Long-acting drugs for the treatment of allergic diseases and their active ingredient chimeric antigen receptors

Abstract

The application provides a chimeric antigen receptor comprising five domain structures of antigen-specific binding, hinge, transmembrane, costimulation and CD3 zeta signaling; specifically binds to an antigen comprising an immunoglobulin IgE EMPD domain through the antigen-specific binding domain; and provides its long-term therapeutic application for IgE-mediated allergic diseases. Compared with the prior art, the application combines anti-IgE monoclonal antibody technology and CAR technology, changes from targeting the produced IgE protein to targeting the IgE-producing B cells, and the IgE CAR-T has been proved to have a killing effect on mIgE + B cells in vitro and in vivo experiments, solving the problems of short half-life of monoclonal antibody drugs and high drug frequency. In addition, the CAR of the application can effectively avoid the interference of free sIgE, and the therapeutic effect of CAR-T is successfully verified through in vivo experiments by constructing a humanized mouse model.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to long-acting drugs for the treatment of allergic diseases and their active ingredient, chimeric antigen receptors. Background Technology

[0002] The prevalence of allergic diseases continues to rise globally, including food allergies, allergic asthma, atopic dermatitis, urticaria, allergic rhinitis, conjunctivitis, and chronic sinusitis. Immunoglobulin E (IgE) is a key molecule in allergic reactions. Various forms of IgE-related allergic diseases affect approximately 30% of the world's population.

[0003] When patients are exposed to allergens, the combined action of dendritic cells, Th2 and Tfh cells, and cytokines such as IL-4, IL-5, and IL-13 stimulates B cell differentiation and the production of IgE antibodies. These IgE antibodies bind to mast cells and basophils, mediating their degranulation reaction and releasing inflammatory mediators, thereby triggering a series of allergic symptoms. Therefore, IgE has become an important target for drug development in allergic diseases. The anti-IgE monoclonal antibody Omalizumab became the first approved targeted therapy, blocking downstream allergic inflammatory responses by neutralizing free IgE. Omalizumab was approved by the FDA in 2003 for the treatment of severe allergic asthma and in 2014 for the treatment of urticaria. Some studies have also shown therapeutic effects on allergic rhinitis, food allergies, and other allergic diseases. Omalizumab treatment can effectively improve the symptoms of allergic asthma and urticaria, improving patients' quality of life. However, as an IgE neutralizing antibody, omalizumab primarily neutralizes existing free IgE, with minimal impact on IgE production. Therefore, omalizumab is not effective in maintaining a low level of free IgE over a long period.

[0004] Following this, Genentech developed the monoclonal antibody 47H4, which recognizes the EMPD domain of mIgE. After humanization, it was named Quilizumab. mIgE is the membrane-bound form of IgE, expressed on the surface of B cells and plasma cells. It is generally believed that mIgE... + Plasma cells and B cells are the main sources of IgE in allergic reactions. 47H4 monoclonal antibody can induce mIgE. +B cell apoptosis. In a phase I clinical trial for allergic rhinitis and a phase II clinical trial for allergic asthma, quilizumab effectively reduced IgE levels in patients, but its improvement in asthma symptoms was low. However, increasing the dosage and frequency of administration had a positive effect on symptom improvement. Quilizumab monoclonal antibody targets mIgE, and although it can induce mIgE to some extent... + B cell apoptosis reduces IgE production, but due to the short half-life of monoclonal antibody drugs, their effectiveness also depends on a certain dosage and frequency of administration.

[0005] To date, monoclonal antibodies remain the primary treatment for IgE-targeting allergic diseases. However, monoclonal antibody drugs have short half-lives, and their efficacy depends on a certain frequency of use, which imposes significant limitations, necessitating more effective treatment methods. Chimeric antigen receptor T-cell (CAR-T) therapy involves genetically modifying human T cells in vitro to recognize specific target antigens, expanding them, and then reinfusing them into the patient to treat the disease. CAR-T technology has achieved remarkable results in treating hematologic malignancies, and the memory function of T cells enables long-term tumor suppression. Therefore, applying CAR-T to the treatment of long-term chronic allergy-related diseases also becomes possible.

[0006] Therefore, current IgE-targeting treatments cannot achieve long-term inhibition of IgE levels in the body or long-term relief of allergy symptoms. Summary of the Invention

[0007] In view of the aforementioned technical limitations, this application proposes a long-acting drug for treating allergic diseases and its active ingredient, a chimeric antigen receptor; which overcomes the deficiencies and defects mentioned in the background art.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] The inventive point of this application is to provide a chimeric antigen receptor, comprising an antigen-specific binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain; the antigen-specific binding domain is capable of specifically binding antigens including an immunoglobulin IgEEMPD domain; the antigen-specific binding domain comprises a single-chain variable fragment scFv consisting of an amino acid sequence as shown in SEQ ID No. 1, or comprises a single-chain variable fragment scFv having more than 80% homology with the amino acid sequence shown in SEQ ID No. 1 and having the same or similar functions.

[0010] Optionally, the chimeric antigen receptor described above, wherein the single-chain variable fragment scFv, which has more than 80% homology with the amino acid sequence shown in SEQ ID No. 1 and has the same or similar function, has the amino acid sequence shown in SEQ ID No. 2.

[0011] Optionally, the chimeric antigen receptor described above may be a polypeptide of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, or a combination thereof.

[0012] Optionally, in the chimeric antigen receptor described above, the hinge domain is selected as the amino acid sequence shown in SEQ ID No. 4 or a hinge domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 4 and having the same or similar functions; the transmembrane domain is selected as the amino acid sequence shown in SEQ ID No. 5 or a transmembrane domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 5 and having the same or similar functions; the CD3ζ signal transduction domain is selected as the amino acid sequence shown in SEQ ID No. 6 or a CD3ζ signal transduction domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 6 and having the same or similar functions; the costimulatory domain is selected as the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No. 8 or a costimulatory domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No. 8 and having the same or similar functions.

