Chimeric antigen receptors, recombinant immune cells and uses

Abstract

The present disclosure provides a kind of chimeric antigen receptor, recombinant immune cell and use.The present application provides a kind of recombinant immune cell, wherein the recombinant immune cell includes: (i) one or more structures for cell adoptive therapy;(ii) the gene regulation system capable of reducing or eliminating the expression and / or function of BCOR gene and ZC3H12A gene in immune cells;Wherein, the structure for cell adoptive therapy specifically binds to the antigen derived from eosinophil.The recombinant immune cell provided by the present application avoids the use of pretreatment with great toxic side effects;Target cells can be effectively eliminated for a long time, and a single treatment can achieve the effect of prevention and long-term cure.And these long-term existing recombinant immune cells in vivo can also act as carriers to secrete proteins with therapeutic effects, including antibodies, polypeptides and hormones, etc.

Description

Technical Field

[0001] This disclosure belongs to the field of cell technology and relates to chimeric antigen receptors, recombinant immune cells, and their uses. More specifically, this disclosure relates to the preparation of chimeric receptor T cells based on IL-5 molecules that simultaneously inhibit or knock out the ZC3H12A and BCOR genes, and to using these cells as cell carriers to release IL-4 mutant proteins with inhibitory functions, and ultimately to construct multi-target cell drugs with long-lasting therapeutic, curative, and preventative effects. Background Technology

[0002] Adoptive cell transfer therapy, including chimeric antigen receptor (CAR) T-cell immunotherapy (CAR-T) and T-cell receptor (TCR) T-cell immunotherapy (TCR-T), has shown remarkable efficacy in the immunotherapy of tumors, particularly lymphocytic leukemia. Furthermore, mounting evidence suggests that cell immunotherapy will also play a significant role in the treatment of autoimmune diseases.

[0003] As is well known, allergic asthma is a typical type 2 immune response-mediated allergic disease, leading to disruption of local immune homeostasis, specifically manifested as a significant increase in eosinophils, ultimately resulting in damage and dysfunction of parenchymal organs. However, the persistently enhanced type 2 immune response further exacerbates the disease progression. Although many chemical and antibody drugs targeting different disease stages have been developed, the gradual development of drug resistance in patients still casts a shadow over treatment. Cell therapy has clearly become one of the more efficient and ideal treatment options. However, unlike cell immunotherapy for tumors, where chemotherapy drugs can be used to enhance the efficacy of CAR-T therapy, this approach would undoubtedly cause unnecessary and additional harm to patients in the treatment of type 2 immune response-mediated inflammatory or allergic diseases, particularly those with eosinophils as effector cells. Therefore, although many CAR molecules targeting different targets have shown the expected effect of killing target cells in vitro, once these CAR-modified T cells are adopted into the body, they exhibit significant defects, failing to proliferate or kill target cells. This situation also makes it impossible to obtain long-term cures for cellular immunotherapy for type 2 immune response-mediated inflammatory or allergic diseases, including allergic asthma.

[0004] Non-patent literature (Chen et al. Cell Discovery (2022) 8:80; https: / / doi.org / 10.1038 / s41421-022-00433-y) constructed an IL-5CAR-T cell and disclosed that after infusing IL-5CAR-T cells into mice and then inducing asthma, IL-5CAR-T cells had a protective effect against asthma in mice, and a decrease in eosinophils was still observed at 3 months compared to the control group without IL-5CAR-T cells. However, cited literature 1 only demonstrated the preventive effect of IL-5CAR-T cells on asthma, and its therapeutic effect is still unclear, let alone the long-term therapeutic effect of IL-5CAR-T cells on type 2 immune response-mediated inflammatory diseases or allergic diseases, and diseases in which eosinophils are effector cells.

[0005] Therefore, adoptive cell therapy for inflammatory or allergic diseases mediated by type 2 immune responses, as well as diseases with eosinophils as effector cells, still requires further development. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] To address the aforementioned problems in existing technologies, this disclosure provides a chimeric antigen receptor, recombinant immune cells, related biomaterials, compositions, and uses. The recombinant immune cells are generated by editing T cells through gene knockout or gene repression, enabling them to target disease-related eosinophils in vivo via a chimeric receptor based on the IL-5 molecule for a long period and with high efficiency. Furthermore, using these cells as a carrier, an inhibitory IL-4 mutant protein is released, effectively suppressing the type 2 immune response. Ultimately, in multiple disease models, these gene-knockout, multi-target-targeting recombinant cells have demonstrated long-term, highly effective therapeutic, curative, and preventative therapeutic effects.

[0008] Solution for solving the problem

[0009] [1]. A recombinant immune cell, wherein the recombinant immune cell comprises:

[0010] (i) one or more structures for adoptive cell therapy;

[0011] (ii) Gene regulatory systems capable of reducing or eliminating the expression and / or function of the BCOR gene and the ZC3H12A gene in immune cells;

[0012] The structure-specific binding of the cell adoptive therapy to the antigen derived from eosinophils is described herein.

[0013] [2]. The recombinant immune cells according to [1], wherein the antigens derived from eosinophils include one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8;

[0014] Preferably, the antigen derived from eosinophils is IL-5Rα.

[0015] [3]. The recombinant immune cells according to [1] or [2], wherein the structure for adoptive cell therapy is selected from one or more of chimeric antigen receptors, T-cell antigen receptors, receptor-binding receptors, and synthetic T-cell receptors and antigen receptors;

[0016] Preferably, the structure used for adoptive cell therapy is a chimeric antigen receptor, comprising:

[0017] (a) A polypeptide derived from IL-5;

[0018] (b) Peptides derived from CD28; and,

[0019] (c) Peptides derived from CD3zeta.

[0020] [4]. According to the recombinant immune cells described in [3], wherein,

[0021] The amino acid sequence of the polypeptide derived from IL-5 is selected from: SEQ ID NO:13 or SEQ ID NO:17; and / or,

[0022] The amino acid sequence of the polypeptide derived from CD28 is selected from: SEQ ID NO:14 or SEQ ID NO:18; and / or,

[0023] The amino acid sequence of the polypeptide derived from CD3zeta is selected from either SEQ ID NO:15 or SEQ ID NO:19.

[0024] [5]. The recombinant immune cells according to [3] or [4], wherein the chimeric antigen receptor comprises one or more of the following sequences:

[0025] (a1) The amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0026] (a2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0027] (a3) An amino acid sequence having one or more amino acid residues truncated, added, substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and having, or partially having, the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16; or,

[0028] (a4) An amino acid sequence encoded by a nucleotide sequence hybridized under stringent conditions with a polynucleotide sequence encoding an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and the amino acid sequence having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, wherein the stringent conditions are moderate stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

[0029] The recombinant immune cells disclosed herein have their BCOR and ZC3H12A genes expressed and / or functioned by a reduction or elimination. These cells are then applied to IL-5CAR-modified T cells to enhance or equip them with in vivo activity.

[0030] [6]. The recombinant immune cells according to any one of [1] to [5], wherein the recombinant immune cells further include:

[0031] (iii) Biomolecules used to treat diseases;

[0032] Optionally, the biomolecules used to treat the disease are selected from: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activating peptides, antibodies or their antigen-binding fragments, or mutants thereof;

[0033] Preferably, the biomolecule used to treat the disease is selected from one or more of the following: IL-23R protein, IL-4R antibody, IFN-α, IFN-β, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-22, IL-23, IL-24, TNF, TNF-α, GM-CSF, CD40L, CTLA-4, FLT3L, TRAIL, LIGHT, GLP1, or mutants thereof.

[0034] More preferably, the biomolecule used to treat the disease is an IL-4 mutant, the amino acid sequence of which is shown in SEQ ID NO:7 or SEQ ID NO:20.

[0035] [7]. The recombinant immune cells according to any one of [1] to [6], wherein the immune cells are derived from mammalian immune cells;

[0036] Optionally, the immune cells are selected from one or more of T cells, B cells, NK cells, mast cells, and tumor-infiltrating lymphocytes;

[0037] Preferably, the immune cells are selected from T cells or NK cells;

[0038] More preferably, the T cells are selected from one or more of CD4+CD8+ T cells, CD8+ T cells, CD4+ T cells, effector T cells, suppressor T cells, primitive T cells, memory T cells, γ-δ T cells, α-β T cells, CD4-CD8- double-negative T cells or NKT cells.

[0039] [8]. Recombinant immune cells according to any one of [1] to [7], wherein the gene regulation system is used to treat the BCOR gene and ZC3H12A gene in the recombinant immune cells by gene knockout technology, gene silencing technology, inactivation mutation technology, PROTAC technology or small molecule inhibitor.

[0040] [9]. A chimeric antigen receptor comprising:

[0041] (a) A polypeptide derived from IL-5;

[0042] (b) Peptides derived from CD28; and,

[0043] (c) Peptides derived from CD3zeta;

[0044] Preferably, the chimeric antigen receptor comprises one or more of the following sequences:

[0045] (a1) The amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0046] (a2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0047] (a3) An amino acid sequence having one or more amino acid residues truncated, added, substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and having, or partially having, the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16; or,

[0048] (a4) An amino acid sequence encoded by a nucleotide sequence hybridized under stringent conditions with a polynucleotide sequence encoding an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and the amino acid sequence having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, wherein the stringent conditions are moderate stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

[0049] This disclosure provides a novel CAR molecule that uses the full length of the IL-5 molecule as the target recognition structure, incorporates CD28 as the extracellular segment near the membrane end, uses the transmembrane end of the CD28 molecule as the transmembrane region of the CAR, and incorporates the intracellular segments of CD28 and CD3zeta as the intracellular signal transduction region of the CAR, ultimately constructing a CAR molecule that can effectively recognize target cells and induce downstream activation signals.

[0050]

[10] . A biomaterial, wherein the biomaterial comprises at least one of the following: b1) to b3):

[0051] b1) Encoding a polynucleotide of the chimeric antigen receptor as described in [9];

[0052] b2) A carrier containing the polynucleotides described in b1);

[0053] b3) Cells containing the carrier described in b2).

[0054]

[11] . A composition comprising recombinant immune cells as described in any one of [1] to [8], a chimeric antigen receptor as described in [9], and / or a biomaterial as described in

[10] ; and, optionally, a pharmaceutically acceptable carrier.

[0055]

[12] . Use of any of the recombinant immune cells described in [1] to [8], the chimeric antigen receptor described in [9], and / or the biomaterial described in

[10] in the preparation of medicaments for the treatment and / or prevention of diseases or conditions;

[0056] The diseases or conditions mentioned are selected from inflammatory diseases or allergic diseases mediated by type 2 immune response, and diseases in which eosinophils are effector cells.

[0057] Optionally, the type 2 immune response-mediated inflammatory or allergic diseases include one or more of the following: asthma, allergic rhinitis, inflammatory dermatitis, and food allergies.

[0058] Optionally, diseases in which eosinophils are effector cells include one or more of the following: acute and chronic asthma, eosinophilia, eosinophilic nasal polyps, eosinophilic enteritis, eosinophilic dermatitis, chronic obstructive pulmonary disease, and eosinophilic leukemia.

[0059]

[13] . Use of recombinant immune cells as described in any of [1] to [8] as carriers for delivering biomolecules for treating diseases.

[0060] The recombinant immune cells disclosed herein serve as vectors for expressing various types of molecules with different activities, including but not limited to the expression of IL-4 mutant proteins with inhibitory functions. This also includes the expression of physiologically active proteins, therapeutically active antibodies, CAR or TCR molecules with other targeting capabilities, cell surface molecules with targeting capabilities, and molecules that regulate intracellular transduction and transcription.

[0061]

[14] . A method for preparing recombinant immune cells as described in any one of [1] to [8], comprising:

[0062] (i) the step of introducing a structure for adoptive cell therapy into immune cells; and,

[0063] (ii) Steps for introducing gene regulatory systems into immune cells;

[0064] And, optional,

[0065] (iii) The step of introducing biomolecules for treating diseases into immune cells.

[0066] In step (ii), the BCOR gene and ZC3H12A gene in the recombinant immune cells may be treated using gene knockout technology, gene silencing technology, inactivation mutation technology, or small molecule inhibitors.

[0067] The effects of the invention

[0068] The experiments disclosed herein demonstrate that the ZC3H12A / BCOR double gene knockout CAR-T cells obtained through gene editing enable the in vivo application of IL-5CAR-modified T cells, which do not possess in vivo cellular activity, exhibiting the following very significant advantages:

[0069] 1) Avoid using pretreatments with extremely toxic side effects;

[0070] 2) It can effectively eliminate target cells, namely eosinophils, over a long period. Genetically edited IL-5 CAR-T cells persist in the body for a long time, which is equivalent to a group of cells being stably implanted in the body for a long time, thus solving the problem of long-term efficacy of CAR-T therapy;

[0071] 3) A single treatment can achieve both preventative and long-term curative effects. Animal experiments have shown that, in both acute and chronic asthma models, recombinant immune cells have demonstrated significant and thorough clearance of eosinophils, remodeling of the local immune environment, and improvement of lung parenchymal organ function, thus achieving a therapeutic and curative effect. Simultaneously, they enhance the body's immune tolerance, effectively preventing strong stimulation from sensitizing agents.

[0072] 4) These long-term IL-5 CAR-T cells can also act as carriers to secrete proteins with therapeutic effects, including antibodies, peptides, and hormones. For example, the expression of the inhibitory IL-4 mutant protein can significantly suppress the type 2 immune response. Attached Figure Description

[0073] Figure 1 Recombinant IL-5 CAR-T cells with Bcor and Zc3h12a knocked out persist in vivo and clear eosinophils. A. Schematic diagram of IL-5CAR-T cells recognizing and killing target cells expressing IL-5Ra; B. Detection of IL-5CAR cell membrane expression level; C. In vitro killing of MC-38 tumor cells expressing IL-5Ra by IL-5CAR-T cells; D. Statistical results of IL-5CAR-T cell killing ability under different effector cell-target cell ratios; E. Statistical results of IL-5CAR-T cell killing of eosinophils; F. Statistical results of IL-5CAR-T cell killing of eosinophils under different effector cell-target cell ratios; Statistical analysis of C and E, n=4, data are mean±SEM, using unpaired student's t-test: NS, no significant difference; ***p<0.001; Statistical analysis of D and F, n=3, data are mean±SEM; G is the proportion of peripheral blood CAR-T cells and eosinophils detected by flow cytometry on day 14; H is the statistical result of the flow cytometry results of G, n=4, data are mean±SEM, using one-way... ANOVA: NS, no significant difference; *p<0.05; ***p<0.001; I<0.5T IF After adoptive transfer into recipient mice, the proportion of peripheral blood CAR-T cells was analyzed by flow cytometry at different time points; J represents 5T cells. IF After adoptive transfer into recipient mice, the proportion of peripheral blood eosinophils was analyzed by flow cytometry at different time points; statistical analysis of I and J, n=4, data are mean±SEM, two-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0074] Figure 2IL-5 CAR-T cells, without pretreatment, neither expanded nor killed target cells after adoptive transfer into mice. A is a schematic diagram of the experimental procedure; B shows the percentage of CAR-T cells in CD8+ cells in mouse peripheral blood cells analyzed by flow cytometry on day 7 after adoptive transfer of IL-5 CAR-T cells. + C represents the proportion of T cells; C is the proportion of eosinophils in mouse peripheral blood cells analyzed by flow cytometry on day 7 after adoptive transfer of IL-5 CAR-T cells; D is the statistical analysis results of B and C, n=4, data are mean±SEM, unpaired student's t-test was used: NS, no significant difference was found; E is the proportion of CAR-T cells in CD8+ cells in peripheral blood on days 7 and 28 after adoptive transfer of different doses of IL-5 CAR-T cells, analyzed by flow cytometry. + The proportion of T cells and the proportion of eosinophils in peripheral blood; F and G are the statistical analysis results of the E results; H is the flow cytometry analysis of the proportion of CAR-T cells in the spleen and bone marrow on day 28 after adoptive transfer of different doses of IL-5 CAR-T cells. + The proportions of T cells and eosinophils; I and J are the statistical analysis results of the H results; statistical analysis of F, G, I, and J, n=4, data are mean±SEM, one-way ANOVA: NS, no significant differences were found.

