Treg can significantly suppress the killing effect of antigen-specific T cells in vivo.

[0102] analyze

[0103] The GPIbαCAAR-Treg cells provided by the present invention exhibited a highly stable inhibitory phenotype during long-term in vitro culture, and their FOXP3, CTLA-4, and Helios expression rates continued to be maintained at a high level, showing good immunoregulatory cell characteristics. In terms of function, the cells can be significantly activated under conditions of specific antigen stimulation (such as hybridoma cells expressing GPIbα or soluble anti-GPIbα antibodies), as shown by a significant increase in the proportion of LAP-GARP-positive cells in the CAR+ population, while there was no significant change in the CAR- population, confirming that they have good antigen specificity. Further inhibition experiments demonstrated that, under stimulation with anti-GPIbα (CD42b) antibodies, GPIbαCAAR-Tregs effectively suppressed the activity of effector T cells (Teff) even at a low effector cell to target cell ratio (E:T), whereas non-transduced Tregs (NTD-Tregs) exhibited inhibitory ability only at higher E:T ratios. Under nonspecific TCR stimulation (CD3 / CD28), there was no significant difference in inhibitory ability between the two, further highlighting the antigen-specific immune regulatory function conferred by the CAAR structure. In in vivo experiments, GPIbαCAAR-Tregs significantly inhibited the cytotoxicity of CAAR-Teff against GPIbα-expressing hybridoma cells, while NTD-Tregs did not show a similar effect. This therapy addresses the challenge of multiple immune disorders coexisting in ITP through a three-level regulatory approach: "precisely eliminating pathogenic T cells → blocking TB cell collaboration → remodeling immune tolerance." Compared with current therapies, its core breakthroughs are: 1) Targetedness: CAR guides Tregs to precisely locate autoreactive B cells, avoiding broad-spectrum immunosuppression; 2) Synergy: Synergistic intervention in T cell-mediated platelet destruction and B cell antibody production; 3) Long-term effectiveness: By reestablishing endogenous immune homeostasis, it is expected to achieve sustained remission after drug discontinuation. From a social and economic perspective, this technology provides a new cell therapy strategy for patients with refractory and recurrent ITP that is precise, efficient, and provides long-term remission, reducing the toxic side effects and recurrence risk associated with long-term use of hormones and immunosuppressants, lowering the medical burden, and improving patients' quality of life. At the same time, this technology platform also has broad application potential and can be expanded to the treatment of other autoimmune diseases, with significant clinical translation prospects and industrial value.

[0104] It will be clear to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. Reference may be made to prior art for any matters not fully detailed herein. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.

Claims

1. A method for constructing GPIbαCAAR, characterized in that: The method comprises the following steps: integrating the GPIbα extracellular ligand binding domain LBD with a sequence of SEQ ID NO.1 into a second-generation CAR structure comprising a CD8α hinge / transmembrane region, a 4-1BB co-stimulatory domain and an intracellular CD3ζ domain to form a GPIbαCAAR with a sequence of SEQ ID NO.

2.

2. The GPIbαCAAR prepared by the construction method of GPIbαCAAR according to claim 1.

3. Use of the GPIbαCAAR according to claim 2 in the preparation of a drug for treating immune thrombocytopenia.

4. A method for constructing a GPIbαCAAR plasmid, characterized in that: The steps include: Construction of GPIbαCAAR: The GPIbα extracellular ligand binding domain (LBD) of SEQ ID NO. 1 was integrated into a second-generation CAR structure comprising a CD8α hinge / transmembrane region, a 4-1BB costimulatory domain, and an intracellular CD3ζ domain to form a GPIbαCAAR of SEQ ID NO. 2; Construction of GPIbαCAAR plasmid: GPIbαCAAR sequence SEQ ID NO.2 was inserted into the vector to form GPIbαCAAR plasmid; Among them, the vector is preferably a lentiviral vector; more preferably, the lentiviral vector is a pLenti-EF1α vector; more preferably, GPIbαCAAR is inserted into the pLenti-EF1α vector to form a GPIbαCAAR plasmid with a sequence of SEQ ID NO.

3.

5. The GPIbαCAAR plasmid prepared by the construction method of the GPIbαCAAR plasmid according to claim 4.

6. Use of the GPIbαCAAR plasmid according to claim 5 in the preparation of a drug for treating immune thrombocytopenia.

7. A method for constructing GPIbαCAAR-Treg, characterized in that: The steps include: Transfecting the GPIbαCAAR plasmid according to any one of claims 4 to 5 into human embryonic kidney cells, collecting and concentrating the viral supernatant, The viral supernatant was co-incubated with activated Treg cells to obtain GPIbαCAAR-Treg cells; Preferably, the activated Treg cells are of human origin, further derived from human peripheral blood mononuclear cells; more preferably, the activated Treg cells are stimulated by CD3 / 28 magnetic beads.

8. GPIbαCAAR-Treg cells prepared by the method for constructing GPIbαCAAR-Treg according to claim 7.

9. Use of the GPIbα CAAR-Treg cells according to claim 8 in the preparation of a drug for treating immune thrombocytopenia.

10. Use of an anti-GPIbα antibody or its secretory cells in the preparation of a product for stimulating and activating GPIbα CAAR-Treg cells.

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