[0013] Optionally, in the chimeric antigen receptor described above, the hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No. 10; the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No. 11; the CD3ζ signal transduction domain is encoded by the nucleic acid sequence shown in SEQ ID No. 12; and the co-stimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No. 13 or SEQ ID No. 14.

[0014] A second objective of this application is to provide an isolated immune cell modified to express a chimeric antigen receptor, the chimeric antigen receptor comprising an antigen-binding domain connected to a co-stimulatory domain and a CD3ζ signaling domain, the antigen-binding domain being capable of specifically binding a single-chain variable fragment scFv comprising an immunoglobulin IgE EMPD domain, the amino acid sequence of the single-chain variable fragment scFv being as shown in SEQ ID No. 1 or SEQ ID No. 2; the immune cell is preferably a T cell.

[0015] Optionally, in the isolated immune cells described above, the co-stimulatory domain is selected as a CD28 or 41BB polypeptide.

[0016] Optionally, the chimeric antigen receptor expressed in the isolated immune cells described above is the aforementioned chimeric antigen receptor.

[0017] Optionally, the isolated immune cells described above are selected as CD4 cells. + T cells, CD8 + T cells or CD4 cells mixed in any proportion + and CD8 + T cells.

[0018] A third objective of this application is to provide an expression vector that encodes the aforementioned chimeric antigen receptor.

[0019] The fourth objective of this application is to provide a host cell comprising the aforementioned expression vector.

[0020] The fifth objective of this application is to provide the use of the above-mentioned chimeric antigen receptor, the above-mentioned isolated immune cells, the above-mentioned expression vector, and the above-mentioned host cells in the preparation of a long-acting medicament for treating allergic diseases.

[0021] Optionally, in the above application, the allergic disease is selected as any one or more of the types of IgE-mediated allergic reactions, preferably allergic asthma, food allergy, or urticaria.

[0022] The sixth objective of this application is to provide a long-acting pharmaceutical composition for treating allergic diseases, the pharmaceutical composition comprising the chimeric antigen receptor described above, the isolated immune cells described above, the expression vector described above, and the host cells described above.

[0023] The core technology of this application combines anti-IgE monoclonal antibodies with CAR-T technology. The IgE monoclonal antibody forms the extracellular domain of the CAR's scFv structure, which targets B cells or plasma cells expressing mIgE. A lentiviral vector containing the IgE scFv fragment is constructed, and lentivirus is packaged by transfection with 293T cells. T cells are isolated from human PBMCs or CBMCs, activated with CD3 / CD28 Dynabeads, and cultured and expanded using IL-2-added medium. Primary T cells are then infected with the IgE CAR lentivirus to construct CAR-T cells. The expanded CAR-T cells can be used in vitro to target mIgE expression. + Cells are killed by mIgE +The cells included the U266 cell line, which expressed low levels of mIgE, and the Daudi-mIgE-mCherry cell line, constructed via lentiviral transduction. This application also validated the therapeutic effect of IgE CAR-T cells in vivo by constructing a humanized mouse HDM-induced asthma model. In this asthma model, the adopted IgE CAR-T cells were also able to kill mIgE cells in vivo. + B cells, reduce IgE levels and maintain this level long-term.

[0024] Compared with the prior art, this application has the following advantages:

[0025] First, this application combines IgE monoclonal antibodies with CAR-T technology, changing the target from the already produced IgE protein to the target of IgE-producing B cells. Both in vitro and in vivo experiments have demonstrated that IgE CAR-T targets mIgE. + This improves the killing effect on B cells, solving the problem of short half-life and high-frequency dosing required by monoclonal antibody drugs.

[0026] Secondly, the monoclonal antibody in this application can specifically target mIgE instead of sIgE, and the resulting CAR-T has been shown to be less susceptible to interference from free sIgE, thus maintaining its targeting to mIgE. + The killing ability of B cells has also been verified.

[0027] Finally, due to significant species differences in key mIgE sequences between humans and mice, it is difficult to conduct in vivo experiments using mouse animal models, and previous techniques have failed to demonstrate in vivo experimental data. This application proposes a scheme for conducting in vivo experiments using humanized mouse models and has successfully verified the therapeutic effects of CAR-T. Attached Figure Description

[0028] Figure 1 A schematic diagram illustrating the construction of mIgECAR-T is shown; in which,

[0029] Figure 1 A is a schematic diagram of plasmid construction; Figure 1 B represents the flow cytometry results after staining CAR-T with protein L, including CAR-T from three donors, indicating CAR membrane surface expression. Figure 1 C represents CD4 in CAR-T. + and CD8 + Statistical graph of cell clustering results; Figure 1 D is a statistical diagram showing the clustering of naive T cells, central memory T cells, effector memory T cells, and effector T cells in CAR-T therapy.

[0030] Figure 2 Displayed as mIgE + Target cell construction and CAR-T against mIgE + A schematic diagram illustrating the results of target cell killing; where,

[0031] Figure 2 A is a statistical graph showing the relative cell count of target cells after co-culturing CAR-T and U266 target cells; Figure 2 B is a schematic diagram of the plasmid used for constructing Daudi target cells; Figure 2 C represents the expression of IgE on the cell membrane surface in U266 and Daudi target cells; Figure 2 D is a statistical graph showing the relative cell number of target cells after co-culturing CAR-T cells with Daudi-mIgE-mCherry, Daudi-sIgE-mCherry, and Daudi-mCherry target cells, respectively (the data shows the mean ± SEM, and the difference was not statistically significant after two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001, nsp>0.05, and the statistical tests in this application were all labeled according to this standard; the p value in the figure is marked as the difference between each group and the UTD group at a 10:1 effector-to-target ratio).