[0075] Figure 3 .5T IF Cells can act as carriers to secrete IL-4 mutant protein with inhibitory functions, thereby suppressing type 2 immune responses. In the diagram, A is a schematic; B shows the ELISA detection of 5T in the culture supernatant. IF The levels of IL-4 mutant protein secreted by 4 cells and their adoption by 5T cells were measured by ELISA. IF Statistical results of the content of IL-4 mutant protein secreted in the serum of recipient mice under homeostatic conditions; C is a schematic diagram of the experimental procedure; D is the statistical results of the IL-13 content in the serum of immunized mice detected by ELISA; E is the statistical results of the total IgE content in the serum of immunized mice detected by ELISA; F is a representative result of the flow cytometry analysis of plasma cell levels in the spleen of immunized mice; G is the statistical results of the flow cytometry analysis of plasma cell levels and absolute numbers in the spleen; Statistical analysis of B, D, E and G, n=4, data are mean±SEM, one-way ANOVA: NS, no significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0076] Figure 4 .5T IF 4 cells have and 5T IF The cells also possess the long-term function of killing target cells. Among them, A represents the construction of 5T cells.IF 4. Flowchart of cell lines; B is a representative graph of IL-5CAR molecular membrane expression level analyzed by flow cytometry; C is the statistical analysis results of IL-5CAR membrane uptake efficiency and expression level analyzed by flow cytometry, n=3, data are mean±SEM, unpaired student's t-test was used: NS, no significant difference; D is the flow cytometry analysis of the proportion of CAR-T cells in CD8+ cells in peripheral blood cells 2 weeks after adoptive transfer. + A representative graph showing the proportion of T cells; E is a representative graph showing the proportion of eosinophils in peripheral blood cells 2 weeks after adoptive transfer, analyzed by flow cytometry; F shows the statistical analysis results of D and E, n=4, data are mean±SEM, one-way ANOVA: NS, no significant difference; **p<0.01.

[0077] Figure 5 .5T IF Cells and 5T IF 4. CAR-T cells can cure OVA antigen-induced acute allergic asthma. A is the flowchart of the experiment; B is the flow cytometry analysis of the proportion of CD8+ CAR-T cells in the lungs. + A representative graph of T cell proportions; C represents the proportion of CAR-T cells in CD8+ in the lungs as analyzed by flow cytometry. + Statistical analysis results of the proportion of T cells and the absolute number of CAR-T cells; D represents the statistical analysis results of HE staining results; G and H represent the statistical results of the absolute number of various blood cells in lung lavage fluid and lungs detected by flow cytometry, including CD45. + All white blood cells, EOS for eosinophils, NEU for neutrophils, MAC for macrophages, T for T cells, and B for B cells; E represents the statistical result of IL-13 content in bronchoalveolar lavage fluid detected by ELISA; F represents the statistical result of total IgE content in serum detected by ELISA; Statistical analysis of C, E, F, G, H (n=4), and statistical analysis of D (n=8), data are presented as mean ± SEM, and one-way ANOVA was used: NS, no significant difference was found; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0078] Figure 6 .5T IF Cells and 5T IF 4. CAR-T cells can cure OVA antigen-induced chronic long-term allergic asthma. A is the flowchart of the experiment; B is the flow cytometry analysis of the proportion of CD8+ CAR-T cells in the lungs. + A representative graph of T cell proportions; C represents the proportion of CAR-T cells in CD8+ in the lungs as analyzed by flow cytometry. +Statistical analysis results of T cell proportion and absolute CAR-T cell count; D shows the statistical analysis results of HE staining and PAS staining; G and H show the statistical results of the absolute number of various blood cells in lung lavage fluid and lungs detected by flow cytometry; E shows the statistical results of IL-13 content in lung lavage fluid detected by ELISA; F shows the statistical results of total IgE content in serum detected by ELISA; Statistical analysis of C, E, F, G, and H, n=4; statistical analysis of D, n=8; data are mean ± SEM, one-way ANOVA: NS, no significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0079] Figure 7 .5T IF Cells and 5T IF 4. Cell-mediated prevention of IL-33-induced asthma. A is the flowchart of the experiment; B is the flow cytometry analysis of the proportion of CAR-T cells in CD8+ in the lungs. + A representative graph of T cell proportions; C represents the proportion of CAR-T cells in CD8+ in the lungs as analyzed by flow cytometry. + Statistical analysis results of the proportion of T cells and the absolute number of CAR-T cells; D is the statistical analysis result of HE staining results; E is the statistical result of IL-13 content in lung lavage fluid detected by ELISA; F and G are the statistical results of the absolute number of various blood cells in lung lavage fluid and lungs detected by flow cytometry; Statistical analysis of C, E, F, G, n=4, statistical analysis of D, n=8, data are mean±SEM, one-way ANOVA: NS, no significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0080] Figure 8 .5T IF Cells and 5T IF 4. Cellular Prevention of HDM-Induced Allergic Asthma. A is the flowchart of the experiment; B is the flow cytometry analysis of the proportion of CAR-T cells in CD8+ in the lungs. + A representative graph of T cell proportions; C represents the proportion of CAR-T cells in CD8+ in the lungs as analyzed by flow cytometry. +Statistical analysis results of the proportion of T cells and the absolute number of CAR-T cells; D is the statistical analysis result of HE staining; E is the statistical result of ELISA detecting the IL-13 content in bronchoalveolar lavage fluid; F and G are the statistical results of flow cytometry analyzing the absolute number of various blood cells in bronchoalveolar lavage fluid and lungs; For the statistical analysis of C, E, F, G, n = 4, for the statistical analysis of D, n = 8, the data are mean ± SEM, and one-way ANOVA is used: NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0081] Figure 9 . Construction of human IL-5 CAR lentiviral vector and identification of human IL-5 CAR-T cells. Among them, A and E are the schematic diagrams of IL-5 CAR lentiviral vector, IL-5 CAR lentiviral vector carrying sgRNA element, and lentiviral vector expressing IL-4 mutant protein; B, detection of IL-5 CAR cell membrane expression level; C, human IL-5 CAR-T cells kill 143B tumor cells expressing IL-5Ra in vitro; D, statistical result of the killing ability detection of human IL-5 CAR-T cells at different effector cell-target cell ratios; F, flow cytometry analysis of the expression analysis of corresponding proteins in human T cells after co-infection with two viruses; G is the statistical result of ELISA detecting the content of IL-4 mutant protein secreted by 5T IF 4 cells; For the statistical analysis of C and G, n = 4, the data are mean ± SEM, and unpaired student’s t-test is used: NS, no significant difference; ***p < 0.001.

[0082] Figure 10 . Human 5T IF 4 cells have significant in vivo long-term efficacy. Among them, A is the construction flow chart; B is the proportion of CAR-T cells and eosinophils in peripheral blood analyzed by flow cytometry 4 weeks after adoptive transfer of different human IL-5 CAR-T cells; C and D are the statistical analysis results of B; E is 5T IF Statistical analysis of the proportion of peripheral blood CAR-T cells analyzed by flow cytometry at different time points after adoptive transfer into recipient mice; F is 5T IF Statistical analysis of the proportion of peripheral blood eosinophils analyzed by flow cytometry at different time points after adoptive transfer into recipient mice; G is the statistical result of ELISA analyzing the IL-4 mutant protein in serum; H is the result diagram of Sanger sequencing analyzing the corresponding gene editing situation; For the statistical analysis of C, D, E, F, G, n = 4, the data are mean ± SEM, and two-way ANOVA is used: *p < 0.05, **p < 0.01, ***p < 0.001.

[0083] Figure 11 .5T IF 4. Schematic diagram of the principles of curing and preventing diseases. Cells carrying IL-5CAR molecules that simultaneously lack ZC3H12A and BCOR, and simultaneously expressing IL-4 mutants, 5T IF 4. It can completely kill eosinophils, while inhibiting the type 2 immune response mediated by IL-4 and IL-13, and ultimately cure or prevent diseases mediated by type 2 immune response and diseases related to eosinophilia. Detailed Implementation

[0084] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments described herein; it should also be understood that the terminology used herein is for description only and not for limiting the specific embodiments.

[0085] [Terminology Definition]

[0086] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0087] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0088] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0089] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0090] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0091] In this specification, the term "and / or" when used to connect two or more options should be understood to mean any one of the options or any two or more of the options.

[0092] According to this disclosure, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0093] According to this disclosure, the terms "nucleic acid molecule," "polynucleotide," "polynucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0094] According to this disclosure, the three-letter and single-letter codes for amino acids used are as described in J. biol. chem, 243, p3558 (1968).

[0095] According to this disclosure, an amino acid "addition" refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to this disclosure, an amino acid "deletion" refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. According to this disclosure, an amino acid "insertion" refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence; the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other.

[0096] According to this disclosure, an amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, "substitution" can be a conserved amino acid substitution.

[0097] According to this disclosure, "conservative modification," "conservative substitution," or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), such that frequent changes can be made without altering the protein's biological activity. Those skilled in the art will appreciate that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to impair biological activity. Exemplary conserved substitutions are described in the following "Exemplary Conservative Amino Acid Substitutions."

[0098] [1] Exemplary amino acid conserved substitution

[0099] Original residues Conservative replacement Ala(A) Gly;Ser Arg(R) Lys;His Asn(N) Gln; His; Asp Asp(D) Glu;Asn Cys(C) Ser;Ala;Val Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn;Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0100] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0101] According to this disclosure, "moderate to very high stringency conditions" includes "moderate stringency conditions," "moderate to high stringency conditions," "high stringency conditions," or "very high stringency conditions," which describe the conditions for nucleic acid hybridization and washing. For guidance on performing hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in that literature, and either can be used. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0102] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic, preventative, or prophylactic measures, research, and diagnostic applications.

[0103] "Treatment" means administering an oral or topical therapeutic agent, such as recombinant immune cells comprising this disclosure, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms.

[0104] In this specification, the term "prevention" refers to preventive treatment for subjects who currently do not have or have not had any disease but are at risk of developing it, or who have had a disease in the past but are currently not at risk of relapse. In some implementations, subjects have a higher risk of developing a disease or a higher risk of disease relapse compared to the average healthy member of the subject population.

[0105] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0106] In this specification, "therapeutic effective amount" is an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutic effective amount refers to the amount of a therapeutic agent, alone or in combination with other therapies, that provides therapeutic benefit in the treatment of a condition. The term "therapeutic effective amount" may include amounts that improve overall therapy; reduce or avoid symptoms, signs, or causes of a condition; and / or enhance the therapeutic efficacy of another therapeutic agent.

[0107] In this specification, "preventive effective amount" is an amount sufficient to prevent the condition or one or more symptoms associated with the condition, or to prevent its recurrence. A preventive effective amount refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides preventive benefit in preventing the condition. The term "preventive effective amount" may also include amounts that improve overall prevention or enhance the preventive efficacy of another preventive agent.

[0108] In this specification, "pharmaceutical composition" or "composition" means containing one or more recombinant immune cells as described herein, as well as other components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0109] In this specification, the term "pharmaceutical acceptable" (or "pharmacologically acceptable", "medicinal") means a molecular entity or composition that, when appropriate, does not produce an adverse reaction, allergic reaction, or other adverse reaction when administered to animals or humans. As used herein, the term "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial agents, isotonic agents and absorption delay agents, buffers, excipients, binders, lubricants, gels, surfactants, etc., that can be used as a medium for pharmaceutically acceptable substances.

[0110] The terms "BCOR gene" and "Bcor gene" can be used interchangeably unless otherwise specified. The "BCOR gene" refers to the BCOR gene of any target subject.

[0111] The terms “ZC3H12A gene” and “Zc3h12a gene” can be used interchangeably unless otherwise specified. The “ZC3H12A gene” refers to the “ZC3H12A gene” of any target subject.

[0112] "Stemness," also known as "stem cell characteristics," refers to the ability of cells to self-renew and differentiate into different cell types.

[0113] "Immortal-like" or "functional" (IF) refers to the characteristic of cells acquiring the ability to continuously grow and proliferate, without the phenotypic features of malignant transformation, tumorigenicity, or the invasiveness and metastasis characteristic of tumor cells. In this article, the subscript IF is used to denote T cells with "immortal-like" properties that have had both Bcor and Zc3h12a knocked out, abbreviated as T cells. IF Including 5T IF 4T IF 4.

[0114] "Subject" or "host" refers to a human or non-human animal, including mammals. Examples include primates (such as humans and monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, and mice. "Subject" or "host" can be therapeutic or non-therapeutic. "Subject" or "host" includes experimental animal models or animals used to produce biomolecules expressing therapeutic diseases; these are "non-therapeutic hosts" or "non-therapeutic subjects."

[0115] In the absence of a specific definition, “molecules related to the treatment of diseases” or “biomolecules related to the treatment of diseases” refer to molecules or biomolecules that are introduced into immune cells through “exogenous genes” and secreted by recombinant immune cells.

[0116] As used herein, "pretreatment" refers to the pretreatment of patients or experimental animals before cell infusion. Conventional CAR-T therapy and other cell therapies require chemotherapy or radiotherapy to remove lymphocytes from the patient's body, providing space and other factors for the infused cells. Without pretreatment, the infused T cells proliferate insufficiently, resulting in limited efficacy. However, pretreatment can lead to many side effects, including immunodeficiency and cytokine storms. The 5T cells prepared in this invention... IF and 4T IF 4 cells can expand in vivo without any pretreatment, kill target cells, and persist for a long time. [Detailed Description of the Invention]

[0118] This disclosure reveals that T cells modified with a chimeric receptor molecule constructed using IL-5 as the recognition structure can target and kill tumor cell lines expressing the IL-5α receptor molecule, and can also target and kill eosinophils isolated from mice. However, T cells modified with this CAR molecule cannot proliferate and kill target cells in vivo.

[0119] Based on this discovery, this disclosure provides recombinant immune cells and their preparation method, a treatment method for asthma, a method for disease prevention, and extends to treatment methods for inflammatory or allergic diseases mediated by type 2 immune responses and diseases with eosinophils as effector cells. By reducing or eliminating the expression and / or biological function of the BCOR gene and ZC3H12A gene, IL-5CAR-modified T cells can be expanded in vivo without pretreatment, persist for a long time, and exert long-term killing function, endowing recombinant immune cells with strong stemness or functional immortality-like properties. Simultaneously, by expressing the IL-4 mutant protein, its therapeutic scope and indications are expanded, enabling it to effectively suppress type 2 immune responses and ultimately be used for disease treatment. In acute and chronic asthma models, recombinant immune cells showed significant therapeutic and curative effects. They also demonstrated a significant therapeutic effect in suppressing type 2 immune responses. The recombinant immune cells provided in this disclosure require only a single infusion to achieve a lifelong cure for asthma and other diseases caused by eosinophilia and type 2 inflammation. Meanwhile, in the sensitizer exposure model, recombinant immune cells endowed the body with a significant ability to resist airway sensitization reactions, thus providing a preventive effect.