[0032] Figure 3 The display shows the kill effect after mutating this portion of the CAR-TscFv; among which...

[0033] Figure 3 A is a schematic diagram of CAR-T plasmid construction, BB-1 is the negative control plasmid, BB-3 is the original CAR-T, and BB-3-m1 is the modified mutant CAR-T; Figure 3 B is a statistical graph showing the relative cell number of target cells after co-culturing CAR-T cells with Daudi-mCherry and Daudi-mIgE-mCherry target cells at effector-to-target ratios of 1:1 and 10:1, respectively.

[0034] Figure 4 This diagram illustrates the activation status of each CAR-T cell after co-culturing with target cells; among them...

[0035] Figure 4 A represents the CD8+ levels of CAR-T cells after co-culturing with Daudi-mIgE-mCherry, U266, Daudi-sIgE-mCherry, and Daudi-mCherry, or after CAR-T cells were cultured alone. + Expression of CD107a, Granzyme B, CD69, CD25, and PD-1 in CAR-T cells, and PD-1 expression. + TIM-3 + Proportional statistics chart; Figure 4B represents the content of TNFα and IFNγ in the culture supernatant after CAR-T cells were co-cultured with Daudi-mIgE-mCherry, U266, Daudi-sIgE-mCherry, and Daudi-mCherry, respectively. Figure 4 C is a flow cytometry plot showing the proportions of PD-1, LAG-3, and TIM-3 expression of the two CAR-T cells in the exhaustion model (BB-1 and 28-1 are CAR-1 groups, and BB-3 and 28-3 are CAR-3 groups, the difference being the antigen-specific binding domain).

[0036] Figure 5 Displayed as CD4 + and CD8 + A diagram illustrating the comparison of CAR-T lethality; where...

[0037] Figure 5 A is CD4 in CAR-T cells after sorting. + and CD8 + Cell ratio; Figure 5 B is CD4 + and CD8 + Statistical graph of relative cell number of target cells after co-culturing the two CAR-T cells with Daudi-mIgE-mCherry target cells.

[0038] Figure 6 The graph shows the relative number of target cells after co-culturing in a CAR-T and Daudi-mIgE-mCherry co-culture system with or without the addition of recombinant sIgE protein. The effector-target ratio is 1:1, and the co-culture time is 24 hours.

[0039] Figure 7 The results are shown for mIgECAR-T therapy in humanized mice with asthma; among them...

[0040] Figure 7 A and Figure 7 B represents the percentage of CAR-T cells (hCD45) in the lungs and spleen of mice after secondary stimulation, respectively. + )Proportion; Figure 7 C and Figure 7 D represents the levels of mIgE in the lungs and spleen of mice after the second challenge. + The proportion of B cells in human immune cells (significance analysis in the figure indicates differences compared to the model group, the same below); Figure 7 E represents the total IgE content in mouse serum after the first challenge. Figure 7 F represents the level of HDM-specific IgE protein in mouse serum after the first challenge. Figure 7 G represents the total IgE content in mouse serum after the second challenge. Figure 7H represents the level of HDM-specific IgE protein in mouse serum after the second challenge. Figure 7 Image I shows H&E staining of mouse lung sections after the second stimulation (scale bar marked 100 μm). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0043] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0044] Example 1

[0045] The chimeric antigen receptor provided in this application includes an antigen-specific binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain. The antigen-specific binding domain is capable of specifically binding antigens including the immunoglobulin IgE EMPD domain. The antigen-specific binding domain includes a single-chain variable fragment scFv composed of the amino acid sequence shown in SEQ ID No. 1, or includes a single-chain variable fragment scFv that has more than 80% homology with the amino acid sequence shown in SEQ ID No. 1 and has the same or similar function, and its amino acid sequence is shown in SEQ ID No. 2.

[0046] The co-stimulatory domains are selected from peptides of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1 or combinations thereof.

[0047] The hinge domain is selected as the amino acid sequence shown in SEQ ID No. 4 or a hinge domain that has more than 80% homology with the amino acid sequence shown in SEQ ID No. 4 and has the same or similar functions.

[0048] The transmembrane domain is selected as the amino acid sequence shown in SEQ ID No. 5 or a transmembrane domain that has more than 80% homology with the amino acid sequence shown in SEQ ID No. 5 and has the same or similar functions.

[0049] The CD3ζ signal transduction domain is selected as the amino acid sequence shown in SEQ ID No. 6 or the CD3ζ signal transduction domain that has more than 80% homology with the amino acid sequence shown in SEQ ID No. 6 and has the same or similar functions.

[0050] The co-stimulatory domain is selected as the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No. 8, or a co-stimulatory domain that has more than 80% homology with the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No. 8 and has the same or similar function.

[0051] The above homology can be selected as 80% homology, 85% homology, 90% homology, 95% homology, or 99% homology.

[0052] The hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No. 10;

[0053] The transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No. 11;

[0054] The CD3ζ signal transduction domain is encoded by the nucleic acid sequence shown in SEQ ID No. 12;

[0055] The co-stimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No. 13 or SEQ ID No. 14.