[0120] Chimeric antigen receptor (CAR)

[0121] In some aspects disclosed herein, a chimeric antigen receptor is provided, comprising:

[0122] (A) An extracellular domain that specifically binds to antigens derived from eosinophils;

[0123] (B) Transmembrane domain;

[0124] (C) Intracellular signal transduction domains.

[0125] In some specific embodiments, this disclosure provides a chimeric antigen receptor comprising:

[0126] (a) A polypeptide derived from IL-5;

[0127] (b) Peptides derived from CD28; and,

[0128] (c) Peptides derived from CD3zeta.

[0129] (Extracellular domain)

[0130] In some embodiments, the eosinophil-derived antigen includes one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8. In some embodiments, the eosinophil-derived antigen includes IL-5Rα. In some more preferred embodiments, the eosinophil-derived antigen is IL-5Rα.

[0131] In some embodiments, the extracellular domain comprises a polypeptide derived from the cytokine interleukin-5 (IL-5) that specifically binds to IL-5Rα. In some specific embodiments, the extracellular domain comprises a full-length polypeptide of the cytokine IL-5 as a target recognition domain.

[0132] In some specific embodiments, the amino acid sequence of the full-length IL-5 polypeptide is shown in SEQ ID NO:13. The IL-5 used as part of the extracellular domain in the chimeric antigen receptor provided by this invention can simultaneously recognize human IL-5Rα and mouse IL-5Rα. In other specific embodiments, the amino acid sequence of the full-length IL-5 polypeptide is shown in SEQ ID NO:17.

[0133] In some embodiments, the extracellular domain further includes the juxtamembrane terminus of CD28, i.e., the juxtamembrane terminus of CD28 is incorporated into the target recognition structure using the full-length polypeptide derived from the aforementioned IL-5 molecule as the extracellular domain (extracellular segment).

[0134] (Transmembrane domain)

[0135] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from the following proteins: the α, β or ζ chain of the T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0136] In some specific implementations, the transmembrane domain includes a transmembrane domain derived from CD28.

[0137] (Intracellular signal transduction domain)

[0138] In some embodiments, intracellular signaling domains may include primary intracellular signaling domains. Example primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, intracellular signaling domains may include co-stimulatory intracellular domains. Example co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may contain a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may contain a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0139] In some implementations, the primary intracellular signaling domain may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, as well as DAP10 and DAP12.

[0140] In this disclosure, “ζ” or alternatively “ζ chain”, “CD3-ζ”, “TCR-ζ” or “CD3 zeta” is defined as a protein provided with GenBank accession number BAG36664.1, or an equivalent residue from a non-human species (e.g., mouse, rabbit, primate, rodent, monkey, ape, etc.), and “ζ-stimulatory domain” or alternatively “CD3-ζ-stimulatory domain” or “TCR-ζ-stimulatory domain” is defined as an amino acid residue from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal necessary for T cell activation.

[0141] In this disclosure, "co-stimulatory molecules" refer to homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0142] The intracellular signal transduction domain of a costimulatory molecule can be the intracellular portion of that molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte-activating molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0143] In some specific implementations, the intracellular domains of CD28 and CD3 zeta are combined as the intracellular signal transduction domain (the intracellular region of signal transduction) of the CAR.

[0144] In this invention, the design of the CD28 molecule as an extracellular hinge segment (or juxtamembrane end or extracellular segment; as part of the extracellular domain), a transmembrane segment (transmembrane domain), and an intracellular segment (as part of the intracellular signal transduction domain) results in a chimeric antigen receptor-mediated signal transduction level that is stronger (compared to other designs) and a more significant killing ability, leading to higher clearance efficiency of target cells. In some specific embodiments of this invention, the sequence of the CD28 molecule is shown in SEQ ID NO:14. In other specific embodiments of this invention, the sequence of the CD28 molecule is shown in SEQ ID NO:18.

[0145] In this invention, the intracellular segment of CD3zeta used in the intracellular signal transduction domain contains three ITAM motifs to maximize signal levels. In some specific embodiments of this invention, the sequence of the CD3zeta molecule is shown in SEQ ID NO:15. In other specific embodiments of this invention, the sequence of CD3zeta is shown in SEQ ID NO:19.

[0146] (IL-5 chimeric antigen receptor)

[0147] In some preferred embodiments of this disclosure, an IL-5 chimeric antigen receptor is provided, which is a CAR molecule capable of effectively recognizing target cells and inducing downstream activation signals. Immune cells / recombinant immune cells (e.g., T cells / recombinant T cells) modified with this molecule can kill target cells in vitro, while adoptive transfer of T cells modified with the IL-5 chimeric antigen receptor into recipient mice did not result in expansion or killing of target cells.

[0148] In some embodiments, the chimeric antigen receptor comprises one or more of the following sequences:

[0149] (a1) The amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0150] (a2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16;

[0151] (a3) An amino acid sequence in which one or more amino acid residues are extracted, added, replaced, deleted, or inserted in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and which has or partially has the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16; or,

[0152] (a4) An amino acid sequence encoded by a nucleotide sequence hybridized under stringent conditions with a polynucleotide sequence encoding an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, and the amino acid sequence having or partially having the function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:16, wherein the stringent conditions are moderate stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

[0153] <Biomaterials>

[0154] In some aspects, this disclosure provides a biomaterial, wherein the biomaterial comprises at least one of the following b1) to b3):

[0155] b1) Encodes a polynucleotide for the chimeric antigen receptor as described above;

[0156] b2) A carrier containing the polynucleotides described in b1);

[0157] b3) Cells containing the carrier described in b2).

[0158] In some embodiments, a polynucleotide is provided that encodes the chimeric antigen receptor disclosed herein.

[0159] The polynucleotides disclosed herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0160] The polynucleotides encoding the chimeric antigen receptor disclosed herein include: a coding sequence that encodes only the chimeric antigen receptor; a coding sequence for the chimeric antigen receptor and various additional coding sequences; a coding sequence for the chimeric antigen receptor (and optional additional coding sequences); and a non-coding sequence.

[0161] The term "polynucleotide encoding a chimeric antigen receptor" can include a polynucleotide encoding the chimeric antigen receptor or it can include additional coding and / or non-coding sequences.

[0162] This disclosure also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This disclosure particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. These stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent conditions.

[0163] In some embodiments, an expression vector is provided that contains the polynucleotides disclosed herein.

[0164] In some embodiments, a cell is provided that contains the expression vector of this disclosure.

[0165] In some specific implementation schemes, the cells are immune cells. The source and types of these immune cells can be found in the detailed description of related immune cells in the recombinant immune cell section below.

[0166] <Recombinant Immune Cells>

[0167] In some aspects of this disclosure, a recombinant immune cell is provided, said recombinant immune cell comprising:

[0168] (i) one or more structures for adoptive cell therapy;

[0169] (ii) Gene regulatory systems capable of reducing or eliminating the expression and / or function of the BCOR gene and the ZC3H12A gene in immune cells;

[0170] The structure-specific binding of the cell adoptive therapy to the antigen derived from eosinophils is described herein.

[0171] (Immune cells)

[0172] In some implementations, the recombinant immune cells are immune cells derived from mammals.

[0173] The mammals mentioned include, but are not limited to, primates (such as humans and monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, etc.

[0174] In this disclosure, there are no specific limitations on the types of immune cells. In some embodiments, the immune cells are selected from one or more of T cells, B cells, NK cells, mast cells, and tumor-infiltrating lymphocytes. In some preferred embodiments, the immune cells are selected from T cells or NK cells. In some specific embodiments, the T cells are selected from CD4+ cells. + CD8 + T cells, CD8 + T cells, CD4 + T cells, effector T cells, suppressor T cells, primitive T cells, memory T cells, γ-δ T cells, α-β T cells, CD4+ - CD8 - One or more of double-negative T cells or NKT cells. In some preferred embodiments, the T cells are CD8+ cells. + T cells.

[0175] In some specific implementations, the recombinant immune cells are recombinant T cells.

[0176] The recombinant T cells described above do not contain the BCOR gene and the ZC3H12A gene, or the biological functions of the BCOR gene products and the ZC3H12A gene products of the recombinant T cells are suppressed.

[0177] The recombinant T cells mentioned above are formed by knocking out the BCOR and ZC3H12A genes of the target T cells and modifying them with IL-5CAR molecules to form the final version of recombinant T cells.

[0178] In the above-mentioned recombinant T cells, the target T cells are CD8 T cells or other types of T cells.

[0179] As will be detailed later, in the above-mentioned recombinant T cells, the knockout refers to knocking out the BCOR gene and ZC3H12A gene of the target T cell by CRISPR-Cas9 or other methods, or inhibiting the function of the BCOR gene product and ZC3H12A gene product by other methods.

[0180] In the aforementioned recombinant T cells, the target sequence targeting the BCOR gene when knocking out the BCOR gene in the target T cells using the CRISPR-Cas9 method is SEQ ID NO:4 or SEQ ID NO:10; the target sequence targeting the ZC3H12A gene when knocking out the ZC3H12A gene in the target T cells using the CRISPR-Cas9 method is SEQ ID NO:5 or SEQ ID NO:11. Further, the recombinant cells are formed by introducing a vector containing the target sequence targeting the BCOR gene during knockout, the target sequence targeting the ZC3H12A gene, and an IL-5CAR expression structure into the target T cells.

[0181] Further detailed description: The recombinant cells are cells obtained by introducing pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR or pHAGE-U6-sgBCOR-U6-sgZC3H12A-SFFV-GFP-P2A-IL-5-CAR into target CD8 T cells. For example, the target CD8 T cells are derived from the spleen of Cas9 transgenic mice (from Jaxson Laboratory, Stock No: 026430).

[0182] (Gene regulatory system)

[0183] The recombinant immune cells contain a gene regulatory system capable of reducing or eliminating the expression and / or function of the BCOR and ZC3H12A genes in immune cells, thereby reducing or eliminating the expression and / or function of the BCOR and ZC3H12A genes in the recombinant immune cells. Exemplarily, the recombinant T cells do not contain the BCOR and ZC3H12A genes, or the biological functions of the BCOR and ZC3H12A gene products of the recombinant T cells are suppressed.

[0184] Exemplary information regarding the BCOR gene and the ZC3H12A gene can be found in Table 1 below.

[0185] Table 1 Information on BCOR and ZC3H12A genes

[0186]

[0187] Specifically, in this disclosure, the human BCOR gene (Gene ID: 54880, updated May 29, 2022, https: / / www.ncbi.nlm.nih.gov / gene / 54880) and the mouse Bcor gene (Gene ID: 71458, updated May 22, 2022, https: / / www.ncbi.nlm.nih.gov / gene / 71458) encode the transcriptional repressor BCOR in cells. The human ZC3H12A gene (Gene ID: 80149, updated May 22, 2022, https: / / www.ncbi.nlm.nih.gov / gene / 80149) and the mouse Zc3h12a gene (Gene ID: 230738, updated May 22, 2022, https: / / www.ncbi.nlm.nih.gov / gene / 230738) encode the protein ZC3H12A, which is involved in mRNA degradation in cells. All of the above genes are incorporated into this disclosure by reference.

[0188] In this disclosure, there are no particular limitations on the methods used by the gene regulation system to reduce or eliminate the expression and / or function of the BCOR and ZC3H12A genes. Exemplarily, in some embodiments, the gene regulation system may employ gene knockout technology, gene silencing technology, inactivation mutation technology, PROTAC technology, or small molecule inhibitors to treat the BCOR and ZC3H12A genes in the recombinant immune cells.

[0189] In some embodiments, compared with unmodified or control immune cells (without a gene regulation system), the gene regulation system in the recombinant immune cells of this disclosure reduces the expression or function of the BCOR gene and the ZC3H12A gene in the immune cells by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, respectively.

[0190] In some specific embodiments, the gene regulation system disclosed herein uses gene knockout technology, gene silencing technology, inactivation mutation technology, or small molecule inhibitors to treat the BCOR gene and ZC3H12A gene in the recombinant immune cells.

[0191] In some implementations, the gene knockout technologies used include CRISPR / Cas technology, artificial zinc finger nucleases (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas technology.

[0192] In some implementations, the gene regulation system comprises nucleic acid molecules and enzyme proteins, wherein the nucleic acid molecules are guide RNA (gRNA) molecules, and the enzyme proteins are Cas proteins or Cas orthologs.

[0193] In some embodiments, the enzyme protein is selected from Cas9, Cas12a, Cas12b, Cas13a, Cas13b, Cas13c, Cas13e or Cas13f proteins or their orthologs.

[0194] In some embodiments, the gene regulation system disclosed herein includes:

[0195] (i) The targeting domain sequence in the BCOR gene-directing RNA (gRNA) complexes with the first Cas endonuclease protein to form the first ribonucleoprotein (RNP) complex; and;

[0196] (ii) The targeting domain sequence in the ZC3H12A gene guide RNA (gRNA) is combined with the second Cas endonuclease protein to form the second ribonucleoprotein (RNP) complex.

[0197] In some implementations, the first ribonucleoprotein (RNP) complex and the second ribonucleoprotein (RNP) complex may be introduced into immune cells simultaneously, sequentially, or sequentially.

[0198] In some embodiments, in the gene regulation system of this disclosure, the nucleic acid binding segment of the BCOR gene guide RNA (gRNA) binds to a target DNA sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the DNA sequence encoded by the subject's BCOR gene (e.g., NCBI Gene ID: 54880 or NCBI Gene ID: 71458); and the nucleic acid binding segment of the ZC3H12A gene guide RNA (gRNA) binds to a target DNA sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the DNA sequence encoded by the subject's ZC3H12A gene (e.g., NCBI Gene ID: 80149 or NCBI Gene ID: 230738).

[0199] In some specific embodiments, in the gene regulation system of this disclosure, the targeting domain of the guide RNA (gRNA) targeting the BCOR gene comprises the sequence ACTGGGCAATACCGCAACAG (SEQ ID NO:4) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:4; the targeting domain of the guide RNA (gRNA) targeting the BCOR gene comprises the sequence GCTGCCACAAGCACTCTAGG (SEQ ID NO:10) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:10; the targeting domain of the guide RNA (gRNA) targeting the ZC3H12A gene comprises the sequence CTAGGGGAATTGGTGAAGCA (SEQ ID NO:5) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:5; the targeting domain of the guide RNA (gRNA) targeting the ZC3H12A gene comprises the sequence CAGGACGCTGTGGATCTCCG (SEQ ID NO:11) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:5. NO:11 is a sequence with at least 85%, 90%, and 95% identity.

[0200] (Structure used for adoptive cell therapy)

[0201] In some embodiments, the recombinant immune cells described in this disclosure include one or more structures for adoptive cell therapy.

[0202] In some implementations, the corresponding structures for adoptive cell therapy are chimeric antigen receptor (CAR) structures, T-cell antigen receptor (TCR) structures, receptor-binding-based structures, or synthetic T-cell receptor and antigen receptor (STAR). For a description of STAR, see WO2020029774A1 and Yue Liu et al. Chimeric STAR receptors using TCR machinerymediate robust responses against solid tumors. Sci Transl Med. 2021 Mar 24; 13(586):eabb5191. doi:10.1126 / scitranslmed.abb5191.

[0203] In some embodiments, the eosinophil-derived antigen includes one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8. In some embodiments, the eosinophil-derived antigen includes IL-5Rα. In some more preferred embodiments, the eosinophil-derived antigen is IL-5Rα.

[0204] In some embodiments, the structure used for adoptive cell therapy is a chimeric antigen receptor (CAR) structure. In some specific embodiments, the structure used for adoptive cell therapy is the chimeric antigen receptor provided above in this disclosure.

[0205] (Biomolecules used to treat diseases)

[0206] In some embodiments, the recombinant immune cells provided in this disclosure also include:

[0207] (iii) Biomolecules used to treat diseases.

[0208] In some specific embodiments, the disease-treating biomolecule is selected from: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activating peptides, antibodies or their antigen-binding fragments, or mutants of the above. Compared with the natural form of the above molecules, the mutants exhibit different biological functions and have potential clinical effects in treating different diseases. Specifically, the disease-treating biomolecule is selected from IL-23R protein, IL-4R antibody, IFN-α, IFN-β, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-22, IL-23, IL-24, TNF, TNF-α, GM-CSF, CD40L, CTLA-4, FLT3L, TRAIL, LIGHT, GLP1, or one or more mutants of the above.

[0209] In some specific implementations, the biomolecule used to treat the disease is an IL-4 mutant with an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:20, which has the function of inhibiting the IL-4 and IL-13 pathways.

[0210] In some embodiments, the recombinant immune cells described herein can be detected in the peripheral blood of subjects at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 2 years, 5 years, 10 years, 20 years, and 40 years after administration. In some embodiments, the recombinant immune cells described herein are immortalized immune cells. These immortalized recombinant immune cells are non-tumor cells.

[0211] In some implementations, after administering the medication to the subject for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months, the proportion of the recombinant immune cells described in this disclosure relative to the total amount of similar immune cells is not less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0212] In other embodiments, after administering the medication to the subject for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months, the proportion of recombinant immune cells described in this disclosure relative to the total number of peripheral blood cells is selected from 1%-35%, 3-30%, or 3-20%; the specific value can be any value within the above range, including but not limited to 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 35%.

[0213] In other embodiments, the recombinant immune cells of this disclosure exhibit increased or prolonged cell viability compared to unmodified immune cells. In such embodiments, the result is an increase in the number of recombinant immune cells of this disclosure present after a given time period compared to unmodified immune cells. For example, in some embodiments, the recombinant immune cells of this disclosure remain viable and persist for 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more times longer than unmodified immune cells.

[0214] In some embodiments, the recombinant immune cells of this disclosure exhibit a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more-fold increase in the production of disease-treating biomolecules (e.g., IL23R, TNF, or IL-5, GLP1, IL-4 mutants) compared to the production of disease-treating biomolecules observed in unmodified immune cell populations.

[0215] The recombinant immune cells provided in this disclosure have increased immune cell stemness, inhibited immune cell exhaustion, promoted immune cell proliferation, endowed immune cells with memory, prolonged immune cell persistence, and increased immune cell self-renewal capacity. As demonstrated in the examples, long-term protection can be achieved in an asthmatic mouse model without pretreatment.

[0216] <Preparation Methods of Recombinant Immune Cells>

[0217] In some aspects, this disclosure provides a method for preparing the above-mentioned recombinant immune cells, the method comprising:

[0218] (i) the step of introducing a structure for adoptive cell therapy into immune cells; and,

[0219] (ii) The steps of introducing gene regulatory systems into immune cells.

[0220] (Steps for introducing gene regulatory systems into immune cells)

[0221] In some embodiments, the gene regulation system can reduce or eliminate the expression and / or function of the BCOR and ZC3H12A genes. Optionally, the gene regulation system may employ gene knockout, gene silencing, inactivation mutation, or small molecule inhibitors to treat the BCOR and ZC3H12A genes in immune cells.

[0222] In some implementations, the gene knockout technology includes CRISPR / Cas technology, artificial zinc finger nucleases (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas technology.

[0223] In some implementations, the CRISPR / Cas technology is selected from CRISPR-Cas3, CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-CasX, or CRISPR-IscB systems. For example, a description of the CRISPR-CasX system can be found in Liu JJ et al., Nature, 2019 or https: / / doi.org / 10.1016 / j.molcel.2022.02.002. A description of the CRISPR-IscB system can be found in Han Altae-Tran et al., Science 374, Vol 374, Issue 6563, 57–65 (2021). DOI:10.1126 / science.abj6856.

[0224] In some implementations, the CRISPR / Cas technology is specifically selected from CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas12b, CRISPR-Cas13a, CRISPR-Cas13b, CRISPR-Cas13c, CRISPR-Cas13e, or CRISPR-Cas13f systems.

[0225] In some implementations, CRISPR / Cas technology uses guide RNA (gRNA) and Cas endonuclease targeting the BCOR gene, and guide RNA (gRNA) and Cas endonuclease targeting the ZC3H12A gene.

[0226] In some implementations, the CRISPR / Cas technology guide RNA (gRNA) includes, simultaneously or separately, a guide RNA (gRNA) targeting the BCOR gene and a guide RNA (gRNA) targeting the ZC3H12A gene.

[0227] This disclosure provides guide RNA (gRNA) for directing site-modified peptides to specific target nucleic acid sequences. The gRNA contains a nucleic acid targeting region and a protein-binding region. The nucleic acid targeting region of the gRNA contains a nucleotide sequence complementary to a sequence in the target nucleic acid sequence. Therefore, the nucleic acid targeting region of the gRNA interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the nucleic acid targeting region determines the location within the target nucleic acid where the gRNA will bind. The nucleic acid targeting region of the gRNA can be modified (e.g., through genetic engineering) to hybridize with any desired sequence within the target nucleic acid sequence.

[0228] The protein-binding segment of the guide RNA interacts with a site-directed modifying polypeptide (e.g., a Cas protein) to form a complex. The guide RNA then directs the bound polypeptide to a specific nucleotide sequence within the target nucleic acid via this nucleic acid-targeting segment. The protein-binding segment of the guide RNA contains two complementary nucleotide fragments that form a double-stranded RNA double helix.

[0229] In some embodiments, gRNA comprises two separate RNA molecules. In such embodiments, each of the two RNA molecules contains a complementary nucleotide segment, such that the complementary nucleotides of the two RNA molecules hybridize to form a double-stranded RNA duplex of a protein-binding segment. In some embodiments, gRNA comprises a single RNA molecule (single guide RNA, sgRNA).

[0230] The specificity of the gRNA to the target locus is mediated by the sequence of a nucleic acid-binding region comprising approximately 20 nucleotides complementary to a target nucleic acid sequence within the target locus. In some embodiments, the corresponding target nucleic acid sequence is approximately 20 nucleotides in length. In some embodiments, the nucleic acid-binding region of the gRNA sequence disclosed herein is at least 90% complementary to the target nucleic acid sequence within the target locus. In some embodiments, the nucleic acid-binding region of the gRNA sequence disclosed herein is at least 95%, 96%, 97%, 98%, or 99% complementary to the target nucleic acid sequence within the target locus. In some embodiments, the nucleic acid-binding region of the gRNA sequence disclosed herein is 100% complementary to the target nucleic acid sequence within the target locus. In some embodiments, the target nucleic acid sequence is an RNA target sequence. In some embodiments, the target nucleic acid sequence is a DNA target sequence.

[0231] In some implementations, the target nucleic acid sequence within the target locus must be altered. For example, the target nucleic acid sequence may change because the Cas protein used changes and the new Cas protein has a different PAM. This specification provides numerous examples of target nucleic acid sequences for gRNAs in the specifications and tables provided herein. Any of these target nucleic acid sequences can be altered by moving the target nucleic acid sequence at the 5' or 3' end within the target locus of a given gene. In some implementations, the target nucleic acid sequence is moved at the 5' or 3' end within the target locus of a given gene by up to 100 bp. In other embodiments, the target nucleic acid sequence moves at a 5' or 3' bp at the target locus within a given gene (e.g., the human or mouse BCOR gene and / or ZC3H12A gene as described in Table 1) for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 bp.

[0232] In some implementations, the nucleic acid-binding segment of the BCOR gene guide RNA (gRNA) binds to a target DNA sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the DNA sequence encoded by the subject's BCOR gene (e.g., NCBI Gene ID: 54880 or NCBI Gene ID: 71458); the nucleic acid-binding segment of the ZC3H12A gene guide RNA (gRNA) binds to a target DNA sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the DNA sequence encoded by the subject's ZC3H12A gene (e.g., NCBI Gene ID: 80149 or NCBI Gene ID: 230738).

[0233] In some implementations, the nucleic acid-binding segment of the ZC3H12A gene-guided RNA (gRNA) binds to a target DNA sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with a DNA sequence defined by a set of genomic coordinates shown in Table 7 or Table 8 of WO2020163365A2. Alternatively, the nucleic acid-binding segment of the ZC3H12A-targeting gRNA molecule binds to a target DNA sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with a target DNA sequence shown in Tables 16 and 17 of WO2020163365A2.

[0234] In some embodiments, the targeting domain of the BCOR gene guide RNA (gRNA) comprises the sequence ACTGGGCAATACCGCAACAG (SEQ ID NO:4) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:4; the targeting domain of the BCOR gene guide RNA (gRNA) comprises the sequence GCTGCCACAAGCACTCTAGG (SEQ ID NO:10) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:10; the targeting domain of the ZC3H12A gene guide RNA (gRNA) comprises the sequence CTAGGGGAATTGGTGAAGCA (SEQ ID NO:5) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:5; the targeting domain of the ZC3H12A gene guide RNA (gRNA) comprises the sequence CAGGACGCTGTGGATCTCCG (SEQ ID NO:11) or a sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:5. IDNO:11 is a sequence with at least 85%, 90%, and 95% identity.

[0235] (The steps involved in introducing structures for adoptive cell therapy into immune cells)

[0236] In some embodiments, the step of introducing a structure for adoptive cell therapy into immune cells involves introducing a sequence of a CAR structure, TCR structure, or other adoptive cell therapy-appropriate structure carrying a target. As previously mentioned, the target may be an antigen derived from eosinophils.

[0237] In some embodiments, the eosinophil-derived antigen includes one or more of IL-5Rα, CRTh2, CCR3, and Siglec-8. In some embodiments, the eosinophil-derived antigen includes IL-5Rα. In some more preferred embodiments, the eosinophil-derived antigen is IL-5Rα.

[0238] In some embodiments, the structure used for adoptive cell therapy is a chimeric antigen receptor (CAR) structure. In some specific embodiments, the structure used for adoptive cell therapy is the chimeric antigen receptor provided above in this disclosure.

[0239] (The steps involved in introducing biomolecules used to treat diseases into immune cells)

[0240] In some embodiments, the method for preparing recombinant immune cells provided in this disclosure further includes:

[0241] (iii) The step of introducing biomolecules for treating diseases into immune cells.

[0242] In some preferred embodiments, the biomolecule used to treat the disease is selected from one or more of the following: IL-23R protein, IL-4R antibody, IFN-α, IFN-β, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-22, IL-23, IL-24, TNF, TNF-α, GM-CSF, CD40L, CTLA-4, FLT3L, TRAIL, LIGHT, GLP1, and mutants of the above.

[0243] (Import Method)

[0244] In this disclosure, there are no particular limitations on the methods for introducing structures for adoptive cell therapy, gene regulatory systems, and biomolecules for treating diseases. For example, nucleotides carrying structures, gene regulatory systems, or biomolecules for treating diseases that can express the structure for adoptive cell therapy, are introduced into immune cells using techniques known to those skilled in the art.

[0245] In some embodiments, the vector used is a viral vector, a viral-like vector, or a non-viral vector. In some embodiments, a recombinant vector containing polynucleotides encoding one or more components of the structure, gene regulatory system described herein for adoptive cell therapy (e.g., components of a gene regulatory system for reducing or eliminating the expression and / or function of the BCOR gene and ZC3H12A gene in immune cells, such as sgRNA, Cas protein, etc.) and / or biomolecules for treating the disease is a viral vector. Suitable viral vectors include, but are not limited to, viral vectors based on: vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Suitable non-viral vectors are selected from transposons, lipid nanoparticles, liposomes, exosomes, attenuated bacteria, or virus-like particles.

[0246] In some embodiments, a polynucleotide sequence encoding one or more components of a structure, gene regulatory system, and / or a biomolecule for treating a disease, as described herein for adoptive cell therapy, is operatively linked to a control element, such as a transcriptional control element, like a promoter. The transcriptional control element may be functional in eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaea cells). In some embodiments, a polynucleotide sequence encoding one or more components of a structure, gene regulatory system, and / or a biomolecule for treating a disease, as described herein, is operatively linked to multiple control elements that allow the polynucleotide to be expressed in both prokaryotic and eukaryotic cells. Depending on the cell type and gene regulatory system used, any of many suitable transcriptional and translational control elements (including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used in the expression vector.

[0247] In some embodiments, non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeat (LTR) sequences from retroviruses, and mouse metallothionein-1. The selection of suitable vectors and promoters is entirely within the capabilities of those skilled in the art. Expression vectors may also contain a ribosome-binding site for translation initiation and a transcription terminator. Expression vectors may also include appropriate sequences for amplifying expression. Expression vectors may also contain nucleotide sequences encoding a protein tag (e.g., a 6xHis tag, a hemagglutinin tag, green fluorescent protein, etc.) that fuses with a site-directed modified polypeptide, thereby producing a chimeric polypeptide.

[0248] In some specific implementations, exemplary examples, the sgRNA expression vectors used include the basic structures of: vector-promoter 1-sgZc3h12a-promoter 2-tag-P2A-therapeutic biomolecular sequence, vector-promoter 1-sgBcor-promoter 2-tag-P2A-therapeutic biomolecular sequence, or vector-promoter 1-sgBcor-promoter 2-sgZc3h12a-promoter 3-tag-P2A-therapeutic biomolecular sequence. The "-" above does not represent a limitation on a specific linking order and should be understood as including the relevant element expression vector. The aforementioned therapeutic biomolecular sequence includes one or more of the sequences of structures used for adoptive therapy in recombinant immune cells described in this disclosure or sequences of biomolecules used for treating diseases. Specifically, the sgRNA expression vector includes the basic structures of pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR, or pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR. Furthermore, the promoters in the expression vector, such as promoter 1, promoter 2, and promoter 3, can be the same or different; the tag may be optionally present or absent; and the biomolecule sequence for treating the disease may be optionally present or absent. In other embodiments, sgBcor, sgZc3h12a, the structure for adoptive therapy, and the biomolecule for treating the disease can also be constructed separately or in any combination thereof in the vector to form multiple expression vectors (such as viral vectors), packaged into corresponding viruses, and co-transduced into immune cells.

[0249] In some embodiments, the method for preparing recombinant immune cells disclosed herein includes the step of introducing an expression vector into the recombinant immune cells. Methods for introducing polynucleotides and recombinant expression vectors into host cells are known in the art, and any known method can be used to introduce components of a gene regulatory system into cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipid transfection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, microfluidic delivery methods, etc. Furthermore, cells can be introduced by administering non-viral delivery media such as transposons, nanoparticles (e.g., lipid nanoparticles), liposomes, exosomes, attenuated bacteria, or virus-like particles.

[0250] (Other steps)

[0251] In some embodiments, the method for preparing recombinant immune cells provided in this disclosure further includes the step of obtaining immune cells. In principle, there are no particular limitations on the method for obtaining immune cells in this disclosure. For example, peripheral blood mononuclear cells can be isolated from the peripheral blood of a subject, and immune cells of a specific phenotype can be separated using techniques such as magnetic bead sorting or flow cytometry sorting.

[0252] In some embodiments, the method for preparing recombinant immune cells provided in this disclosure further includes a step of culturing recombinant immune cells. In principle, there are no particular limitations on the method for culturing immune cells in this disclosure. In some embodiments, recombinant immune cells can be implanted into a subject for expansion, and the expanded recombinant immune cells can be obtained in vivo. The recombinant immune cells obtained from the expansion of a first-generation subject can be used for the subject's autologous treatment or for allogeneic treatment of other subjects. In some embodiments, the immune cells are the subject's own immune cells or allogeneic immune cells.