[0056] In addition to the sequences shown above, the chimeric antigen receptor in this application may also have the following sequences selected for each domain:

[0057] A signal peptide may or may not be provided upstream of the chimeric antigen receptor. The signal peptide may be CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0058] The hinge domain of the chimeric antigen receptor can also be selected as CD8, CD28, CD137, or a combination thereof;

[0059] The transmembrane domain of the chimeric antigen receptor may also be selected as CD3epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof;

[0060] The co-stimulatory domain of the chimeric antigen receptor may also be selected as OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, or a combination thereof;

[0061] The 2A selection for the chimeric antigen receptor is T2A, P2A, E2A, or F2A.

[0062] This application also provides isolated immune cells, namely T cells, which are modified to express the aforementioned chimeric antigen receptor, and the T cells are selected to be CD4+. + T cells, CD8 + T cells or CD4 cells mixed in any proportion + and CD8 + T cells.

[0063] This application also provides an expression vector capable of encoding the aforementioned chimeric antigen receptor.

[0064] This application also provides a host cell including the aforementioned expression vector.

[0065] This application also provides the use of chimeric antigen receptors, isolated T cells, expression vectors, and host cells in the preparation of long-acting drugs for the treatment of allergic diseases.

[0066] Allergic diseases are selected as any one or more of the types of allergic reactions mediated by IgE, preferably allergic asthma, food allergy, and urticaria.

[0067] This application also provides a long-acting pharmaceutical composition for treating allergic diseases, comprising the above-described chimeric antigen receptor, isolated T cells, expression vector, and host cells.

[0068] Example 2

[0069] 1. Experimental materials and methods:

[0070] 1) CAR-T construction:

[0071] First, a lentiviral plasmid targeting mIgE was synthesized. Its coding region includes a signal peptide gene fragment, an anti-mIgE single-chain antibody gene fragment, a transmembrane region gene fragment, and an intracellular signal region gene fragment, with or without a T2A-linked EGFP tag gene fragment. Following this protocol, CAR plasmids containing different single-chain antibodies and a negative control plasmid without single-chain antibodies were constructed. Each CAR lentivirus was packaged using 293T cells.

[0072] The constructed CAR plasmids contain different single-chain antibodies or co-stimulatory domains. Figure 1BB-3 and 28-3 are different co-stimulatory domains; Figure 3 BB-3 and BB-3-m1 are different single-chain antibodies, and their structural profiles are shown below:

[0073] CD8 leader (leading strand domain, CD8-SP) — single-chain antibody gene fragment scFv — CD8-hinge — CD8-TM — 4-1BB or CD28 — CD3zeta — T2A EGFP;

[0074] Negative control CAR plasmid ( Figure 1 The structure of BB-1 is as follows:

[0075] CD8 leader—CD8-hinge—CD8-TM—4-1BB or CD28—CD3zeta—T2AEGFP.

[0076] The sequences of each part are as follows:

[0077] (1) The sequence of the CD8 leader peptide is shown in SEQ ID No. 3:

[0078] MALPVTALLLPLALLLHAARP;

[0079] (2) When the single-chain antibody gene fragment scFv is selected from 47H4 ( Figure 1 (BB-3 or 28-3), the sequence of which is shown in SEQ ID No. 1:

[0080] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFISDLAYTIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWDAMDYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRSSQSLVHNNGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQNTLVPWTFGQGTKVEIKR;

[0081] When the single-chain antibody gene fragment scFv is selected as scFv-3-m1 ( Figure 3 The sequence of BB-3-m1 is shown in SEQ ID No. 2:

[0082] EVQLVQSGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWIAFISDLAYTIYYADTVTGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARDNWDAMDYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSSLSASVGDRVTLTCRSSQSLVHNNGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPTRFSGSGSGTDFTLTISSLQPEDFATYYCSQNTLVPWTFGQGTKVEIKR.

[0083] (3) The sequence of the CD8 hinge (CD8-hinge) is shown in SEQ ID No. 4:

[0084] AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0085] (4) The sequence of the CD8 transmembrane region (CD8-TM) is shown in SEQ ID No. 5:

[0086] IYIWAPLAGTCGVLLLSLVITLYC;

[0087] (5) The sequence of the CD3ζ signal transduction structure (CD3zeta) is shown in SEQ ID No. 6:

[0088] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR;

[0089] (6) When the co-stimulatory structure is selected as 4-1BB, its sequence is shown in SEQ ID No. 7:

[0090] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0091] When the co-stimulatory structure is CD28, its sequence is shown in SEQ ID No. 8:

[0092] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;

[0093] (7) The sequence of the tracer structure (T2AEGFP) is shown in SEQ ID No. 15:

[0094] EGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0095] The single-chain antibody gene fragment scFv is linked to the CD8 leader peptide via the short peptide SR, and to the CD8 hinge domain via the short peptide PGAAA. The negative control CAR-1 plasmid does not contain scFv; its CD8 leader peptide is linked to the CD8 hinge domain via a linker peptide (SRLVLEHMHPGVAA).

[0096] The nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 3, which encodes the leading strand domain, is shown in SEQ ID No. 9;

[0097] SEQ ID No. 9:

[0098] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg.

[0099] The nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4, which encodes the hinge domain, is shown in SEQ ID No. 10.

[0100] SEQ ID No. 10:

[0101] gcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccc tgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat.

[0102] The nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 5, which encodes the transmembrane domain, is shown in SEQ ID No. 11;

[0103] SEQ ID No. 11:

[0104] atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc.

[0105] The nucleic acid sequence encoding the CD3ζ signal transduction domain of SEQ ID No. 6 is shown in SEQ ID No. 12;

[0106] SEQ ID No. 12:

[0107] agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa ggcctgtacaatgaactgcagaaagataagatggcggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc.

[0108] The nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 7 or SEQ ID No. 8, which encodes the co-stimulatory domain, is shown in SEQ ID No. 13 or SEQ ID No. 14;

[0109] SEQ ID No. 13:

[0110] aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagg aagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg.

[0111] SEQ ID No. 14:

[0112] cgaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggaccaaccaga aagcactatcagccttacgctcctcctcgggacttcgccgcctatagatct.

[0113] The nucleotide sequence encoding the tracer structure (T2AEGFP) SEQ ID No. 15 is shown in SEQ ID No. 21;

[0114] SEQ ID No. 21:

[0115] gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa。

[0116] As can be seen from the above description, the specific structural sequences of CAR plasmids containing different single-chain antibodies are as follows:

[0117] Negative control CAR plasmid ( Figure 1 BB-1 in it) has the sequence shown in SEQ ID No. 16:

[0118] SEQ ID No. 16:

[0119] MALPVTALLLPLALLLHAARPSRLVLEHMHPGVAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

[0120] Negative control CAR plasmid ( Figure 1 in 28-1), has the sequence shown in SEQ ID No. 17:

[0121] SEQ ID No. 17:

[0122] MALPVTALLLPLALLLHAARPSRLVLEHMHPGVAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTR AEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0123] The constructed CAR plasmids containing different single-chain antibodies ( Figure 1 BB-3, 28-3 and Figure 3 BB-3-m1), each having the sequences shown in SEQ ID No. 18 to SEQ ID No. 20;

[0124] BB-3 (SEQ ID No. 18):

[0125] MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFISDLAYTIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQSLVHNNGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQNTLVPWTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

[0126] 28-3(SEQ ID No.19):

[0127] MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFISDLAYTIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQSLVHNNGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQNTLVPWTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

[0128] BB-3-m1(SEQ ID No.20):

[0129] MALPVTALLLPLALLLHAARPSREVQLVQSGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWIAFISDLAYTIYYADTVTGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARDNWDAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTLTCRSSQSLVHNNGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPTRFSGSGSGTDFTLTISSLQPEDFATYYCSQNTLVPWTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK。

[0130] The nucleic acid sequence encoding the amino acid sequence of the negative control CAR plasmid ( Figure 1 BB-1 in it, SEQ ID No. 16) is shown as SEQ ID No. 22;

[0131] SEQ ID No. 22:

[0132]

[0133] Encoding negative control CAR plasmid ( Figure 1 The nucleic acid sequence of the amino acid sequence (28-1, SEQ ID No. 17) is shown in SEQ ID No. 23;

[0134] SEQ ID No. 23:

[0135]

[0136] CAR plasmids encoding different single-chain antibodies ( Figure 1 The nucleic acid sequence of BB-3 (SEQ ID No. 18) is shown in SEQ ID No. 24;

[0137] SEQ ID No. 24:

[0138]

[0139] CAR plasmids encoding different single-chain antibodies ( Figure 1 The nucleic acid sequence of the amino acid sequence (28-3, SEQ ID No. 19) is shown in SEQ ID No. 25;

[0140] SEQ ID No. 25:

[0141]

[0142] CAR plasmids encoding different single-chain antibodies ( Figure 3 The nucleic acid sequence of BB-3-m1 (SEQ ID No. 20) is shown in SEQ ID No. 26.

[0143] SEQ ID No. 26:

[0144]

[0145] T cells were isolated from human mononuclear cells using a human T cell negative selection kit (Medrin) and cultured in X VIVO15 medium (supplemented with 2.5% human AB serum, 1% penicillin-streptomycin, and 100 IU / mL IL-100). -2 The cells were cultured and stimulated with CD3 / CD28 dynabeads (Invitrogen) for 3 days. During this period, T cells were infected with CAR lentivirus to construct CAR-T cells. The positivity rate was detected on days 7-10 of culture, and sorting was performed using EGFP fluorescent labeling.

[0146] 2) Flow cytometry:

[0147] Antibodies used PE / Cyanine7 anti-human CD45, Brilliant Violet 605anti-humanCD3, PE / Cyanine5 anti-human CD4, Brilliant Violet 421anti-human CD8, APC anti-human CD69, Brilliant Violet anti-human CD25, Alexa Fluor 700anti-human PD-1, PE / Cyanine7 anti-human LAG-3, Brilliant Violet 750anti-human TIM-3, APC anti-human CD19, PE anti-human IgE, Brilliant Violet 510anti-human IgM, Brilliant Violet 421anti-human IgD, Brilliant Violet 605anti-human CD107a, PE / Cyanine7anti-human Granzyme B, PE protein L, Brilliant Violet 711anti-human CD62L, PE / Cyanine7 anti-human Cells were stained with CD45RA (all purchased from BioLegend). For intracellular staining, cells were treated with cell fixation / permeability buffer (BD Bioscience) before antibody staining. Analysis was performed using a BD Fortessa flow cytometer, or sorting was performed using a BD Fusion flow cytometer. Flow cytometry data were analyzed using Flowjo software.

[0148] 3) T-cell killing efficiency assay:

[0149] CAR-T cells or UTDs (UTDs, meaning untransduced cells; in cell therapy, UTD cells refer to cells that have not undergone specific gene transduction) and target cells were co-cultured in 96-well U-bottom plates at an effector-to-target ratio (E:T) using RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Separate wells for target cells were also included, with the same cell count as the co-culture wells. Daudi-mIgE-mCherry, Daudi-sIgE-mCherry, and Daudi-mCherry target cells were all fluorescently labeled with mCherry. U266 target cells were stained with CellTracker CMTPX (Invitrogen) before co-culture. At the end of co-culture, Daudi wild-type cells were stained with CellTracker CM-Dil (Daudi-Dil) as quantification. Equal amounts of Daudi-Dil cells were added to each co-culture well or individual culture well, and the proportion of each cell population in each well was detected by flow cytometry. The relative number of target cells was calculated as follows: (proportion of target cells in co-culture wells / proportion of Daudi-Dil cells in co-culture wells) / (proportion of target cells in individual culture wells / proportion of Daudi-Dil cells in individual culture wells).