[0253] <Composition>

[0254] In other aspects, this disclosure provides a composition for treating a disease. In some embodiments, "composition" refers to an formulation of genetically regulated and / or modified recombinant immune cells, chimeric antigen receptors, and / or biological materials provided in this disclosure, which can be administered or delivered to a subject or cells. A "therapeutic composition" or "pharmaceutical composition" (which may be used interchangeably herein) is a composition comprising genetically regulated and / or modified recombinant immune cells, chimeric antigen receptors, and / or biological materials provided in this disclosure, which can be administered to a subject to treat a specific disease or condition.

[0255] In some embodiments, the therapeutic composition comprises the recombinant immune cells described in any of the above embodiments. In some embodiments, the therapeutic composition comprises the chimeric antigen receptor described in any of the above embodiments. In some embodiments, the therapeutic composition comprises the biological material described in any of the above embodiments.

[0256] In some alternative embodiments, the composition for treating the disease also includes a pharmaceutically acceptable carrier.

[0257] <Methods for treating diseases or symptoms>

[0258] In some embodiments, this disclosure provides a method for treating a disease or condition in a subject in need. The method includes administering to the subject the recombinant immune cells described in any of the above embodiments, the chimeric antigen receptor described in any of the above embodiments, the biomaterial described in any of the above embodiments, and / or the composition described in any of the above embodiments.

[0259] In some embodiments, this disclosure provides the recombinant immune cells, the chimeric antigen receptor, the biomaterial, and / or the composition described in any of the above embodiments for the treatment and / or prevention of diseases or conditions in subjects.

[0260] In some implementations, the disease or condition includes inflammatory or allergic diseases mediated by type 2 immune response, and diseases in which eosinophils are effector cells.

[0261] In some specific embodiments, the type 2 immune response-mediated inflammatory or allergic diseases include asthma, allergic rhinitis, inflammatory skin diseases, food allergies, etc. IL-13 and IL-4 are both important effector molecules involved in this disease process and are also therapeutic targets of recombinant immune cells in some embodiments of the present invention.

[0262] In some specific implementation schemes, diseases in which eosinophils are the (primary) effector cells include: acute and chronic asthma, eosinophilia, eosinophil-induced nasal polyps, eosinophil-induced enteritis, eosinophilic dermatitis, chronic obstructive pulmonary disease, and eosinophilic leukemia.

[0263] In some preferred embodiments, when the recombinant immune cells described in any of the above embodiments are administered to the subject, the subject does not need to undergo pretreatment.

[0264] <Pharmaceutical Uses and Other Uses>

[0265] In some respects, this disclosure provides an use in preparing a medicine.

[0266] In some embodiments, this disclosure provides the use of the recombinant immune cells, chimeric antigen receptors, biomaterials, and / or the composition described in any of the above embodiments in the preparation of a medicament for treating and / or preventing a disease or condition in a subject in need.

[0267] In some implementations, the disease or condition includes inflammatory or allergic diseases mediated by type 2 immune response, and diseases in which eosinophils are effector cells.

[0268] In other aspects, this disclosure provides for the non-therapeutic use of the recombinant immune cells described in any of the above embodiments.

[0269] In some embodiments, the non-therapeutic purpose includes using recombinant immune cells for the preparation of DNA-encoded proteins or for the preparation of therapeutic compositions. Preferably, the DNA-encoded proteins include any one or more disease-treating biomolecules described in any of the above embodiments.

[0270] <Applications of Delivery Carriers>

[0271] In another embodiment, this disclosure provides the use of recombinant immune cells as carriers for the stable delivery of biomolecules for treating diseases.

[0272] In some embodiments, the recombinant immune cells are the recombinant immune cells described in any of the above embodiments. The disease-treating biomolecules are selected from any one or more disease-treating biomolecules described in any of the above embodiments.

[0273] In some embodiments, the biomolecule used to treat the disease is selected from: cytokines, hormones, growth factors, coagulation factors, chemokines, co-stimulatory molecules, activating peptides, antibodies or their antigen-binding fragments, or mutants of the above. Specifically, the biomolecule used to treat the disease is selected from IL-23R protein, IL-4R antibody, IFN-α, IFN-β, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-22, IL-23, IL-24, TNF, TNF-α, GM-CSF, CD40L, CTLA-4, FLT3L, TRAIL, LIGHT, GLP1, or one or more mutants of the above.

[0274] To make the objectives and technical solutions of this disclosure clearer, the implementation schemes of this disclosure are described in detail below with reference to embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this disclosure.

[0275] All reagents and instruments used in the examples are commercially available conventional products. Unless otherwise specified, all procedures were performed under standard conditions or conditions recommended by the manufacturer.

[0276] Example 1: Preparation of recombinant IL-5 CAR-T cells with Bcor and / or Zc3h12a knocked out

[0277] 1. Construction of IL-5CAR expression vector

[0278] This embodiment designed and constructed a retrovirus-based IL-5 CAR expression vector, namely pMSCV-EFS-Thy1.1-P2A-IL-5-CAR: the pMSCV vector is derived from Addgene#52107, and the IL-5 CAR includes a mouse IL-5 molecule, a CD28 molecule (including the extracellular segment, transmembrane segment, and intracellular signal transduction region of the CD28 molecule), and a CD3zeta molecule. In the constructed IL-5 CAR molecule, the extracellular segment is the full-length IL-5 molecule derived from humans or mice, and the mouse-derived CD28 extracellular segment serves as the extracellular structure of the CAR molecule. The intracellular segment consists of the mouse-derived CD28 and CD3zeta signal transduction domains, which are ultimately fused with the Thy1.1 tag for expression.

[0279] The amino acid sequence of IL-5CAR is (SEQ ID NO:1):

[0280]

[0281] The single underlined portion represents IL-5 (SEQ ID NO:13); the double underlined portion represents CD28 molecule (SEQ ID NO:14); and the dotted underlined portion represents CD3zeta molecule (SEQ ID NO:15).

[0282] 2. Construction of the gene knockout IL-5 CAR vector

[0283] Based on the IL-5CAR expression vector constructed in section 1 above, this embodiment constructs retrovirus-based sgRNA expression vectors, namely pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR, pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR, pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, and pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR;

[0284] in:

[0285] The vector pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR (SEQ ID NO:2), wherein positions 242-261 are random sequences that do not target any gene (SEQ ID NO:3), serving as a control without any gene knockout;

[0286] The vector pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR (this vector sequence is obtained by replacing the 242nd to 261st positions of SEQ ID NO:2 with SEQ ID NO:4, while keeping the other sequences unchanged. SEQ ID NO:4 is the target sequence recognition region of sgBcor used to knock out Bcor, which is used to knock out Bcor);

[0287] The vector pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR (this vector sequence is obtained by replacing positions 242-261 of SEQ ID NO:2 with SEQ ID NO:5, while keeping the other sequences unchanged. SEQ ID NO:5 is the target sequence recognition region of sgZc3h12a used to knock out Zc3h12a;)

[0288] pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR (constructed based on pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR, using molecular cloning and restriction enzyme ligation methods, where hU6-sgBcor from vector pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR and hU6-sgZc3h12a from vector pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR are PCR-amplified and spliced, and then inserted into the hU6-sgControl position), where positions 242-261 are the target sequence recognition region for sgBcor in mice with Bcor knockout (SEQ ID). NO:4), positions 687-706 are the target sequence recognition region of sgZc3h12a used to knock out mouse Zc3h12a (SEQ ID NO:5) to simultaneously knock out mouse Bcor and mouse Zc3h12. All vectors were obtained through whole-genome synthesis.

[0289] The sgRNAs used above are shown in Table 2 below:

[0290] Table 2 Target sequence recognition regions of sgRNA

[0291]

[0292] 3. Isolation and activation of naïve CD8T cells

[0293] Initial CD8 T cells were isolated from the spleen of Cas9 transgenic mice (from Jaxson Laboratory, #026430) using magnetic bead sorting. The cells were then sorted into 10... 6 Cells were seeded at a density of 1 μg / ml in 12-well culture dishes coated with anti-CD3 antibody (CD3ε, BioXcell#BE0001-1), and 2 ml of RPMI 1640 medium (containing 5% fetal bovine serum and interleukin-2) was added. At the same time, 1 μg / ml of anti-CD28 antibody (BioXcell#BE0015-1) was added for in vitro activation. The cells were then incubated in a 5% CO2 incubator at 37°C for viral infection.

[0294] 4. Construction of IL-5 CAR-T cells

[0295] 1) Preparation of retroviruses

[0296] 10 6 Phoenix-Eco cells (ATCC#CRL-3214) were cultured adherently for 24 hours. Then, 20 μg of the IL-5CAR expression vector pMSCV-EFS-Thy1.1-P2A-IL-5CAR prepared in step 1 above and 60 μg of the packaging plasmid pCL-Eco (purchased from Addgene#12371) were co-transfected by calcium phosphate precipitation. 48 hours after transfection, the supernatant containing the packaging virus was harvested. The viral supernatant was the retrovirus carrying IL-5CAR.

[0297] 2) Retroviral infection

[0298] The above three in vitro culture activation cultures were 1×10 6 Add 1 ml of the retrovirus supernatant obtained in step 1) to a quantity of CD8 T cells, mix well, and centrifuge at 2000g for 2 hours at room temperature. Then incubate in a CO2 incubator for 4 hours, replace with 2 ml of fresh RPMI 1640 medium (containing 5% fetal bovine serum and 2 ng / ml interleukin-2) and continue culturing (this time is recorded as the post-infection time). Use flow cytometry to sort out Thy1.1 positive cells (Thy1.1-biotin, BioLegend#202510), thus obtaining IL-5 CAR-T cells.

[0299] 5. Construction of gene knockout IL-5 CAR-T cells

[0300] The only difference from "4. Construction of IL-5CAR-T cells" is that "pMSCV-EFS-Thy1.1-P2A-IL-5CAR" is replaced with an sgRNA expression vector that knocks out the corresponding gene, such as "pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR", "pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR", "pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR", and "pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR" without knocking out the specific gene. All other steps remain the same. Ultimately, IL-5 CAR-T cells with simultaneous knockout of Bcor and Zc3h12a (denoted as sgBcor / Zc3h12a) were obtained and named 5T. IF IF represents I mmortal-like and F The term "unctional" is translated into Chinese as "immortalized T-cells". The other two types of cells are named sgBcor IL-5CAR-T cells, sgZc3h12a IL-5CAR-T cells, and sgControl IL-5CAR-T cells, respectively.

[0301] 6. Phenotypic identification of IL-5 CAR-T cells

[0302] 1) Detection of IL-5CAR molecular membrane expression

[0303] Two days after CD8 T cells were infected with the IL-5CAR retrovirus mentioned in section 4 above, the cells expanded and cultured in vitro were analyzed by flow cytometry to detect the Thy1.1 positivity rate and IL-5 positivity rate. The results are as follows: Figure 1 As shown in B, the vast majority of cells carrying the Thy1.1 tag, i.e., those successfully transduced into CAR vectors, successfully expressed IL-5 molecules.

[0304] 2) Detection of IL-5 CAR-T cells killing target cells in vitro

[0305] On day 3 after CD8 T cells were infected with the aforementioned 3IL-5CAR retrovirus, a co-incubation killing experiment was conducted with MC-38 cells, a murine tumor cell line expressing IL-5Rα and not expressing IL-5Rα, at a ratio of effector cells to target cells of 1:1. Anti-EGFR CAR-T cells were used as a control. Results are as follows... Figure 1The C-cell curve shows that IL-5CAR-T cells can significantly kill target cells expressing the target gene, but do not exhibit a killing effect on cells not expressing the target gene. Compared with EGFR CAR-T cells, IL-5CAR-T cells can kill target cells very significantly, demonstrating the specificity of IL-5CAR-T cells. Furthermore, we compared the killing ability of IL-5CAR-T cells under different effector cell to target cell ratios. The results are as follows... Figure 1 The results showed that even at a ratio of 1:8, IL-5CAR-T cells still exhibited a significant killing effect. Finally, we isolated eosinophils from mouse lungs after OVA antigen induction as target cells and conducted the same experiment. The results consistently showed that IL-5CAR-T cells possess the ability to specifically kill target cells (see [reference]). Figure 1 (E and F in the text).

[0306] 7. IL-5 CAR-T cells, when reinfused into the body without pretreatment, cannot proliferate or kill target cells such as eosinophils.

[0307] Cell reinfusion process as follows Figure 2 As shown in A: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 (method as in Example 1, step 3); then, the activated CD8 T cells were infected with a retrovirus obtained by transfecting pMSCV-EFS-Thy1.1-P2A-IL-5-CAR to obtain IL-5CAR-T cells. The cells obtained 24 hours after infection were then intravenously injected into C57bl / B6 mice (hereinafter referred to as B6 mice). The specific injection method is as follows:

[0308] Six- to eight-week-old B6 mice weighing 20-25g were divided into two groups: the PBS group (n=4) and the IL-5CAR-T group (n=4).

[0309] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0310] IL-5CAR-T group: IL-5CAR-T cells prepared according to the method in the example were prepared into a cell suspension with PBS and reinfused into each mouse via tail infusion, with each mouse receiving 4 × 10⁶ cells. 5 CAR-T cells;

[0311] On day 7 post-infusion, the proportion of infused IL-5 CAR-T cells to total CD8 T cells in the peripheral blood of each mouse was analyzed by flow cytometry using Thy1.1 antibody (CAR vector tagged with Thy1.1). Simultaneously, the proportion of eosinophils as target cells in the peripheral blood of each mouse was analyzed by flow cytometry using Siglec-F antibody (a surface marker molecule for eosinophils).

[0312] The results are as follows Figure 2 B and Figure 2 As shown in D in the figure, no CAR-T cells were detected in the PBS group (represented by "PBS" in the figure), serving as a control; and no expanded CAR-T cells were detected in the IL-5CAR group (represented by "IL-5CAR" in the figure) which received IL-5CAR-T cell infusion. Meanwhile, the results are as follows... Figure 2 C and Figure 2 As shown in Figure D, both the PBS group and the IL-5CAR-T cell group showed an equivalent number of eosinophils, and statistical results indicated no significant difference between the two groups. These results suggest that IL-5CAR-T cells cannot proliferate or kill target cells without any treatment of the recipient mice.

[0313] To gain a clearer understanding of the in vivo phenotype of IL-5CAR-T cells, we conducted another experiment, obtaining the same IL-5CAR-T cells using the same method as in the previous examples. The amount of cells infused was increased in a gradient, as detailed below:

[0314] B6 mice aged 6-8 weeks with a weight of 20-25g were divided into 4 groups: the PBS group (4 mice), and 3 groups with different doses of cells, with 4 mice in each group.

[0315] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0316] IL-5CAR-T group: IL-5CAR-T cells prepared according to the method described in the example were prepared into a cell suspension with PBS and reinfused into each mouse in each group via tail infusion. The tail infusion dose per mouse was 1×10⁻⁶. 6 3×10 6 5×10 6 cell.

[0317] On days 7 and 28 post-infusion, the proportion of infused IL-5 CAR-T cells to total CD8 T cells in the peripheral blood of each mouse was analyzed by flow cytometry using Thy1.1 antibody (CAR vector tagged with Thy1.1). Simultaneously, the proportion of target eosinophils in the peripheral blood of each mouse was analyzed by flow cytometry using Siglec-F antibody (a surface marker molecule for eosinophils). On day 28 post-infusion, mice were euthanized, and the spleen and bone marrow were harvested for flow cytometry analysis of the proportion of IL-5 CAR-T cells to total CD8 T cells and the proportion of target eosinophils in these two organs.