[0150] 4) ELISA:

[0151] The levels of TNFα and IFNγ in the cell co-culture supernatant were detected using the Human TNFα Deluxe Kit and the Human IFNγ Deluxe Kit (BioLegend), respectively. The procedures were performed according to the kit instructions.

[0152] 5) Humanized mouse asthma model:

[0153] Humanized Hu-HSC-NPG-GM3 mice, constructed by Vitronic, were used as experimental material. The degree of humanization of their immune systems was measured to be over 40%. A humanized mouse asthma model was established by intratracheal administration of HDM (GREER, XPB91D3A2.5). Sensitization was achieved by intratracheal administration of 100 μg / 40 μL HDM on days 0 and 7. Simultaneously, IgE CAR-T cells were constructed using T cells isolated from the same donor's umbilical cord blood from the humanized mice, and CAR-T adoptive transfer was performed on day 12. HDM stimulation was repeated on day 14. HDM-specific IgE levels were analyzed by tail vein blood collection on day 17. Relapse was induced by repeated administration of 50 μg / 20 μL HDM on days 32, 33, and 34. Mice were sacrificed on day 36, and spleen, lung tissue, and peripheral blood samples were collected for analysis.

[0154] 2. Experimental Results:

[0155] 1) Constructing IgE CAR-T:

[0156] To test whether CAR-T therapy targeting mIgE could be used to treat allergic diseases, the inventors first selected the mIgE-targeting monoclonal antibody 47H4 to form the scFv fragment, as shown in the figure. Figure 1 A) Construct lentiviral plasmids and transduce human PBMC-derived T cells using lentivirus. Currently, the commonly used co-stimulatory domains for second-generation CAR-T are 4-1BB or CD28. Depending on the co-stimulatory domain, the constructed CAR-T cells are designated as BB-3 or 28-3, respectively. BB-1 and 28-1 are CAR-T controls that do not contain scFv.

[0157] Protein L staining and GFP positivity analysis showed that CARs were normally expressed on the cell membrane in both BB-3 and 28-3 cells, with a positive rate of over 60%. Figure 1 B). CAR-T cell typing is shown in the figure ( Figure 1 C Figure 1 D), CD8 was measured at 14-20 days of culture. + The proportion of T cells was approximately 50%, and the proportion of effector T cells was approximately 10%-20%, with no significant difference between the two CAR-T cells.

[0158] 2) Both BB-3 and 28-3 are effective against mIgE. + The target cells have a specific killing effect:

[0159] To detect the cytotoxic effect of IgE CAR-T on target cells in vitro, the inventors used the U266 cell line, which naturally expresses low levels of mIgE, as target cells. IgE CAR-T cells were co-cultured with the CAR-T cells in vitro to assess the cytotoxic effect. The experiment showed that while BB-3 and 28-3 cells did not significantly kill U266 cells within 24 hours, a relatively low efficacy-to-target ratio of 1:1 allowed for sustained cytotoxicity over 5 days. After 5 days, they reduced U266 cells to below 60%, indicating similar cytotoxicity between BB-3 and 28-3. Figure 2 A). To further demonstrate the effects of BB-3 and 28-3 on mIgE + To further enhance the killing effect on target cells, the inventors also constructed a human mIgE inhibitor. + Daudi-mIgE-mCherry cells, and two mIgE strains - Cells, namely Daudi-sIgE-mCherry expressing sIgE and negative control Daudi-mCherry ( Figure 2 B. Figure 2 C). At different effector-to-target ratios, IgE CAR-T cells were co-cultured with three types of target cells for 24 hours, and the remaining target cell count was measured. Figure 2 D) It was found that co-culturing BB-3 and 28-3 with Daudi-mIgE-mCherry significantly reduced the survival rate of the target cells, and the killing effect increased with the increase of the effector-to-target ratio. At an effector-to-target ratio of 1:1, both BB-3 and 28-3 killed less than 70% of Daudi-mIgE-mCherry cells; at an effector-to-target ratio of 10:1, both BB-3 and 28-3 killed less than 10% of the target cells. Similar to the killing effect on U266, the killing abilities of the two were quite similar. Compared with the killing effect on Daudi-mIgE-mCherry, BB-3 and 28-3 had no significant killing effect on Daudi-sIgE-mCherry and Daudi-mCherry. These results indicate that BB-3 and 28-3 target mIgE... + The cells all possess strong specific killing ability.

[0160] 3) BB-3 variants against mIgE + The target cells have a specific killing effect:

[0161] The inventors further mutated the 47H4scFv sequence to construct a variant CAR-T of BB-3: BB-3-m1. Figure 3 A). Similarly, CAR-T cells were co-cultured with Daudi-mIgE-mCherry and Daudi-mCherry at different effector-to-target ratios for 24 hours, and the remaining target cells were detected. Figure 3 B). The results showed that, at both the 1:1 and 10:1 effective-target ratios, BB-3-m1 exhibited significantly higher lethality against Daudi-mIgE-mCherry than Daudi-mCherry, with the 10:1 ratio showing even stronger lethality than the 1:1 ratio. Furthermore, it was observed that the lethality of BB-3-m1 was essentially equivalent to that of BB-3. These results indicate that the BB-3 variant CAR-T still exhibits lethality against mIgE. + The target cells have a specific killing effect.

[0162] 4) IgE CAR-T cells are activated upon stimulation by target cells:

[0163] Flow cytometry staining analysis was performed on CAR-T cells at the end of co-culture. Figure 4A) Stimulation with Daudi-mIgE-mCherry significantly increased the proportions of CD107a, Granzyme B, CD69, CD25, and PD-1 positive cells in both BB-3 and 28-3, with the most significant increase observed in 28-3. CD107a and Granzyme B reflect the intensity of degranulation and cytotoxicity; the positive rate of CD107a in BB-3 was approximately 40%, while in 28-2 it was approximately 60%. The positive rate of Granzyme B in BB-3 was approximately 8%, while in 28-3 it reached 15%. CD69 and CD25 reflect cell activation levels; the positive rates in both BB-3 and 28-3 were significantly higher than in the UTD group, and 28-3 was also significantly higher than BB-3. PD-1 can also reflect cell activation levels to some extent; 28-3 was also significantly higher than BB-3. Similarly, when U266 was used as the co-culture target cell, the expression levels of CD107a, Granzyme B, CD69, CD25, and PD-1 in BB-3 and 28-3 cells showed similar trends. In contrast, BB-3 and 28-3 cells showed different expression levels for the two mIgE inhibitors. - The cells showed no obvious response to stimulation. Cytokine levels were detected in the supernatant of the co-culture system. Figure 4 B) The results showed that BB-3 and 28-3 cells significantly increased the levels of TNFα and IFNγ secreted by Daudi-mIgE-mCherry cells, while showing a lower increase in U266 cells. Regarding mIgE... - Cellular stimulation did not elicit a significant response. The above data indicate that BB-3 and 28-3 play a role in mIgE activity. + Specific activation occurs upon stimulation of target cells, with 28-3 cells showing a higher level of activation.

[0164] It is generally believed that the CD28 co-stimulatory domain is more likely to lead to CAR-T depletion than 4-1BB, therefore the inventors also compared the depletion of CAR-T cells. At the end of the 24-hour co-culture, PD-1 in CAR-T cells was detected. + TIM-3 + Cell ratio analysis revealed that 28-3 was significantly higher than BB-3, suggesting that CD28, while inducing higher CAR-T activation levels, also caused stronger exhaustion. To further demonstrate this, the following exhaustion model was introduced: CAR-T cells were repeatedly stimulated with Daudi-mIgE-mCherry target cells for a prolonged period (10 days) at a low target-to-cell ratio (1:10), and the expression of three exhaustion markers, PD-1, LAG-3, and TIM-3, was detected to indicate their exhaustion level. The results showed that after stimulation, although BB-3 and 28-3 had similar PD-1 expression, the expression of exhaustion markers LAG-3 and TIM-3 in 28-3 was higher than that in BB-3. Figure 4 C) further proves that CD28 exacerbates the depletion of 28-3.

[0165] The above data indicate that BB-3 and 28-3 can specifically target mIgE. + Cells exert a killing effect, with 28-3 exhibiting more intense activation and depletion.

[0166] 5) CD4 + and CD8 + CAR-T all have lethal capabilities:

[0167] To compare CD4 + and CD8 + The effect of CAR-T, separating CD4 from CAR-T cells + and CD8 + Cell population ( Figure 5 A) Its killing ability was detected by co-culturing with Daudi-mIgE-mCherry target cells for 24 hours. (See figure) Figure 5 B), CD4 + and CD8 + CAR-T cells were all able to effectively kill Daudi-mIgE-mCherry target cells within 24 hours, and the killing effect increased with the effector-to-target ratio. In comparison, CD8... + CAR-T vs CD4 + CAR-T has slightly stronger lethality, with all CD8s having a 10:1 effectiveness-to-target ratio. + CAR-T cells can kill less than 10% of target cells, while CD4... + CAR-T can also reduce damage to below 20%. Regardless of CD4... + Still CD8 + The BB-3 and 28-3 have very similar damage output. Meanwhile, CD8... + In CAR-T therapy, the control group showed non-specific killing effects of BB-1, 28-1, and UTD relative to CD4. + CAR-T is lower. Overall, CD4 + and CD8 + CAR-T cells can effectively kill target cells. Therefore, it can be concluded that any combination of CD4+... + and CD8 + The CAR-T products of the cell population also have an effect on mIgE. + The target cells have the ability to kill.

[0168] 6) The effect of sIgE on IgE CAR-T:

[0169] To simulate the in vivo environment and investigate the effect of free sIgE on the cytotoxic effect of IgE CAR-T, recombinant sIgE protein was added to a co-culture system of CAR-T and Daudi-mIgE-mCherry. It was found that compared to the control group without sIgE, BB-3 and 28-3 slightly reduced the cytotoxic effect of Daudi-mIgE-mCherry, but still maintained a high cytotoxic level. Figure 6 The result indicates that its killing effect is minimally affected by free sIgE. This result can be inferred to be related to its targeting of mIgE rather than sIgE. Therefore, using CAR-T combinations with different scFvs may have certain advantages.

[0170] 7) Therapeutic effects of IgE CAR-T in humanized mouse asthma models:

[0171] To investigate the therapeutic effect of IgE CAR-T on allergic diseases, the inventors established a humanized mouse asthma model. Since the EMPD domain of mIgE exists only in primates, it was not possible to directly construct a disease model using ordinary mice; instead, Hu-HSC-NPG-GM3 humanized mice were used. Hematopoietic stem cells (hCD34) derived from human umbilical cord blood were transferred to immunodeficient mice. + This allows for the reconstruction of a human immune system in mice, with a humanization rate exceeding 40% in this experiment.