[0318] The results are as follows Figure 2 E in Figure 2 F and Figure 2 As shown in G in the figure, the PBS group (represented by "PBS" in the figure) did not contain any CAR-T cells and served as a control; while the IL-5CAR group, which received different doses of IL-5CAR-T cells (represented by "1×10" in the figure), showed no detectable CAR-T cells. 6 3×10 6 5×10 6 No expanded CAR-T cells were detected in the PBS group (indicated by the IL-5 CAR-T cell group). Meanwhile, eosinophils were detected in the same order of magnitude in both the PBS group and the different doses of IL-5 CAR-T cell groups, and statistical results showed no significant difference between the groups. These results indicate that increasing the number of IL-5 CAR-T cells reinfused without any treatment of the recipient mice does not improve cell proliferation or cytotoxicity. Similar results are shown in... Figure 2 H in Figure 2 I and Figure 2 As shown in J, no CAR-T cells were detected in the main target cell organs and organs where CAR-T cells accumulated, and eosinophils of the same order of magnitude were also detected in all groups. Statistical results showed no significant differences between the groups.

[0319] The above results indicate that although IL-5CAR-T cells have a significant ability to kill target cells in vitro, they cannot expand and exert their killing function after being reinfused into the body.

[0320] 8. Phenotypic identification of IL-5 knockout CAR-T cells

[0321] Cell reinfusion process as follows Figure 2As shown in A: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice and activated for 24 hours using CD3 / CD28 to obtain activated CD8 T cells (method as in Example 1, step 3). Then, the activated CD8 T cells were infected with retroviruses obtained by transfecting pMSCV-hU6-sgBcor-EFS-Thy1.1-P2A-IL-5-CAR, pMSCV-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, and pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR, respectively, to obtain IL-5CAR-T cells with the corresponding gene knockout. These were named sgBcor, sgBcor, and sgBcor, respectively. IL-5CAR-T cells, sgZc3h12a IL-5CAR-T cells, and IL-5CAR-T cells with simultaneous knockout of Bcor and Zc3h12a (denoted as sgBcor / Zc3h12a) are named 5T. IF sgControl IL-5 CAR-T cells. The cells obtained 24 hours after infection were intravenously transfused into C57bl / B6 mice (hereinafter referred to as B6 mice) via the tail vein. The specific transfusion method is as follows:

[0322] Six- to eight-week-old B6 mice weighing 20-25g were randomly divided into two groups: the PBS group (n=4), the sgControl group (n=4), the sgZc3h12a group (n=4), the sgBcor group (n=4), and the sgBcor / Zc3h12a group (n=5). IF Group (4 animals)

[0323] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0324] sgControl group: sgControl IL-5 CAR-T cells prepared by the method in Example 1, section 5 were prepared into a cell suspension with PBS and reinfused into each mouse via tail infusion, with each mouse receiving 4 × 10⁶ cells. 5 CAR-T cells;

[0325] sgBcor group: sgBcor IL-5 CAR-T cells prepared by the method in Example 1, section 5 were prepared into a cell suspension with PBS and reinfused into each mouse via tail infusion, with each mouse receiving 4 × 10⁶ cells. 5 CAR-T cells;

[0326] sgZc3h12a group: sgZc3h12a IL-5 CAR-T cells prepared by the method in Example 1, section 5 were prepared into a cell suspension with PBS and reinfused into each mouse via tail infusion, with each mouse receiving 4 × 10⁶ cells. 5 CAR-T cells;

[0327] sgBcor / Zc3h12a group (5T) IF Group): The sgBcor / Zc3h12a IL-5CAR-T (5T) prepared by the method in Example 1, Section 5 IF The cells were prepared into a cell suspension using PBS and reinfused into each mouse via the tail, with each mouse receiving 4 × 10⁻⁶ cells. 5 One CAR-T cell.

[0328] Two weeks after transfusion into mice, flow cytometry analysis was performed on the proportion of IL-5 CAR-T cells to total CD8 T cells in the peripheral blood of each group, and the proportion of eosinophils, the target cells, in the peripheral blood of each group. Results are as follows: Figure 1 The G in and the H in 1 show that only in the sgBcor / Zc3h12a IL-5 group (5T) IF Significantly expanded IL-5CAR-T cells were detected in the sgBcor / Zc3h12a IL-5CAR-T group. Meanwhile, compared to other groups, the sgBcor / Zc3h12a IL-5CAR-T group (5T...) showed significantly increased IL-5CAR-T cell counts. IF Eosinophils, the target cells, were almost undetectable in the peripheral blood of mice. These results indicate that only IL-5CAR-T cells with both BCOR and ZC3h12a knocked out can significantly expand and kill target cells, making the IL-5CAR molecule, which originally only functions in vitro, a true cell drug applicable in vivo.

[0329] 9. In vivo long-term phenotypic identification of IL-5 knockout CAR-T cells

[0330] Cell reinfusion process as follows Figure 2 As shown in A: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice and activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); then, the activated CD8 T cells were infected with a retrovirus obtained by transfecting pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR, and IL-5CAR-T cells with Bcor and Zc3h12a knocked out (represented as sgBcor / Zc3h12a) were named 5T. IFThe cells obtained 24 hours after infection were intravenously injected into C57bl / B6 mice (hereinafter referred to as B6 mice) via the tail vein. The specific injection method is as follows:

[0331] Six- to eight-week-old B6 mice weighing 20-25g were divided into two groups: the PBS group (n=4) and the sgBcor / Zc3h12asgBcor / Zc3h12a group (n=5). IF Group (4 animals)

[0332] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0333] sgBcor / Zc3h12a group (5T) IF Group): The sgBcor / Zc3h12a IL-5CAR-T (5T) prepared by the method in Example 1, Section 5 IF The cells were prepared into a cell suspension using PBS and reinfused into each mouse via the tail, with each mouse receiving 4 × 10⁻⁶ cells. 5 One CAR-T cell.

[0334] After 24 weeks of transfusion into mice, flow cytometry analysis was performed on the proportion of IL-5 CAR-T cells in total peripheral blood cells and the proportion of eosinophils, the target cells, in the peripheral blood of each group. Results are as follows: Figure 1 I and Figure 1 The J-value in the data shows that 5T can be detected in peripheral blood at different time points. IF Cellular activity peaked at week 4 after infusion, and the proportion of CAR-T cells in peripheral blood eventually stabilized. Meanwhile, compared to the PBS group, 5T cells were infused. IF Eosinophils were almost undetectable in mice for an extended period of time. These results indicate that 5T cells... IF The cells can not only expand significantly, but also persist in mice for a long time, while effectively killing target cells.

[0335] Example 2: 5T expressing inhibitory IL-4 mutant protein IF Recombinant cells can effectively suppress type 2 immune responses.

[0336] 1. Construction of a vector for the inhibitory IL-4 mutant protein

[0337] In this embodiment, an expression vector for the retrovirus-based IL-4 mutant protein, namely pMSCV-EFS-GFP-P2A-IL-4Mutant (SEQ ID NO:6), was designed and constructed. This vector includes a full-length molecule in which glutamic acid at position 116 of the murine IL-4 is mutated to aspartic acid, and tyrosine at position 119 is mutated to aspartic acid.

[0338] IL-4 mutant amino acid sequence (SEQ ID NO:7):

[0339] HIHGCDKNHLREIIGILNEVTGEGTPCTEMDVPNVLTATKNTTESELVCRASKVLRIFYLKHGKTPCLKKNSSVLMELQRLFRAFRCLDSSISCTMNESKSTSLKDFLESLKSIMDMDDS

[0340] GFP amino acid sequence (SEQ ID NO:8):

[0341] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0342] 2. 5T cells expressing inhibitory IL-4 mutant protein IF Cells (i.e., 5T) IF Construction of 4 cells

[0343] 1) Preparation of retroviruses

[0344] 10 6Phoenix-Eco cells (ATCC#CRL-3214) were cultured adherently for 24 hours. Then, 20 μg of either the gene knockout IL-5 CAR expression vector pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR prepared in Example 1, or the pMSCV-EFS-GFP-P2A-IL-4Mutant (SEQ ID NO:6) expression vector prepared in Example 2, were co-transfected with 60 μg of the packaging plasmid pCL-Eco (purchased from Addgene#12371). Forty-eight hours after transfection, the supernatant containing the packaging virus was harvested. The viral supernatant was filtered through a 0.45 μm filter to remove dead cells and impurities, yielding retroviral supernatants: a retrovirus with both genes knocked out and carrying IL-5 CAR, and a retrovirus expressing the IL-4 mutant protein.

[0345] 2) Retroviral infection

[0346] After 36 hours of in vitro culture activation, 1×10⁻⁶ of the three cultures from Example 1 were cultured. 6 A quantity of CD8 T cells were added to 1 ml of the supernatant of the two retroviruses obtained in step 1) of Example 2 (2), mixed 1:1, and centrifuged at 2000g for 2 hours at room temperature. The cells were then incubated in a CO2 incubator for 4 hours, and then the medium was replaced with 2 ml of fresh RPMI 1640 medium (containing 5% fetal bovine serum and 2 ng / ml interleukin-2) for further culture (this time is recorded as the post-infection time). Flow cytometry was used to sort out new Thy1.1 and GFP double-positive cells (Thy1.1-biotin, BioLegend#202510), thus obtaining IL-5 CAR-T cells expressing the IL-4 mutant protein and with the two genes BCOR and ZC3h12a knocked out, named 5T. IF 4.

[0347] 3. Detection and functional verification of inhibitory IL-4 mutant protein

[0348] like Figure 4 In method A, two retroviruses were co-infected and flow cytometry-sorted to obtain double-positive cells, which were then cultured. After 48 hours of continued culture, the culture supernatant was collected. Simultaneously, viral supernatant from a CAR vector infected only with pMSCV-hU6-sgBcor-hU6-sgZc3h12a-EFS-Thy1.1-P2A-IL-5-CAR virus and uninfected cell supernatant (as controls) were collected. The supernatant was recovered by centrifugation. The IL-4 protein content was detected using ELISA. Results are as follows: Figure 3 The B in the image shows 5T IF4. Cells can successfully secrete inhibitory mutant proteins.

[0349] Cell reinfusion process as follows Figure 2 As shown in A in Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by method 2 in Example 2. IF 4. Cells. The obtained cells were intravenously injected into C57bl / B6 mice (hereinafter referred to as B6 mice) via the tail vein. The specific injection method is as follows:

[0350] Six- to eight-week-old B6 mice weighing 20-25g were divided into two groups: the PBS group (n=4) and the 5T group. IF Group (4 animals), 5T IF 4 groups (4 animals)

[0351] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0352] 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 One CAR-T cell.

[0353] 5T IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0354] Five weeks after cell reinfusion, mice were euthanized, blood was collected, cells were centrifuged, and the supernatant serum was collected. The serum content of 5T cells was then measured using ELISA in a non-disease model. IF The levels of IL-4 mutant protein in peripheral blood in the four groups. Results are as follows: Figure 3 As shown in B, compared to the PBS group (control) and 5T IF The recipient mice in the group were infused with 5T IF In mice with 4 cells, there was a significantly elevated level of the mutant IL-4 protein.

[0355] The above results indicate that 5T IF 4. Cells can be used as carriers to express exogenous functional proteins.

[0356] 4. Functional verification of inhibitory IL-4 mutant protein

[0357] Cell reinfusion process as follows Figure 2 As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4. Cells. The obtained cells were intravenously injected into C57bl / B6 mice (hereinafter referred to as B6 mice) via the tail vein. The specific injection method is as follows:

[0358] Six- to eight-week-old B6 mice weighing 20-25g were divided into two groups: the PBS group (n=4) and the 5T group. IF Group (4 animals), 5T IF 4 groups (4 animals)

[0359] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0360] 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 One CAR-T cell.

[0361] 5T IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0362] Subsequently, as Figure 3 The flowchart shown in section C describes the immunization of mice that received three different infusions of substances: intraperitoneal injection of a mixture of OVA protein and aluminum adjuvant at one-week intervals. One week after the second immunization, the mice were euthanized, and serum and spleen were collected. Single-cell suspensions of the spleen were prepared, and the ratio of plasma cells to plasmablasts was detected by flow cytometry using anti-B220 and anti-CD138 antibodies. Serum cytokine IL-13 and total IgE were detected by ELISA.

[0363] like Figure 3 As shown in D, 5T IF 4 cells compared to 5T IF Cells can significantly inhibit IL-13 levels. Meanwhile, such as Figure 3 As shown in E, 5T IF 4 cells and 5TIF Compared to the control group, the cells showed significantly suppressed IgE levels, while 5T cells... IF The inhibitory effect on cells 4 was more significant. And as... Figure 3 F in Figure 3 In the G-cell analysis, the analysis of B cell subsets in the spleen showed that 5T IF 4 cells compared to 5T IF The cells can effectively inhibit the production of plasma cells.

[0364] The above results consistently indicate that 5T IF The released IL-4 mutant protein can effectively inhibit the type 2 immune response induced by OVA protein.

[0365] Example 3, 5T IF 4. Recombinant cells possess the same long-lasting expansion and target cell killing functions.

[0366] Cell reinfusion process as follows Figure 2 As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4 cells.

[0367] A portion of the two cell types obtained were cultured in vitro, and the membrane deposition efficiency and expression level of CAR molecules were detected by flow cytometry. The results are as follows: Figure 4 B and Figure 4 The C in the text indicates that 5T IF Cells and 5T IF The four cells showed no significant difference in CAR molecule membrane loading efficiency, and the levels of CAR molecule expression were consistent between the two groups.

[0368] Another portion of the obtained cells was directly injected into C57bl / B6 mice (hereinafter referred to as B6 mice) via the tail vein, as follows:

[0369] Six- to eight-week-old B6 mice weighing 20-25g were divided into two groups: the PBS group (n=4) and the 5T group. IF Group (4 animals), 5T IF 4 groups (4 animals)

[0370] PBS group: Mice were infused with an equal volume of PBS, and 200 μl of PBS was infused into each mouse in the PBS group via the tail.

[0371] 5T IF Group: Prepared 5T IFCells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 One CAR-T cell.

[0372] 5T IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0373] Two weeks after reinfusion into mice, flow cytometry analysis was performed on the proportion of reinfused IL-5 CAR-T cells to total CD8 T cells in the peripheral blood of each group, and the proportion of eosinophils, the target cells, in the peripheral blood of each group. Results are as follows: Figure 4 D in Figure 4 E and Figure 4 The F in the image shows that, compared to the 5T IF 5T IF 4. It also possesses the ability to proliferate and kill target cells over a long period. The above results indicate that the expression of exogenous proteins does not affect their own function of long-term killing of target cells, and that exogenous proteins can perform independent functions in a biologically orthogonal manner.

[0374] Example 4: In an OVA antigen-induced acute asthma model, 5T IF and 5T IF 4. It showed a healing effect.

[0375] 1. Construction of an OVA asthma model

[0376] For example Figure 5 The model was constructed using method A in the above method as follows: Six- to eight-week-old B6 mice weighing 20-25g were selected, and each mouse was sensitized and immunized with 40μg of OVA protein mixed with aluminum adjuvant each time. On days 0 and 7, mice were immunized once each via intraperitoneal injection. Then, from days 16 to 20, mice were immunized via nasal drops at a dose of 40μg per mouse for antigen exposure. On day 21, 24 hours after the last immunization, the mice were euthanized, and blood was collected to separate serum. Bronchoalveolar lavage fluid was obtained via the airway, and lung tissue was isolated to obtain single-cell suspensions for subsequent flow cytometry analysis.