[0172] An asthma model was established using humanized mice. Mice were sensitized with house dust mites (HDM), then adopted with donor-derived BB-3 or UTD cells, followed by asthma challenge (first challenge) using HDM. Serum IgE levels after challenge were measured, revealing increased total IgE and HDM-specific IgE levels in both the model group and the UTD adoptive group compared to the untreated group (NC), indicating successful model establishment. Figure 7 E, Figure 7 F). The total IgE level and HDM-specific IgE level in the BB-3 treatment group were significantly lower than those in the model group. Figure 7 E, Figure 7 F).

[0173] Two weeks later, mice were subjected to a second asthma challenge using HDM, after which they were sacrificed for further analysis. The composition of immune cells in mouse tissues was examined, and the results showed that the BB-3 adoptive group had a certain proportion of CAR-T cells in both the lungs and spleen, accounting for approximately 0.5%-2% of all human-derived immune cells. Figure 7 A, Figure 7 B). Detection of mIgE + B cell percentage, results showed in the BB-3 treatment group, mIgE+ B cells account for a significant portion of total hCD45 in the lungs and spleen. + The proportion of immune cells was significantly lower in both the NC group and the model group. Figure 7 C Figure 7 D). Serum total IgE and HDM-specific IgE levels were measured, and the results showed that both levels in the BB-3 treatment group were still significantly lower than those in the model group. Figure 7 G, Figure 7 H). These data indicate that BB-3CAR-T can survive in vivo for a long time and effectively kill mIgE. + B cells reduce IgE production.

[0174] H&E staining was performed on lung sections of mice after the second asthma excitation. Figure 7 I) The results showed that compared with the untreated group, the alveolar shape of the model group changed, the septa thickened, showing fibrotic characteristics, and there was a large amount of inflammatory cell infiltration. In contrast, the degree of pulmonary fibrosis in the BB-3 treatment group was significantly improved compared with the model group, and there was less inflammatory cell infiltration, which proved the therapeutic effect on asthma.

[0175] In summary, using a humanized mouse asthma model, we have demonstrated that BB-3 CAR-T has a therapeutic effect on asthma, and that BB-3 treatment can kill mIgE. + The reduction in B cell levels in the body also decreases the levels of total IgE and antigen-specific IgE, and can also reduce inflammatory cell infiltration and fibrosis in the lungs. More importantly, this effect lasts from the first asthma excitation to the second asthma excitation, up to 24 days after CAR-T adoptive therapy, while the half-life of monoclonal antibodies (such as Omalizumab) in the body is only 14 days, demonstrating the superiority of CAR-T therapy.

[0176] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes an antigen-specific binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain; the antigen-specific binding domain is capable of specifically binding antigens including immunoglobulin IgE EMPD domains; the antigen-specific binding domain includes a single-stranded variable fragment scFv composed of an amino acid sequence as shown in SEQ ID No. 1 or SEQ ID No.

2. The chimeric antigen receptor targets B cells or plasma cells that express mIgE; The structure of the chimeric antigen receptor consists of a leader chain domain CD8-SP, an antigen-specific binding domain, a hinge domain CD8-hinge, a transmembrane domain CD8-TM, a co-stimulatory domain 4-1BB or CD28, a CD3ζ signal transduction domain CD3zeta, and a tracer structure T2A EGFP connected in sequence.

2. The chimeric antigen receptor according to claim 1, characterized in that, The co-stimulatory domain is selected from polypeptides of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1 or combinations thereof.

3. The chimeric antigen receptor according to any one of claims 1 or 2, characterized in that, The hinge domain is selected as the amino acid sequence shown in SEQ ID No. 4; the transmembrane domain is selected as the amino acid sequence shown in SEQ ID No. 5; the CD3ζ signal transduction domain is selected as the amino acid sequence shown in SEQ ID No. 6; and the co-stimulatory domain is selected as the amino acid sequence shown in SEQ ID No. 7 or SEQ ID No.

8.

4. The chimeric antigen receptor according to claim 3, characterized in that, The hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No. 10; the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No. 11; the CD3ζ signal transduction domain is encoded by the nucleic acid sequence shown in SEQ ID No. 12; and the co-stimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No. 13 or SEQ ID No.

14.

5. An isolated immune cell, characterized in that, The isolated immune cells are modified to express a chimeric antigen receptor, which includes an antigen-binding domain connected to a co-stimulatory domain and a CD3ζ signaling domain. The antigen-binding domain is capable of specifically binding to a single-chain variable fragment scFv that includes an immunoglobulin IgE EMPD domain. The amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID No. 1 or SEQ ID No.

2.

6. The isolated immune cells according to claim 5, characterized in that, The immune cells mentioned are T cells.

7. The isolated immune cells according to claim 6, characterized in that, The co-stimulatory domain is selected as either CD28 or 41BB peptide.

8. The isolated immune cells according to any one of claims 5-7, characterized in that, The chimeric antigen receptor is selected as the chimeric antigen receptor described in claim 3 or claim 4.

9. The isolated immune cells according to claim 8, characterized in that, The T cells were selected as CD4. + T cells, CD8 + T cells or CD4 cells mixed in any proportion + Cells and CD8 + T cells.

10. An expression carrier, characterized in that, The expression vector encodes the chimeric antigen receptor according to any one of claims 1-4.

11. A host cell, characterized in that, The host cell includes the expression vector according to claim 10.

12. The use of the chimeric antigen receptor according to any one of claims 1-4, the isolated immune cells according to any one of claims 5-9, the expression vector according to claim 10, and the host cell according to claim 11 in the preparation of a long-acting medicament for treating allergic diseases, wherein the allergic disease is selected as IgE-mediated allergic asthma.

13. A long-acting pharmaceutical composition for treating allergic diseases, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 1-4, the isolated immune cells according to any one of claims 5-9, the expression vector according to claim 10, and the host cell according to claim 11.

Images