[0377] In another batch of experiments, mice were perfused with their hearts until their lungs turned white. Finally, the lungs were separated and fixed with paraformaldehyde for pathological sections and staining.

[0378] 2. Cell reinfusion and therapy

[0379] Cell reinfusion process as follows Figure 2As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4 cells.

[0380] The cells obtained above will be processed at a specified time after the OVA (e.g., ...). Figure 5 As shown in A, on day 9, the mice were directly injected via the tail vein into C57bl / B6 mice (hereinafter referred to as B6 mice). The specific injection method is as follows:

[0381] Mice immunized with OVA were divided into three groups: the PBS group (n=4), the 5T group, and the 5T group. IF Group (4 animals), 5T IF Four groups (n=4 each) of PBS groups: Mice were re-infused with an equal volume of PBS, and 200 μl of PBS was re-infused into each mouse in the PBS group via the tail. 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 5 CAR-T cells. IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0382] 3. Efficacy testing

[0383] 1) Flow cytometry analysis

[0384] The single-cell suspension obtained in step 1 was stained with an antibody panel consisting of anti-CD45, anti-CD3e, anti-B220, anti-CD11b, anti-CD11c, anti-Siglec-F, and anti-Iy6G antibodies against cells in the bronchoalveolar lavage fluid and lung cells, and then analyzed by multicolor flow cytometry. CAR-T cells infiltrating the lungs were detected using anti-Thy1.1 and anti-CD8 antibodies. Subsequently, a quantitative number of cells were stained with anti-CD45 antibody and counted by flow cytometry.

[0385] The results are as follows Figure 5 B and Figure 5 C, 5T IF and 5T IF 4 cells can significantly infiltrate the lungs, but their absolute numbers do not differ significantly.

[0386] Analysis of other immune cells showed that, for example Figure 5 G and Figure 5 H, 5T IF and 5T IF 4. Cell therapy significantly inhibited the infiltration of total blood cells in bronchoalveolar lavage fluid and lungs. Both significantly inhibited the infiltration of eosinophils, the main pathogenic cells, in both lavage fluid and lungs, but showed no significant difference in their ability to inhibit cell infiltration. These results indicate that both cell therapies can almost completely eliminate eosinophils infiltrating the lungs.

[0387] 2) ELISA detection

[0388] The cellular supernatant from the bronchoalveolar lavage fluid obtained in step 1 was transferred into clean EP tubes, and the IL-13 content was detected using an ELISA kit. The serum obtained in step 1 was transferred into clean EP tubes, and the total IgE content was detected using an ELISA kit.

[0389] The results are as follows Figure 5 E and Figure 5 The F in the display indicates 5T IF and 5T IF 4 can significantly inhibit the production of IL-13, while 5T IF The inhibitory effect of 4 is more significant. Similarly, 5T IF and 5T IF 4 can significantly inhibit IgE production, while 5T IF 4. The inhibitory effect on IgE production is more significant. In summary, 5T IF 4. It showed better efficacy in inhibiting cytokine and antibody production.

[0390] 3) Pathological staining

[0391] The lungs fixed overnight were embedded in paraffin and then sectioned and mounted. The prepared pathological sections were stained with hematoxylin and eosin (HE), and the slides were photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring system.

[0392] Pathological analysis results Figure 5 The D in the data indicates that, because both are very significant in killing target cells and inhibiting immune cell infiltration, the pathological staining results also show that 5T... IF and 5T IF Both of these drugs can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between them.

[0393] Based on the above results, 5T IF and 5T IF4. Cell therapy can significantly eliminate pathogenic eosinophils while inhibiting the infiltration of more immune cells, thus alleviating the inflammatory response in pathological tissues. Furthermore, 5T... IF 4. Cells are more effective at inhibiting cytokines and antibodies.

[0394] Example 5: In a chronic asthma model induced by OVA antigen and with repeated antigen exposure, 5T IF and 5T IF 4. Demonstrated a curative effect. 1. Construction of the OVA asthma model.

[0395] For example Figure 6 The model was constructed using method A in the above method as follows: 6-8 week old B6 mice weighing 20-25g were selected. Each mouse was sensitized and immunized with 40μg of OVA protein mixed with aluminum adjuvant per immunization. On days 0 and 10, mice were immunized once each via intraperitoneal injection. From day 12 onwards, mice were immunized twice weekly via nasal drops at a dose of 40μg per mouse, with monthly booster immunizations via intraperitoneal OVA. From days 176 to 179, mice were immunized via nasal drops at a dose of 40μg per mouse for terminal antigen exposure. On day 180, 24 hours after the final immunization, mice were euthanized, serum was collected, and bronchoalveolar lavage fluid was obtained via the airway. Lung tissue was isolated, and single-cell suspensions were obtained for subsequent flow cytometry analysis.

[0396] In another batch of experiments, mice were perfused with their hearts until their lungs turned white. Finally, the lungs were separated and fixed with paraformaldehyde for pathological sections and staining.

[0397] 2. Cell reinfusion and therapy

[0398] Cell reinfusion process as follows Figure 2 As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4 cells.

[0399] The cells obtained above will be processed at a specified time after the OVA (e.g., ...). Figure 6 As shown in A, on day 12, the mice were directly injected via the tail vein into C57bl / B6 mice (hereinafter referred to as B6 mice). The specific injection method is as follows:

[0400] Mice immunized with OVA were divided into three groups: the PBS group (n=4), the 5T group, and the 5T group. IF Group (4 animals), 5T IFFour groups (n=4 each) of PBS groups: Mice were re-infused with an equal volume of PBS, and 200 μl of PBS was re-infused into each mouse in the PBS group via the tail. 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 5 CAR-T cells. IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0401] 3. Efficacy testing

[0402] 1) Flow cytometry analysis

[0403] The single-cell suspension obtained in step 1 was stained with an antibody panel consisting of anti-CD45, anti-CD3e, anti-B220, anti-CD11b, anti-CD11c, anti-Siglec-F, and anti-Iy6G antibodies against cells in the bronchoalveolar lavage fluid and lung cells, and then analyzed by multicolor flow cytometry. CAR-T cells infiltrating the lungs were detected using anti-Thy1.1 and anti-CD8 antibodies. Subsequently, a quantitative number of cells were stained with anti-CD45 antibody and counted by flow cytometry.

[0404] The results are as follows Figure 6 B and Figure 6 C, 5T IF and 5T IF Four types of cells could significantly infiltrate the lungs, with no significant difference in absolute numbers. Furthermore, the total cell count in both cases showed a significant decreasing trend compared to Example 4, indicating a gradual slowdown in the CAR-T response.

[0405] Analysis of other immune cells showed that, for example Figure 6 G and Figure 6 H, 5T IF and 5T IF 4. Cell therapy significantly inhibited the infiltration of total blood cells in bronchoalveolar lavage fluid and lungs. Both significantly inhibited the infiltration of eosinophils, the main pathogenic cells, in both lavage fluid and lungs, but there was no significant difference in their inhibitory effects. Both also significantly inhibited the infiltration of T cells and B cells in the lungs. These results indicate that both cell therapies can almost completely eliminate eosinophils infiltrating the lungs.

[0406] 2) ELISA detection

[0407] The cellular supernatant from the bronchoalveolar lavage fluid obtained in step 1 was transferred into a clean EP tube, and the IL-13 content was detected using an ELISA kit. The serum obtained in step 1 was transferred into a clean EP tube, and the total IgE content was detected using an ELISA kit.

[0408] The results are as follows Figure 6 The E and F in the display indicate that 5T IF 4 can significantly inhibit the production of IL-13. Meanwhile, 5T IF It can significantly inhibit IgE production. In summary, 5T IF 4. It showed better efficacy in inhibiting cytokine and antibody production.

[0409] 3) Pathological staining

[0410] The lungs fixed overnight were embedded in paraffin and then sectioned and mounted. The prepared pathological sections were stained with hematoxylin and eosin (HE) and PAS, and the slides were photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring system.

[0411] Pathological analysis results Figure 6 The D in the data indicates that, because both are very significant in killing target cells and inhibiting immune cell infiltration, the pathological staining results also show that 5T... IF and 5T IF Both 4 significantly inhibited the infiltration and distribution of immune cells in the lungs, and there was no significant difference between the two. However, the results of PAS staining clearly showed that 5T... IF 4 compared to 5T IF It significantly inhibits the increase and proliferation of PAS-positive goblet cells.

[0412] Based on the above results, 5T IF and 5T IF 4. Cell therapy can significantly eliminate pathogenic eosinophils while inhibiting the infiltration of more immune cells, thus alleviating the inflammatory response in pathological tissues. Furthermore, 5T... IF 4 cells were more effective at inhibiting cytokines and antibodies. Meanwhile, 5T cells... IF 4. Cells have a more significant effect in alleviating pathological changes in the lungs.

[0413] Example 6, 5T IF and 5T IF 4. It can effectively prevent IL-33-induced asthma.

[0414] 1. IL-33 respiratory sensitization model

[0415] For example Figure 7The model was constructed using method A in the above method, as follows: Six- to eight-week-old mice with a B6 genetic background and weighing 20-25g were selected and CAR-T cells were reinfused into them. Five weeks after infusion, the mice were sensitized with IL-33, i.e., immunized once each via intranasal drip from day 0 to day 3. On day 4, 24 hours after the last intranasal immunization, the mice were euthanized, and bronchoalveolar lavage fluid was obtained via the airway. The lungs were isolated, and single-cell suspensions were obtained for subsequent flow cytometry analysis.

[0416] In another batch of experiments, mice were perfused with their hearts until their lungs turned white. Finally, the lungs were separated and fixed with paraformaldehyde for pathological sections and staining.

[0417] 2. Cell reinfusion and therapy

[0418] Cell reinfusion process as follows Figure 2 As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4 cells.

[0419] The obtained cells will be processed at a specified time (e.g., Figure 7 The B6 mouse (shown as A in the diagram) was input as follows:

[0420] The mice were divided into three groups: the PBS group (n=4), the 5T group, and the 5T group. IF Group (4 animals), 5T IF Four groups (n=4 each) of PBS groups: Mice were re-infused with an equal volume of PBS, and 200 μl of PBS was re-infused into each mouse in the PBS group via the tail. 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 5 CAR-T cells. IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0421] 3. Prevention effect testing

[0422] 1) Flow cytometry analysis

[0423] The single-cell suspension obtained in step 1 was stained with an antibody panel consisting of anti-CD45, anti-CD3e, anti-B220, anti-CD11b, anti-CD11c, anti-Siglec-F, and anti-Iy6G antibodies against cells in the bronchoalveolar lavage fluid and lung cells, and then analyzed by multicolor flow cytometry. CAR-T cells infiltrating the lungs were detected using anti-Thy1.1 and anti-CD8 antibodies. Subsequently, a quantitative number of cells were stained with anti-CD45 antibody and counted by flow cytometry.

[0424] The results are as follows Figure 7 B and Figure 7 C, 5T IF and 5T IF 4 cells can significantly infiltrate the lungs, indicating that local cytokines can also elicit a strong CAR-T immune response.

[0425] Analysis of other immune cells showed that, for example Figure 7 F and Figure 7 G, 5T IF and 5T IF Four cell therapies significantly inhibited the infiltration of total blood cells in bronchoalveolar lavage fluid and lungs. Both significantly inhibited the infiltration of eosinophils, the main pathogenic cells, in both lavage fluid and lungs, but there was no significant difference in their inhibitory effects. Both also significantly inhibited the infiltration of T cells and B cells in the lungs. These results indicate that both cell therapies can almost completely eliminate eosinophils infiltrating the lungs.

[0426] 2) ELISA detection

[0427] The pulmonary lavage fluid obtained in step 1 was decellularized and transferred into a clean EP tube; at the same time, blood was collected from the mice, and serum was separated and the IL-13 content was detected using an ELISA kit.

[0428] The results are as follows Figure 7 The E in the text indicates that 5T IF 4. It has a significant inhibitory effect on the production of IL-13.

[0429] 3) Pathological staining

[0430] The lungs fixed overnight were embedded in paraffin and then sectioned and mounted. The prepared pathological sections were stained with hematoxylin and eosin (HE), and the slides were photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring system.

[0431] Pathological analysis result 7, specifically D, indicates that because both are highly effective in killing target cells and inhibiting immune cell infiltration, the pathological staining results also show that 5T...IF and 5T IF Both of these drugs can significantly inhibit the infiltration and distribution of immune cells in the lungs, and there is no significant difference between them.

[0432] Based on the above results, the 5T return transmission IF and 5T IF 4-cell antibodies can significantly clear pathogenic eosinophils and inhibit the infiltration of more immune cells, preventing the inflammatory response in pathological tissues. Furthermore, 5-T antibodies... IF 4 cells were more effective at inhibiting cytokines. 5T cells were reinfused. IF and 5T IF The 4-cell mice remodeled their peripheral tolerance environment, including the tolerance environment of local organs, which showed tolerance to strong stimulation of sensitizing cytokines and had an anti-inflammatory effect.

[0433] Example 7, 5T IF and 5T IF 4. It can effectively prevent HDM-induced asthma.

[0434] 1. HDM respiratory sensitization exposure model

[0435] For example Figure 8 The model was constructed using method A in the above method as follows: Six- to eight-week-old mice with a B6 genetic background and weighing 20-25g were selected and CAR-T cells were reinfused into them. Ten weeks after reinfusion, the mice were sensitized with house dust mite (HDM) through intranasal immunization, specifically from day 7 to day 11. On day 14, 72 hours after the final intranasal immunization, the mice were euthanized, and bronchoalveolar lavage fluid was collected via the airway. The lungs were isolated, and single-cell suspensions were obtained for subsequent flow cytometry analysis.

[0436] In another batch of experiments, mice were perfused with their hearts until their lungs turned white. Finally, the lungs were separated and fixed with paraformaldehyde for pathological sections and staining.

[0437] 2. Cell reinfusion and therapy

[0438] Cell reinfusion process as follows Figure 2 As shown in A of Example 1: CD8 T cells were isolated from the spleen and lymph nodes of Cas9 transgenic mice, activated by CD3 / CD28 for 24 hours to obtain activated CD8 T cells (method as in Example 1, step 3); 5T cells were obtained using the method in Example 1, step 5. IF Cells; 5T were obtained by means of method 2 in Example 2. IF 4 cells.

[0439] The obtained cells will be processed at a specified time (e.g., Figure 8As shown in A in the figure, the mice were directly injected into the tail vein of C57bl / B6 mice (hereinafter referred to as B6 mice). The specific injection method is as follows:

[0440] The mice were divided into three groups: the PBS group (n=4), the 5T group, and the 5T group. IF Group (4 animals), 5T IF Four groups (n=4 each) of PBS groups: Mice were re-infused with an equal volume of PBS, and 200 μl of PBS was re-infused into each mouse in the PBS group via the tail. 5T IF Group: Prepared 5T IF Cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁶ cells via tail infusion. 5 5 CAR-T cells. IF Group 4: Prepared 5T IF 4 cells were prepared into a cell suspension using PBS and reinfused into each mouse via tail, with each mouse receiving 4 × 10⁴ cells via tail infusion. 5 One CAR-T cell.

[0441] 3. Prevention effect testing

[0442] 1) Flow cytometry analysis

[0443] The single-cell suspension obtained in step 1 was stained with an antibody panel consisting of anti-CD45, anti-CD3e, anti-B220, anti-CD11b, anti-CD11c, anti-Siglec-F, and anti-Iy6G antibodies against cells in the bronchoalveolar lavage fluid and lung cells, and then analyzed by multicolor flow cytometry. CAR-T cells infiltrating the lungs were detected using anti-Thy1.1 and anti-CD8 antibodies. Subsequently, a quantitative number of cells were stained with anti-CD45 antibody and counted by flow cytometry.

[0444] The results are as follows Figure 8 B and Figure 8 C, 5T IF and 5T IF 4 cells could significantly infiltrate the lungs, with no significant difference in absolute numbers. Furthermore, the total cell count in both groups was significantly lower than that in the previous examples under immunization, indicating that 5T cells... IF and 5T IF 4 cells prevented local inflammatory response and the corresponding CAR-T immune response tended to decrease, but significant infiltration was still observed.

[0445] Analysis of other immune cells showed that, for example Figure 8 F and Figure 8 G, 5T IF and 5T IF4. Cell therapy significantly inhibited the infiltration of total blood cells in bronchoalveolar lavage fluid and lungs. Both significantly inhibited the infiltration of eosinophils, the main pathogenic cells, in both lavage fluid and lungs, although no statistically significant difference was shown in their inhibitory effects. IF 4 showed a more pronounced inhibitory trend. Both significantly inhibited the infiltration of T cells and B cells in the lungs. In summary, these results indicate that both cell therapies can almost completely eliminate eosinophils infiltrating the lungs.

[0446] 2) ELISA detection

[0447] The pulmonary lavage fluid obtained in step 1 was decellularized and transferred into a clean EP tube; at the same time, blood was collected from the mice, and serum was separated and the IL-13 content was detected using an ELISA kit.

[0448] The results are as follows Figure 8 The E in the text indicates that 5T IF and 5T IF All four cell lines significantly inhibited IL-13 production, and 5T cells also showed this effect. IF The inhibitory effect is more significant.

[0449] 3) Pathological staining

[0450] The lungs fixed overnight were embedded in paraffin and then sectioned and mounted. The prepared pathological sections were stained with hematoxylin and eosin (HE), and the slides were photographed under a high-power microscope. The pathological tissues were scored using a 1-4 grade pathological scoring system.

[0451] Pathological analysis results Figure 8 The D in the data indicates that, because both are very significant in killing target cells and inhibiting immune cell infiltration, the pathological staining results also show that 5T... IF and 5T IF All four cells could significantly inhibit the infiltration and distribution of immune cells in the lungs, and there was no significant difference between them.

[0452] Based on the above results, the 5T return transmission IF and 5T IF 4-cell antibodies can significantly clear pathogenic eosinophils and inhibit the infiltration of more immune cells, preventing the inflammatory response in pathological tissues. Furthermore, 5-T antibodies... IF 4 cells were more effective at inhibiting cytokines. 5T cells were reinfused. IF and 5T IF The 4-cell mice remodeled their peripheral tolerance environment, including the tolerance environment of local organs, which showed tolerance to strong stimulation of sensitizing cytokines and had an anti-inflammatory effect.

[0453] Example 8, Human h5T IF 4 cells can continuously kill target cells in NSG mice.

[0454] 1. Construction of human IL-5CAR, human gene-editing IL-5CAR vector and human IL-4 mutant protein vector.

[0455] This embodiment designed and constructed a lentivirus-based IL-5 CAR expression vector, namely pHAGE-SFFV-GFP-P2A-IL-5-CAR: the vector is derived from Addgene#117055, and the amino acid sequence of the IL-5 CAR is shown in SEQ ID NO:1 (as mentioned above, the IL-5 used in this invention can simultaneously recognize human IL-5Rα and mouse IL-5Rα). Figure 9 A in the middle.

[0456] In this embodiment, an expression vector for expressing lentiviral sgRNA, namely pHAGE-U6-sgBCOR-U6-sgZC3H12A-SFFV-GFP-P2A-IL-5-CAR (SEQ ID NO:9), was constructed based on the human IL-5CAR vector. In this vector, positions 2474-2493 are the target sequence recognition region for sgBCOR used to knock out human BCOR (SEQ ID NO:10), and positions 2915-2935 are the target sequence recognition region for sgZC3H12A used to knock out human ZC3H12A (SEQ ID NO:11). This vector is used to simultaneously knock out human BCOR and human ZC3H12A. All vectors were obtained through whole-genome synthesis.

[0457] The sgRNAs used above are shown in Table 3 below:

[0458] Table 3 Target sequence recognition regions of sgRNA

[0459] sgBCOR GCTGCCACAAGCACTCTAGG(SEQ ID NO:10) sgZC3H12A CAGGACGCTGTGGATCTCCG(SEQ ID NO:11)

[0460] In this embodiment, an expression vector for the lentivirus-based IL-4 mutant protein, namely pHAGE-SFFV-GFP-P2A-IL-4Mutant (SEQ ID NO:12), was designed and constructed. The amino acid sequence of the IL-4 mutant is shown in SEQ ID NO:7, and the amino acid sequence of the GFP is shown in SEQ ID NO:8.

[0461] 2. Isolation (resuscitation), activation, and lentiviral transduction of human PBMCs

[0462] 1) Isolation and resuscitation of human PBMCs: Human PBMCs were isolated using Ficoll lymphocyte separation medium. Peripheral blood samples were diluted 1:1 with PBS; lymphocyte separation medium was added at a 2:1 ratio; gradient centrifugation was performed at 800g for 25 minutes; after centrifugation, the intermediate layer was transferred to a new centrifuge tube, and PBS was added for washing twice; the cells were resuspended in PBS, counted, centrifuged, and then resuspended in 100% serum and stored at -80℃. The frozen human PBMCs were rapidly resuscitated at 37℃, and the cells were resuspended in human T cell culture medium and incubated at 37℃ for 6–12 hours in a CO2 incubator.

[0463] 2) Lentiviral packaging and concentration: 10 6 Lenti-X cells (ATCC#CRL-3214) were cultured adherently for 20 hours. Then, 30 μg of the expression vector prepared in step 1 above, 20 μg of packaged psPAX2 (Addgene#12260), and 10 μg of packaged pMD2.G (Addgene#12259) were co-transfected using calcium phosphate precipitation. Forty-eight hours after transfection, the supernatant containing the packaged virus, i.e., the retroviral supernatant, was harvested. The obtained supernatant was concentrated by ultracentrifugation.

[0464] 3) Activation and transduction of human T cells: Fully resuscitated human PBMCs were added to cell culture plates coated with anti-CD3 antibody and retrotronectin and activated for 24-36 hours; after activation, concentrated virus was added and the cells were left to incubate for 24-36 hours; routine culture was then performed.

[0465] 3. Building H5T via electro-transfer IF 4. Cells and Adoptive Cell Transfer

[0466] 1) Electroporation of human T cells: Cultured human T cells were washed three times with PBS; electroporation was performed at a concentration of 2.5–4 × 10⁻⁶ cells / mL. 6 For each well of electroporation reaction, add 2 μl of Cas9 mRNA (Abixin), mix well, and let stand for 2 minutes. Perform electroporation in a Lonza electroporator using the EO-115 program. After electroporation, incubate the cells in a CO2 incubator for resuscitation, then transfer them to 48-well plates for further culture. Figure 10 A.

[0467] 2) The cells will be cultured for another 48 hours, washed twice with PBS, resuspended in clean sterile PBS, and then adopted into NSG mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ; whose genetic background is NOD / ShiLtJ mice, and which have the immunodeficiency characteristics of SCID mice and IL2rgnull gene-deficient mice).

[0468] 4. Human IL-5 CAR-T cells and h5T IF 4. Identification of in vitro functional characteristics of cells

[0469] 1) Identification of the in vitro killing ability of human IL-5 CAR-T cells: First, the expression of CAR molecules in human IL-5 CAR-T cells was analyzed by FACS. The results showed that... Figure 9 The killing effect of IL-5Ra on tumor cells (human osteosarcoma cells, from ATCC) was assessed by co-incubation for 24 hours. The results showed that... Figure 9 In the study, human IL-5-based CAR-T cells effectively killed target cells. Furthermore, by varying the ratio of CAR-T cells to target cells, the killing level of CAR-T cells at different E:T ratios was detected, and the results are presented as statistical findings. Figure 9 D in the middle.

[0470] 2) Identification of the expression levels of the viral co-infection system and the IL-4 mutant protein: through expression vectors, such as... Figure 9 Two lentiviruses were constructed using the E gene. Through co-infection, both viruses were simultaneously added to the culture medium of activated human T cells. Co-infection was detected by FACS at 36 or 48 hours after static infection. The results are shown below. Figure 9 F. Cell culture supernatant was collected, and the expression level of IL-4 was detected by ELISA kit. The statistical results are shown in the figure. Figure 9 G in the middle.

[0471] 3) Detection of gene editing level: Hours before adoptive transplantation into NSG cells, a portion of cells were collected, DNA was extracted, and specific gene-edited sites were amplified using PCR. The products were purified, recovered, and sequenced. Representative gene results are shown in the following diagram to assess gene editing status. Figure 10 H

[0472] 5. h5T IF 4. In vivo functional verification of cells

[0473] 1)h5T IF 4. In vivo expansion and killing capacity assay of cells: At week 4 after adoptive transplantation, blood samples were collected and FACS was used to detect the proportion of CAR-T cells and eosinophils in peripheral blood. The results showed that compared with untransfected IL-5 CAR-T cells, h5T cells had significantly higher cytotoxicity. IF 4 cells can significantly proliferate and kill target cells, as shown in the following results. Figure 10 B, C, and D.

[0474] 2)h5T IF4. Kinetic analysis of cell proliferation and killing capacity: Blood samples were collected at weeks 2, 4, and 8 post-adoptive transplantation, and the proportions of CAR-T cells and eosinophils in peripheral blood were detected by FACS. Statistical graphs of dynamic changes were plotted, such as... Figure 10 E and F. The results showed that, starting in the second week post-transplantation, h5T IF Four CAR-T cells were sufficient to kill target cells, and this effect persisted until the experimental endpoint at week 8. The results also showed that the proportion of CAR-T cells peaked at week 4 post-transplantation and remained there until week 8. At the endpoint of week 8, mice were euthanized, serum samples were collected, and h5T levels were detected using ELISA. IF The levels of IL-4 released by 4 cells were as follows: Figure 10 G.

[0475] In addition, this embodiment also constructed a fully humanized IL-5CAR, the sequence of which is (SEQ ID NO:16):

[0476]

[0477] The single underlined portion represents IL-5 (SEQ ID NO:17); the double underlined portion represents CD28 molecule (SEQ ID NO:18); and the dotted underlined portion represents CD3zeta molecule (SEQ ID NO:19).

[0478] Simultaneously, this embodiment also constructed a fully humanized IL-4 mutant protein, the sequence of which is (SEQ ID NO:20): HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGA TAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMDEKDSKCSS*

[0479] They were placed on the same expression vector, namely pHAGE-SFFV-human IL-4mutant-P2A-humanIL-5CAR, whose sequence is SEQ ID NO:21.

[0480] The homology between SEQ ID NO:16 and SEQ ID NO:1, as well as between SEQ ID NO:20 and SEQ ID NO:7, is highly conserved, suggesting that they have the same expected therapeutic effects.

[0481] vector sequence

[0482] SEQ ID NO:2(pMSCV-hU6-sgControl-EFS-Thy1.1-P2A-IL-5-CAR)

[0483] TTCTGGGCCCTGGTGGTGGTGGCCGGCGTGCTTGTTCTGTTACGGCCTGCTGGT CACAGTGGCCCTGTGCGTGATCTGGACC

[0484] SEQ ID NO:6(pMSCV-EFS-GFP-P2A-IL-4Mutant)

[0485]

[0486] SEQ ID NO:9(pHAGE-U6-sgBCOR-U6-sgZC3H12A-SFFV-GFP-P2A-IL-5-CAR)

[0487]

[0488] SEQ ID NO:12(pHAGE-SFFV-GFP-P2A-IL-4Mutant)

[0489]

[0490] SEQ ID NO:21(pHAGE-SFFV-human IL-4 mutant-P2A-human IL-5 CAR)

[0491]

Claims

1. A recombinant immune cell, wherein, The recombinant immune cells include: (i) One or more structures for adoptive cell therapy; (ii) Gene regulatory systems capable of reducing or eliminating the expression and / or function of the BCOR gene and the ZC3H12A gene in immune cells; Wherein, the structure-specific binding antigen derived from eosinophils for adoptive cell therapy is IL-5Rα; The structure used for adoptive cell therapy is a chimeric antigen receptor; The recombinant immune cells also include: (iii) Biomolecules used to treat diseases; The biomolecule used to treat the disease is an IL-4 mutant, whose amino acid sequence is shown in SEQ ID NO:7 or SEQ ID NO:20; The gene regulation system uses gene knockout, gene silencing, or inactivation mutation techniques to treat the BCOR and ZC3H12A genes in the recombinant immune cells.

2. The recombinant immune cells according to claim 1, wherein, The chimeric antigen receptor includes: (a) A polypeptide derived from IL-5; (b) Peptides derived from CD28; and, (c) Peptides derived from CD3zeta.

3. The recombinant immune cells according to claim 2, wherein, The amino acid sequence of the IL-5-derived polypeptide is selected from: SEQ ID NO:13 or SEQ ID NO:17; and / or, The amino acid sequence of the CD28-derived polypeptide is selected from: SEQ ID NO:14 or SEQ ID NO:18; and / or, The amino acid sequence of the polypeptide derived from CD3zeta is selected from SEQ ID NO:15 or SEQ ID NO:

19.

4. The recombinant immune cells according to claim 2 or 3, wherein, The chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

16.

5. The recombinant immune cells according to any one of claims 1 to 3, wherein, The immune cells mentioned are derived from mammalian immune cells.

6. The recombinant immune cells according to claim 5, wherein, The immune cells are selected from one or more of T cells, B cells, NK cells, mast cells, and tumor-infiltrating lymphocytes.

7. The recombinant immune cells according to claim 5, wherein, The immune cells mentioned are selected from T cells or NK cells.

8. The recombinant immune cells according to claim 6, wherein, The T cells mentioned are selected from CD4. + CD8 + T cells, CD8 + T cells, CD4 + T cells, effector T cells, suppressor T cells, primitive T cells, memory T cells, γ-δ T cells, α-β T cells, CD4+ - CD8 - One or more of double-negative T cells or NKT cells.

9. A composition comprising recombinant immune cells as described in any one of claims 1 to 8.

10. The composition of claim 9, wherein the composition comprises a pharmaceutically acceptable carrier.

11. Use of the recombinant immune cells as described in any one of claims 1 to 8 in the preparation of a medicament for treating and / or preventing a disease or condition; in, The disease or condition is selected from inflammatory or allergic diseases mediated by type 2 immune response, and diseases in which eosinophils are effector cells.

12. The use according to claim 11, wherein, The type 2 immune response-mediated inflammatory or allergic diseases include one or more of the following: asthma, allergic rhinitis, inflammatory skin diseases, and food allergies.

13. The use according to claim 11, wherein, Diseases that use eosinophils as effector cells include one or more of the following: acute and chronic asthma, eosinophilia, eosinophil-induced nasal polyps, eosinophil-induced enteritis, eosinophilic dermatitis, chronic obstructive pulmonary disease, and eosinophilic leukemia.

14. Use of the recombinant immune cells as described in any one of claims 1 to 8 in the preparation of a carrier for delivering biomolecules for treating diseases.

15. A method for preparing recombinant immune cells according to any one of claims 1 to 8, comprising: (i) The step of introducing a structure for adoptive cell therapy into immune cells; and, (ii) The steps for introducing gene regulatory systems into immune cells; as well as, (iii) Steps for introducing biomolecules for treating diseases into immune cells.

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